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.

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: 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.

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: 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: 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: 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.

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: 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: 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: 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: 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: 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: 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: 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.

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.

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 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: 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: 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: 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 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.

Abstract: Patients presenting with Acquired Immune Deficiency Syndrome (AIDS) are predisposed to co-infection with Mycobacterium avium. The management of such patients is problematic due to underlying immuno-incompetence and the high resistance of M. avium to most non-toxic compounds. Therefore, the need for effective agents is obvious. Because phenothiazines, especially the relatively mild thioridazine, have significant activity against Mycobacterium tuberculosis, we investigated the in vitro activity of chlorpromazine, thioridazine, promazine, promethazine and desipramine against a reference and clinical strains of M. avium. The results obtained show that whereas all of the phenothiazines employed in this study had an minimum inhibitory concentration (MIC) against the strains studied that ranged from ca. 10 to > 50 mg/L, as was previously shown for M. tuberculosis, thioridazine was the most active of the group against M. avium.

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: Multidrug-resistant Mycobacterium tuberculosis (MDRTB) and antibiotic-resistant Plasmodium falciparum are the major global lethal infections accounting for over 4 million deaths per year. Methicillin-resistant Staphylococcus aureus (MRSA) is the major global nosocomial infection and resistance to vancomycin is evident and may become common. This review provides the scientific and medical basis that support the use of one particular group of compounds, the phenothiazines, and in particular thioridazine, for the management of the above antibiotic-resistant infections. Because thioridazine, a relatively mild neuroleptic as compared to its parental compound chlorpromazine, kills intracellular MDRTB and MRSA at clinical concentrations, its use for the management of these infections may be considered. The review also discusses the activity of phenothiazines against protozoa and parasites, the mechanisms by which phenothiazines promote their antimicrobial effects, their potential for regulating efflux pumps that are a cause for mono or multidrug resistance, as well as their potential for the therapy of problematic infections caused by bacteria that have acquired plasmid-antibiotic-resistant genes.

Abstract: Chlorpromazine (CPZ) is concentrated by human macrophages where it kills intracellular mycobacteria when the concentration outside the macrophage is sub-clinical. We have previously demonstrated that thioridazine (TZ), a much milder phenothiazine, has similar activity and kills intracellular methicillin-susceptible S. aureus at sub-clinical concentrations. We have extended this latter study to include methicillin-resistant S. aureus (MRSA) and show that TZ kills intracellular MRSA at clinically relevant concentrations. The ultrastructure of MRSA exposed to in vitro concentrations of TZ just below its MIC and that of MRSA phagocytosed by macrophages previously exposed to a clinically relevant concentration of TZ was also studied. TZ inhibits the replication of phagocytosed MRSA, affecting the structure of the cell envelope, resulting in lysis of the bacterium 6 hours post-phagocytosis. These ultrastructural changes are identical to those produced in vitro by a TZ concentration that is just below the MIC. Because macrophage intracellular MRSA is not killed by the macrophage and its intracellular location protects it from antibiotics that are unable to reach that site, recurrent infections which result may be successfully managed with the use of TZ.

Abstract: Although alkaloids from the family Aizoaceae have anticancer activity, species of this family have received little attention. Because these alkaloids also exhibit properties normally associated with compounds that have activity at the level of the plasma membrane, a methanol extract of Carpobrotus edulis, a common plant found along the Portuguese coast, was studied for properties normally associated with plasma membrane active compounds. The results of this study show that the extract is non-toxic at concentrations that inhibit a verapamil sensitive efflux pump of L5178 mouse T cell lymphoma cell line thereby rendering these multi-drug resistant cells susceptible to anticancer drugs. These non-toxic concentrations also prime THP-1 human monocyte-derived macrophages to kill ingested Staphylococcus aureus and to promote the release of lymphokines associated with cellular immune functions. The extract also induces the proliferation of THP-1 cells within 1 day of exposure to quantities normally associated with phytohaemagglutinin. The potential role of the compound(s) isolated from this plant in cancer biology is intriguing and is currently under investigation. It is supposed that the resistance modifier and immunomodulatory effect of this plant extract can be exploited in the experimental chemotherapy of cancer and bacterial or viral infections.

Abstract: Changes in protein kinase C (PKC) activity influence the progression of meiosis; however, the specific function of the various PKC isoforms in female gametes is not known. In the current study, the protein expression and subcellular distribution profile of PKC-delta (PKC-delta), a novel isoform of the PKC family, was determined in mouse oocytes undergoing meiotic maturation and following egg activation. The full-length protein was observed as a doublet (76 and 78 kDa) on Western blot analysis. A smaller (47 kDa) carboxyl-terminal fragment, presumably the truncated catalytic domain of PKC-delta, was also strongly expressed. Both the full-length protein and the catalytic fragment became phosphorylated coincident with the resumption of meiosis and remained phosphorylated throughout metaphase II (MII) arrest. Immunofluorescence staining showed PKC-delta distributed diffusely throughout the cytoplasm of oocytes during maturation and associated with the spindle apparatus during the first meiotic division. Discrete foci of the protein also localized with the chromosomes in some mature eggs. Following the completion of meiosis, PKC-delta became dephosphorylated within 2 h of in vitro fertilization or parthenogenetic activation. The protein also accumulated in the nuclei of early embryos and was phosphorylated during M-phase of the initial mitotic cleavage division. By the two-cell stage, expression of the truncated catalytic fragment was minimal. These data demonstrate that the subcellular distribution and posttranslational modification of PKC-delta is cell cycle dependent, suggesting that its activity and/or function likely vary with the progression of meiosis and egg activation.

Abstract: The phenothiazines chlorpromazine (CPZ) and thioridazine (TZ) have equal in vitro activities against antibiotic-sensitive and -resistant Mycobacterium tuberculosis. These compounds have not been used as anti-M. tuberculosis agents because their in vitro activities take place at concentrations which are beyond those that are clinically achievable. In addition, chronic administration of CPZ produces frequent severe side effects. Because CPZ has been shown to enhance the killing of intracellular M. tuberculosis at concentrations in the medium that are clinically relevant, we have investigated whether TZ, a phenothiazine whose negative side effects are less frequent and serious than those associated with CPZ, kills M. tuberculosis organisms that have been phagocytosed by human macrophages, which have nominal killing activities against these bacteria. Both CPZ and TZ killed intracellular antibiotic-sensitive and -resistant M. tuberculosis organisms when they were used at concentrations in the medium well below those present in the plasma of patients treated with these agents. These concentrations in vitro were not toxic to the macrophage, nor did they affect in vitro cellular immune processes. TZ thus appears to be a serious candidate for the management of a freshly diagnosed infection of pulmonary tuberculosis or as an adjunct to conventional antituberculosis therapy if the patient originates from an area known to have a high prevalence of multidrug-resistant M. tuberculosis isolates. Nevertheless, we must await the outcomes of clinical trials to determine whether TZ itself may be safely and effectively used as an antituberculosis agent.

Abstract: Mechanisms of antibiotic resistance of bacteria include efflux pumps which extrude the antibiotic prior to reaching its target. Phenothiazines inhibit the activity of some efflux pumps thereby altering the susceptibility of bacteria. This study demonstrated that chlorpromazine and thioridazine reduce the susceptibility of methicillin-resistant strains (MRSA) but not that of methicillin-susceptible Staphylococcus aureus (MSSA) strains to oxacillin (MIC of oxacillin reduced from >500 to 10 mg/l). Reserpine, an inhibitor of antibiotic efflux pumps also reduced the resistance of MRSA strains to oxacillin suggesting the presence of an efflux pump that contributes to antibiotic resistance of MRSA strains.

Abstract: Phenothiazines have activity against Schistosoma mansoni, Trypanosoma brucei, Trypanasoma gambiensi, Molinema dessetae, Leishmania spp., Plasmodium falciparum and free-living protozoa. These organisms and other parasitic infections are prevalent in HIV-infected humans. These infections are becoming more frequently resistant to commonly employed antibiotics, and due to the absence of economic motivation, new and effective compounds against these infections are not anticipated in the near future. Resistance of prokaryotes and eukaryotes to antibiotics is now known to be also due to the presence of efflux pumps that extrude the antibiotic prior to the agent reaching its target. Because phenothiazines are known to inhibit some efflux pumps and therefore alter the susceptibility of the organism to an antibiotic to which it is resistant, and also because of the sensitivity of the above parasites to phenothiazines, efflux pumps may play a role in emerging antibiotic resistance of these organisms. Furthermore, their prevalence is known to be greatest in areas that have high rates of HIV infection; therefore, it would be necessary that these agents should receive close scrutiny. This review concerns the attributes afforded by phenothiazines related to their effective activity against a wide range of parasites. Because these agents are inexpensive and many are no longer protected by patent, they may be exploited as anti-parasitic agents in the poorer areas of the world.

Abstract: The demonstration of the existence of active efflux pumps in mycobacteria raises the question of whether or not these can increase in number and activity rendering wild-type mycobacteria increasingly resistant to a given antibiotic. This could be a mechanism by which mutated resistant strains become better fit to the selective environment. Mycobacterium tuberculosis genome analysis reveals several genes encoding putative drug efflux pumps. During the course of tuberculosis chemotherapy many of these pumps might play a role in the survival of the mycobacterial populations. Compounds capable of inactivating these pumps could improve anti-tuberculous therapeutics.

Abstract: An American Type Culture Collection reference strain and eight clinical strains of Mycobacterium tuberculosis, all of which were susceptible to isoniazid (INH) (mean MIC, 0.06 mg/liter) and negative for the Ser315Thr katG mutation, were left in their BACTEC 12B vials (for use with the BACTEC 460-TB method) containing 0.1 mg of INH per liter for periods of up to 28 days after the completion of the antibiotic susceptibility test. Each eventually grew to levels compatible with those of INH-resistant strains. Successive passages in INH-containing BACTEC 12B vials and onto solid media showed that the resistance noted above was maintained. Successive passages of these M. tuberculosis strains in which INH resistance had been induced into BACTEC 12B vials or solid media containing stepwise increases in INH concentrations eventually yielded organisms resistant to 20 mg of INH per liter. Transfer of cells in which INH resistance had been induced to drug-free medium followed by repeated passages in that medium eventually yielded organisms whose susceptibility to INH was identical to that of the original parent strains. The cycle of induced INH resistance could be repeated with these now INH-susceptible cells. The use of M. tuberculosis identification probes and IS6110-based restriction fragment length polymorphism analyses of cultures throughout the induction of INH resistance and the reversal of resistance in drug-free medium eliminated the possibility that the culture was contaminated or that the initial specimen had a mixed type of infection. Induced high-level resistance to INH (20 mg/liter) could be reduced 100-fold with a subinhibitory concentration of reserpine but not with verapamil. These results collectively suggest that high-level resistance to INH can be induced in INH-susceptible M. tuberculosis strains by the induction of a reserpine-sensitive efflux mechanism.

Abstract: The effect of thioridazine (TZ) was studied on the killing activity of human peripheral blood monocyte derived macrophages (HPBMDM) and of human macrophage cell line THP-1 at extracellular concentrations below those achievable clinically. These macrophages have nominal killing activity against bacteria and therefore, would not influence any activity that the compounds may have against intracellular localised Staphylococcus aureus. The results indicated that whereas TZ has an in vitro minimum inhibitory concentration (MIC) against the strains of S. aureus of 18, 0.1 mg/l of TZ in the medium completely inhibits the growth of S. aureus that has been phagocytosed by macrophages. The latter concentration was non-toxic to macrophages, did not cause cellular expression of activation marker CD69 nor induction of CD3+ T cell production of IFN-gamma, but blocked cellular proliferation and down-regulated the production of T cell-derived cytokines (IFN-gamma, IL-5). These results suggest that TZ induces intracellular bactericidal activities independent of the capacity to generate Type 1 responses against S. aureus.

Abstract: Chlorpromazine (CPZ) has in vitro antimicrobial activity against Staphylococcus aureus at concentrations that greatly exceed those achieved clinically. It is concentrated by tissues that are rich in macrophages and it is active against phagocytosed mycobacteria when the concentration in the medium is compatible with that achieved clinically. In this report we show that nontoxic concentrations of CPZ below clinical levels have killing activity against S. aureus phagocytosed by human monocyte-derived macrophages that have nominal killing activity against these bacteria. Little or no resistance to the antimicrobial activity of this compound is anticipated to result because of its large number of cellular targets. Therefore, CPZ may have a role in the management of intracellular staphylococcal infections that normally require the use of antibiotics whose potential toxicity exceeds that associated with short-term management with CPZ.

Abstract: The in vitro and in vivo anti-mycobacterial activities of a number of phenothiazine compounds are reviewed. These compounds, normally employed for the management of psychosis, inhibit the growth in vitro of Mycobacterium tuberculosis at concentrations that are significantly greater than those that can safely be achieved in a patient harbouring these infections. Nevertheless, one of these phenothiazines, chlorpromazine, is concentrated by human macrophages to 10-100 times its concentration in plasma, and has activity against mycobacteria that have been phagocytosed by these cells. Phenothiazines have significant in vitro activity against susceptible, polydrug- and multidrug-resistant strains of M. tuberculosis, as well as enhancing the activity of some agents employed for first-line treatment. Because thioridazine, the very mild anti-psychotic agent whose most common side effect is drowsiness, has equal anti-tuberculosis properties in vitro to chlorpromazine, we recommend that thioridazine be studied as an adjuvant to the four- or five-drug regimens employed for the management of a freshly diagnosed tuberculosis infection of unknown antibiotic susceptibility, at least during the period required for the assessment of antibiotic susceptibility. Because it also enhances the activity of rifampicin and streptomycin, antibiotics that frequently have adverse effects, additional studies evaluating the use of thioridazine as an adjuvant may eventually allow a reduction in the dosages of these antibiotics and result in a decreased frequency of adverse effects. It is important to note that whereas the management of patients with thioridazine for periods in excess of many months will result in the appearance of some undesirable side effects, its use for a limited period of 2-3 months should not produce side effects that are more severe than simple drowsiness. Nevertheless, further in vitro and in vivo studies are essential before thioridazine may be recommended for the management of select cases of pulmonary tuberculosis.

Abstract: Phenothiazines have been shown to inhibit the in vitro growth of multi-drug resistant (resistant to rifampicin and isoniazid) Mycobacterium tuberculosis (MDRTB). They have been considered as potential adjuvants to regimens employing four or more antibiotics for the management of freshly diagnosed infections of M. tuberculosis in patients from areas known to have a high prevalence of MDRTB. Chlorpromazine has been shown to enhance the activity of antibiotics (except ethambutol) to which M. tuberculosis is susceptible. This might result in a reduction in the dose of some or all of the antibiotics employed without sacrificing the integrity of treatment. Chlorpromazine, thioridazine and promethazine were shown to enhance the activity of rifampicin and streptomycin when used in combinations at concentrations that are minimally effective when employed separately against clinical strains of M. tuberculosis resistant to two or more antibiotics (poly-drug resistant MTB). The phenothiazines had no effect on the activity of isoniazid against poly-drug resistant MTB.

Abstract: The in vitro and in vivo activity of phenothiazines against antibiotic susceptible and antibiotic resistant Mycobacterium tuberculosis and malaria-causing Plasmodia is reviewed. Given the facts that pulmonary tuberculosis and malaria are the major causes of death in developing countries, that both of these infections continue to escalate in their resistance to antibiotics, that the cost for the management of these infections is beyond that afforded by most developing nations, and lastly, that new and effective agents are not forthcoming from the pharmaceutical industry, the scientific rationale for the potential use of select phenothiazines for the management of these infections is presented.

Abstract: SETTING: Multidrug-resistant tuberculosis (MDR-TB) mainly among human immunodeficiency virus (HIV) seropositive patients in Lisbon hospitals in 1996-1997. OBJECTIVE: Detection of transmission of MDR-TB strains and epidemic outbreaks in several hospital units in the city of Lisbon, including a prison hospital. DESIGN: Use of restriction fragment length polymorphism (RFLP) to fingerprint isolates of Mycobacterium tuberculosis resistant to isoniazid, rifampicin, and one other drug. RESULTS: A total of 43 MDR-TB strains were typed. Sixty-seven per cent of the patients were HIV positive, 12% were HIV negative, and the remainder had unknown HIV status. About 88% of the isolates were grouped in three genetically similar clusters, suggesting possible recent transmission. A predominant cluster (cluster A), corresponding to 72% of the cases, was found, 45% of which came from the prison hospital. Strains from this cluster were resistant to isoniazid, rifampicin, streptomycin, and sometimes ethambutol. A retrospective epidemiological investigation was conducted with respect to all patients in cluster A, and epidemiological links were established between them. CONCLUSION: Our results suggest recent transmission of MDR-TB, mainly in HIV-positive patients, in Lisbon hospitals. Moreover, the predominant MDR-TB clustered strains were not confined to HIV-infected individuals, as they were also isolated in some immunocompetent patients.

Abstract: SETTING: Egas Moniz Hospital, Lisbon, Portugal. OBJECTIVE: To evaluate the Ligase Chain Reaction (LCx) Mycobacterium tuberculosis Assay for the direct detection of M. tuberculosis complex in respiratory specimens after smear observation, and its suitability for non-respiratory clinical specimens. DESIGN: Analysis of 156 specimens collected from 123 patients with pulmonary tuberculosis and/or extrapulmonary involvement. RESULTS: Among 93 pulmonary secretions and 63 extra-pulmonary samples and after resolution of discrepancies based on clinical and laboratory findings, two pulmonary samples from a patient with a diagnosis of sarcoidosis, four samples of cerebrospinal and one of seminal fluid were considered as false positives. Two tissue biopsy samples, one pericardial effusion and one pulmonary secretion from patients strongly suspected of having tuberculosis were considered as false negatives for the assay, without inhibition of amplification. All specimens yielding M. avium on culture were LCx negative. CONCLUSION: The LCx Mycobacterium tuberculosis Assay was found to be useful for the rapid identification of M. tuberculosis complex in all types of specimens. It revealed a high specificity both in pulmonary and extrapulmonary products, and a sensitivity of 97% for the pulmonary secretions and of 75% for the extra-pulmonary specimens, independently of the bacilloscopy results.

Abstract: A mycobacteriophage D29 DNA fragment cloned in pRM64, a shuttle plasmid that transforms Mycobacterium smegmatis, was sequenced. The determined sequence was 2592 nucleotides long and had a mean G+C content of 63.7 mol%, similar to that of mycobacterial DNA. Four ORFs were identified: one with strong homology to dCMP deaminase genes; one homologous to mycobacteriophage L5 gene 36, whose function is unknown; one encoding a possible excisase; and one encoding an integrase. The intergenic region between the putative excisase gene and the integrase gene had a lower than average G+C content and showed the presence of the same attP core sequence as mycobacteriophage L5. Transformation experiments using subclones of pRM64 indicated that the integrase gene and all the intergenic region were essential for stable transformation. A subclone containing the integrase gene and the core attP sequence was able to transform but recombinants were highly unstable. Southern analysis of total DNA from cells transformed with pRM64 and its derivatives showed that all the plasmids were integrated at one specific site of the bacterial chromosome. A recombinant exhibiting a high level of resistance to the selective drug kanamycin had two plasmids integrated at different sites. These results demonstrated that the D29 sequences contained in pRM64 were integrative, indicating that the generally hold view of D29 as a virulent phage must be reviewed.