Leonard Amaral

Abstract: In this Letter, we describe a small plasmid carried by a methicillin-resistant Staphylococcus aureus strain of clinical origin, which carries the determinant for the efflux pump Smr and demonstrate the role played by this plasmid and the Smr pump it encodes on the resistance showed by this MRSA strain to quaternary ammonium compounds, including several biocides used in the clinical practice. We believe the findings described in this Letter strengthen the importance of plasmidencoded efflux pumps in the maintenance of S. aureus strains in the hospital environment and the importance of improving our knowledge on these elements.

Abstract: Resistance mediated by efflux has been recognized in Staphylococcus aureus in the last decades, although its clinical relevance is only recognized recently. The existence of only few studies on the individual and overall contribution of efflux to resistance phenotypes associated with a need of well established methods to assess efflux activity in clinical isolates contributes greatly to the lack of solid knowledge of this mechanism in S. aureus. This study aims to provide information on approaches useful to the assessment and characterization of efflux activity as well as contributing to our understanding of the role of efflux to phenotypes of antibiotic resistance and biocide tolerance in S. aureus clinical isolates. The results described show that efflux is an important contributor to fluoroquinolone resistance in S. aureus and suggest it as a major mechanism in the early stages of resistance development. We also show that efflux plays an important role on the reduced susceptibility to biocides in S. aureus, strengthening the importance of this so-long neglected resistance mechanism to the persistence and proliferation of antibiotic/biocide-resistant S. aureus in the hospital environment.

Abstract: The use of light-activated bactericidal agents � photobactericides � is suggested in local infection in order to conserve conventional antibacterials for more systemic disease. Local administration of a photobactericide such as methylene blue coupled with locally-targeted red light illumination ensures a rapid and localised antibacterial response, regardless of the conventional resistance status. Mechanisms of photobactericidal uptake and action are discussed.

Abstract: Phenothiazines have their primary effects on the plasma membranes of prokaryotes and eukaryotes. Among the components of the prokaryotic plasma membrane affected are efflux pumps, their energy sources, energy providing enzymes, such as ATPase and genes that regulate and code for the permeability aspect of a bacterium. The response of multidrug and extensively drug resistant tuberculosis to phenothiazines shows an alternative therapy for treatment of these dreaded diseases that is claiming more and more lives every year throughout the world. Many phenothiazines have shown synergistic activity with several antibiotics thereby lowering the doses of antibiotics administered to patients suffering from specific bacterial infections. Trimeprazine is synergistic with trimethoprim. Fp has been found to be synergistic with penicillin and CPZ plus some antibiotics are also synergistic. Along with antibacterial action described in this review, many phenothiazines possess plasmid curing activities which render the bacterial carrier of the plasmid sensitive to antibiotics. Thus simultaneous applications of a phenothiazine like TZ would not only act as an additional antibacterial agent but also would help to eliminate drug resistant plasmid from the infectious bacterial cells.

Abstract: The emergence of infections caused by multi- or pan-resistant bacteria in the hospital or in the community settings is an increasing health concern. Albeit there is no single resistance mechanism behind multiresistance, multidrug efflux pumps, proteins that cells use to detoxify from noxious compounds, seem to play a key role in the emergence of these multidrug resistant (MDR) bacteria. During the last decades, experimental data has established their contribution to low level resistance to antimicrobials in bacteria and their potential role in the appearance of MDR phenotypes, by the extrusion of multiple, unrelated compounds. Recent studies suggest that efflux pumps may be used by the cell as a first-line defense mechanism, avoiding the drug to reach lethal concentrations, until a stable, more efficient alteration occurs, that allows survival in the presence of that agent. In this paper we review the current knowledge on MDR efflux pumps and their intricate regulatory network in Staphylococcus aureus, a major pathogen, responsible from mild to life-threatening infections. Particular emphasis will be given to the potential role that S. aureus MDR efflux pumps, either chromosomal or plasmid-encoded, have on resistance towards different antimicrobial agents and on the selection of drug - resistant strains. We will also discuss the many questions that still remain on the role of each specific efflux pump and the need to establish appropriate methodological approaches to address all these questions.

Abstract: Chlorpromazine (CPZ) was exposed to a 266 nm laser beam for varying periods of time that ranged from minutes to 24 hrs. At intervals, the products from irradiation were evaluated by thin layer chromatography (TLC) and evaluated for activity against mycobacteria of human interest (Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intercellare and their corresponding reference strains or clinical isolate. With the exception of the M. avium 47/07 clinical strain, the products produced from the irradiation of CPZ for 4 hrs had greater activity against M. intracellulare ATCC, M. avium ATCC, H37Rv and the MDR TB strains as opposed to that produced by the un-irradiated control CPZ. The level of activity after the 4 hr exposure of CPZ remained the same throughout the next 20 hrs of irradiation of CPZ. Of significant note is that the products of irradiation of CPZ by the 266 nm laser beam expressed lower in vitro cytotoxicity against human cells suggesting that the approach used may be useful for the development of more bioactive compounds than that expressed by the parental species.

Abstract: Cancer grows at the expense of the host as a parasite or superprasite following the second law of thermodynamics (conservation of energy). When the cancer cell progresses via replication to the special state called �spheroid�, a new phase begins with its intimiate interaction and development of responses from the stroma which together assist in the formation of a full blown cancer. Among the processes involved are the development of blood vessels and lymphatic channels which are essential for maintenance and further growth of the cancer mass. In this way the condition of �parasitism� is completed with simultaneous suppression of the immune response of the host to the histoincompatability of the tumor mass. Stroma/parenchyma promotes cancer invasion by feeding cancer cells and inducing immunetolerance. The dynamic changes in composition of stroma and biological consequences as feeder of cancer cells and immuntolerance can give a perspective for rational drug design in anti-stromal therapy. There are differences between normal and cancer cells at subcellular level such as compartmentalzation and structure of cytoskeleton and energy distribution ( that is low generally, but locally high in normal cells),. In cancer cannibalism of normal cells, the growing cancer mass is a factor for progression and invasion. Cancer cells have been shown to kill normal cells and the products of cell death used for progression of growth of the cancer cell. Serum and growth factors produced by tumor stroma also provide the needed nutrients and conditions for further tumor growth. Cancer cannot feed off other cancer cells and therefore grow poorly. Probably, although not yet proven, the inability of cancer to �parasitise� other cancer cell types is probably due to some kind of competition or interference. The tumor is in charge of its own development due to its induction proteinases, lipid mobilization factors and angiogenetic factors as well as its ability to negate immune responses of the host response to what is in essence a foreign body. In our review co-existence of normal and cancer cells in tumor with the growth promoting factors, and the immuntolerance mediating factors produced in the stromal and cancer cells/tissues will be discussed with perspective of the stroma targeted therapy.

Abstract: Introduction: Therapy with some phenothiazines such as chlorpromazine produces phototoxicity. It was thought that phototoxicity was produced by exposure of the subcutaneous localized phenothiazine to light which generated toxic substances. Phenothiazines when exposed to white light or to wavelengths in the UV spectral range are known to undergo a variety of reactions that result in the degradation of the parental compound and the formation of new species. This process of �photolysis� is slow and may be sped up with exposure to high energy light such as that produced by a laser. Indeed, exposure of the phenothiazine chlorpromazine (CPZ) to �flash photolysis� yields in nanoseconds products which have been deduced to be sulfoxide radicals. Our prior studies showed that exposure of CPZ to a pulsed laser beam yielded consistent changes in the molecule and that the bioactivity of the products of irradiation was increased. Methods: Varying concentrations of CPZ ranging from 2 to 20 mg/L were exposed to a 266 nm laser beam for varying periods of time that ranged from 1 to 24 hrs. At distinct intervals the products of irradiation were evaluated by spectrophometry in the spectral range 200 to 1500 nm, by thin layer chromatography (TLC), by High Pressure Liquid Chromatograpy - Diode Array Detection (HPLC-DAD) and for activity against the CPZ sensitive test organism ATCC 25923 Staphylococcus aureus. Results. Exposure of CPZ to the 266 nm laser beam of given energy level yielded species derived from the CPZ whose number increased with duration of exposure. Although the major species produced were Promazine (PZ), hydroxypromazine or PZ sulfoxide, and CPZ sulfoxide, over two hundred compounds were generated with exposure of 20 mg/L of CPZ for 24 hrs. Evaluation of the products of irradiation indicated that the bioactivity against the test organism increased despite the total disappearance of CPZ. Because PZ has far less activity against the test organism than CPZ, the increased activity noted by laser exposed CPZ is due, most probably, to one or more new species that as of yet remain unidentified. Conclusions. Exposure of a biologically active phenothiazine such as CPZ to a relatively high energy (6.5mJ) 266 nm laser beam yields very rapidly a large number of new and stable species. The process is reproducible and because the concentration of compound to be exposed, the wavelength of the laser beam, its energy level, and the amount of time of exposure are variables that can be controlled, the end point of the process, namely, the production of new species and their number can be readily reproduced. Because the process is �clean� and rapid, it may offer certain advantages over the pyrogenically based methods for the production of derivatives. Lastly, the method offers opportunities to modify a parental compound and evaluate the product for any changes in its biological activity.

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Abstract: Morpholino-disiloxane (ALIS-409) and piperazinodisiloxane (ALIS-421) compounds were developed as inhibitors of multidrug resistance of various types of cancer cell. In the present study, the effect of ALIS-409 and ALIS-421 compounds were investigated on cancer promotion and on co-existence of tumor and normal cells. The two compounds were evaluated for their inhibitory effects on Epstein-Barr virus immediate early antigen (EBV-EA) expression induced by tetradecanoylphorbol- acetate (TPA) in Raji cell cultures. The method is known as a primary screening test for antitumor effect, below the (IC50) concentration. ALIS-409 was more effective at inhibition of EBV-EA at 100 μg/ml and tumor promotion than ALIS-421 in the concentration range up to 1000 μg/ml. However, neither of the compounds was able to reduce tumor promotion significantly, expressed as inhibition of TPA-induced tumor antigen activation. Based on the in vitro results, the two disiloxanes were investigated in vivo for their effects on mouse skin tumors in a two-stage mouse skin carcinogenesis study. The application of dimethyl-benzanthracene (DMBA; 390 nmol) as tumor initiator was followed by exposure to TPA (1.7 nmol/l) as tumor promoter. The experiments showed that ALIS-409 at a concentration of 85 nmol/l had a weak EBV-EA inhibitory effect in vitro and a moderate antitumor activity, compared to the positive control of DMBA plus TPA-treated mice. Flow cytometry by differential staining demonstrated interactions in co-cultures of MCF7 breast cancer and MRC5 human lung fibroblasts. The growth rate of tumor cells in mixed population of MCF7 breast cancer and MRC5 normal fibroblasts cells was reduced in the presence of ALIS-409, as compared to the control non-treated cell populations. The two disiloxanes were moderately effective in chemoprevention in DMBA induced and TPA-promoted in vivo tumor formation. Authors suggest that the inhibition of tumor cell and fibroblast interaction by ALIS409 might has some perspective in the development of anti-stromal therapy.

Abstract: Varying concentrations of Chlorpromazine Hydrochloride (CPZ) were exposed to a 266 nm laser beam for varying periods of time ranging from 4 to 24 hrs and the products of irradiation were evaluated for activity against a panel of bacteria that consisted of representatives of Gram-positives and Gram-negatives that expressed different degrees of efflux pump activity, and compared to the parental unexposed compound. With prolonged irradiation, whereas the antibacterial activity of the product against Staphylococcus aureus and Escherichia coli strains was many folds greater, no activity against their efflux pumps was noted. The activity of the products of irradiation against Salmonella enterica serovar Enteritidis was slight. However, the products of prolonged irradiation of CPZ produced increasingly significant concentration dependent inhibition of efflux by the salmonella strains.

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Abstract: It is known that bacteria showing a multi-drug resistance phenotype use several mechanisms to overcome the action of antibiotics. As a result, this phenotype can be a result of several mechanisms or a combination of thereof. The main mechanisms of antibiotic resistance are: mutations in target genes (such as DNA gyrase and topoisomerase IV); over-expression of efflux pumps; changes in the cell envelope; down regulation of membrane porins, and modified lipopolysaccharide component of the outer cell membrane (in the case of Gram-negative bacteria). In addition, adaptation to the environment, such as quorum sensing and biofilm formation can also contribute to bacterial persistence. Due to the rapid emergence and spread of bacterial isolates showing resistance to several classes of antibiotics, methods that can rapidly and efficiently identify isolates whose resistance is due to active efflux have been developed. However, there is still a need for faster and more accurate methodologies. Conventional methods that evaluate bacterial efflux pump activity in liquid systems are available. However, these methods usually use common efflux pump substrates, such as ethidium bromide or radioactive antibiotics and therefore, require specialized instrumentation, which is not available in all laboratories. In this review, we will report the results obtained with the Ethidium Bromide-agar Cartwheel method. This is an easy, instrument-free, agar based method that has been modified to afford the simultaneous evaluation of as many as twelve bacterial strains. Due to its simplicity it can be applied to large collections of bacteria to rapidly screen for multi-drug resistant isolates that show an over-expression of their efflux systems. The principle of the method is simple and relies on the ability of the bacteria to expel a fluorescent molecule that is substrate for most efflux pumps, ethidium bromide. In this approach, the higher the concentration of ethidium bromide required to produce fluorescence of the bacterial mass, the greater the efflux capacity of the bacterial cells. We have tested and applied this method to a large number of Gram-positive and Gram-negative bacteria to detect efflux activity among these multi-drug resistant isolates. The presumptive efflux activity detected by the Ethidium Bromide-agar Cartwheel method, was subsequently confirmed by the determination of the minimum inhibitory concentration for several antibiotics in the presence and absence of known efflux pump inhibitors.

Abstract: Organosilicon compounds are efflux pump inhibitors (EPIs) with potency as anti-tuberculosis drugs. Of the organosilicon compounds tested, SILA 421 has the highest potency as an anti-tuberculosis drug [1]. It shares common pathways for antimycobacterial killing with other EPIs: it revealed direct in vitro activity against Mycobacterium tuberculosis [1], has been shown to modify resistance by inhibiting mdr-1 efflux pumps and has been shown to enhance killing of M. tuberculosis by macrophages [1]. However, the activity of SILA 421 has only been tested against the M. tuberculosis H37Rv type strain and one clinical extensively drug resistant M. tuberculosis isolate so far [1]. We therefore assessed the antimycobacterial activity of SILA 421 against an extended set of M. tuberculosis strains with varying drug susceptibility profiles. Second, we compared the antimycobacterial activity of SILA 421 with that of thioridazine and other phenothiazines.

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Abstract: Bacteria have the capacity, as all living cells, to escape harm from a noxious agent by extruding the agent before it reaches its target and harms to the cell. This initial response is intrinsic and involves plasma membrane bound efflux pumps which have the capacity to recognise and extrude a large variety of structurally unrelated molecules. When the concentration of the agent is progressively increased, the number of efflux pumps is also progressively increased as a consequence of over-expression of genes that regulate and code for the synthesis of these pumps. Often, when the bacterium is transferred to drug free medium, the number of pump units returns to baseline levels. However, when the concentration of agent is maintained over a prolonged period of time, mutations in genes that code for essential proteins which are usual targets of antibiotics, begin to accumulate and the expression of genes that code for the efflux pumps decreases, oftentimes reaching wild type levels. When the patient is treated with an antibiotic over a prolonged period of time and initial therapy is ineffective, the bacterium most likely escapes via its own intrinsic efflux pump system and with time, it is expected that the number of efflux pump units progressively increase rendering the clinical isolate resistant to the given antibiotic. The amount of energy needed to maintain a high level of efflux pump activity is great and at the expense of other activities that are needed for survival and replication. When this point is reached, and following the second law of thermodynamics, the bacterium goes through changes to survive at low energy costs (for example: switches its mutator gene and a number of predicted mutations of essential proteins take place). These changes render the bacterium permanently resistant to given antibiotics, the need for efflux is no longer and the number of efflux pump units returns to baseline levels. This review will discuss the structure, genetic regulation, physiology of efflux pumps and the means by which a clinical isolate can be characterized for the components that contribute to its resistance during therapy, namely, evaluation of efflux pump activity versus mutations. The ability of a laboratory to perform these evaluations will go a long way toward the selection of effective antibiotic therapy on a real-basis.

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Abstract: Objective. Evaluate a new series of 16 hydantoin derivatives for activity against the intrinsic and over-expressed efflux pumps of the ATTC 25923 Staphhylococcus aureus and the clinical Staphhylococcus aureus HPV-107 strain, respectively.Methods. The hydantoin compounds evaluated for activity against the efflux pumps of the ATTC 25923 Staphhylococcus aureus and the clinical Staphhylococcus aureus HPV-107 strains by the aid of the automated ethidium bromide method. Compounds that inhibited the efflux pumps of either strain were evaluated for ability to reduce or reverse resistance of these strains to oxacillin. Results. Although most of the hyandoins inhibited the efflux pumps of the ATTC strain, none reduced the resistance of this strain to oxacillin. In contrast, although the inhibition of the Qac efflux pump present in HPV-107 could be inhibited to some degree by much higher concentrations of the hydantoin compounds than that needed for similar activity against the ATTC strain, only hydantoin PI8a significantly reduced the MIC of oxacillin against the HPV-107 strain. Conclusions. Hydantoin compound PI8a may have a potential for therapy of an MRSA infection whose multi-drug resistant phenotype is due to the presence of an over-expressed efflux pump.

Abstract: A series of new arylidene imidazolone derivatives of hydroxyethylpiperazine 3-9 (Fig. 2) were investigated for their cancer efflux pump (PgP) inhibitor properties. New compounds were synthesized and were tested for their efflux modulating effects on T-lymphoma cancer cells using fluorescence activated cell sorting and real-time fluorimetry. The modulation of intracellular drug accumulation was evaluated by flow cytometry using rhodamine 123 accumulation assay and structure-activity relationship defined. Compounds 3-9 displayed weak cancer efflux pump inhibitors properties. (Z)-5-(2,4-dichlorobenzylidene)-2-(4-(2-hydroxyethyl)piperazin-1-yl)-1H-imidazol-4(5H)-one hydrochloride (7) was the most promising one which displayed inhibitory activity against Pgp1 on a concentration dependent mannner. Results of SAR-studies indicated that hydrophobic aromatic moieties (Ar) were the most favourable features for cancer EPIs activity, whereas hydrophilic hydroxyl groups (HYL) caused a significant decrease of the activity.

Abstract: Previously, the antipsychotic, non-antibiotic compound flupenthixol dihydrochloride (Fp) was shown to exhibit distinct in vitro antibacterial activity against 14 Gram-positive and Gram-negative bacteria and to significantly protect Swiss albino mice challenged with a known mouse virulent salmonella. The present study was designed to ascertain whether this drug could efficiently augment the action of an antibiotic or a non-antibiotic when tested in combination. A total of 12 bacterial strains belonging to various genera were selected for this study and were sensitive to the antibiotics penicillin (Pc), ampicillin, chloramphenicol, tetracycline, streptomycin, gentamicin, erythromycin, ciprofloxacin, and to the non-antibiotics methdilazine, triflupromazine, promethazine, and Fp. Pronounced and statistically significant synergism (p<0.01) was observed when Fp was combined with Pc following the disc diffusion assay system. With the help of the checkerboard method, the fractional inhibitory concentration (FIC) index of this pair was found to be 0.375, confirming synergism. This pair of Fp+ Pc was then subjected to in vivo experiments in mice challenged with Salmonella enterica serovar Typhimurium NCTC 74. Statistical analysis of the mouse protection test suggested that this combination was highly synergistic (p<0.001, Chi-squared analysis). Fp also revealed augmentation of its antimicrobial property when combined with streptomycin, gentamicin, ciprofloxacin, and the non-antibiotic methdilazine. The results of this study may provide alternatives for the therapy of problematic infections such as those associated with Salmonella spp.

Abstract: Multidrug resistant (MDR) tuberculosis is caused by Mycobacterium tuberculosis resistant to isoniazid and rifampicin, the two most effective drugs used in tuberculosis therapy. Here, we investigated the mechanism by which resistance towards isoniazid develops and how overexpression of efflux pumps favors accumulation of mutations in isoniaz id targets, thus establishing a MDR phenotype. The study was based on the in vitro induction of an isoniazid resistant phenotype by prolonged serial exposure of M. tuberculosis strains to the critical concentration of isoniazid employed for determination of drug susceptibility testing in clinical isolates. Results show that susceptible and rifampicin monoresistant strains exposed to this concentration become resistant to isoniazid after three weeks; and that resistance observed for the majority of these strains could be reduced by means of efflux pumps inhibitors. RT-qPCR assessment of efflux pump genes expression showed overexpression of all tested genes. Enhanced real-time efflux of ethidium bromide, a common efflux pump substrate, was also observed, showing a clear relation between overexpression of the genes and increased efflux pump function. Further exposure to isoniazid resulted in the selection and stabilization of spontaneous mutations and deletions in the katG gene along with sustained increased efflux activity. Together, results demonstrate the relevance of efflux pumps as one of the factors of isoniazid resistance in M. tuberculosis. These results support the hypothesis that activity of efflux pumps allows the maintenance of an isoniazid resistant population in a sub-optimally treated patient from which isoniazid genetically resistant mutants emerge. Therefore, the use of inhibitors of efflux should be considered in the development of new therapeutic strategies for preventing the emergence of MDR-TB during treatment.

Abstract: Quorum Sensing (QS) systems of bacteria consist of a producer of the QS signal and the responder. The generation of a QS signal provides the means by which a population can behave in a concerted manner such as swarming, swimming and secretion of biofilm, etc.. Because concerted bahaviour bestows protection to the bacterial species, and hence factors involved in the severity of an infection such as virulence are products of QS systems, compounds that inhibit the QS system have significant clinical relevance. Recent evidence suggests that the secretion of QS signals takes place via the efflux pump system of the producer of the signal. Interestingly, compounds such as phenothiazines and trifluoromethyl ketones (TFs) that inhibit proton motive force (PMF) activities such as swarming and swimming also inhibit the PMF dependent efflux pump systems of bacteria and their QS systems. This review discusses the relationship between the efflux pump, the QS system and the compounds that affect both. Lastly, suggestions are made regarding classes of compounds that have been shown to inhibit PMF dependent efflux pumps and the need to evaluate them for QS inhibitory properties.

Abstract: Background: Protoapigenone (PA), a natural flavonoid possessing an unusual p-quinol moiety on its B ring, is a prospective novel lead compound against cancer currently in development, together with WYC0209, a potent synthetic PA analog. Structure activity relationships (SAR) concerning different 1�-O-alkyl side-chains were also studied on two sets of derivatives. Materials and Methods: Fifteen 1�-O-alkyl protoflavone derivatives were synthesized from genkwanin or 4�-hydroxy-6-methylflavone, thirteen of which are new compounds. All compounds were tested for their cytotoxic effect on four human cancer cell lines, such as HepG2 and Hep3B (hepatic), A549 (lung) and MDA-MB-231 (breast) cell lines, with doxorubicin as a positive control. All compounds, as well as PA, WYC0209 and fourteen of their previously reported analogs were also tested on a multidrug resistant (MDR) sub-cell line of L5178 mouse T-cell lymphoma and on its parental counterpart (PAR). Results: In general, derivatives bearing a free hydroxyl group at C-1� exerted the strongest activities, while C-1� substituted compounds were found to be much weaker. Derivatives of 6-methylflavone exhibited mild, but statistically significant selectivity towards the MDR cell line. Conclusion: The results are in agreement with our previous findings for fundamental SAR of protoflavones. 6-Methylated protoflavones may serve as valuable leads for developing selective compounds against MDR cancer. Identical activity of other derivatives on the PAR and MDR cell lines suggests that cancer cells cannot exhibit resistance to protoflavones by ABCB1 efflux pump overexpression.

Abstract: Tuberculosis is one of the major causes of infection globally. The emergence of multi, extensively and totally-drug resistant strains contributes to the lack of therapeutic options available. The mechanisms associated with this resistance could involve mutations of several gene targets, decreased permeability, increased efflux, etc. Resistance-mediated by efflux systems has become more relevant since these systems help the bacteria to extrude antibiotics, until relevant mutations emerge and establish in the population. Therefore, compounds that inhibit these transport systems are of major importance and have been studied in the last few years. Not only these compounds act on the bacterial efflux systems but they have also been explored for their dual role as boosters of the macrophage infected cells. The search for novel compounds or combinations of adjuvant compounds and antibiotics to treat mycobacterial multi-drug resistant infections has become a major goal in the treatment of these diseases.

Abstract: This mini-review provides the entire experimental history of the development of the old neuroleptic thioridazine (TZ) for therapy of antibiotic resistant pulmonary tuberculosis infections. TZ is effective when used in combination with antibiotics to which the initial Mycobacterium tuberculosis was resistant. Under proper cardiac evaluation procedures, the use of TZ is safe and does not produce known cardiopathy such as prolongation of QT interval. Because TZ is cheap, it should be considered for therapy of XDR and TDR-Mtb patients.

Abstract: Thioridazine (TZ)-induced accumulation of the universal efflux pump substrate ethidium bromide and its subsequent efflux by Salmonella strains with various degrees of overexpressed efflux pumps takes place automatically at pH 7.4, is independent of a metabolic source, is not affected by a proton ionophore and is precluded by palmitic acid. Salmonella enterica serotype Enteritidis cultured in medium containing increasing concentrations of TZ does not grow during the first 6�8 h, after which time its growth is similar to unexposed controls. At the end of a 16-h exposure period, the organism is resistant to >250 mg/L TZ. Parallel assessment by real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) of the activity of genes that regulate and code for the AcrB transporter of the main efflux pump (AcrAB) of the organism at periodic intervals suggests a sequence of activation beginning with the stress gene soxS, followed by the global regulator ramA, then by the local regulator marA and then by the transporter acrB. These activations take place during the period of no growth. By the end of a 16-h culture period, only the acrB transporter gene is still highly overexpressed. Assessment of the activity of genes of the twocomponent regulon PmrA/B indicates that TZ also activates this regulon. Because activation of pmrA/B also activates acrB, development of high resistance to TZ during a 16-h culture period is in part due to activation of the two-component regulon.

Abstract: Background: Multidrug resistance (MDR) is one of the major concerns in treatment of cancer and one of the major causes of its failure. The over-expression of an ABC transporter, the ABCB1, is often associated with MDR in cancer. Previously it was observed that hydantoin compounds can modulate the activity of the ABCB1 pump. Methods: Fourteen hydantoin derivatives were synthetized and studied for their capacity to increase accumulation of ethidium bromide (EB) by mouse lymphoma cancer cells that were transfected with the human mdr1 gene and over-express the human ABCB1 pump. Results: It was observed that the accumulation of EB by the cells in presence of four of the newly synthetized hydantoins is strongly increased. Similar but milder effects were also observed for the other seven hydantoins and the remaining three had no activity. Conclusion: The 14 hydantoin compounds studied belong to 3 different structural groups. Structure activity relationships were studied and important molecule substituents that were possibly responsible for increased the activity of the molecules were identified. This important information may lead to the continuation of our work and to the future synthesis of more active compounds.

Abstract: The antimicrobial action of a number of non-antibiotic drugs has been demonstrated in recent years, for example the antimycobacterial activity associated with the phenothiazine neuroleptic class. However, as with other classes of non-antibiotic, such activity may be traced back to common precursor structures such as the phenothiazinium dye, methylene blue. Such relationships may be traced for synthetic antimicrobial agents such as the sulphonamides, the antimalarial quinolines and the quinolones. While the rising tide of antimicrobial drug resistance has nullified the effects of many conventional agents, several of the original lead compounds and their non-antibiotic derivatives have a new part to play in the chemotherapy of infectious disease.

Abstract: Gram-negative bacteria that live in the wild are equipped with two mechanisms that allow them to survive in an environment that contains a noxious agent, provided that the concentration of this agent is below that which kills the organism. When a given Gram-negative bacterium infects a human and that human is treated ineffectively with an antibiotic or antimicrobial agent, the bacterium can rapidly invoke the two mechanisms that render it resistant to the therapeutic agent as well as to other non-related therapeutic agents. The two mechanisms that render the bacterium multidrug resistant (MDR) are the downregulation of porins (Viveiros et al., 2007; Davin- Regli et al., 2008) and upregulation of efflux pumps (Viveiros et al., 2007; Gootz, 2010). Porins are tribarrel structures that traverse the cell envelope and permit the penetration of hydrophilic compounds (nutrients) as well as hydrophilic antibiotics and antimicrobial agents from the environment to the cytoplasm (Bolla et al., 2004; Kumar et al., 2010). Efflux pumps are structures that traverse the cell envelope and recognize structurally unrelated noxious compounds, including antibiotics or antimicrobial agents, that have penetrated into the periplasm or cytoplasm, and extrude these agents to the outside of the cell before they reach their intended targets (Blair and Piddock, 2009; Husain and Nikaido, 2010). In this chapter, porins will not be further discussed. Instead, we will focus on AcrAB�TolC, the main efflux pump of the pathogenic Gram-negative bacteria Escherichia coli, Salmonella sp. and Enterobacter aerogenes (Bolla et al., 2004), describing its structure and function and the effects that phenothiazines have on the genes that regulate and code for the AcrAB�TolC efflux pump and on the activity of the pump itself.

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Abstract: Bacterial infections and cancer continue to increase their resistance to chemotherapeutics as a consequence of therapy. Whenever studied, refractory response to chemotherapy is due to the over-expression of efflux pumps that render the bacterium or cancer cell resistant not only to the agent used for therapy, but to many, if not all other agents as well. Control over the efflux pump that bestows multi-drug resistance has been a goal during the past decade. As a consequence of this search for inhibitors of efflux pumps, it has been noted that many agents which affect the efflux pump system of bacteria also have similar activity against efflux pumps of drug resistant cancer cells. This review aims to identify such agents.

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Abstract: Background. Photochemical transformation of certain bioactive compounds for the purpose of obtaining derivatives with increased bioactivity is a prospective area of synthetic chemistry. Ecdysteroids, analogs of the insect molting hormone, which can also exert several beneficial effects in mammals including humans, express an enone moiety in their B ring, and, as such, are good candidates for photochemical transformations. Method. 20-hydroxyecdysone (20E), the most common ecdysteroid in Nature, and the easy to obtain derivative 20-hydroxyecdysone 2,3;20,22-diacetonide (20ED), were exposed to a 266 nm laser beam at an energy level of 6.5 mJ at varying concentrations for varying periods of time and evaluated for fluorescence emission during the process of irradiation. The products of irradiation were scanned from 200 to 1500 nm and then subjected to one dimension and two-dimension thin layer chromatography (TLC). Results. During irradiation, progressive significant changes in the fluorescence emission spectra were noted for both compounds with time that were accompanied with changes in their UV-Vis scans. Full conversion of both compounds was reached within 14 minutes, and both compounds yielded a few major products and several minor ones representing a wide polarity range. Conclusions. The photo-transformation system described here was proven to be a useful and flexibly adjustable tool for the laser-catalyzed conversion of bioactive compounds. Due to the MDR reversal activity of the less polar ecdysteroids, several new products are promising for being tested against various cancer cell lines. Fractionation, isolation and characterization of the irradiated products are currently in process.

Abstract: The recently released WHO global Tuberculosis (TB) report, pictures an optimistic view on the developments in TB control: the incidence of new cases has shown a decrease for several years now, and fell in the period 2010/2011 with 2.2%. The TB mortality rate has decreased with 41% since 1990, and the world is on track to meet the target of a 50% reduction in the global incidence, by the year 2015. Mortality and incidence rates are currently falling in all six WHO region,s and in most of the 22 high-burden countries that account for over 80% of the world�s TB cases [1]. Nevertheless in 2011, there were still an estimated 8.7 million new cases of TB, of which 13% co-infected with HIV and 1.4 million people died from TB. But, this was less than previously expected by the WHO. The development of the Multi-drug Resistant TB (MDR-TB) problem, however, shows little progress in comparison to the previous report [2], and moreover, the currently published rates may only be the tip of the iceberg, as drug resistance testing is still rare in many high prevalence settings. In any case, the WHO report shows that the level of resistance in MDR-TB cases has increased significantly since the last worldwide survey, primarily in Eastern Europe, Central Asia, China and India, where 9- 32% of all new cases of pulmonary Tuberculosis are resistant to, at least, INH and rifampicin. Comparison of this data to the previous WHO report even indicates a 50% increase in the overall notification of MDR-TB. Strikingly, in the report nothing was mentioned on the occurrence of Extensively Drug Resistance (XDR-TB), or on the ill-defined Totally Drug Resistant TB (TDR-TB). Therefore, the increase in the level of resistance in MDR-TB cases so far remains hidden. Although, it is clear from the WHO 2012 report that some progress has been made, with respect to worldwide overall incidence rates and mortality, effective therapy in MDR-TB cases is still a rarity in most hot spot regions, and restricted to efficient Stop TB and The TB Alliance programmes. Even more alarming is the situation in particular large countries that contribute significantly to the worldwide MDR-TB notification, like India. The latter country, together with China and the Former Soviet Union States, account for almost 70% of the MDR-TB cases that occur wordwide annually. The WHO report estimated that approximately 64,000 cases (44,000 - 84,000) of MDR-TB emerge annually among the pulmonary TB cases in India, and although this in absolute terms represents a high number of cases, in comparison to the total load of TB in this huge country, it may give the comfortable impression that TB control is on the right track. The report also indicates on the basis of drug resistance surveys in the states of Gujarat, Maharashtra, and Andhra Pradesh that the estimated prevalence of MDR-TB in India was 2-3% in new cases, and 12-17% in retreatment cases. Moreover, the report states the prevalence of MDR-TB is not increasing in India. However, it is conceivable that the problem of MDR-TB in India is much larger than assumed, and in fact, may be overlooked because it is doubtful whether the official Indian MDR-TB data are representative of the country. First of all, the WHO data is based on a very small sample of sentinel centers where program performance presumably exceeds those in routine locations, by far. Moreover, there is an important political factor influencing the low estimate of the MDR-TB problem in India; the figure of 3% MDR-TB among new cases, and 15% in retreatment cases have almost become part of a policy dogma. Many physicians actually involved in treatment of MDR-TB would beg to work in a situation with such a low prevalence of MDR-TB situation in their setting. In reality, most feel overwhelmed by a huge burden of complicated MDR-TB cases. Harper rightly questioned the reliability of the current figures in India and referred to the term �statistico-tuberculosis�. The Indian program and the politicians welcome the theoretical low prevalence figures because of the �feel good effect�, as they comply with �manageable� global levels. Any deviation from these levels is therefore, dismissed as unrealistic. Data on MDR prevalence from other regions in India, not included in the WHO estimations, reveals completely different figures, with MDR-TB prevalence rates ranging from 20-50% in new and retreated patients. At our institution, a tertiary TB hospital with a modern laboratory and located in the metropol of Mumbai, as many as 30% of the new cases, and 60% of the retreated cases, are currently labeled as MDR-TB. This true and valid data are understandably critiqued as unrepresentative, due to the purposive sampling of patients from non-responders accessing our referral hospital. However, already the fact that even a single center routinely sees MDR-TB rates of such magnitude belies the official data that the MDR-TB problem is under control in India. A recent study by D�souza et al. [3], was the first representative study, attempting to determine the true prevalence of MDR-TB in Mumbai. She selected four centrally located TB wards in Mumbai, covering a total of 38 DOTS centers and a three million population, to establish the MDR rates both in new- and treatment failure patients. In total, 724 sputum positive patients were studied between 2004-2007. Whilst 231 were first line treatment failures with positive smears, despite five months of treatment, 493 representated new patients with their initial sputum studied, and prior to initiation of TB treatment. A major strength of this study was that the quality was assured by all samples being sent single blinded to the WHO SNRL in Sweden, for external quality assurance. Alarmingly high levels of resistance were observed in this study, with a 41% MDR-TB rate in first line treatment failures. More worryingly, a 24% MDR rate was encountered in newly diagnosed, previously untreated patients. Whilst indeed this data, coming from a hyper-endemic area like Mumbai will not represent the average situation in the diverse and huge India, it does serve to point out that MDR-TB rates in such pockets in the country exceed the national averages depicted in the WHO report for the whole of India. Knowing there are multiple metropolises like Mumbai in India with similar human density and health situations, it is conceivable that a considerable part of the population in India unfortunately suffers from a much higher rate of MDR-TB, than reflected in the current WHO document. This is apart from the fact that for several reasons, there may be a major part of the Indian population not seeking adequate health care, when confronted with symptoms of TB. What is urgently needed is a national and representative resistance survey in India, as has been conducted in China, where a first nationwide survey was organized in 2007, and confirmed previously published estimates based on extrapolation from sub national level data [4]. Whether the RNTCP (Revised National TB Control Program) will have the political courage and the funds to undertake this huge undertaking is unclear, but without this, the true extent of India�s MDR-TB crisis will remain obfuscated. Moreover, the magnitude of the resistance problem in India may not be restricted to MDR-TB. In 2011, TB patients with an extreme form of drug-resistance were encountered in our MDR-TB clinic at the Hinduja Hospital and Research Center in Mumbai, and provisionally indicated as Totally Drug Resistant TB (TDR-TB). The first four of these cases were reported in a short letter to Clinical Infectious Disease [5], as the Mycobacterium tuberculosis isolates of the respective patients were resistant to all the 12 drugs our mycobacterial laboratory was performing DST on: isoniazid, rifampicin, ethambutol, pyrazinamide, streptomycin, both the older and newer fluoroquinolones (ofloxacin and moxifloxacin), three injectable second-line drugs (kanamycin, amikacin, capreomycin), and the two most commonly used Group 4 drugs: ethionamide and Paraamino Salicylic acid (PAS). Second-line drugs had been misused in India for several decades, hence, this amplified form of drug resistance was not unexpected, but yet confronting. It was the choice of the author�s terminology �Totally Drug Resistant� that stirred up an unprecedented storm of international attention and controverise in the lay press and the medical literature. However, such cases of �extremely� or �totally� drug resistant TB were not really new. In practice, the terminology follows the actual situation with a delay of several years. While in 2006, the term �Extensively Drug Resistant TB� (XDR-TB) was introduced as a new phenomenon in Europe. In the period 2003-2006, already 10% of the MDR-TB cases were already XDR-TB, as retrospective analysis confirmed [6]. Even before that, in 2006, Migliori already reported two TDR-TB cases, like the one reported in India in 2011 [7], in immigrants to Italy [8]. Both had failed to respond to any drug and died after a protracted course. The next description of cases with this pattern of resistance was from Velayati, who reported 15 cases in Teheran, Iran, of whom 43% were immigrants [9]. No details are available on the outcome of these cases. The Indian government, initially, very critical and then defensive about these TDR-TB cases, was eventually galvanized into action, partly as a response to all the international attention this topic had been receiving in the press. They put into motion a number of promising measures. Notification of Tuberculosis became compulsory from May 2012. The staff and budget for TB control in Mumbai increased sixfold. The Union TB budget increased by 70%, and large sums of money were spent to upgrade infrastructure, and to strengthen the laboratory capacity in the city and the state of Maharashtra. The WHO response was even more immediate. Within a week after the publication on TDR-TB, they published a set of frequently asked questions and answers on this new terminology, on their web site. A few months later, at a meeting of experts in Geneva, they reached the consensus that whilst patterns of TB with resistance worse than XDR-TB, were clearly increasing and represent a formidable challenge, a new definition of resistance beyond XDR could not be recommended, in view of the huge impact this would have on lab capacity. Moreover, also the definition of XDRTB in itself was already doubtful, as the extent of drug resistance in such cases and the prognosis of the concerned patients already differs significantly [10]. They also stressed that the reliability, reproducibility of DST and correlation with the in vivo consequences was unclear. In fact, the magnitude of the problem of increasing drug resistance in TB cases in hot spots worldwide is therefore, not monitored and will remain hidden, until we will be forced into investment in visualizing the cruelty of this vastly incurable disease, by increasing mortality. After all, this may be a natural, evolutionary response of M. tuberculosis to the increased and uncontrolled exposure to anti tuberculosis drugs, and once created, this monster may be hard to control [11]. Currently, we have a cohort of 16 patients with TDR-TB in our hospital in Mumbai. They are all from poorer walks of life, and at with an average age of 32, they are the young bread earners of their families. They are male and female in equal ratio, and have seen an average of four doctors in the public and private sectors, and received a mean of 9.33 drugs for an average period of 26 months, before being labelled �TDR-TB� by us. Indeed, each TDR-TB patient holds a mirror to the way MDR-TB is mismanaged in India. Each TDR-TB patient represents a collective failure of all the players involved in health care in the country. The WHO report also presents MDR-TB therapies championed by the Stop TB and TB Alliance, none of which are completely effective. However, there is a cheap, safe and inexpensive drug, which has the promise to cure any pulmonary TB infection, regardless of its degree of antibiotic resistance. This drug is the old neuroleptic phenothiazine, Thioridazine (TZ), which has been confirmed to have in vitro activity against MDR [12,13], and XDR M. tuberculosis clinical isolates [14]. Although the in vitro concentrations of TZ that completely inhibit replication of M. tuberculosis are well beyond clinical reach, oncentrations that are quite low and routinely present in the serum of psychotic patients moderately treated with TZ, promote the killing of intracellular M. tuberculosis by non-killing macrophages [14-16]. Moreover, when used as a sole drug, TZ significantly reduces the number of pansusceptible and MDR-TB bacilli in pulmonary infections, in a well established mouse model [17,18]. In addition, it enhances the effectiveness of first line antibiotics in the same model, when used in combination [18]. Morover, TZ has also been successfully applied in compassionate use in 17 XDR-TB patients in Buenos Aires, in combination with other drugs [19]. When used as a sole drug, it rapidly improved the quality of life of an XDR-TB patient [11], suggesting that TZ should be considered as a �salvage drug�. Both studies have shown that no dramatic side effects theoretically could be induced by TZ therapy, namely cardio-pathology, have been noted [11,19]. Thereby, TZ in combination with antibiotics to which the initial infecting micro-organisms were resistant to seems to increase the intracellular concentration of these compounds to a level intracellular killing can be achieved [14-16]. Because of its dual action of TZ that enhances killing of intracellular M. tuberculosis via the indirect activation of phagolysosomal hydrolases [15,16], and its ability to inhibit efflux pumps of these organisms, which extrude the antibiotic(s), prior to reaching their intended targets [20], this drug seems highly valuable, especially in the therapy of TDR-TB, in which almost all other drugs fail [21-23]. Targeting the macrophage for activation of its killing machinery is believed to by-pass any mutational response by the micro-organism [21-23]. As of this writing, the WHO, neither TB Alliance nor Stop TB organizations, have shown any direct interest in pursuing the possibility of using TZ as an effective adjunct in the therapy of antibiotic resistant pulmonary TB infections. Rather, the therapies presented in the 2012 WHO report, are limited to those of the latter two organizations alone. This we believe is a disservice to those patients who are non-responsive to the available therapeutic modalities, especially when these afflicted cases are in regions of the world that are economically limited. An effective anti-MDR/XDR and probably TDR drug that is extremely cheap and safe to use in hands of a competent physician, could shortly have a major impact on the survival rates. How many more TB patients will needlessly suffer and die from an infection that in fact can be cured by an existing, cheap drug? References 1. Global tuberculosis report 2012 (2012) World Health Organisation (WHO), Geneva, Switzerland. 2. WHO Report 2010-Global Tuberculosis Control (2010). World Health Organisation (WHO), Geneva, Switzerland. 3. Almeida D, Rodrigues C, Udwadia ZF, Lalvani A, Gothi GD, et al. (2003) Incidence of multidrug-resistant tuberculosis in urban and rural India and implications for prevention. Clin Infect Dis 36: e152-e154. 4. D�souza DT, Mistry NF, Vira TS, Dholakia Y, Hoffner S, et al. (2009) High levels of multidrug resistant tuberculosis in new and treatment-failure patients from the Revised National Tuberculosis Control Programme in an urban metropolis (Mumbai) in Western India. BMC Public Health 9: 211. 5. http://www.flutrackers.com/forum/showthread.php?t=25164. 6. Devaux I, Manissero D, Fernandez de la Hoz K, Kremer K, van Soolingen D (2010) Surveillance of extensively drug-resistant tuberculosis in Europe, 2003- 2007. Euro Surveill 15. 7. Udwadia ZF, Amale RA, Ajbani KK, Rodrigues C (2012) Totally drug-resistant tuberculosis in India. Clin Infect Dis 54: 579-581. 8. Migliori GB, De Iaco G, Besozzi G, Centis R, Cirillo DM (2007) First tuberculosis cases in Italy resistant to all tested drugs. Euro Surveill 12: E070517. 9. Velayati AA, Masjedi MR, Farnia P, Tabarsi P, Ghanavi J, et al. (2009) Emergence of new forms of totally drug-resistant tuberculosis bacilli: super extensively drug-resistant tuberculosis or totally drug-resistant strains in iran. Chest 136: 420-425. 10. van Ingen J, de Lange WC, Boeree MJ, Iseman MD, Daley CL, et al. (2011) XDR tuberculosis. Lancet Infect Dis 11: 585. 11. Udwadia ZF, Sen T, Pinto LM (2011) Safety and efficacy of thioridazine as salvage therapy in Indian patients with XDR-TB. Recent Pat Antiinfect Drug Discov 6: 88-91. 12. Amaral L, Kristiansen JE, Abebe LS, Millett W (1996) Inhibition of the respiration of multi-drug resistant clinical isolates of Mycobacterium tuberculosis by thioridazine: potential use for initial therapy of freshly diagnosed tuberculosis. J Antimicrob Chemother 38: 1049-1053. 13. van Ingen J, van der Laan T, Amaral L, Dekhuijzen R, Boeree MJ, et al. (2009) In vitro activity of thioridazine against mycobacteria. Int J Antimicrob Agents 34: 190-191. 14. Martins M, Viveiros M, Ramos J, Couto I, Molnar J, et al. (2009) SILA 421, an inhibitor of efflux pumps of cancer cells, enhances the killing of intracellular extensively drug-resistant tuberculosis (XDR-TB). Int J Antimicrob Agents 33: 479-482. 15. Ordway D, Viveiros M, Leandro C, Bettencourt R, Almeida J, et al. (2003) Clinical concentrations of thioridazine kill intracellular multidrug-resistant Mycobacterium tuberculosis. Antimicrob Agents Chemother 47: 917-922. 16. Amaral L, Viveiros M, Kristiansen JE (2006) �Non-Antibiotics�: alternative therapy for the management of MDRTB and MRSA in economically disadvantaged countries. Curr Drug Targets 7: 887-891. 17. Martins M, Viveiros M, Kristiansen JE, Molnar J, Amaral L (2007) The curative activity of thioridazine on mice infected with Mycobacterium tuberculosis. In Vivo 21: 771-775. 18. van Soolingen D, Hernandez-Pando R, Orozco H, Aguilar D, Magis-Escurra C, et al. (2010) The antipsychotic thioridazine shows promising therapeutic activity in a mouse model of multidrug-resistant tuberculosis. PLoS One 5. 19. Abbate E, Vescovo M, Natiello M, Cufré M, García A, et al. (2012) Successful alternative treatment of extensively drug-resistant tuberculosis in Argentina with a combination of linezolid, moxifloxacin and thioridazine. J Antimicrob Chemother 67: 473-477. 20. Amaral L, Martins M, Viveiros M (2007) Enhanced killing of intracellular multidrug-resistant Mycobacterium tuberculosis by compounds that affect the activity of efflux pumps. J Antimicrob Chemother 59: 1237-1246. 21. Amaral L, Udwadia Z, Abbate E, van Soolingen D (2012) The added effect of thioridazine in the treatment of drug-resistant tuberculosis [Correspondence]. Int J Tuberc Lung Dis 16: 1706-1708. 22. Amaral L, Molnar J (2012) Why and how the old neuroleptic thioridazine cures the XDR-TB patient. Pharmaceuticals 5: 1021-1031. 23. Amaral L, Molnar J (2012) Why and how thioridazine in combination with antibiotics to which the infective strain is resistant will cure totally drug-resistant tuberculosis. Expert Rev Anti Infect Ther 10: 869-873.

Abstract: Resistance to isoniazid (INH), one of the main drugs used in tuberculosis (TB) therapy, is mostly due to chromosomal mutations in target genes. However, approximately 20-30% of INH resistant Mycobacterium tuberculosis isolates do not have mutations in any of the genes associated with INH resistance. This suggests that other mechanism(s) may be involved, namely efflux pump systems capable of extruding the drug to the exterior of the cell. In a previous work, we have induced clinical INH susceptible M. tuberculosis isolates and the H37Rv reference strain to high-level resistance to INH, by gradual exposure to increasing concentrations of this drug. In the present study, we have characterized these strains and M. bovis BCG induced to INH resistance with respect to their efflux activity and its contribution to INH resistance using the following approach: determination of the susceptibility to INH in the presence and absence of the efflux inhibitors (EIs) chlorpromazine, thioridazine and verapamil; evaluation of efflux activity by a semi-automated fluorometric method; and quantification of the expression level of genes coding for efflux pumps by real-time RT-qPCR. The EIs decreased INH resistance in the INH induced strains, in particular verapamil promoted a reversal of resistance in some of the strains tested. The induced strains presented an increased efflux activity that was inhibited by the EIs and showed overexpression of the efflux pump genes efpA, mmpL7, mmr, p55 and the Tap-like gene Rv1258c. Altogether, these results correlate efflux activity with INH resistance and demonstrate that efflux pumps play an important role in acquired INH resistance in M. tuberculosis complex. The development of EIs that can restore the antimicrobial activity of the antibiotic subject to efflux is an approach that can be useful in order to prevent the emergence of this resistance and guide the development of new effective anti-TB therapeutical approaches.

Abstract: The review by J. H. Grosset, T. G. Singer, W. R. Bishai �New drugs for the treatment of tuberculosis: hope and reality� in the Int J Tuberculosis and Lung Disease 2012; 16:1005-1014, is highly interesting and rightly pictures a horizon on improved treatment of tuberculosis. However, there are alternative compounds that on the short term can already provide an important relieve as compassionate application in MDR/XDR-TB treatment, and may on the long run offer an important additional effect in treatment regimens because of their special working mechanism; efflux pump inhibition. The review for instance did not discuss the successful use of the neuroleptic thioridazine in MDR/XDR-TB treatment, although sufficiently examined and published. Therefore, our correspondence addresses this omission and summarizes the in vitro, ex vivo, and mouse studies, as well as the use of this drug in treatment of TB patients as a mono therapy or an adjunct in combination with antibiotics to which the causative Mycobacterium tuberculosis (Mtb) was initially even resistant.

Abstract:

Abstract: Globally, tuberculosis infections continue to increase their resistance to antibiotics. Multidrug resistant tuberculosis infections (MDR TB) have progressed to extensively drug resistance status (XDR TB) and the latter have evolved in some parts of the world to totally drug resistant (TDR TB) infections. MDR TB is difficult to treat successfully, and when therapy is ineffective, a single case can cost almost $500,000. When the infection is XDR TB therapy is mostly unsuccessful and is accompanied with high mortality. TDR TB-a yet to be defined infection, is resistant to all forms of therapy and mortality is almost certain. We have, over a period of 14 years, studied thioridazine (TZ) an old neuroleptic that we have shown to: i) have in vitro activity against all antibiotic resistant forms of Mycobacterium tuberculosis; ii) have activity against intracellular Mycobacterium tuberculosis regardless of its antibiotic resistance status; iii) cure the infected mouse of an antibiotic susceptible and MDR TB infections; and, iv) when used in combination with antibiotics used for therapy of tuberculosis, would render the organism significantly more susceptible. These studies have guided our Argentinian colleagues to treat successfully XDR TB infections with thioridazine in combination with three antibiotics to which the infection was initially resistant. This mini review will describe our further work and the mechanisms by which TZ alone and in combination with antibiotics cures an XDR TB infection and why it is expected to cure TDR TB infections as well. The concepts presented are totally new and because they focus on the activation of killing by non-killing macrophages where Mycobacterium tuberculosis normally resides during infection, and coupled to the inhibition of efflux pumps which contribute to the antibiotic resistant status, effective therapy of any antibiotic resistant TB infection is possible. Because TZ is cheap and therefore affordable to any economically disadvantaged country, and will produce no harm when appropriate measures are taken, it is the ideal drug for immediate use in countries that have high frequencies of MDR, XDR and TDR TB infections.

Abstract: Multidrug-resistant tuberculosis (MDRTB) infections that continue to increase in frequency globally have progressed to become extremely drug-resistant tuberculosis (XDRTB). The therapeutic problems associated with MDRTB pale in comparison to those for XDRTB where mortality is high. This mini-review highlights the evidence that supports the use of the phenothiazine neuroleptic thioridazine for the therapy of XDRTB. Although thioridazine does produce some serious side-effects, albeit rarely, the poor prognosis associated with an XDRTB infection of a patient that presents with AIDS merits that the use of thioridazine for therapy of XDRTB is seriously considered. A recommended protocol is presented.

Abstract: N-dienylphenothiazines synthesized from tetrazole[1,5-a]pyridinium salts by treatment with phenothiazine were subjected to catalytic hydrogenation to yield N-butylphenothiazines, whereas transformation of these dienes with borane/dimethylsulfide resulted in hydroboration to give 2-hydroxybutylphenothiazines. Position of the hydroxyl group was supported by NMR spectroscopy and verified by X-ray analysis. Comparison of MDR modulatory activity of the new derivatives revealed that the hydroxybutyl compounds are promising candidates as MDR inhibitors.

Abstract: Globally, tuberculosis infections continue to increase their resistance to antibiotics. Multidrug resistant tuberculosis infections (MDR TB) have progressed to extensively drug resistance status (XDR TB) and the latter have evolved in some parts of the world to totally drug resistant (TDR TB) infections. MDR TB is difficult to treat successfully, and when therapy is ineffective, a single case can cost almost $500,000. When the infection is XDR TB therapy is mostly unsuccessful and is accompanied with high mortality. TDR TB-a yet to be defined infection, is resistant to all forms of therapy and mortality is almost certain. We have, over a period of 14 years, studied thioridazine (TZ) an old neuroleptic that we have shown to: i) have in vitro activity against all antibiotic resistant forms of Mycobacterium tuberculosis; ii) have activity against intracellular Mycobacterium tuberculosis regardless of its antibiotic resistance status; iii) cure the infected mouse of an antibiotic susceptible and MDR TB infections; and, iv) when used in combination with antibiotics used for therapy of tuberculosis, would render the organism significantly more susceptible. These studies have guided our Argentinian colleagues to treat successfully XDR TB infections with thioridazine in combination with three antibiotics to which the infection was initially resistant. This mini review will describe our further work and the mechanisms by which TZ alone and in combination with antibiotics cures an XDR TB infection and why it is expected to cure TDR TB infections as well. The concepts presented are totally new and because they focus on the activation of killing by non-killing macrophages where Mycobacterium tuberculosis normally resides during infection, and coupled to the inhibition of efflux pumps which contribute to the antibiotic resistant status, effective therapy of any antibiotic resistant TB infection is possible. Because TZ is cheap and therefore affordable to any economically disadvantaged country, and will produce no harm when appropriate measures are taken, it is the ideal drug for immediate use in countries that have high frequencies of MDR, XDR and TDR TB infections.

Abstract: Thioridazine in combination with antibiotics to which the XDR TB patient is initially resistant yields a cure. The reason that the cure is achieved is that thioridazine enhances the killing of intracellular Mycobacterium tuberculosis by non-killing macrophages; inhibits the genetic expression of efflux pumps of Mycobacterium tuberculosis that extrude antibiotics prior to reaching their intended targets; and, inhibits the activity of existing efflux pumps that contribute to the mdr phenotype of Mycobacterium tuberculosis. The combination of these thioridazine effects, probably contributes to the successful recent cures of XDR TB cases when the phenothiazine is used in combination with antibiotics to which the XDR TB patient was initially unresponsive.

Abstract:

Abstract: Objective: Development of a non-toxic, fluorescent-based, agar system for the screening of over-expressed bacterial efflux pump systems with common, inexpensive UV accessories. Methods: Wild type Gram-negative and positive bacteria expressing intrinsic efflux pumps and their progeny that over-express a specific efflux pump were selected for evaluation of efflux pump activity in an Mueller-Hinton agar containing increasing concentrations of the non-toxic fluorescent chromophore acridine orange (AO). The method is based on the same principle of its first-generation ethidium bromide method that the concentration of the fluorescent dye that first produces fluorescence of the over-lying bacterial colony represents the maximum concentration of the dye that the bacterium can extrude. The higher the concentration needed to produce fluorescence, the greater the ability of the bacterium to extrude the dye. Results: Progeny of Escherichia coli, Salmonella enterica serovar Enteriditis and Staphylococcus aureus that over-expressed a given efflux pump fluoresced (accumulated AO) at concentrations of AO that were very much greater than that required for the emission of fluorescence by their corresponding wild type that expressed an intrinsic efflux pump. Conclusions: The AO-agar method readily identifies strains of Gram-negative and Gram-positive bacteria that over-express efflux pump systems over that of their wild-type progeny.

Abstract: Background: To determine whether tetracycline competes with the ethidium bromide (EB), used as substrate of efflux pumps, for extrusion by the efflux pump system of Salmonella Enteritidis NCTC 13349 reference strain. Materials and Methods: Evaluation of efflux of EB in competition with tetracycline was conducted with the aid of an automated EB method and by EB agar method. Results: The EB Agar method demonstrated that the accumulation of EB by S. Enteritidis NCTC 13349 reference strain is reduced by high concentrations of tetracycline and that tetracycline accumulates within the cells. The use of the automated EB method confirmed the EB agar results and demonstrated that accumulation of EB is not affected by tetracycline unless the concentration of the antibiotic exceeds 100 mg/l. Conclusion: These results suggest that tetracycline is preferentially retained and that EB is preferentially extruded.

Abstract: Multi-drug resistance (MDR) of Gram-negative and Gram-positive bacteria is strongly associated with a modification of membrane permeability that alters the intracellular concentration of antimicrobial agents such as antibiotics, antiseptics and biocides. In order to combat this bacterial strategy, coordinated efforts from diverse and complementary scientific areas are essential - from clinical bacteriology, bacterial physiology, metabolic regulation, structural biology, biochemistry, biophysics, molecular modeling, chemical synthesis and systems biology. The COST (European Cooperation in the field of Scientific and Technical research) Action network BM0701 designated ATENS (Antibiotic Transport and Efflux: New Strategies to combat bacterial resistance) was initiated in 2008. The main aim of ATENS is to provide a framework, through collaboration, to increase knowledge and understanding of the efflux-mediated resistance in bacterial pathogens and to translate this knowledge into the development of novel antimicrobials. Its subobjectives are to determine prevalence and evolution of bacterial drug efflux mechanisms, identify risk factors, decipher genetic regulation of this mechanism, elucidate the functional and structural bases of efflux resistance, and to synthesize and evaluate molecules that obviate efflux-mediated resistance. More than 35 teams belonging to 20 member states constitute 4 Working Groups: Clinical and Veterinary Bacteriology; Molecular Basis of Drug Efflux; Structural Genomics, Bioinformatics and Molecular Modelling; they are also involved in the production of new molecules, chemosensitizers or inhibitory agents. This multi- and interdisciplinary approach should identify new targets and generate effective agents against efflux mechanisms in MDR bacterial pathogens. ATENS has already accomplished the following: produced more than 50 joint publications involving several BM0701 teams, including special issues focused on antibiotic resistance like the articles in this issue; organized five european COST meetings; and funded several students exchanges between partners to promote an efficient and fruitful transfer of knowledge. In addition, several COST members have contributed to International meetings in the field. We believe that our joint efforts will initiate new discoveries and strategies to overcome multidrug resistance.

Abstract: New and active infections of tuberculosis continue to increase globally. Although antibiotic susceptible infections can be readily cured with isoniazide and rifampicin, infections resistant to these two antibiotics, named Multi-Drug Resistant TB (MDR TB), are problematic for therapy, extol high costs in terms of human suffering and finances, and when these MDR infections progress to Extensive Drug Resistant TB (XDR TB) status, they are not only difficult to treat, they produce high levels of mortality regardless of therapeutic modality employed. The neuroleptic thioridazine (TZ) hasbeen shown to have wide spectrum in vitro and ex vivo activities against antibiotic susceptible, MDR and XDR strains, and has been successfully used for curing mice of active tuberculosis produced by antibiotic susceptible and MDR strains, and has cured 10 out of 12 XDR TB patients when used in combination with three antibiotics to which the XDR TB patients were non-responsive. Mycobacterium tuberculosis TZ has been recommended for �Compassionate Therapy� of MDR/XDR TB infections whose prognoses are significantly serious and anticipated to result in mortality. This review of TZ activity and its potential to cure MDR/XDR TB supports the contention that this neuroleptic offers patenting opportunities for �New Use�. The motivation for patents therefore is expected to rapidly bring TZ to the forefront for therapy of MDR/XDR TB and therefore, the striving for new patents is expected to contribute to the prevention of new infections of antibiotic resistant tuberculosis.

Abstract: Use of the old antipsychotic phenothiazine thioridazine (THZ) for therapy of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) infection is now being seriously considered. It is reported that THZ primarily acts on enzymes involved in fatty acid metabolism and membrane proteins, particularly efflux pumps, as well as oxidoreductases and proteins involved in aerobic respiration that overlap with a number of conventional antituberculous drugs. It targets the products of the Rv3160c-Rv3161c operon, which are perhaps required for the detoxification of THZ, members of the sigma factor SigB regulon that play a crucial role in protecting the pathogen against cell envelope damage, and Rv2745c, a transcription factor that regulates ATP-dependent proteolysis. Some of these genes have been shown to be essential for the survival or persistence of Mycobacterium tuberculosis in the infected host. Since THZ targets multiple pathways, including those involved in cell wall processes and respiratory chain components, it may serve as a model for multi-target drug development, as well as constitute a highly potent addition to a combination of antituberculous drug regimens. The discussion of some of the patents relevant to thioridazine to combat tuberculosis is also included in the present manuscript.

Abstract: The worldwide dissemination of «multi-drug resistant» (MDR) pathogens has severely reduced the efficacy of our antibiotic arsenal and increased the frequency of therapeutic failure. MDR bacteria over-express efflux pumps and this active mechanism can extrude all classes of antibiotics from the cell. It is necessary to clearly decipher the genetic, structural and functional aspects of this transport system in order to combat this polyselective mechanism. By understanding how efflux pumps work we may be able to develop a new group of antibacterial agents, collectively termed efflux reversals, including membrane permeabilisers, efflux pump inhibitors and flux-competitive agents, specific blockers, energy poisons, etc. Several chemical families of efflux pump inhibitors have been described and characterized. Among them several inhibitor compounds demonstrate efficient blocking of the efflux pump activity involved in the MDR phenotype as observed in many Gram-negative clinical isolates. This new family of molecules represents the first antibacterial class of compound specifically targeting active transport in the bacterial cell.

Abstract: Mycobacteria are responsible for some of the oldest diseases known to Man, usually associated with high morbility and mortality rates. An example is tuberculosis (TB), a major public health problem that accounts for eight million new cases each year. Furthermore, the increase of multidrug and extremely-drug resistance seriously threatens the success of the TB control programmes. Resistance to anti-mycobacterial drugs is often due to spontaneous mutations in target genes, followed by selection of resistant mutants during treatment. However, this does not explain all cases of drug resistance and other mechanism(s) may be involved, namely efflux pumps that extrude the drug to the exterior of the cell. Efflux pumps are becoming attractive drug targets for the development of new anti-mycobacterial compounds and several efflux inhibitors have been developed and published in patent applications (i.e., WO2004062674, US2004204378, US2003118541, WO2008141012, WO2009110002, WO2010054102). However, none of these inhibitors is used in clinical practice. This review will focus on the potential use of efflux inhibitors as adjuvants of the anti-mycobacterial therapy, an approach that may restore the activity of antibiotics that are subject to efflux and render the mycobacteria more susceptible to drugs transported by these pumps.

Abstract: Pulmonary tuberculosis infections caused by multi-drug resistant Mycobacterium tuberculosis has progressed to extensively drug resistant status (XDR-TB). XDR-TB is very difficult to treat successfully and results in high mortality. Globally, XDR-TB is now a major threat, especially to India and countries that were once part of the Soviet Union. There is a potential alternative to current ineffective therapy that is solidly supported by in vitro, ex vivo and in vivo studies. It has reported successful therapies in 10 out of 12 non-responsive XDR-TB patients. That therapy is thioridazine, and it is the purpose of this mini-review to provide the rationale for thioridazine therapy, especially for compassionate reasons, when all other therapies have failed, depicting on extremely poor prognosis.

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Abstract: BACKGROUND: Antimicrobial resistance mediated by efflux systems is still poorly characterized in Staphylococcus aureus, despite the description of several efflux pumps (EPs) for this bacterium. In this work we used several methodologies to characterize the efflux activity of 52 S. aureus isolates resistant to ciprofloxacin collected in a hospital in Lisbon, Portugal, in order to understand the role played by these systems in the resistance to fluoroquinolones. RESULTS: Augmented efflux activity was detected in 12 out of 52 isolates and correlated with increased resistance to fluoroquinolones. Addition of efflux inhibitors did not result in the full reversion of the fluoroquinolone resistance phenotype, yet it implied a significant decrease in the resistance levels, regardless of the type(s) of mutation(s) found in the quinolone-resistance determining region of grlA and gyrA genes, which accounted for the remaining resistance that was not efflux-mediated. Expression analysis of the genes coding for the main efflux pumps revealed increased expression only in the presence of inducing agents. Moreover, it showed that not only different substrates can trigger expression of different EP genes, but also that the same substrate can promote a variable response, according to its concentration. We also found isolates belonging to the same clonal type that showed different responses towards drug exposure, thus evidencing that highly related clinical isolates may diverge in the efflux-mediated response to noxious agents. The data gathered by real-time fluorometric and RT-qPCR assays suggest that S. aureus clinical isolates may be primed to efflux antimicrobial compounds. CONCLUSIONS: The results obtained in this work do not exclude the importance of mutations in resistance to fluoroquinolones in S. aureus, yet they underline the contribution of efflux systems for the emergence of high-level resistance. All together, the results presented in this study show the potential role played by efflux systems in the development of resistance to fluoroquinolones in clinical isolates of S. aureus.

Abstract: Background: Hydantoin derivatives have important biochemical and pharmacological properties. In the present study, 23 hydantoin compounds were evaluated for their efflux-modulating effects in Salmonella enterica serovar Enteritidis NCTC 13349 using real-time fluorimetry based on the intracellular accumulation of ethidium bromide (EB), a universal substrate of efflux pumps. Materials and Methods: Twenty-three hydantoin derivatives were tested for intrinsic efflux pump modulation in Salmonella Enteritidis NCTC 13349 by an automated real-time fluorimetric method that monitors the intracellular accumulation of ethidium bromide. Results: None of the compounds were found to have antibacterial activities at concentrations as high as 200 mg/l. Among the hydantoin derivatives tested in our study, only compound SZ7 showed efflux modulating activity on Salmonella Enteritidis NCTC 13349 in the assays that contained or omitted glucose as the singular source of metabolic energy. Conclusion: SZ7 is shown to be non-toxic and it could be evaluated to cure an efflux mediated MDR Salmonella infection, furthermore a quantitative activity structural relationship is also planned in order to determine the site of the SZ7 molecule responsible for its efflux pump inhibitory activity.

Abstract: The main efflux pump of Salmonella enterica serotype Enteritidis, which obtains its energy for the extrusion of noxious agents from the proton-motive force, was studied with the aid of an ethidium bromide (EtBr) semi-automated method under conditions that define the role of metabolic energy, ions and pH in the extrusion of the universal substrate EtBr. The results obtained in this study indicate that in minimal medium containing sodium at pH 5 efflux of EtBr is independent of glucose, whereas at pH 8 metabolic energy is an absolute requirement for the maintenance of efflux. In deionised water at pH 5.5, metabolic energy is required for the maintenance of efflux. The inhibitory effect of the ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) on efflux is shown to be minimised by low pH, and at high pH by metabolic energy. Similarly, thioridazine, an inhibitor of metabolic enzymes, inhibits efflux of EtBr only at pH 8 and the degree of inhibition is lessened by the presence of metabolic energy.

Abstract: Background: Because phenothiazines inhibit efflux pumps of bacteria, forty new phenothiazine derivatives were tested for their inhibition of the efflux pump systems of Grampositive and Gram-negative pathogenic bacteria. Materials and Methods: Detection of efflux pump activity was conducted by a previously described automated fluorimetric method. Results: Although many of the compounds significantly inhibited efflux by distinct bacteria, four compounds had exceptional activity against the efflux pump systems of the pathogenic wild type bacteria Escherichia coli, Salmonella Enteritidis, Enterococcus faecalis and Staphylococcus aureus. These four compounds were then evaluated for ability to reduce or reverse resistance of multidrug resistant members of Escherichia coli, Salmonella and Staphylococcus aureus whose MDR phenotypes are mediated by specific over-expressed efflux pumps. One of the compounds, 2173, significantly reduced resistance of MDR Staphylococcus aureus. Conclusion: These results suggest possible use of compound 2173 for therapy of infections caused by this organism.

Abstract: Most multi-drug resistant (MDR) clinical isolates of Gram-negative bacteria owe their MDR phenotypes to the over-expression of their main efflux pump AcrAB-TolC. The development of an MDR phenotype makes therapy problematic. If this MDR phenotype is to be addressed for the selection of effective therapy it is essential that the regulation and control of the acrAB-TolC efflux pump be fully understood. This review focuses on very recent information that defines the physiological functioning of the AcrAB-TolC efflux of Escherichia coli and Salmonella sp. and relates it to the physical chemistry involved in securing the sources of energy required for its function. Because one specific agent, the phenothiazine thioridazine has certain properties that indirectly inhibit the AcrAB-TolC efflux pump, its mechanism of action is described. Lastly, new methods for screening for over-expressed efflux pumps by MDR strains and for the assessment of efflux pumps on a real-time basis are described.

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Abstract: Several compounds isolated from the plant Carpobrotus edulis were evaluated for their activity against multidrug-resistant (MDR) bacteria and their efflux pump systems. Amongst the compounds isolated, six compounds were tested, namely uvaol, -amyrin, oleanolic acid, catechin, epicatechin and monogalactosyldiacylglycerol. Oleanolic acid presented high antibacterial activity against a large number of bacterial strains. The triterpene uvaol was the most active compound for modulation of efflux activity by MDR Gram-positive strains.

Abstract: One of the alternatives to the existing medicines and treatment procedures in fighting multi drug resistance (MDR) is strengthening the effects of medicines by modifying their molecular structures through exposure to laser radiation. A method associated with this, is the generation of micro-droplets which contain medicines solutions; the droplets are utilized/produced as vectors to transport the medicines to targets. In our studies we try to combine these two methods in order to obtain a new technique to deliver the efficient medicines to targets that can be applied for a relative large number of chemicals. For this purpose we have developed an experimental set-up containing a liquid droplets generator, a tunable laser source used to irradiate droplets, a subunit to measure the laser induced fluorescence (LIF) signals and a real time recording system for droplet image analysis. Measurements on different probes, like ultrapure water, commercial grade medicines, newly developed medicines and laser dyes were performed.. All these measurements were performed on water-based solutions. We present in this paper the laser induced fluorescence measurements results on medicine solutions (in bulk or in a micro-droplet form) that exhibit important modifications after the exposure at laser radiation. It was evidenced that the exposures to laser beams/coherent optical radiation of some medicines solutions in ultrapure water may produce molecular modifications in solutions. These slight modifications of the molecules made them more efficient against bacteria strains.

Abstract: Six cucurbitane-type triterpenoids (1-6) isolated from the aerial parts of Momordica balsamina were evaluated for their ability to inhibit the activity of bacterial efflux pumps of methicillin-resistant Staphylococcus aureus (MRSA) COL(OXA), Enterococcus faecalis ATCC 29212, Salmonella enterica subsp. I serovar Typhimurium 5408 and S. Typhimurium 5408CIP strains. The latter strain overproduces the AcrB transporter of the AcrAB-TolC efflux pump six-fold compared with its parent. Compounds 4-6 were also tested for similar activity against Escherichia coli AG100 wild-type strain and E. coli AG100TET8 that overproduces the AcrAB-TolC efflux pump. Evaluation of efflux activity was performed using a semi-automated method that measures accumulation of the universal efflux pump substrate ethidium bromide (EtBr). Some of the compounds significantly inhibited efflux of EtBr by MRSA COL(OXA) and E. faecalis ATCC 29212. A correlation between activity and the topological polar surface area of the compounds was found for MRSA COL(OXA).

Abstract: BACKGROUND/AIM: Bacterial multidrug resistance may be mediated by the overexpression of efflux pumps. Conventional evaluation of efflux activity using efflux pump substrates, such as ethidium bromide, requires specialised instrumentation. The agar-based method, previously reported, has been modified to evaluate as many as twelve bacterial strains and has been termed the ethidium bromide-agar cartwheel method. MATERIALS AND METHODS: Agar plates containing different concentrations of ethidium bromide were swabbed with bacterial cultures. The cell efflux capacity increased with increasing ethidium bromide concentration, which produced fluorescence of the bacterial mass. RESULTS: The method was shown to be useful for the detection of efflux activity among multidrug-resistant Gram-negative and Gram-positive clinical isolates, as confirmed by the determination of minimum inhibitory concentration for several antibiotics in the presence of known efflux pump inhibitors. CONCLUSION: This method may be adapted to the clinical laboratory for the presumptive identification of multidrug-resistant isolates that overexpress efflux pump systems.

Abstract: Efflux of antibiotics by Escherichia coli AG100 is performed by a variety of efflux pumps, ensuring survival of the bacterium in widely diverse media. At pH 5, efflux is independent of metabolic energy during the period of time the assay is conducted; at pH 8 it is totally dependent upon metabolic energy. Because calcium ions (Ca(2+)) are important for membrane transport channels and the activity of ATPases that provide energy functions, the role of Ca(2+) in the extrusion of an efflux pump substrate under conditions that challenge the bacterium was investigated. Real-time accumulation and efflux of ethidium bromide (EtBr) by E. coli K-12 AG100 strain [argE3 thi-1 rpsL xyl mtl �(gal-uvrB) supE44] was determined by a semi-automated fluorometric method in the presence and absence of Ca(2+) and agents that are known to inhibit access of calcium to enzymes that provide energy. Chlorpromazine (CPZ), an inhibitor of calcium binding to proteins (calcium-dependent enzymes), and ethylene diamine tetra-acetic acid (EDTA), a chelator of Ca(2+), increased accumulation and efflux of EtBr at pH 8 but not at pH 5. Ca(2+) reverses these effects when the assay is conducted at pH 8. In conclusion, the activity of the efflux pump system of E. coli is dependent upon metabolic energy at pH 8. Because at pH 8 hydrolysis of ATP is favoured and contributes protons for activation of the AcrAB-TolC efflux pump, CPZ is suspected of having its effects on accumulation/efflux of EtBr by indirectly affecting ATPase activity that is dependent upon Ca(2+).

Abstract: A quorum is the smallest number of people able to organize the decisions concerning functional activity. Similarly microbes use chemical signal molecules to make population size-dependent �decisions� by changing their gene regulations. The inhibition of quorum sensing (QS) by phenothiazines and structurally related molecules, e.g. amitriptyline, promethazine, acridine orange, imipramine, promazine, diethazine, desipramine, desertomycin and 5-fluorouracil as positive control was studied with Chromobacterium violaceum 026 as a sensor strain, which detects short carbon chain AHLs by the development of a purple pigment. The AHL was produced by Novospingobium Ezf 10-17, and the antibiotic-resistant clinical isolates, E. coli 31298. The QS was demonstrated as a signal transmission between the two bacterial strains. The most effective inhibitors of QS were amitriptyline, promethazine, acridine orange and desertomycin. Imipramine and diethazine were moderately active, while chlorprothixene was ineffective relative to 5-fluorouracil as positive control. The direct complex formation between AHL and QS inhibitors markedly reduced the QS in a chromogenic test. The AHL-neutralizing effect of the related compounds was shown by chromogenic method. The inhibition of QS signal transmission appears to be related to the quasi-planar structure and electron donor capacity of the conjugated �-electron system of the tricyclic framework. The results can be exploited in rational drug design as a new way to reduce the QS mediated processes eg. virulence of pathogens, to vary the formation of biofilms and to modify antibiotic resistance.

Abstract: BACKGROUND: Active efflux systems and reduced cell-wall permeability are considered to be the main causes of mycobacterial intrinsic resistance to many antimicrobials. In this study, we have compared the Mycobacterium smegmatis wild-type strain mc2155 with knockout mutants for porins MspA (the main porin of M. smegmatis) and MspC, the efflux pump LfrA (the main efflux pump system of M. smegmatis) and its repressor LfrR for their ability to transport ethidium bromide (EtBr) on a real-time basis. This information was then correlated with minimum inhibitory concentrations (MICs) of several antibiotics in the presence or absence of the efflux inhibitors chlorpromazine, thioridazine and verapamil. RESULTS: In the absence of porins MspA and MspC, accumulation of ethidium bromide decreased and the cells became more resistant to several antibiotics, whereas the knockout mutant for the LfrA pump showed increased accumulation of EtBr and increased susceptibility to EtBr, rifampicin, ethambutol and ciprofloxacin. Moreover, the efflux inhibitors caused a reduction of the MICs of streptomycin, rifampicin, amikacin, ciprofloxacin, clarithromycin and erythromycin in most of the strains tested. CONCLUSIONS: The methodology used in this study demonstrated that porin MspA plays an important role in the influx of quaternary ammonium compounds and antibiotics and that efflux via the LfrA pump is involved in low-level resistance to several antimicrobial drugs in M. smegmatis. The results obtained with this non-pathogenic mycobacterium will be used in future studies as a model for the evaluation of the activity of the same efflux inhibitors on the susceptibility of multidrug resistant strains of Mycobacterium tuberculosis to isoniazid and rifampicin.

Abstract: Whereas exposure of combinations of a phenothiazine and bacterium to incoherent UV increases the activity of the phenothiazine, exposure of the phenothiazine alone does not yield an increase of its activity. Because the laser beam energy is greater than that produced by the incoherent UV sources, exposure of phenothiazines to specific lasers may yield molecules with altered activities over that of the unexposed parent. Chlorpromazine, thioridazine and promethazine active against bacteria were exposed to two distinct lasers for varying periods of time. Absorption and fluorescence spectra were conducted prior to and post-exposure and the products of laser exposure evaluated for activity against a Staphylococcus aureus ATCC strain via a disk susceptibility assay. Exposure to lasers alters the absorption/fluorescence spectra of the phenothiazines; reduces the activity of thioridazine against the test bacterium; produces a highly active chlorpromazine compound against the test organism. Exposure of phenothiazines to lasers alters their structure that results in altered activity against a bacterium. This is the first report that lasers can alter the physico-chemico characteristics to the extent that altered bioactivity results. Exposure to lasers is expected to yield compounds that are difficult to make via chemical manipulation methods. A survey of selected patents of interest, even co-lateral for the subject of this article is shortly made.

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Abstract: Background: Hydantoin derivatives are very promising candidates to improve the efficacy of anticancer chemotherapy. Previously, we demonstrated that eight hydantoin derivatives inhibited the P-glycoprotein (ABCB1) efflux pump of mouse T-lymphoma cells as well as acting synergistically with the anticancer drug doxorubicin. Materials and Methods: The activity of the hydantoin derivatives were investigated in another MDR cancer model, namely Colo 205/S sensitive and Colo 320/R resistant colon carcinoma cells respectively, having normal or overexpressed ABCB1 systems. Results: Among the hydantoin derivatives evaluated, BS-1, MN-3 and JH-63 were the most effective ABCB1 transporter inhibitors at the concentration of 4 mg/l on the Colo 320/R cells, compared to the positive control, verapamil. Conclusion: The derivatives did not induce apoptosis of Colo 320/R resistant colon carcinoma cells, indicating that these hydantoin compounds are potent efflux pump inhibitors (EPI) without affecting the signalling pathways that regulate apoptosis.

Abstract: Multidrug resistance (MDR) to antibiotics presents a serious therapeutic problem in the treatment of bacterial infections. The importance of this mechanism of resistance in clinical settings is reflected in the increasing number of reports of multidrug resistant isolates. In Salmonella enterica, the most common etiological agent of food borne salmonellosis worldwide, MDR is becoming a major concern. In Salmonella the main mechanisms of antibiotic resistance are mutations in target genes (such as DNA gyrase and topoisomerase IV) and the over-expression of efflux pumps. However, other mechanisms such as changes in the cell envelope; down regulation of membrane porins; increased lipopolysaccharide (LPS) component of the outer cell membrane; quorum sensing and biofilm formation can also contribute to the resistance seen in this microorganism. To overcome this problem new therapeutic approaches are urgently needed. In the case of efflux-mediated multidrug resistant isolates, one of the treatment options could be the use of efflux pump inhibitors (EPIs) in combination with the antibiotics to which the bacteria is resistant. By blocking the efflux pumps resistance is partly or wholly reversed, allowing antibiotics showing no activity against the MDR strains to be used to treat these infections. Compounds that show potential as an EPI are therefore of interest, as well as new strategies to target the efflux systems. Quorum sensing (QS) and biofilm formation are systems also known to be involved in antibiotic resistance. Consequently, compounds that can disrupt or inhibit these bacterial �communication systems� will be of use in the treatment of these infections.

Abstract: The worldwide dissemination of «multi-drug resistant» (MDR) pathogens has severely reduced the efficacy of our antibiotic arsenal and increased the frequency of therapeutic failure. MDR bacteria over-express efflux pumps and this active mechanism can extrude all classes of antibiotics from the cell. It is necessary to clearly decipher the genetic, structural and functional aspects of this transport system in order to combat this polyselective mechanism. By understanding how efflux pumps work we may be able to develop a new group of antibacterial agents, collectively termed efflux reversals, including membrane permeabilisers, efflux pump inhibitors and flux-competitive agents, specific blockers, energy poisons, etc. Several chemical families of efflux pump inhibitors have been described and characterized. Among them several inhibitor compounds demonstrate efficient blocking of the efflux pump activity involved in the MDR phenotype as observed in many Gram-negative clinical isolates. This new family of molecules represents the first antibacterial class of compound specifically targeting active transport in the bacterial cell.

Abstract: Background: Hydantoin derivatives are very promising candidates to improve the efficacy of anticancer chemotherapy. Previously, we demonstrated that eight hydantoin derivatives inhibited the P-glycoprotein (ABCB1) efflux pump of mouse T-lymphoma cells as well as acting synergistically with the anticancer drug doxorubicin. Materials and Methods: The activity of the hydantoin derivatives were investigated in another MDR cancer model, namely Colo 205/S sensitive and Colo 320/R resistant colon carcinoma cells respectively, having normal or overexpressed ABCB1 systems. Results: Among the hydantoin derivatives evaluated, BS-1, MN-3 and JH-63 were the most effective ABCB1 transporter inhibitors at the concentration of 4 mg/l on the Colo 320/R cells, compared to the positive control, verapamil. Conclusion: The derivatives did not induce poptosis of Colo 320/R resistant colon carcinoma cells, indicating that these hydantoin compounds are potent efflux pump inhibitors (EPI) without affecting the signalling pathways that regulate apoptosis.

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Abstract: Meticillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial bacterium for which prevention and control measures consist mainly of the application of biocides with antiseptic and disinfectant activity. In this study, we demonstrated the presence of the plasmid-located efflux pump gene qacA in MRSA strain HPV107, a clinical isolate representative of the MRSA Iberian clone. The existence of efflux activity in strain HPV107 due to the QacA pump was also established and this QacA efflux activity was linked with a phenotype of reduced susceptibility towards several biocide compounds. No association could be made with antibiotic resistance. This work emphasises the potential of QacA pump activity in the maintenance and dissemination of important MRSA strains in the hospital setting and, increasingly, in the community.

Abstract: Multidrug- and extensively drug-resistant tuberculosis have emerged as grave threats to public health worldwide. Very few active drugs are available or likely to become available soon. To address these problems we revisited a classical observation, the applicability of phenothiazines as antimicrobial drugs. Within this pharmacological class we selected thioridazine, which is most efficacious and least toxic, when used as an antipsychotic drug. We tested thioridazine monotherapy in the Balb/c mouse model for its activity to treat both susceptible and multidrug-resistant tuberculosis by a two months daily oral administration of 32 and 70 mg/kg. In addition, we tested its additive value when combined with a standard first-line regimen for susceptible tuberculosis. Thioridazine treatment resulted in a significant reduction of colony-forming-units of the susceptible (-4.4 log CFU, p<0.05) and multidrug-resistant tuberculosis bacilli (-2.4 log CFU, p<0.009) in the lung both at one and two months after infection, compared to controls. Moreover, when thioridazine was added to a regimen containing rifampicin, isoniazid and pyrazinamide for susceptible tuberculosis, a significant synergistic effect was achieved (-6.2 vs -5.9 log CFU, p<0.01). Thioridazine may represent an effective compound for treatment of susceptible and multidrug-resistant tuberculosis. The phenothiazines and their targets represent interesting novel opportunities in the search for antituberculosis drugs.

Abstract: BACKGROUND: Most clinical isolates that exhibit a multi-drug resistant phenotype owe that resistance to over-expressed efflux pumps. Compounds that are efflux pump inhibitors (EPIs) reduce or reverse resistance to antibiotics to which the bacterial strain is initially resistant. We have evaluated non-antibiotics to reduce resistance of commonly encountered bacterial pathogens to antibiotics. MATERIALS AND METHODS: The effect of non-antibiotics on the susceptibility of bacteria to antibiotics was conducted by minimum inhibition concentration determinations of the antibiotic in the absence and presence of the non-antibiotic. RESULTS: Non-antibiotics such as chlorpromazine, amitryptiline and trans-chlorprothixene are shown to reduce or reverse resistance of a variety of bacteria to antibiotics. CONCLUSION: The results suggest that non-antibiotics may serve as adjuncts to conventional antibiotics for the therapy of problematic antibiotic infections caused by bacteria that owe their resistance to over-expressed efflux pumps.

Abstract: Mycobacterium tuberculosis that is resistant to Isoniazid (INH) and Rifampin (Rif) and hence, multi-drug resistant (MDR) has progressed to extensive drug resistant (XDR) status. XDR strains of Mycobacterium tuberculosis (XDR Mtb) are resistant, in addition to INH and Rif, to any fluoroquinolone, streptomycin and to any of the injectable anti-TB drugs kanamycin, amikacin and capreomycin. Therapy of the XDR TB patient, even under the best conditions, is problematic and at least 20% of XDR TB patients die within one year after diagnosis. Mortality among XDR TB patients co-infected with HIV or presenting with AIDS is considerably higher reaching levels of 80% or higher. Drugs that are to prove effective against XDR Mtb must be able to reach the organism at the site where it mainly resides-the pulmonary macrophage. However, experience tells us that no matter how effective a drug may be, it will be followed by resistance. We have been able to demonstrate that thioridazine, a neuroleptic in safe use for over forty years, enhances the killing of phagocytosed Mycobacterium tuberculosis regardless of its antibiotic susceptibility profile and cure the mouse of a Mycobacterium tuberculosis pulmonary infection. Most recently, others have employed our studies for the therapy of the XDR TB patient with thioridazine and cured 10 out of 12 XDR TB patients of an XDR Mtb infection. Although thioridazine is beyond patent protection, its use for the therapy of XDR TB is new and therefore, a patent may be sought for "use as an anti-XDR TB agent". PMID: 20156179 [PubMed - in process]

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Abstract: Amongst the three series of quinazoline derivatives synthesised and studied in this work, some molecules increase the antibiotic susceptibility of Gram-negative bacteria presenting multidrug-resistant phenotypes. N-alkyl compounds induced an increase in the activity of chloramphenicol, nalidixic acid and sparfloxacin, which are substrates of the AcrAB-TolC and MexAB-OprM efflux pumps in clinical isolates. These molecules are able to increase the intracellular concentration of chloramphenicol in efflux pump-overproducing strains. Their activity depends on the antibiotic structure, suggesting that different sites may be involved for the recognition of substrates by a given efflux pump. Quinazoline molecules exhibiting a nitro functional group are more active, and structure-activity relationship studies may be undertaken to identify the pharmacophoric group involved in the AcrB and MexB affinity sites. Copyright (c) 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. PMID: 20494558 [PubMed - in process]

Abstract: When administered to mice at doses of 100microg/mouse and 200microg/mouse, thioridazine (TDZ) significantly protected animals from the lethality produced by a virulent strain of Salmonella enterica serovar Typhimurium and reduced the number of bacteria retrieved from the spleen, liver and heart blood. The protection conferred by TDZ against a virulent Salmonella infection is hypothesised to be due to a reduction in the 55kDa virulence protein of the outer membrane of the organism, as this protein is almost totally absent when the organism is exposed to the phenothiazine. It is further hypothesised that the reduction in the 55kDa virulence factor renders the organism susceptible to the action of hydrolytic enzymes of the neutrophil phagolysosome, whereas in the absence of exposure to TDZ intracellular ingestion and localisation of the phagocytosed bacterium does not result in killing owing to rapid induction of the two-step PmrA/B regulon that results in the eventual synthesis and insertion of lipid A into the nascent lipopolysaccharide layer of the outer membrane. Copyright 2009 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. PMID: 20005679 [PubMed - indexed for MEDLINE]

Abstract: Enterobacter aerogenes predominates amongst Enterobacteriaceae species that are increasingly reported as producers of extended-spectrum beta-lactamases. Although this mechanism of resistance to beta-lactams is important, other mechanisms bestowing a multidrug-resistant (MDR) phenotype in this species are now well documented. Amongst these mechanisms is the overexpression of efflux pumps that extrude structurally unrelated antibiotics prior to their reaching their targets. Interestingly, although knowledge of the genetic background behind efflux pumps is rapidly advancing, few studies assess the physiological nature of the overall efflux pump system of this, or for that matter any other, bacterium. The study reported here evaluates physiologically the efflux pump system of an E. aerogenes ATCC reference as well as two strains whose MDR phenotypes are mediated by overexpressed efflux pumps. The activities of the efflux pumps in these strains are modulated by pH and glucose, although the effects of the latter are essentially restricted to pH 8, suggesting the presence of two general efflux pump systems, i.e. proton-motive force-dependent and ABC transporter types, respectively.

Abstract: BACKGROUND: Hydantoin derivatives possess a variety of biochemical and pharmacological properties. Although hydantoin compounds are studied extensively, there are not many studies that investigate their anticancer properties. MATERIALS AND METHODS: Thirty hydantoin compounds were evaluated for their efflux modulating effects in cancer cells using a rhodamine 123 accumulation assay and real-time fluorometry based on the intracellular accumulation of ethidium bromide. RESULTS: The 30 derivatives were screened by real-time fluorometry for rhodamine 123 accumulation. Among the selected derivatives, compounds SZ-7, LL-9, BS-1, MN-3, P3, RW-15b, AD-26, RW-13, AD-29 and KF-2 significantly increased the retention of rhodamine 123. Compounds AD-26, AD-29, RW-13, KF-2, BS-1, MN-3, RW-15b and JH-63 showed synergistic effect with doxorubicin on mouse lymphoma cells. Furthermore, compound SZ-7 had indifferent effect with doxorubicin. CONCLUSION: These results indicated the role of chemical modifications within the hydantoin ring for its potential inhibition of the ABCB1 transporter. The most active structures contained aromatic substituents as well as some tertiary amine fragments.

Abstract: Ninety-three Salmonella isolates recovered from commercial foods and exotic animals in Colombia were studied. The serotypes, resistance profiles and where applicable the quinolone resistance genes were determined. Salmonella Anatum (n=14), Uganda (19), Braenderup (10) and Newport (10) were the most prevalent serovars, and resistance to tetracycline (18.3%), ampicillin (17.2%) and nalidixic acid (14%) was most common. Nalidixic acid-resistant isolates displayed minimum inhibitory concentrations ranging from 32 to 1024 μg mL(-1) . A Thr57�Ser substitution in ParC was the most frequent (12 of the 13 isolates). Six isolates possessed an Asp87�Tyr substitution in GyrA. No alterations in GyrA in a further seven nalidixic acid-resistant isolates were observed. Of these, four serovars including two Uganda, one Infantis and a serovar designated 6,7:d:-, all carried qnrB19 genes associated with 2.7 kb plasmids, two of which were completely sequenced. These exhibited 97% (serovar 6,7:d:- isolate) and 100% (serovar Infantis isolate) nucleotide sequence identity with previously identified ColE-like plasmids. This study demonstrates the occurrence of the qnrB19 gene associated with small ColE plasmids hitherto unrecognized in various Salmonella serovars in Colombia. We also report unusual high-level quinolone resistance in the absence of any DNA gyrase mutations in serovars S. Carrau, Muenchen and Uganda.

Abstract: Two simple, instrument-free, user-friendly methods that can readily be implemented by a routine microbiology laboratory are described for the detection of multi-drug-resistant (MDR) isolates that overexpress efflux pump (EP) systems responsible for the MDR phenotype. The first method employs the universal EP substrate ethidium bromide (EB) at varying concentrations in agar-containing plates upon which the contents of an overnight culture are swabbed as spokes of a wheel. In this method, named the EB-agar cartwheel method, it is assumed that the smallest concentration of EB that produces fluorescence of the bacterial mass represents the highest concentration of EB that the bacteria can exclude. Consequently, as the efflux system(s) of a given MDR clinical bacterial isolate is overexpressed relative to that of a reference strain, the minimal concentration of EB producing fluorescence is significantly greater. A simple formula is provided which affords the ranking of MDR clinical isolates with respect to the degree of their efflux capacity. The second method, which can be used after the first one, determines whether the MDR phenotype is based upon an overexpressed efflux system. This method employs a 24-well microplate with separate wells containing or lacking an efflux pump inhibitor (EPI) and Kirby-Bauer discs that correspond to the antibiotics to which the MDR strain is resistant. After the wells are inoculated with the MDR clinical isolate, the plate is incubated overnight and each well is evaluated by eye for evidence of growth. Comparison of growth to the relevant control enables the observer to determine the following outcomes: no growth produced by the EPI-antibiotic combination (i.e., reversal of antibiotic resistance); reduced growth produced by the EPI-antibiotic combination; no difference in growth, i.e., EPI does not affect the resistance to the given antibiotic. If the first method showed that there was a significant difference between the minimum concentrations of EB in agar that produced fluorescence for the clinical isolate and its reference strain, then one can conclude that if the EPI had no effect on reducing antibiotic resistance, the differences in the EB concentrations that produced fluorescence are probably due to differences in the permeability of the strain to EB, reflecting a downregulation of porins if the clinical isolate is a Gram-negative bacterium. PMID: 20401592 [PubMed - indexed for MEDLINE]

Abstract: A bioassay-guided separation protocol, including the testing of the extracts, fractions and pure compounds for their ability to inhibit P-glycoprotein (the efflux pump responsible for the multidrug resistance of the used cell line) of mouse lymphoma cells containing the human efflux pump gene MDR1, led to the isolation of seven compounds from the chloroform and ethyl acetate soluble fractions of the methanolic extract of Carpobrotus edulis. The compounds were identified by 1D, 2D NMR and MS investigations as triterpens (beta-amyrin, uvaol and oleanolic acid), monogalactosyldiacylglycerol, catechin, epicatechin and procyanidin B5. Uvaol was the most effective and promising compound in the reversal of multidrug resistance in MDR mouse lymphoma cell line. PMID: 20393003 [PubMed - indexed for MEDLINE]

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Abstract: Clin Microbiol Infect 2010; 16: 1161-1164 Abstract Nontuberculous mycobacteria (NTM) are being increasingly isolated in clinical laboratories and present technical and therapeutic challenges. In the present study, we report our experience with the identification of NTM received from 12 Lisbon hospitals over a 3-year period using GenoType Mycobacterium (CM/AS) assays (HAIN Lifescience GmbH, Nehren, Germany). Together, the two kits identified 96.6% of all NTM isolates tested. Among the 18 NTM species identified, Mycobacterium avium complex was the most frequent, although it accounted for only 34% of all NTM. Introducing these methods for the rapid identification of NTM highlights the importance of NTM as potential pathogens and assisted the selection of adequate therapy. PMID: 19832711 [PubMed - in process]

Abstract: Phenothiazines have their primary effects on the plasma membrane of prokaryotes and eukaryotes. Among the components of the prokaryotic plasma membrane affected are efflux pumps, their energy sources, energy providing enzymes such as ATPases, and genes that regulate and code for permeability aspects of the bacterium. The responses of multi-drug (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis to the neuroleptic phenothiazine thioridazine are reviewed. The information collated suggests that this phenothiazine has the potential to cure XDR and MDR tuberculosis infections, a potential that has been recently demonstrated by its ability to cure 10 patients who presented with XDR TB infections. The mechanism by which this phenothiazine produces the desired effects within the infected macrophage is also discussed. PMID: 20668307 [PubMed - in process]

Abstract: The role of quorum sensing (QS) is well known in microbial pathogenicity and antibiotic resistance. QS is responsible for motility, swarming, and biofilm production based on the signal molecules, e.g., acylated homoserine lactones (AHLs) produced by micro-organisms above certain population density. The inhibition of QS may reduce pathogenicity, antibiotic resistance and biofilm formation in systemic and local infections. The homoserine lactones and other transmitters contribute to antibiotic resistance and pathogenicity of several bacteria; consequently the inhibition of QS signals reduces the problem of resistance and virulence. Due to the increasing number of persistent non-treatable infections, there is an urgent need to develop new strategies to combat infections that destabilize bacterial communities in the host. The effect of essential oils on bacterial growth and QS were evaluated using the sensor strain Chromobacterium violaceum CV026 and N-acyl homoserine lactone (AHL) producing Escherichia coli ATTC 31298 and the grapevine colonizing Ezf 10-17 strains. Of the tested oils, rose, geranium, lavender and rosemary oils were the most potent QS inhibitors. Eucalyptus and citrus oils moderately reduced pigment production by CV026, whereas the chamomile, orange and juniper oils were ineffective. Copyright (c) 2009 John Wiley & Sons, Ltd. PMID: 19827025 [PubMed - indexed for MEDLINE]

Abstract: Tuberculosis (TB) is mainly an intracellular infection of the lung alveolar macrophages, and any anti-TB agent must therefore be active at the macrophage. Among the available therapies, isoniazid and rifampicin are the most effective drugs against susceptible Mycobacterium tuberculosis, but they are ineffective against multidrug-resistant TB (MDRTB) strains. Rates of MDRTB in Portugal are the highest in Western Europe, demanding effective measures for their control. Our application of molecular techniques for the early identification of MDRTB assisted in the reduction of these rates. Further examination revealed that a large number of MDRTB cases were extensively-drug resistant (XDRTB), providing evidence for the urgent need of new and effective anti-MDRTB/XDRTB therapeutic strategies. This review describes in detail: the characteristics of the main M. tuberculosis strains circulating in Portugal; the creation of a Task Force for TB control, based on molecular tools that allow 1-day identification of an MDRTB patient; the usefulness of evaluating the ex vivo activity of anti-tubercular agents against the M. tuberculosis isolated from the patient's sputum; and the mode of action by which phenothiazines have been shown to promote the killing of intracellular MDRTB/XDRTB by nonkilling macrophages. PMID: 20377340 [PubMed - indexed for MEDLINE]

Abstract: Thioridazine (TDZ) has been shown to have in vitro activity against multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis, to promote the killing of intracellular MDR and XDR strains and to cure the mouse of antibiotic-susceptible and -resistant pulmonary tuberculosis (TB) infections. Recently, TDZ was used to cure 10 of 12 XDR-TB patients in Buenos Aires, Argentina. At the time of writing, it is being used for the therapy of non-antibiotic-responsive terminal XDR-TB patients in Mumbai, India, on the basis of compassionate therapy and although it is too early to determine a cure, the patients have improved appetite, weight gain, are afebrile and free of night sweats, and their radiological picture shows great improvement. Because XDR-TB is essentially a terminal disease in many areas of the world and no new effective agents have yet to yield successful clinical trials, global clinical trials for the therapy of XDR-TB are urgently required. 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. PMID: 20188526 [PubMed - indexed for MEDLINE]

Abstract: A semi-automated method that uses the common efflux pump (EP) substrate ethidium bromide (EB) is described for the assessment of EP systems of bacteria. The method employs the Rotor-Gene(TM) 3000 thermocycler (Corbett Research) for the real-time assessment of accumulation and efflux of EB in Phosphate-Buffered Solution (PBS) under varying physiological conditions, such as temperature, pH, presence and absence of the energy source, and presence of efflux pumps inhibitors (EPIs). The method is sufficiently sensitive to characterize intrinsic EP systems of reference strains, a prime necessity if there is a need for assessment of EP-mediated multi-drug resistance (MDR). The method has been successfully applied by us to characterize intrinsic and over-expressed EP systems of Escherichia coli, Salmonella Enteritidis, Enterobacter aerogenes, Enterococcus faecalis and Enterococcus faecium, Staphylococcus aureus, and Mycobacterium smegmatis and Mycobacterium avium, suggesting that if the organism can be maintained in PBS, the system described may suffice for the evaluation and assessment of its EP system. PMID: 20401593 [PubMed - indexed for MEDLINE]

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Abstract: BACKGROUND: Cancer cells become refractory to chemotherapy as a consequence of their overexpressing ABC transporters that extrude not only the therapeutic agent but other unrelated compounds such as chemotoxins and biocides before they can reach their intended targets. A compound that can inhibit the activity of these transporters may find use as an adjunct to chemotherapy that had been rendered ineffective. MATERIALS AND METHODS: Four curcubitane-type triterpenes isolated from Momordica balsamina Linn. (Cucurbitaceae), a plant from Mozambique were evaluated for their inhibition of the ABC transporter P-glycoprotein coded by the human ABCB1 gene transfected into mouse lymphoma cells. The evaluation was conducted by flow cytometry using rhodamine 123 and real-time fluorometry assessing accumulation of ethidium bromide (EB) on a real-time basis. RESULTS: Among the compounds isolated, the most active was 7-methoxycucurbita-5,24-diene-3beta,23(R)-diol, which inhibited the efflux of ethidium bromide (EB) and rhodamine 123 from the ABCB1-transfected mouse lymphoma cell. CONCLUSION: Real-time fluorometry replicated the flow cytometric results with significant advantages for the evaluation of efflux pump inhibitors. The substitution of side groups on the cucurbitane skeleton appears to be significant in the inhibition of ABCB1 activity. PMID: 19846941 [PubMed - indexed for MEDLINE]

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Abstract: BACKGROUND: Resistance Nodulation Division (RND) efflux pumps of Escherichia coli extrude antibiotics and toxic substances before they reach their intended targets. Whereas these pumps obtain their energy directly from the proton motive force (PMF), ATP-Binding Cassette (ABC) transporters, which can also extrude antibiotics, obtain energy from the hydrolysis of ATP. Because E. coli must pass through two pH distinct environments of the gastrointestinal system of the host, it must be able to extrude toxic agents at very acidic and at near neutral pH (bile salts in duodenum and colon for example). The herein described study examines the effect of pH on the extrusion of ethidium bromide (EB). METHODOLOGY/PRINCIPAL FINDINGS: E. coli AG100 and its tetracycline induced progeny AG100(TET) that over-expresses the acrAB efflux pump were evaluated for their ability to extrude EB at pH 5 and 8, by our recently developed semi-automated fluorometric method. At pH 5 the organism extrudes EB without the need for metabolic energy (glucose), whereas at pH 8 extrusion of EB is dependent upon metabolic energy. Phe-Arg beta-naphtylamide (PAbetaN), a commonly assumed inhibitor of RND efflux pumps has no effect on the extrusion of EB as others claim. However, it does cause accumulation of EB. Competition between EB and PAbetaN was demonstrated and suggested that PAbetaN was preferentially extruded. A K(m) representing competition between PAbetaN and EB has been calculated. CONCLUSIONS/SIGNIFICANCE: The results suggest that E. coli has two general efflux systems (not to be confused with a distinct efflux pump) that are activated at low and high pH, respectively, and that the one at high pH is probably a putative ABC transporter coded by msbA, which has significant homology to the ABC transporter coded by efrAB of Enterococcus faecalis, an organism that faces similar challenges as it makes its way through the toxic intestinal system of the host. PMCID: PMC2722724 PMID: 19684858 [PubMed - indexed for MEDLINE]

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Abstract: The activities of cell surface serine proteases are markedly enhanced in malignant tumours. Proteolytic degradation of the extracellular matrix and basal membrane of normal cells is an important event for tumour cell growth and invasion. Two well-known broad-spectrum inhibitors of serine protease, Foy-305 and Ono-3403, were evaluated for their ability to affect the growth rate and survival of MCF7 breast cancer cells co-cultured with MRC5 lung fibroblasts as feeder cells in the absence of serum. Flow cytometry and differential staining demonstrated that in the mixed culture, the rate of tumor growth was dependent upon the presence of the feeder MRC5 lung fibroblasts and could be obviated by the additional presence of the inhibitors of serine proteases. PMID: 19779105 [PubMed - indexed for MEDLINE]

Abstract: In this study, we aimed to answer the following question: 'How does a bacterium become so resistant to a given antibiotic even though the levels of antibiotic to which it has become resistant remained constant in the patient?'Escherichia coli AG100 strain induced to high-level resistance due to overexpression of an AcrAB efflux pump was serially cultured in 10mg/L tetracycline for 60 passages. Between each passage it became increasingly resistant to tetracycline, beta-lactams and quinolones with concomitant restoration of wild-type AcrAB activity. Because the multidrug-resistant phenotype could not be reversed with transfer to drug-free medium or with efflux pump inhibitors, it may have resulted from activation of a 'mutator gene' system that reduced the 'energy consumption' associated with an overexpressed efflux pump system. PMID: 19734019 [PubMed - indexed for MEDLINE]

Abstract: Vitamin B1 is in its active form thiamine pyrophosphate (TPP), an essential cofactor for several key enzymes in the carbohydrate metabolism. Mammals must salvage this crucial nutrient from their diet in order to complement the deficiency of de novo synthesis. In the human pathogenic bacterium Staphylococcus aureus, two operons were identified which are involved in vitamin B1 metabolism. The first operon encodes for the thiaminase type II (TenA), 4-amino-5-hydroxymethyl-2-methylpyrimidine kinase (ThiD), 5-(2-hydroxyethyl)-4-methylthiazole kinase (ThiM) and thiamine phosphate synthase (ThiE). The second operon encodes a phosphatase, an epimerase and the thiamine pyrophosphokinase (TPK). The open reading frames of the individual operons were cloned, their corresponding proteins were recombinantly expressed and biochemically analysed. The kinetic properties of the enzymes as well as the binding of TPP to the in vitro transcribed RNA of the proposed operons suggest that the vitamin B1 homeostasis in S. aureus is strongly regulated at transcriptional as well as enzymatic levels. PMCID: PMC2766623 PMID: 19888457 [PubMed - indexed for MEDLINE]

Abstract: BACKGROUND: We have developed a semi-automated fluorometric method that utilizes ethidium bromide (EB), a common substrate of bacterial efflux pumps. The method is sufficiently sensitive to characterize the efflux pump systems of bacteria. Because EB is also recognized and extruded by ATP-binding cassette (ABC) transporters and these have similarity to P-glycoprotein (P-gp), the method has been extended for the evaluation of agents that can inhibit the extrusion of EB on a real-time basis by mouse lymphoma cells containing the human ABCB1 (mdr1) gene. MATERIALS AND METHODS: Monitoring of uptake and extrusion of EB was assessed using the Rotor-Gene 3000 (Corbett Research) under different conditions. RESULTS: Whereas extrusion of EB took place readily, the addition of known inhibitors of efflux pumps (verapamil, reserpine) caused retention of EB. CONCLUSION: This method is inexpensive and allows the detection of neoplastic cells with increased efflux activity as well as the screening of large numbers of compounds for inhibition of the P-gp. PMID: 19528478 [PubMed - indexed for MEDLINE]

Abstract: BACKGROUND: Efflux pump activity has been associated with multidrug resistance phenotypes in bacteria, compromising the effectiveness of antimicrobial therapy. The development of methods for the early detection and quantification of drug transport across the bacterial cell wall is a tool essential to understand and overcome this type of drug resistance mechanism. This approach was developed to study the transport of the efflux pump substrate ethidium bromide (EtBr) across the cell envelope of Escherichia coli K-12 and derivatives, differing in the expression of their efflux systems. RESULTS: EtBr transport across the cell envelope of E. coli K-12 and derivatives was analysed by a semi-automated fluorometric method. Accumulation and efflux of EtBr was studied under limiting energy supply (absence of glucose and low temperature) and in the presence and absence of the efflux pump inhibitor, chlorpromazine. The bulk fluorescence variations were also observed by single-cell flow cytometry analysis, revealing that once inside the cells, leakage of EtBr does not occur and that efflux is mediated by active transport. The importance of AcrAB-TolC, the main efflux system of E. coli, in the extrusion of EtBr was evidenced by comparing strains with different levels of AcrAB expression. An experimental model was developed to describe the transport kinetics in the three strains. The model integrates passive entry (influx) and active efflux of EtBr, and discriminates different degrees of efflux between the studied strains that vary in the activity of their efflux systems, as evident from the calculated efflux rates: = 0.0173 +/- 0.0057 min-1; = 0.0106 +/- 0.0033 min-1; and = 0.0230 +/- 0.0075 min-1. CONCLUSION: The combined use of a semi-automated fluorometric method and an experimental model allowed quantifying EtBr transport in E. coli strains that differ in their overall efflux activity. This methodology can be used for the early detection of differences in the drug efflux capacity in bacteria accounting for antibiotic resistance, as well as for expedite screening of new drug efflux inhibitors libraries and transport studies across the bacterial cell wall. PMCID: PMC2774284 PMID: 19835592 [PubMed]

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Abstract: Although many compounds have been described to inhibit the replication of drug-susceptible and drug-resistant strains of Mycobacterium tuberculosis, most of these studies only evaluate their in vitro activity. There is a lack of studies that show whether any of these agents can kill these organisms at the site where they normally reside post infection, namely, the macrophage of the lung parenchyma. It is the aim of this mini-review to identify agents that have been shown to enhance the killing of intracellular drug-susceptible, multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant TB (XDR-TB) strains by non-killing macrophages. Because these agents appear to promote their activity by affecting the transport of K(+) and Ca(2+) from the phagolysosome containing the bacteria, and thereby promoting its acidification and activation of hydrolases that will eventually kill the organism, the authors suggest that compounds that are known to affect the transport of K(+) and Ca(2+) should be considered for possible activity against intracellular MDR- and XDR-TB.

Abstract: Chemoresistance presents a general health problem concerning the therapy of infectious disease and cancer. In this context, the worldwide dissemination of "multidrugresistant" (MDR) pathogens has severely reduced the efficacy of our antimicrobial weapons and dramatically increased the frequency of therapeutic failure. Because MDR bacterial infections involve the over-expression of efflux pumps that expel unrelated antibiotics before they can reach their targets, it is necessary to clearly define the molecular and genetic bases of the MDR mechanisms in order to combat these infectious diseases. This characterization of efflux pumps allows the definition of an original anti-resistance weapon, the efflux pump inhibitor (EPI). Several chemical families of EPIs have been now described and characterized. Among them several inhibitor compounds display an efficient activity and inhibit the major AcrAB-TolC and MexAB-OprM efflux systems which are the major efflux pumps responsible for MDR Gram negative clinical isolates. The use of these EPIs induces a significant reduction of resistance to one or more antibiotics to which these isolates were initially resistant. Hence, the EPI when used as an adjuvant to the given antibiotic, restores the activity of the antibiotic. The description of the responsible efflux mechanism at its structural and physiological level will make it possible to develop along intelligent lines an improved new generation of EPIs that can readily be added to the armamentarium of current and past "fallen by the wayside" antibiotic therapies.

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Abstract: The flavonoid fraction isolated from the ethyl acetate fraction (BF-1) of Butea frondosa (L.) stem bark exhibited distinct antimicrobial activity when tested against 129 bacterial strains belonging to 9 different genera of both gram-positive and gram-negative types. Minimum inhibitory concentration (MIC) of the fraction BF-1 was determined following NCCLS guidelines using the agar dilution method. Twenty-four out of 36 strains of Staphylococcus aureus were inhibited by 50-200 mg/l of the fraction. This fraction also inhibited strains of Bacillus spp., Shigella spp., Salmonella spp. and even a few Pseudomonas at concentrations between 50-200 mg/l. Other bacteria including Escherichia coli, Vibrio cholerae and V. parahaemolyticus were moderately sensitive to BF-1. In the in vivo studies, this fraction offered significant protection to Swiss albino mice at a concentration of 80 microg/mouse (p<0.001) when they were challenged with 50 median lethal dose of Salmonella enteritidis NCTC 74. A fraction named BF-1 that was isolated from an ethyl acetate fraction of Butea frondosa provided protection against an infection from a Salmonella enteritidis NCTC strain. PMID: 19368121 [PubMed - indexed for MEDLINE]

Abstract: The essential oil of Helichrysum italicum significantly reduces the multidrug resistance of Enterobacter aerogenes, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. Combinations of the two most active fractions of the essential oil with each other or with phenylalanine arginine beta-naphthylamide yield synergistic activity. Geraniol, a component of one fraction, significantly increased the efficacy of beta-lactams, quinolones, and chloramphenicol. PMCID: PMC2681508 PMID: 19258278 [PubMed - indexed for MEDLINE]

Abstract: Enterococcus faecalis is recognized as a multidrug-resistant nosocomial pathogen. The phenotypic basis for this is largely uncharacterized. The intrinsic efflux system of the antibiotic-susceptible E. faecalis ATCC29212 strain was studied using a semi-automated method that assesses accumulation and efflux of the universal efflux pump substrate ethidium bromide (EB). The results show that the intrinsic efflux system of this Enterococcus strain is controlled by energy derived from the catabolism of glucose and the proton concentration of the medium. At pH 5, agents that inhibit efflux pumps in Gram-positive organisms and the proton gradient un-coupler CCCP do not increase accumulation nor inhibit efflux of EB. In contrast, at pH 8, where the proton concentration is 1,000-fold lower, these agents increase accumulation and efflux of EB. These results are relevant to infections produced by E. faecalis and subsequent antibiotic therapy with antibiotics to which the organism is known to be intrinsically resistant. PMID: 19368129 [PubMed - indexed for MEDLINE]

Abstract: Multidrug-resistant and extensively drug-resistant tuberculosis (MDR/XDR-TB) are problematic to manage, especially in patients with acquired immune deficiency syndrome (AIDS). There is therefore a dire need for effective anti-MDR/XDR-TB agents. We have previously shown that agents that inhibit the efflux pumps of MDR bacteria and cancer cells also enhance killing of intracellular mycobacteria, possibly by increasing the availability of K(+) and Ca(2+) needed for the activation of lysosomal enzymes of the phagolysosomal unit. In this study, the newly synthesised and recently patented SILA 409 and 421 were tested for in vitro and ex vivo activity against XDR-TB. The minimum inhibitory concentration (MIC) of SILA compounds was determined by the BACTEC 460 TB system. The effect of each compound on the killing activity of human macrophages infected with XDR-TB was determined by exposing the macrophage that had phagocytosed the bacterium to the compounds and assessing the killing activity by colony-forming unit counting. Amongst the two compounds tested, SILA 421 was shown to have in vitro activity against XDR-TB (MIC<3.5mg/L) and to transform non-killing macrophages into effective killers of phagocytosed bacteria, without any cytotoxic activity. Because SILA 421 revealed good in vitro and ex vivo activities and is devoid of any cytotoxic activity, it is a potential candidate as an anti-MDR/XDR-TB drug. PMID: 19155160 [PubMed - indexed for MEDLINE]

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Abstract: Global rates of pulmonary tuberculosis (TB) continue to increase. Moreover, resistance of the causative organism Mycobacterium tuberculosis to the two most effective anti-TB medications continue to rise. Now, multi-drug resistant TB (MDR-TB) has progressed to extensively drug resistant TB (XDR-TB) - a M. tuberculosis organism that is resistant to the most effective second line drugs available for the treatment of TB. This review provides detailed, significant evidence that supports the use of an old neuroleptic compound, thioridazine (TZ), for the management of MDR-TB and XDR-TB infections and which has been shown to inhibit efflux pumps of bacteria. The argument has been previously presented but no one seems to be listening - and the disease continues unabated when there is a very good probability that the suggested drug will prove to be effective. When the prognosis is poor, available therapy predictably ineffective and death is inevitable, compassionate therapy with TZ should be contemplated. The risks are small and the rewards great.

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Abstract: OBJECTIVES: Therapy of AIDS patients infected with Mycobacterium avium is problematic due to its intrinsic resistance to antibiotics. We have characterized the efflux pump activity of M. avium wild-type strain through an automated fluorometric method and correlated it with intrinsic resistance to antibiotics. METHODS: M. avium ATCC 25291(T) and Mycobacterium smegmatis mc(2)155 were evaluated for accumulation and efflux of ethidium bromide in the presence or absence of the efflux pump inhibitors (EPIs) thioridazine, chlorpromazine, verapamil and the proton uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). For this purpose, a new automated fluorometric method was used that separately assesses accumulation and extrusion of ethidium bromide. RESULTS: The automated fluorometric method described in this paper allowed the detection and quantification of ethidium bromide transport across M. avium and M. smegmatis cell walls. Accumulation of ethidium bromide was found to be temperature-dependent and significantly increased by EPIs thioridazine, chlorpromazine, verapamil and CCCP in a concentration-dependent manner. Efflux of ethidium bromide under optimum conditions of temperature and glucose is inhibited by the above agents. At half their intrinsic MICs, both thioridazine and chlorpromazine, similarly to verapamil and CCCP, significantly increased the susceptibility of M. avium to erythromycin, suggesting an effect upon an efflux pump with ethidium bromide and erythromycin as substrates. A similar effect was observed for M. smegmatis with verapamil only. CONCLUSIONS: M. avium and M. smegmatis intrinsic resistance is affected by EPIs such as thioridazine or chlorpromazine, an effect that might be important in research and development of new, more effective antimycobacterial therapies.

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Abstract: Multidrug resistance in Gram-negative bacteria is now known to be primarily caused by overexpression of efflux pumps that extrude unrelated antibiotics from the periplasm or cytoplasm of the bacterium prior to their reaching their intended target. This review focuses on a variety of agents that have been shown to be efflux pump inhibitors (EPIs) and which, if used as 'helper compounds' in combination with antibiotics to which the organism is initially resistant, may produce the required cure. Although not all of the EPIs may serve a helper role owing to their toxicity, they may nevertheless serve as lead compounds.

Abstract: Demonstration of efflux of ethidium bromide (EtBr) has been made for over 30 bacterial species, usually by showing enhanced efflux in multidrug-resistant strains that was then abolished by inactivating efflux pumps. Here we present a relatively simple automated method that employs EtBr as an efflux pump substrate for the demonstration of intrinsic efflux activity in Escherichia coli K-12 AG100. The method uses the Rotor-Gene 3000 instrument for real-time fluorometric measurement of EtBr accumulation under conditions that limit energy (absence of glucose, low temperature) and of EtBr extrusion under optimum conditions. The method can be used for screening compound libraries for efflux inhibiting capacity.

Abstract: Therapy of multidrug-resistant (MDR)-TB is highly problematic; that of extensively drug-resistant (XDR)-TB even more so. Both infections result in high mortality, especially if the patient is coinfected with HIV or presents with AIDS. Selection of therapy for these infections is limited and, for most situations, it is performed 'blind'. However, there is a solution for the selection of effective therapy and this is presented herein. Ideal therapy of the patient infected with MDR-TB or XDR-TB can be determined a priori by the mycobacteriology laboratory. This would involve the isolation of the patient's macrophages, the phagocytosis of the mycobacterial isolate and the presentation of the antitubercular agent to the macrophage-bacterium complex. This system is reviewed in its entirety and its potential and feasibility are supported by hard experimental demonstrations.

Abstract: We have developed a number of methods that identify efflux pump mediated multi-drug resistant bacteria, characterize efflux systems and screen for inhibitors of efflux pumps. These approaches were complemented by the quantification of the expression of genes that regulate and code for constituents of efflux pumps. The methods described are easy to use, reproducible and for the most part, require instrumentation normally present in a clinical bacteriology laboratory. Because each method provides good reproducibility, they lend themselves for inter-laboratory use.

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Abstract: OBJECTIVES: By adapting an antibiotic-susceptible Staphylococcus aureus strain to increasing concentrations of ethidium bromide, a known substrate of efflux pumps (EPs), and by phenotypically and genotypically analysing the resulting progeny, we characterized the molecular mechanisms of S. aureus adaptation to ethidium bromide. METHODS: S. aureus ATCC 25923 was grown in increasing concentrations of ethidium bromide. The MICs of representatives of eight classes of antibiotics, eight biocides and two dyes against ATCC 25923 and its ethidium bromide-resistant progeny ATCC 25923(EtBr) were determined with or without six efflux pump inhibitors (EPIs). Efflux activity in the presence/absence of EPIs was evaluated by real-time fluorometry. The presence and expression of eight EP genes were assayed by PCR and quantitative RT-PCR (qRT-PCR), respectively. Mutations in grlA, gyrA and norA promoter regions were screened by DNA sequencing. RESULTS: Compared with its parental strain, ATCC 25923(EtBr) was 32-fold more resistant to ethidium bromide and also more resistant to biocides and hydrophilic fluoroquinolones. Resistance to these could be reduced by the EPIs chlorpromazine, thioridazine and reserpine. Increased efflux of ethidium bromide by ATCC 25923(EtBr) could be inhibited by the same EPIs. qRT-PCR showed that norA was 35-fold over-expressed in ATCC 25923(EtBr), whereas the remaining EP genes showed no significant increase in their expression. Sequencing of the norA promoter region revealed a 70 bp deletion in ATCC 25923(EtBr). CONCLUSIONS: Exposure of S. aureus to quaternary compounds such as ethidium bromide results in decreased susceptibility of the organism to a wide variety of compounds, including quinolones and biocides through an efflux-mediated response, which for strain ATCC 25923 is mainly NorA-mediated. This altered expression may result from alterations in the norA promoter region.

Abstract: BACKGROUND: Human monocyte-derived macrophages that have little killing activity of their own kill intracellular Staphylococcus aureus when cultured in the presence of inhibitors of calcium and potassium efflux pumps. The aim of this study was to evaluate the effect of inhibitors such as ouabain, reserpine and verapamil in the killing activity of macrophages infected with Mycobacterium tuberculosis. MATERIALS AND METHODS: Macrophages obtained from peripheral blood were infected with M. tuberculosis ATCC27294 H37Rv and treated with reserpine, ouabain and verapamil. RESULTS: After three days post-infection, macrophages treated with the inhibitors demonstrated an enhancement of the killing activity destroying the internalized bacteria. CONCLUSION: Whereas drugs that target the bacterium are predicted to lose effectiveness due to mutation of the bacterial target, drugs that enhance killing by macrophages that normally do not kill mycobacteria may yield a more effective form of infections therapy caused by multidrug resistant M. tuberculosis.

Abstract: Pulmonary tuberculosis (TB) has again become a global problem: it infects 2.2 billion people world-wide, caused the deaths of over 3 million last year and will produce over 8 million new cases of TB this coming year. Although effective therapy is widely available for antibiotic susceptible strains of Mycobacterium tuberculosis, current drugs are relatively useless against multi-drug resistant infections (MDRTB). Mortality is almost complete within two years regardless of therapy, and in the case of co-infection with HIV/AIDS, mortality is 100% within a few months of diagnosis especially the M. tuberculosis strain in XDRTB. As of the time of this writing no new effective anti-TB drugs have been made available by the pharmaceutical industry and XDRTB. Because TB is an intracellular infection of the non-killing macrophage of the lung, any agent that is to prove effective must have activity against MDRTB and XDRTB strains that have been phagocytosed by the human macrophage. This review intents to provide cogent in vitro, ex vivo and in vivo evidence that supports the use of a variety of commonly available phenothiazines for the therapy of MDRTB and XDRTB, especially when the prognosis of the infection is poor and the use of the recommend agents can take place along lines of "compassionate therapy". In addition, we will describe the macrophage assay as indispensable when an agent is to be further studied for its effectiveness as an anti-TB drug. In vitro studies if not complemented by ex vivo studies will for the most part be dead-ended since few agents that have activity in vitro have any activity against phagocytosed M. tuberculosis.

Abstract: Patented SILA compounds 409 and 421, previously shown to inhibit the efflux pumps of bacteria and cancer cells, have been studied for their ability to reduce or eliminate the presence of plasmids from Escherichia coli strains that have been induced to high level resistance to tetracycline by gradual exposure to increasing concentrations of the antibiotic. The results demonstrate that SILA compound 421, which has greater efflux pump inhibitory activity than its parent SILA compound 409, can reduce plasmid loads by 5 logs, over that present in the absence of the drug. The ability of the SILA compound to eliminate much larger plasmids is substantially lower. Because in vivo studies have shown that these compounds are not toxic to the mouse, the results obtained in our study suggest a potential role for SILA compound 421 as an adjunct for the therapy of antibiotic-resistant E. coli infections whose resistance is plasmid-mediated. In addition, because plasmid-mediated resistance is often found in tetracycline-treated cattle, SILA compound 421 may have potential as an adjunct during the time that the cattle are maintained on tetracycline prior to slaughter.

Abstract: Ergosterol peroxide, cycloart-23-en-3beta,25-diol, vanillin and 4-hydroxybenzaldehyde have been isolated and characterized from a crude methanol extract of Euphorbia lagascae. Previous studies have shown contradictory results about the antibacterial activity of ergosterol peroxide against Mycobacterium tuberculosis. In order to clarify this question, the activity of this compound was tested against Mycobacterium tuberculosis H37Rv ATCC 27294 strain using two different systems: BACTEC 460TB (Bactec 460) and BACTEC MGIT 960 system (Bactec 960). The results obtained show that significant activity was demonstrable only with the Bactec 460 system. The lack of activity noted with the Bactec 960 system appears to be due to the much faster growth rate of the organism in the medium of this system as opposed to that of the Bactec 460 system. Ergosterol peroxide is also shown by the current study to be devoid of any activity against an antibiotic sensitive ATCC strain of Staphylococcus aureus.

Abstract: Thioridazine (TZ) has previously been shown by us to have in vitro and ex vivo activity against antibiotic-susceptible and multidrug-resistant Mycobacterium tuberculosis (MDRTB). Because current therapy of MDRTB is highly problematic even when all five 'first line of defence' drugs are employed, there is a need for effective antituberculosis drugs. New derivatives of TZ were synthesised and their in vitro activity against a reference strain of M. tuberculosis was evaluated with the aid of the BACTEC 460 system. Derivatives that presented significant activity were evaluated by ex vivo studies and were shown to enhance the killing of intracellular M. tuberculosis.

Abstract: Whereas human neutrophils are effective and efficient killers of bacteria, macrophages such as those derived from monocytes are almost devoid of killing activity. Nevertheless, monocytes can be transformed into effective killers of mycobacteria or staphylococci when exposed to clinical concentrations of a phenothiazine or to inhibitors of efflux pumps (reserpine and verapamil), or to ouabain, an inhibitor of K(+) transport. Because the rates of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) continue to escalate globally, and because no new effective drug has been made available for almost 40 years, compounds that enhance the killing activity of monocytes against MDR-TB are obviously needed. This review covers the specific characteristics of MDR-TB, identifies a variety of agents that address these characteristics and therefore have potential for managing MDR-TB. Because the mechanism by which these agents enhance the killing of intracellular bacteria is important for the intelligent design of new anti-tubercular agents, the review correlates the mechanisms by which these agents manifest their effects. Lastly, a model is presented which describes the mechanisms by which distinct efflux pumps of the phagosome-lysosome complex are inhibited by agents that are known to inhibit K(+) flux. The model also predicts the existence of a K(+) activated exchange (pump) that is probably located in the membrane that delineates the lysosome. This putative pump, which is immune to inhibitors of K+ flux, is identified as being the cause for the acidification of the lysosome thereby activating its hydrolytic enzymes. Because the non-killer macrophage can be transformed into an effective killer by a variety of compounds that inhibit K(+) transport, perhaps it would be wise to develop drugs that enhance the killing activity of these cells inasmuch as this approach would not be subject to any resistance, as is the eventual case for conventional antibiotics.

Abstract: Our previous studies demonstrated that exposure of a bacterium to increasing concentrations of an antibiotic would increase resistance to that antibiotic as a consequence of activating efflux pumps. This study utilises the same approach; however, it employs the methicillin-resistant Staphylococcus aureus (MRSA) COL strain, which is highly resistant to oxacillin (OXA). MRSA COL was adapted to 3200 mg/L of OXA. Changes in resistance to other antibiotics were evaluated and efflux pump activity during the adaptation process was determined. MRSA COL was exposed to stepwise two-fold increases of OXA. At the end of each step, minimum inhibitory concentration determination for erythromycin (ERY) and other antibiotics was conducted. Reserpine (RES) was employed to evaluate whether resistance to ERY was dependent on efflux pump activity. Efflux pump activity was also evaluated using the ethidium bromide (EB) assay. DNA typing of the products of each culture step was conducted to assess purity. Serial exposure of MRSA COL to increasing concentrations of OXA resulted in increased resistance to ERY, which could be eliminated with RES. Evaluation of efflux pump activity by the EB method indicated increased efflux activity. Resistance to ERY was accompanied by resistance to kanamycin, amikacin, ofloxacin, norfloxacin, ciprofloxacin and rifampicin. This is the first time that a multidrug-resistant phenotype has been experimentally produced as a consequence of exposure of the organism to an antibiotic to which it is initially highly resistant.

Abstract: BACKGROUND: Membrane permeability is the first step involved in resistance of bacteria to an antibiotic. The number and activity of efflux pumps and outer membrane proteins that constitute porins play major roles in the definition of intrinsic resistance in Gram-negative bacteria that is altered under antibiotic exposure. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe the genetic regulation of porins and efflux pumps of Escherichia coli during prolonged exposure to increasing concentrations of tetracycline and demonstrate, with the aid of quantitative real-time reverse transcriptase-polymerase chain reaction methodology and western blot detection, the sequence order of genetic expression of regulatory genes, their relationship to each other, and the ensuing increased activity of genes that code for transporter proteins of efflux pumps and down-regulation of porin expression. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that, in addition to the transcriptional regulation of genes coding for membrane proteins, the post-translational regulation of proteins involved in the permeability of Gram-negative bacteria also plays a major role in the physiological adaptation to antibiotic exposure. A model is presented that summarizes events during the physiological adaptation of E. coli to tetracycline exposure.

Abstract: Bacteria and cancer cells develop resistance to more than one agent as a consequence of being exposed to ineffective levels of the agent for a prolonged period of time. The resistance of these cells is mediated by over-expressed efflux pumps that have the ability to extrude a large variety of unrelated chemicals. This review discusses the main types of multidrug resistant (MDR) efflux systems of bacteria and cancer cells, and shows the similarity of specific efflux systems between them with respect to given agents that inhibit efflux, thus rendering these cells once more susceptible to agents to which they had developed MDR.

Abstract: Diclofenac sodium (Dc), an anti-inflammatory agent, has remarkable inhibitory action both against drug-sensitive and drug-resistant clinical isolates of various Gram-positive and Gram-negative bacteria. The aim of this study was to determine the ability of Dc to protect mice from a virulent Salmonella infection. Dc injected at 1.5 microg/g and 3.0 microg/g mouse body weight significantly protected animals from the lethality of Salmonella infection. As was the case for the in vitro interaction, Dc in combination with streptomycin was even more effective. The non-antibiotic drug Dc has potential for the management of problematic antibiotic-resistant bacterial infections.

Abstract: BACKGROUND: The aim of the study was to evaluate the effectiveness of thioridazine (TZ) at different dose levels on mice that had been infected intraperitoneally (i.p.) with a high dose of the Mycobacterium tuberculosis ATCC H37Rv strain. SUBJECTS AND METHODS: Groups of five female BALB/C mice were infected i.p. with 10(6) colony forming units/mL. After thirty days, treatment with TZ was initiated, except for the control group. Mice were treated with TZ at equivalent concentrations to that used in the humans (1200 mg/day), ranging from 0.05 to 0.5 mg/day. RESULTS: The results demonstrated that a daily dose of 0.5 mg/day of TZ reduced the number of colony forming units retrieved from the lungs of infected mice within one month. CONCLUSION: By the end of 300 days of therapy, although mycobacteria were still retained their presence, in comparison to that of the control was 8 orders of magnitude lower.

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Abstract: The aim of the study was to develop a simple, inexpensive, reproducible ethidium bromide (EB)-agar based method that is independent of any specialized instrumentation, for the demonstration of efflux pump activity, which is responsible for antibiotic resistance of bacteria. MATERIALS AND METHODS: A series of agar plates containing varying concentrations of EB were swabbed with strains of Escherichia coli or Staphylococcus aureus, which differed with respect to efflux pump activity. The plates were incubated at different temperatures and time periods and the measurements of fluorescence were used to evaluate the efflux activity of each culture. RESULTS: This simple assay allowed us to identify the efflux of EB in different bacteria following an overnight incubation. The minimal concentration of EB that produced fluorescence was significantly greater at 37 degrees C than at 4 degrees C, suggesting the presence of an energy-dependent pump. The method was shown to simultaneously identify strains of a mixed culture that differed from each other with respect to the activity of their efflux pumps. CONCLUSION: The method, in conjunction with the use of antibiotic-containing disks, provides an additional advantage for the easy identification and selection of colonies that differ with respect to antibiotic susceptibility and degree of efflux pump activity. Because the method is very reproducible it may form the basis for interlaboratory standardization of efflux pump activity of multi-drug resistant (MDR) clinical isolates.

Abstract: Previous studies suggested that the phenothiazine chlorpromazine (CPZ) could reverse or reduce the antibiotic resistance of bacteria. In some areas of the world, the majority of Staphylococcus aureus isolates are now resistant to methicillin, prompting this study to see whether such resistance can be altered by phenothiazine thioridazine (TZ), an agent with equal antibacterial activity, which is free of the severe side-effects associated with chronic administration of CPZ. The results indicated that, whereas methicillin-sensitive strains of Staphylococcus aureus (MSSA) were not rendered more susceptible to oxacillin, resistance to oxacillin by highly-resistant strains (MRSA) could be significantly reduced by sub-inhibitory concentrations of TZ. Reserpine, an inhibitor of efflux pumps, was also shown to reduce the resistance of MRSA strains to oxacillin in a concentration-dependent manner. The phenothiazines have been shown, by others, to inhibit the efflux pumps of bacteria and the mechanism by which MRSA are rendered more susceptible to oxacillin in the presence of TZ is believed to be due to a similar efflux pump.

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Abstract: The antibiotic resistance is now common place throughout the globe. Two highly problematic antibiotic resistant infections are those produced by multi-drug resistant Mycobacterium tuberculosis (MDRTB) and methicillin resistant Staphylococcus aureus (MRSA). Although vancomycin is useful for therapy of MRSA, there is now evidence that resistance to this antibiotic is taking place. Intracellular infections of MRSA are very difficult to manage and are recurrent especially when invasive prosthetic devices are employed. This mini-review provides cogent evidence that both intracellular MDRTB and intracellular MRSA can be killed by concentrations of the non-antibiotic phenothiazine, Thioridazine, at concentrations in the medium that are below those present in the plasma of patients treated with this agent. Although thioridazine has been claimed to cause arrhythmias and even sudden death, the frequencies of these episodes are rare and when present, they are related to the patients underlying cardiac status as opposed to the direct effect of the agent itself. The authors do not suggest that thioridazine be used indiscriminately for MDRTB or intracellular infections produced by MRSA. However, there are circumstances where there are no alternative forms of therapy and the patient faces an unfavourable prognosis. For these highly selective and controlled situations, the use of thioridazine in the manner employed for the therapy of psychosis is recommended (compassionate therapy).

Abstract: Bacterial plasmids have a major impact on metabolic function. Lactose fermentation of E. coli or hemolysin B transporter expressed by the plasmids that carry these respective genes could be readily obviated by heterocyclic compounds that readily bind to plasmid DNA. These compounds could also reverse the resistance to antibiotics of E. coli, Enterobacter, Proteus, Staphylococcus and Yersinia strains by eliminating plasmids. However, the frequency and extent of this effect was significantly less than might have been expected based on a complex interaction with plasmid DNA. The effects of heterocyclic compounds on the plasmids responsible for the virulence of Yersinia and A. tumefaciens, or on nodulation, nitrogen fixation of Rhizobia accounted for the elimination of 0.1 to 1.0 % of plasmids present in the populations studied. Bacterial plasmids can be eliminated from bacterial species grown as pure or mixed bacterial cultures in the presence of sub-inhibitory concentrations of non-mutagenic heterocyclic compounds. The antiplasmid action of the compounds depends on the chemical structure of amphiphillic compounds having a planar ring system with substitution in the L-molecular region. A symmetrical pi-electron conjugation at the highest occupied molecular orbitals favours the antiplasmid effect. The antiplasmid effect of heterocyclic compounds is expressed differentially in accordance with the structural form of the DNA to which they bind. In this manner "extrachromosomal" plasmid DNA that exists in a superhelical state binds more compound than its linear or open-circular form; and least to the chromosomal DNA of the bacterium, that carries the plasmid. It can also be noted that these compounds are not mutagenic and their antiplasmid effects correlate with the energy of HOMO-orbitals. Plasmid elimination is considered also to take place in ecosystems containing numerous bacterial species. This opens up a new perspective in rational drug design against bacterial plasmids. The inhibition of conjugational transfer of antibiotic resistance plasmid can be exploited to reduce the spread of antibiotic resistance plasmid in the ecosystem. Inhibition of plasmid replication at various stages, as shown in the "rolling circle" model (replication, partition, conjugal transfer) may also be the theoretical basis for the elimination of bacterial virulence in the case of plasmid mediated pathogenicity and antibiotic resistance. The large number of compounds tested for antiplasmid effects provides opportunities for QSAR studies in order to find a correlation between the antiplasmid effect and the supramolecular chemistry of these plasmid curing compounds. Plasmid elimination in vitro provides a method of isolating plasmid free bacteria for biotechnology without any risk of inducing mutations.

Abstract: A proton pump-deleted mutant E. coli, AG100 A, had greater sensitivity to ampicillin, tetracycline and erythromycin than the wild-type parent E. coli AG100 containing the proton pump. This antibiotic sensitivity was further increased by resistance modifiers such as the Ca2+ channel blocker (+/-) verapamil (VP) and the calmodulin antagonist promethazine (PMZ). Whereas the newly-synthesized trifluoromethyl-ketone (TF) enhanced the activity of these antibiotics against the wild-type strain, it did not enhance the activity of ampicillin against the proton pump-deleted mutant. These results suggested that TF14 had an inhibitory effect on the proton pump. Elimination of plasmids from another strain of E. coli, K12, was promoted by PMZ and 9-amino-acridine (9-AA), but not by TF14 alone. However, combinations of TF14 with either PMZ or 9-AA enhanced the plasmid elimination capacity of the latter compounds. The combination of TF14, PMZ and VP proved that the Ca2+ channel blocker was not effective by itself These results collectively suggest that TF14 inhibited the proton pump of E. coli and that it was this pump which, when inhibited by TF14, allowed more PMZ to reach its plasmid elimination target.

Abstract: The phenothiazinium salt methylene blue [3,7-bis(dimethylamino)phenothiazinium chloride] is the oldest known synthetic antimalarial drug, its clinical efficacy having been reported in 1891. The role of methylene blue in the evolution of the modern antimalarial armoury is often unappreciated, yet it can be linked directly to standard drugs such as chloroquine and its congeners. Also, in the face of increasing plasmodial resistance to modern antimalarials, phenothiazinium derivatives have again featured as lead compounds in drug research. The precise mode of action of methylene blue and its commercial analogues against Plasmodium spp. remains a cause for conjecture, having been variously described as nucleic acid intercalation, food vacuole basification, parasite redox cycle interference and haem polymerization inhibition. That the activity of the series may be due to more than one route - i.e. a multifactorial activity - underlines the utility of these compounds in antimalarial research either as single drugs or as adjuvants (partners in a drug combination), particularly in the face of resistant parasitic strains.

Abstract: This study evaluated T cell immune responses to purified protein derivative (PPD) and Mycobacterium tuberculosis (Mtb) in health care workers who remained free of active tuberculosis (HCWs w/o TB), health care workers who went on to develop active TB (HCWs w/TB), non-health care workers who were TB free (Non-HCWs) and tuberculosis patients presenting with minimal (Min TB) or advanced (Adv TB) disease. Peripheral blood mononuclear cells (PBMC) were stimulated with Mtb and PPD and the expression of T cell activation markers CD25+ and HLA-DR+, intracellular IL-4 and IFN-gamma production and cytotoxic responses were evaluated. PBMC from HCWs who developed TB showed decreased percentages of cells expressing CD8+CD25+ in comparison to HCWs who remained healthy. HCWs who developed TB showed increased gammadelta TCR+ cell cytotoxicity and decreased CD3+gammadelta TCR- cell cytotoxicity in comparison to HCWs who remained healthy. PBMC from TB patients with advanced disease showed decreased percentages of CD25+CD4+ and CD25+CD8+ T cells that were associated with increased IL-4 production in CD8+ and gammadelta TCR+ phenotypes, in comparison with TB patients presenting minimal disease. TB patients with advanced disease showed increased gammadelta TCR+ cytotoxicity and reduced CD3+gammadelta TCR- cell cytotoxicity. Our results suggest that HCWs who developed TB show an early compensatory mechanism involving an increase in lytic responses of gammadelta TCR+ cells which did not prevent TB.

Abstract: The Carpobrotus edulis methanol extract, inactive against the methicillin-resistant Staphylococcus aureus or the multidrug-resistant Mycobacterium tuberculosis, does inhibit the growth of these two bacteria once they are phagocytosed by monocyte derived human macrophages.

Abstract: Expression of eight transporter genes of Escherichia coli K-12 and its DeltaacrAB mutant prior to and after induction of both strains to tetracycline resistance and after reversal of induced resistance were analyzed by quantitative reverse transcriptase PCR. All transporter genes were overexpressed after induced resistance with acrF being 80-fold more expressed in the DeltaacrAB tetracycline-induced strain.

Abstract: The INNO-LiPA Rif.TB assay for the identification of Mycobacterium tuberculosis complex strains and the detection of rifampin (RIF) resistance has been evaluated with 360 smear-positive respiratory specimens from an area of high incidence of multidrug-resistant tuberculosis (MDR-TB). The sensitivity when compared to conventional identification/culture methods was 82.2%, and the specificity was 66.7%; the sensitivity and specificity were 100.0% and 96.9%, respectively, for the detection of RIF resistance. This assay has the potential to provide rapid information that is essential for the effective management of MDR-TB.