Abstract: Vesicles shed by cancer cells are known to mediate several tumor-host interactions. Tumor microenvironment may, in turn, influence the release and the activity of tumor-shed microvesicles. In this study, we investigated the molecular mediators of the pH-dependent proinvasive activity of tumor-shed vesicles. Gelatinase zymography showed increased microvesicle activity of matrix metalloproteinases 9 and 2 as a result of acid exposure (pH 5.6) compared to pH 7.4. Thus, we reasoned that the cysteine protease cathepsin B might play a role in mediating the pH-dependent activation of gelatinases. Cathepsin B expression in tumor-shed microvesicles was confirmed by Western blot analysis and zymography. The activity of vesicle-associated cathepsin B measured using Z-Arg-Arg-pNA as substrate was significantly increased at acidic pH values. Inhibition of protease activity by the cysteine protease inhibitor, E-64, and treatment of ovarian cancer cells with small interfering RNA against cathepsin B suppressed the ability of tumor-shed microvesicles to stimulate both gelatinase activation and the invasiveness of endothelial cells observed at low pH values. We conclude that microvesicle shedding is a major secretory pathway for cathepsin B release from tumor cells. Hence, the acidic microenvironment found in most solid tumors may contribute to cathepsin B-mediated proinvasive capabilities of tumor-shed vesicles.
Abstract: By computer modelling and protein engineering we have investigated changes in two amino acid residues located in the coenzyme pocket of the yeast Kluyveromyces lactis mitochondrial alcohol dehydrogenase III. These two residues, Gly 225 and Ala 274, were hypothesized to be involved in the enzyme discrimination between NAD(H) and NADP(H). Upon changing Gly 225 to Ala we produced an enzyme (mutant G225A) showing very little difference from the wild-type. On the contrary, change at position 274 of Phe instead of Ala (mutant A274F) caused a significant increase of K(m) values for NAD(P) and for NADPH and even a more marked decrease in catalytic activity. The k(cat)/K(m) rates for NADP(H) were also decreased in this mutant. Enzymes with the double changes at 225 and 274 (mutant G225A-A274F) showed, apart the substantial low K(m) value for NADPH and its high catalytic efficiency, kinetic parameters relative to coenzymes which were not additive over the single substitutions. Surprisingly, enzymes with changes at the two positions reduced efficiently acetaldehyde, displaying a K(m) value 10-fold lower and a catalytic efficiency sevenfold higher with respect to parent or singularly mutated enzymes. None of the engineered enzymes would convert formaldehyde, glutaraldehyde or aromatic aldehydes but all enzymes reduced propionaldehyde and butyraldehyde at relative reaction rates approximately half of that exhibited by acetaldehyde. Interestingly only mutant A274F was able to oxidize methanol almost as well as ethanol. In addition, this mutant was capable to convert secondary and cyclic alcohols, at a rate not detected in the other isoforms. These results are in general agreement with the prediction that increasing the size of amino acids in the proximity of the coenzyme pocket would hamper the accommodation of NADP but discord the increased affinity for NADPH as well as for alcoholic or aldehydic substrates with high steric hindrance.
Abstract: Three mutants of the extended-spectrum beta-lactamase TEM-60, the P51L, K104E, and S164R mutants, were constructed by site-directed mutagenesis. The kinetic parameters of the mutated enzymes and interactions of inhibitors were significantly different from those of TEM-60, revealing that the L51P mutation plays an important role in enzyme activity and stability in the TEM-60 background.
Abstract: The antimicrobial susceptibility of 103 clinical isolates of Enterobacteriaceae to 11 antibiotics, was investigated, using a conventional inoculum size (5 x 10(5) CFU) and a higher inoculum size (5 x 10(8) CFU). All the isolates produced complex beta-lactamase patterns, including an extended-spectrum beta-lactamase (ESBL) of the TEM- or SHV-type plus other enzymes (a TEM-type or an SHV-type non-ESBL and/or a class C enzyme). The following repertoire of ESBLs was produced by the isolates: TEM-15, TEM-19, TEM-26, TEM-52, TEM-72, TEM-87, TEM-92, SHV-2a, SHV-5 and SHV-12, as assessed by sequencing. Production of the other enzymes was showed by analytical isoelectric focusing. Overall, meropenem was the most active agent and less influenced by inoculum size, while other beta-lactams showed a lower activity and a significant inoculum size effect. In conclusion, from its in vitro performance, meropenem could be considered as the last resource drug against strains producing complex beta-lactamase patterns including an ESBL.
Abstract: The CphA metallo-beta-lactamase produced by Aeromonas hydrophila exhibits two zinc-binding sites. Maximum activity is obtained upon binding of one zinc ion, whereas binding of the second zinc ion results in a drastic decrease in the hydrolytic activity. In this study, we analyzed the role of Asn116 and Cys221, two residues of the active site. These residues were replaced by site-directed mutagenesis and the different mutants were characterized. The C221S and C221A mutants were seriously impaired in their ability to bind the first, catalytic zinc ion and were nearly completely inactive, indicating a major role for Cys221 in the binding of the catalytic metal ion. By contrast, the binding of the second zinc ion was only slightly affected, at least for the C221S mutant. Mutation of Asn116 did not lead to a drastic decrease in the hydrolytic activity, indicating that this residue does not play a key role in the catalytic mechanism. However, the substitution of Asn116 by a Cys or His residue resulted in an approximately fivefold increase in the affinity for the second, inhibitory zinc ion. Together, these data suggested that the first zinc ion is located in the binding site involving the Cys221 and that the second zinc ion binds in the binding site involving Asn116 and, presumably, His118 and His196.
Abstract: The crystal structure of the class-B beta-lactamase, BlaB, from the pathogenic bacterium, Chryseobacterium meningosepticum, in complex with the inhibitor, d-captopril, has been solved at 1.5-A resolution. The enzyme has the typical alphabeta/betaalpha metallo-beta-lactamase fold and the characteristic two metal binding sites of members of the subclass B1, in which two Zn2+ ions were identified. d-Captopril, a diastereoisomer of the commercial drug, captopril, acts as an inhibitor by displacing the catalytic hydroxyl ion required for antibiotic hydrolysis and intercalating its sulfhydryl group between the two Zn2+ ions. Interestingly, d-captopril is located on one side of the active site cleft. The x-ray structure of the complex of the closely related enzyme, IMP-1, with a mercaptocarboxylate inhibitor, which also contains a sulfhydryl group bound to the two Zn2+ ions, shows the ligand to be located on the opposite side of the active site cleft. A molecule generated by fusion of these two inhibitors would cover the entire cleft, suggesting an interesting approach to the design of highly specific inhibitors.
Abstract: The interaction between meropenem and class A, B, C and D beta-lactamases was studied by a spectrophotometric method. Class A, C and D beta-lactamases were unable to confer in vitro resistance to carbapenems. Surprisingly, several class B metallo-beta-lactamases expressed in Escherichia coli failed to confer resistance when a conventional inoculum (105 cfu/mL) was used.
Abstract: A new natural TEM derivative, named TEM-87, was identified in a Proteus mirabilis isolate from an Italian hospital. Compared to TEM-1, TEM-87 contains the following mutations: E104K, R164C, and M182T. Kinetic analysis of TEM-87 revealed extended-spectrum activity against oxyimino cephalosporins (preferentially ceftazidime) and aztreonam. Expression of blaTEM-87 in Escherichia coli decreased the host susceptibility to these drugs.
Abstract: Beta-lactams represent one of the most important class of antibiotics for the treatment of infectious diseases due to pathogenic bacteria. The selective pressure exerted from the wide spread use of third generation cephalosporins generated mutant beta-lactamases belonging mainly to the TEM or SHV family that are able to extend the activity spectrum of hydrolysis. Moreover, extended spectrum cephalosporins are often a good choice in clinical practice towards Enterobacteriaceae. Here we report a comparative analysis of stability between cefotaxime, desacetyl-cefotaxime and ceftazidime with some common TEM-derivatives extended spectrum beta-lactamases.
Abstract: Stenotrophomonas maltophilia is a nosocomial pathogen with an intrinsic broad-spectrum resistance to beta-lactam compounds and other antibacterial agents. It produces two chromosomal beta-lactamases: a clavulanic acid-sensitive class A (L2) and a tetrameric carbapenemase (L1 or BlaS). We screened 40 S. maltophilia multidrug-resistant clinical isolates recovered between 1995 and 1998 in the Varese Hospital (Italy) for the presence of the metallo-beta-lactamase. The isolates were investigated by phenotypic profiling (enzymatic activity and antibiotic resistance pattern) and molecular methods such as PCR and pulsed-field gel electrophoresis (PFGE) to reveal intraspecies diversity. For the tested S. maltophilia strains, we showed that the beta-lactamase production could be induced by the presence of imipenem (50 microg/ml) in the culture media. Addition of 1 mM dipicolinic acid completely inhibited the hydrolysis of imipenem but decreased that nitrocefin in a strain-dependent manner. Full activity of crude extract towards imipenem could be restored by addition of 1 mM ZnCl2. Finally, the gene encoding the carbapenem-hydrolyzing beta-lactamase from S. maltophilia ULA-511 and 39/95, a clinical strain, were isolated and sequenced. These two strains have a different profile of multidrug resistance. The two metallo-beta-lactamases were found to be isologous. The difference of sensitivity of these two strains was associated to the level of production of the metallo-beta-lactamase.
Abstract: The BlaB metallo-beta-lactamase of Chryseobacterium meningosepticum CCUG4310 was overproduced in Escherichia coli by means of a T7 promoter-based expression system. The overproducing system, scaled up in a 15-liter fermentor, yielded approximately 10 mg of BlaB protein per liter, mostly released in the culture supernatant. The enzyme was purified by two ion-exchange chromatographic steps with an overall yield of 66%. Analysis of the kinetic parameters revealed efficient activities (k(cat)/K(m) ratios of >10(6) M(-1) s(-1)) toward most penam and carbapenem compounds, with the exception of the 6-alpha-methoxypenam derivative temocillin and of biapenem, which were poorer substrates. Hydrolysis of cephalosporins was overall less efficient, with a remarkable variability that was largely due to variable affinities of the BlaB enzyme for different compounds. BlaB was also able to hydrolyze serine-beta-lactamase inhibitors, including beta-iodopenicillanate, sulbactam and, although less efficiently, tazobactam.
Abstract: We have investigated the influence of substrate binding on the zinc ion affinity of representatives from the three metallo-beta-lactamase subclasses, B1 (BcII from Bacillus cereus and BlaB from Chryseobacterium meningosepticum), B2 (CphA from Aeromonas hydrophila), and B3 (L1 from Stenotrophomonas maltophilia). By competition experiments with metal-free apoenzymes and chromophoric zinc chelators or EDTA, we determined the dissociation constants in the absence and presence of substrates. For the formation of the monozinc enzymes we determined constants of 1.8, 5.1, 0.007, and 2.6 nm in the absence and 13.6, 1.8, 1.2, and 5.7 pm in the presence of substrates for BcII, BlaB, CphA, and L1, respectively. A second zinc ion binds in the absence (presence) of substrates with considerably higher dissociation constants, namely 1.8 (0.8), 0.007 (0.025), 50 (1.9), and 0.006 (0.12) microm for BcII, BlaB, CphA, and L1, respectively. We have concluded that the apo form might be the prevailing state of most of the metallo-beta-lactamases under physiological conditions in the absence of substrates. Substrate availability induces a spontaneous self-activation due to a drastic decrease of the dissociation constants, resulting in the formation of active mononuclear enzymes already at picomolar free zinc ion concentrations. In the presence of substrates, the binuclear state of the enzymes only exists at unphysiologic high zinc concentrations and might be of no biological relevance. From the competition experiments with EDTA it is further concluded that the reactivation rate does not depend on the pool of free zinc ions but proceeds via the EDTA-Zn(II)-enzyme ternary complexes.
Abstract: Ceftibuten is an oral third-generation cephalosporin active against a wide range of bacteria and shows an improved stability to hydrolysis by several beta-lactamases because of the carboxyethilidine moiety at position 7 of the ss-acyl side chain. The kinetic interactions between ceftibuten and active-site serine and metallo-ss-lactamases were investigated. The activity of several TEM-derived extended spectrum beta-lactamases (ESbetaLs) against ceftibuten, cefotaxime and ceftazidime was compared using K(m), K(cat) and K(cat)/K(m). Ceftibuten behaved as a poor substrate for class A and B beta-lactamases compared with cefotaxime. The chromosomal class C beta-lactamase from Enterobacter cloacae 908R gave a high K(cat) value (21 s(-1)), whereas there was poor activity with enzymes from Acinetobacter baumannii and Morganella morganii and ceftibuten. Ceftibuten resists hydrolysis in the presence of typical respiratory or urogenital-tract pathogens producing beta-lactamases.
Abstract: A class D beta-lactamase determinant was isolated from the genome of Legionella (Fluoribacter) gormanii ATCC 33297(T). The enzyme, named OXA-29, is quite divergent from other class D beta-lactamases, being more similar (33 to 43% amino acid identity) to those of groups III (OXA-1) and IV (OXA-9, OXA-12, OXA-18, and OXA-22) than to other class D enzymes (21 to 24% sequence identity). Phylogenetic analysis confirmed the closer ancestry of OXA-29 with members of the former groups. The OXA-29 enzyme was purified from an Escherichia coli strain overexpressing the gene via a T7-based expression system by a single ion-exchange chromatography step on S-Sepharose. The mature enzyme consists of a 28.5-kDa polypeptide and exhibits an isoelectric pH of >9. Analysis of the kinetic parameters of OXA-29 revealed efficient activity (k(cat)/K(m) ratios of >10(5) M(-1) x s(-1)) for several penam compounds (oxacillin, methicillin, penicillin G, ampicillin, carbenicillin, and piperacillin) and also for cefazolin and nitrocefin. Oxyimino cephalosporins and aztreonam were also hydrolyzed, although less efficiently (k(cat)/K(m) ratios of around 10(3) M(-1) x s(-1)). Carbapenems were neither hydrolyzed nor inhibitory. OXA-29 was inhibited by BRL 42715 (50% inhibitory concentration [IC(50)], 0.44 microM) and by tazobactam (IC(50), 3.2 microM), but not by clavulanate. It was also unusually resistant to chloride ions (IC(50), >100 mM). Unlike OXA-10, OXA-29 was apparently found as a dimer both in diluted solutions and in the presence of EDTA. Its activity was either unaffected or inhibited by divalent cations. OXA-29 is a new class D beta-lactamase that exhibits some unusual properties likely reflecting original structural and mechanistic features.
Abstract: Ten quinolone-resistant mutants of Citrobacter freundii, which were selected in vitro with fluoroquinolones from two clinical isolates, were studied. The parent isolates were susceptible to quinolones in spite of showing a single substitution in the GyrB (His-417 --> Leu). No change was observed in the outer membrane proteins or in the lipopolysaccharide in any of the ten mutants studied with respect to their parent isolates. The development of quinolone resistance in selected mutants was associated with the appearance of a substitution in the GyrA (Thr-83 --> Ile) in nine of the ten mutants plus enhanced active efflux in all of them.
Abstract: Levofloxacin has been reported to have in vitro activity against both gram-positive and gram-negative bacteria. A recent survey carried out at our Institution showed clinical isolates of Pseudomonas aeruginosa to be more susceptible to levofloxacin than to ciprofloxacin. The in vitro activity of the two fluoroquinolones was evaluated further by looking at their bactericidal activity against two strains of each of the following antibio-phenotypes of P. aeruginosa: levofloxacin- and ciprofloxacin-susceptible, levofloxacin-susceptible/ciprofloxacin-resistant, levofloxacin-susceptible/ciprofloxacin-susceptible and ceftazidime-resistant, (National Committee for Clinical Laboratory Standards susceptibility breakpoints were used). MIC and MBC values were measured and time-kill experiments were carried out. Drugs were used at susceptibility or resistance breakpoint concentrations in the time-kill experiments and results were recorded over 12 h in an attempt to link in vitro results with the clinical situation The polypeptide profiles of outer membrane preparations of the six strains were examined by gel electrophoresis. Levofloxacin was shown to be more bactericidal than ciprofloxacin in the time-kill experiments. No differences were observed between the outer membrane proteins of the six strains. Levofloxacin showed greater bactericidal activity against P. aeruginosa clinical isolates than ciprofloxacin.
Abstract: Aeromonas spp. are increasingly being recognized as human pathogens. The presence of metallo-beta-lactamases in these organisms represents a potential problem in antimicrobial therapy. Mechanism-based inactivators of beta-lactamases are used to overcome the resistance of clinical pathogens to beta-lactam antibiotics, but no clinical useful inhibitors of the metallo-beta-lactamases are presently known. Studying the interaction between cefotetan and Aeromonas spp. producing metallo-beta-lactamase activity, we observed that cefotetan behaved as a transient inactivator for both the crude extracts of Aeromonas strains and the purified enzymes from Aeromonas hydrophila AE036 and Aeromonas schubertii MNSA20. The direct hydrolysis of cefotetan showed that it was a poor substrate for both purified enzymes. In view of the minimum inhibitory concentrations, cefotetan shows to be a useful antimicrobial agent against Aeromonas spp.
Abstract: VIM-1 is a new group 3 metallo-beta-lactamase recently detected in carbapenem-resistant nosocomial isolates of Pseudomonas aeruginosa from the Mediterranean area. In this work, VIM-1 was purified from an Escherichia coli strain carrying the cloned bla(VIM-1) gene by means of an anion-exchange chromatography step followed by a gel permeation chromatography step. The purified enzyme exhibited a molecular mass of 26 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an acidic pI of 5.1 in analytical isoelectric focusing. Amino-terminal sequencing showed that mature VIM-1 results from the removal of a 26-amino-acid signal peptide from the precursor. VIM-1 hydrolyzes a broad array of beta-lactam compounds, including penicillins, narrow- to expanded-spectrum cephalosporins, carbapenems, and mechanism-based serine-beta-lactamase inactivators. Only monobactams escape hydrolysis. The highest catalytic constant/K(m) ratios (>10(6) M(-1). s(-1)) were observed with carbenicillin, azlocillin, some cephalosporins (cephaloridine, cephalothin, cefuroxime, cefepime, and cefpirome), imipenem, and biapenem. Kinetic parameters showed remarkable variability with different beta-lactams and also within the various penam, cephem, and carbapenem compounds, resulting in no clear preference of the enzyme for any of these beta-lactam subfamilies. Significant differences were observed with some substrates between the kinetic parameters of VIM-1 and those of other metallo-beta-lactamases. Inactivation assays carried out with various chelating agents (EDTA, 1,10-o-phenanthroline, and pyridine-2,6-dicarboxylic acid) indicated that formation of a ternary enzyme-metal-chelator complex precedes metal removal from the zinc center of the protein and revealed notable differences in the inactivation parameters of VIM-1 with different agents.
Abstract: The metallo-beta-lactamase determinant of Acinetobacter baumannii AC-54/97, a clinical isolate from Italy that was previously shown to produce an enzyme related to IMP-1, was isolated by means of a PCR methodology which targets amplification of gene cassette arrays inserted into class 1 integrons. Sequencing revealed that this determinant was an allelic variant (named bla(IMP-2)) of bla(IMP) found in Japanese isolates and that it was divergent from the latter by 12% of its nucleotide sequence, which evidently had been acquired independently. Similar to bla(IMP), bla(IMP-2) was also carried by an integron-borne gene cassette. However, the 59-base element of the bla(IMP-2) cassette was unrelated to those of the bla(IMP) cassettes found in Japanese isolates, indicating a different phylogeny for the gene cassettes carrying the two allelic variants. Expression of the integron-borne bla(IMP-2) gene in Escherichia coli resulted in a significant decrease in susceptibility to a broad array of beta-lactams (ampicillin, carbenicillin, cephalothin, cefoxitin, ceftazidime, cefepime, and carbapenems). The IMP-2 enzyme was purified from an Escherichia coli strain carrying the cloned determinant, and kinetic parameters were determined with several beta-lactam substrates. Compared to IMP-1, the kinetic parameters of IMP-2 were similar overall with some beta-lactam substrates (cefoxitin, ceftazidime, cefepime, and imipenem) but remarkably different with others (ampicillin, carbenicillin, cephaloridine, and meropenem), revealing a functional significance of at least some of the mutations that differentiate the two IMP variants. Present findings suggest that the environmental reservoir of bla(IMP) alleles could be widespread and raise a question about the global risk of their transfer to clinically relevant species.
Abstract: The interaction between tazobactam and several chromosome- and plasmid-encoded (TEM, SHV, PSE types) class A and C beta-lactamases was studied by spectrophotometry. Tazobactam behaved as a competitive inhibitor or inactivator able to restore in several cases the efficiency of piperacillin as a partner beta-lactam. A detailed kinetic analysis permitted measurement of the acylation efficiency for some cephalosporinases and broad-spectrum beta-lactamases; the presence of a turn-over of acyl-enzyme complex was also evaluated.
Abstract: A national survey on susceptibility patterns of 334 Pseudomonas aeruginosa isolates from intensive care units and hematology and oncology wards from 13 Italian hospitals compared the in vitro activity of levofloxacin, an injectable oral fluoroquinolone, to those of ciprofloxacin, ofloxacin, ceftazidime, imipenem, amikacin, and gentamicin. Amikacin and imipenem had the best susceptibility profiles. The activity of levofloxacin was superior to those of the other quinolones and was comparable to that of ceftazidime. The effect of levofloxacin in vitro on P. aeruginosa clinical isolates suggests that further clinical investigations are warranted.
Abstract: In addition to the BlaB metallo-beta-lactamase, Chryseobacterium (Flavobacterium) meningosepticum CCUG 4310 (NCTC 10585) constitutively produces a 31-kDa active-site serine beta-lactamase, named CME-1, with an alkaline isoelectric pH. The blaA(CME) gene that encodes the latter enzyme was isolated from a genomic library constructed in the Escherichia coli plasmid vector pACYC184 by screening for cefuroxime-resistant clones. Sequence analysis revealed that the CME-1 enzyme is a new class A beta-lactamase structurally divergent from the other members of this class, being most closely related to the VEB-1 (also named CEF-1) and PER beta-lactamases and the Bacteroides chromosomal cephalosporinases. The blaA(CME) determinant is located on the chromosome and exhibits features typical of those of C. meningosepticum resident genes. The CME-1 protein was purified from an E. coli strain that overexpresses the cloned gene via a T7-based expression system by means of an anion-exchange chromatography step followed by a gel permeation chromatography step. Kinetic parameters for several substrates were determined. CME-1 is a clavulanic acid-susceptible extended-spectrum beta-lactamase that hydrolyzes most cephalosporins, penicillins, and monobactams but that does not hydrolyze cephamycins and carbapenems. The enzyme exhibits strikingly different kinetic parameters for different classes of beta-lactams, with both K(m) and k(cat) values much higher for cephalosporins than for penicillins and monobactams. However, the variability of both kinetic parameters resulted in overall similar acylation rates (k(cat)/K(m) ratios) for all types of beta-lactam substrates.
Abstract: The blaIMP gene coding for the IMP-1 metallo-beta-lactamase produced by a Pseudomonas aeruginosa clinical isolate (isolate 101/1477) was overexpressed via a T7 expression system in Escherichia coli BL21 (DE3), and its product was purified to homogeneity with a final yield of 35 mg/liter of culture. The structural and functional properties of the enzyme purified from E. coli were identical to those of the enzyme produced by P. aeruginosa. The IMP-1 metallo-beta-lactamase exhibits a broad-spectrum activity profile that includes activity against penicillins, cephalosporins, cephamycins, oxacephamycins, and carbapenems. Only monobactams escape its action. The enzyme activity was inhibited by metal chelators, of which 1,10-o-phenanthroline and dipicolinic acid were the most efficient. Two zinc-binding sites were found. The zinc content of the P. aeruginosa 101/1477 metallo-beta-lactamase was not pH dependent.
Abstract: OBJECTIVE: The matrix metalloproteinases (MMPs) play a key role in the extracellular matrix turnover. This protein family has been involved in some ocular pathologies such as glaucoma, diabetic retinopathy, macular degeneration, vitreous degeneration and corneal stroma ulceration cleaving all the matrix components. In the present study we evaluated the action of leucocyanidin from Vitis vinifera seeds as non toxic inhibitor of these proteinases. MATERIALS AND METHODS: To this purpose we used a fluorimetric method to evaluate the effect of this substance on the collagenase activity. We evaluated "in vitro" the inhibitory potency of the tested drug on type III collagenase activity, and the recover of the metalloprotease activity upon removal by dialysis of the inhibitor. RESULTS: The leucocyanidines extract (minimum procyanidines value of 95.0) resulted to be a good collagenase activity inhibitor showing an inhibition constant value, Ki, of 82 microM, evident index of affinity between the extract and the enzyme. Furthermore, the dialysis experiments demonstrated that the inhibitory effect persisted 24 h later, probably because the extract forms a stable complex with the enzyme. CONCLUSIONS: These results should be related to the pharmacokinetic profile of leucoanthocyanins, a family of natural polyphenols belonging to the class of bioflavonoids of grape seds extract (Vitis vinifera L.).
Abstract: The interaction between tazobactam and several chromosome- and plasmid-encoded (TEM, SHV, PSE types) class A and C beta-lactamases was studied by spectrophotometry. Tazobactam behaved as a competitive inhibitor or inactivator able to restore in several cases the efficiency of piperacillin as a partner beta-lactam. A detailed kinetic analysis permitted measurement of the acylation efficiency for some cephalosporinases and broad-spectrum beta-lactamases; the presence of a turn-over of acyl-enzyme complex was also evaluated.
Abstract: The effect of fluoroquinolones in Citrobacter freundii strains that results in a decreased expression of cephalosporin-hydrolysing beta-lactamases was studied. Resistance to broad-spectrum cephalosporins and penicillins in two C. freundii clinical isolates was associated with moderate production of chromosomal AmpC-type-beta-lactamase in addition to changes in the outer membrane proteins profile with respect to wild-type C. freundii strains. Ten quinolone-resistant mutants were derived from the two clinical isolates using increasing fluoroquinolone concentrations. The level of susceptibility to cephalosporins and meropenem of these 10 mutants was increased and was associated with a 3.6-32% diminution in the hydrolyzing activity of their periplasmic extracts containing beta-lactamases on cephaloridine as compared with those from their parent strains. Susceptibility to cephalosporins and meropenem, as well as the expression of chromosomal AmpC-type-beta-lactamase in C. freundii strains, was influenced by the exposure to quinolones.
Abstract: PURPOSE: Matrix metalloproteinases (MMPs) play an important role in the degradation of articular cartilage in several diseases, including osteorthritis and rheumatoid arthritis. Aiming at developing new drugs with selective inhibiting action against enzyme damaging the extracellular matrix, research is mainly directed towards the: 1) development of new drugs with specific inhibitory effect on MMPs; 2) better understanding of the pharmacologic profile of drugs already used in the treatment of rheumatic diseases, in order to identify those having an inhibiting action on degradative enzymes. MATERIALS AND METHODS: The interaction between rifamycins and collagenase type XI were studied using a fluorogenic substrate MOCAc-Pro-Leu-Gly-Leu-A2pr(Dnp)-Ala-Arg-NH2. RESULTS: In our experimental conditions rifamycins showed a marked inhibition capacity with a IC50 ranging from 13 to 20.7 microM. This inhibition was reversible after extensive dialysis. CONCLUSIONS: Our results indicate that the effects of rifamycins in rheumatoid arthritis may correlate to the inhibitory activity of these molecules on collagenase activity.
Abstract: A plasmid-encoded beta-lactamase produced from a clinical strain of Providencia stuartii has been purified and characterized. The gene coding for the beta-lactamase was cloned and sequenced. It appears to be a new natural TEM-derived enzyme, named TEM-60. Point mutations (Q39K, L51P, E104K, and R164S) are present with respect to the TEM-1 enzyme; the mutation L51P has never been previously reported, with the exception of the chromosomally encoded extended-spectrum beta-lactamase PER-1. Kinetic parameters relative to penicillins, cephalosporins, and monobactams other than mechanism-based inactivators were related to the in vitro susceptibility phenotype.
Abstract: The metallo-beta-lactamase produced by Chryseobacterium (formerly Flavobacterium) meningosepticum, which is the flavobacterial species of greatest clinical relevance, was purified and characterized. The enzyme, named BlaB, contains a polypeptide with an apparent Mr of 26000, and has a pI of 8.5. It hydrolyses penicillins, cephalosporins (including cefoxitin), carbapenems and 6-beta-iodopenicillanate, a mechanism-based inactivator of active-site serine beta-lactamases. The enzyme was inhibited by EDTA, 1-10 phenanthroline and pyridine-2,6-dicarboxylic acid, with different inactivation parameters for each chelating agent. The C. meningosepticum blaB gene was cloned and sequenced. According to the G+C content and codon usage, the blaB gene appeared to be endogenous to the species. The BlaB enzyme showed significant sequence similarity to other class B beta-lactamases, being overall more similar to members of subclass B1, which includes the metallo-enzymes of Bacillus cereus (Bc-II) and Bacteroides fragilis (CcrA) and the IMP-1 enzyme found in various microbial species, and more distantly related to the metallo-beta-lactamases of Aeromonas spp. (CphA, CphA2 and ImiS) and of Stenotrophomonas maltophilia (L1).
Abstract: From May 1996 to September 1997, 615 Pseudomonas aeruginosa strains isolated from patients in intensive care units collected from different Italian laboratories were studied. The susceptibility of piperacillin/tazobactam, in comparison with other antipseudomonal antibiotics, to their NCCLS breakpoints was evaluated: amikacin 79. 6%, carbenicillin 67.0%, ceftazidime 73.4%, ciprofloxacin 55.8%, imipenem 64.1%, piperacillin 88.1%, piperacillin/tazobactam 92.4% and ticarcillin/clavulanic acid 69.0%. Seventy-three strains were selected because of their resistance to piperacillin and the mechanisms underlying such a resistance were investigated. Isoelectric focusing and hydrolysis assays revealed the presence of 15 plasmid-mediated beta-lactamases. Chromosomal beta-lactamase derepression was demonstrated in 34 isolates. The remaining 24 piperacillin-resistant strains did not produce beta-lactamases and an 'intrinsic mechanism' of resistance was inferred. The piperacillin/tazobactam combination restored resistance in 25 piperacillin strains. Nine of these were derepressed for chromosomal beta-lactamase, 8 showed impermeability and 8 showed plasmid enzymes.
Abstract: The in-vitro effects of some non-steroidal anti-inflammatory drugs and some analgesic drugs on collagenase activity were studied by use of a self-quenched fluorogenic esapeptide as substrate. The increased fluorescence signal arising as a result of peptide cleavage by collagenase was recorded and related to the inhibitory potency of the drugs. The effective concentrations in collagenase modulation were also correlated with the levels of the drugs in the plasma and synovial fluids of patients receiving therapeutic doses. Six of the tested drugs, nimesulide, piroxicam, tolmetin, meloxicam, sulindac and sodium meclofenamate, inhibited enzyme activity with IC50 values (concentrations resulting in 50% inhibition) ranging from 1.9 to 28.2 microM and Ki (apparent inhibition constant) ranging from 0.83 to 21.8 microM. Much of the activity was restored after dialysis of the enzyme-drug complex, demonstrating the reversibility of the effect. Although these results indicate that some anti-inflammatory drugs could modulate enzymatic activity involved in the degradation of the extracellular matrix, their possible pharmacological involvement as collagenase inhibitors in collagen degradative diseases remains to be assessed by clinical studies.
Abstract: Ceftriaxone and ceftriaxone S-oxide behaved as inactivators against the metallo-beta-lactamase of Aeromonas hydrophila AE036 and as substrates for the zinc beta-lactamase produced by Bacillus cereus (569/H/9) and Stenotrophomonas maltophilia ULA 511. Moreover, RO 09-1428, a catechol-cephalosporin, was not recognized by the A. hydrophila enzyme. Panipenem, cephalosporin C, cephalosporin C-gamma-lactone, and loracarbef were substrates for the three studied beta-lactamases.
Abstract: The beta-lactamase of the soil-borne strain 108 (parental strain) of Nocardia asteroides is a non-inducible enzyme mainly associated with the cells; it can be efficiently extracted by ultrasonication and SDS treatment. Crude enzyme preparations showed penicillinase and cephalosporinase activity. The kinetics of beta-lactamase production and in-vitro susceptibility to combinations of beta-lactam antibiotics plus beta-lactamase inhibitors have been studied in two stable overproducer mutants (A14 and B1) obtained by mutagenization of the parental strain with nitrosoguanidine. The cell-associated enzyme increased with bacterial growth in parental and mutant strains and was particularly abundant in stationary phase cells. The beta-lactamase inhibitors sulbactam and clavulanic acid decreased MIC values of penicillins more efficiently in the parental strain than in mutants, thus indicating some involvement of the enzyme in the resistance of N. asteroides strain 108 to beta-lactam antibiotics.
Abstract: A natural TEM variant beta-lactamase was isolated from an epidemic strain of Serratia marcescens. Nucleotide gene sequencing revealed multiple point mutations located in the 42-to-44 tripeptide and positions 145 to 146, 178, and 238. In addition, a glutamic acid 212 deletion was also found. The purified enzyme was studied from a kinetic point of view, revealing the highest catalytic efficiency (k[cat]/Km) values for ceftazidime and aztreonam compared with the TEM-1 prototype enzyme. The in vitro resistance correlated with kinetic parameters, and the enzyme also mediated resistance to some penicillins and an ampicillin-clavulanic acid combination. The mutational and kinetic changes are discussed in relation to the three-dimensional crystallographic structure of the wild-type TEM-1 enzyme.
Abstract: Adenosine analogs previously reported as reversible inhibitors of mammalian S-adenosylhomocysteine hydrolase (SAHase) have been found to exert similar effects on Acinetobacter calcoaceticus ULA-501 enzyme. In addition, two metal coordination compounds, cis-platinum diammine dichloride (cis-DDP) and its trans isomer (trans-DDP), the former well known for its employment in anticancer chemotherapy, were assayed on both bacterial and mammalian SAHases. In our conditions, trans-DDP appeared to be the strongest inhibitor toward both SAHases. Finally, treatment of the bacterial enzyme with a mixture of ATP-Mg acetate and KC1 caused only a slight reversible inhibition, in contrast to the complete inactivation exerted on the mammalian SAHase.
Abstract: The Aeromonas hydrophila CphA metallo-beta-lactamase was overexpressed in a soluble secreted form in Escherichia coli using a T7 RNA polymerase-based expression system, and a simple protocol based on a single cation-exchange chromatographic step was developed, which is suitable for rapid purification of the overexpressed enzyme from E. coli lysates. A yield of up to 30 micrograms of purified enzyme per milliliter of culture was obtained. The purified enzyme preparation showed properties identical to those previously reported in the literature.
Abstract: Mycobacterium fortuitum is a fast-growing Mycobacterium species which produces a beta-lactamase involved in the intrinsic resistance of the microorganism to beta-lactam antibiotics. An anti-beta-lactamase serum against the purified enzyme was raised in rabbits. Antibody binding was specific for native beta-lactamase, and enzyme activity was partially inhibited by the serum; furthermore, cross-reactions with denatured class A beta-lactamases were observed. This serum was used as a probe in immunogold labeling for the localization of the cell-bound beta-lactamase in both the low-level producer ATCC 19542 (parental strain) and the overproducer mutant D316. By the combination of preembedding immunogold labeling and replica technique, it was shown that the beta-lactamase was uniformly distributed on the whole external cell surface, where it appeared to be associated with a Tween 80-removable capsule-like material. Compared with the parental strain, a much higher level of expression of surface enzyme was observed in strain D316. Surface labeling was more intense in the stationary phase of growth than in exponentially growing cells. The data obtained are interpreted in the context of the intrinsic resistance of M. fortuitum to beta-lactam antibiotics.
Abstract: Biapenem, formerly LJC 10,627 or L-627, a carbapenem antibiotic, was studied in its interactions with 12 beta-lactamases belonging to the four molecular classes proposed by R. P. Ambler (Philos. Trans. R. Soc. Lond. Biol. Sci. 289:321-331, 1980). Kinetic parameters were determined. Biapenem was readily inactivated by metallo-beta-lactamases but behaved as a transient inhibitor of the active-site serine enzymes tested, although with different acylation efficiency values. Class A and class D beta-lactamases were unable to confer in vitro resistance toward this carbapenem antibiotic. Surprisingly, the same situation was found in the case of class B enzymes from Aeromonas hydrophila AE036 and Bacillus cereus 5/B/6 when expressed in Escherichia coli strains.
Abstract: Benzylpenicillin, amoxicillin, amoxicillin plus clavulanic acid, cephalothin, cephaloridine, cefotaxime, imipenem, erythromycin, clarithromycin, azithromycin, amikacin, ciprofloxacin and trimethoprim-sulfamethoxazole were tested in vitro by the agar dilution method against eleven strains of Nocardia asteroides isolated both from AIDS and other immunocompromised patients. Imipenem, amikacin and trimethoprim-sulfamethoxazole were shown to be the most active drugs with minimum inhibitory concentrations (MIC) values nearly always lower than concentrations achievable in blood. Ciprofloxacin, cephaloridine and cefotaxime were moderately active, while the remaining drugs were totally ineffective. When susceptibility was assessed by the radiometric method the MIC90 values were uniformly lower than those in the agar method, possibly due to lower inactivation of drugs during incubation. The two methods showed a good correlation only for imipenem, amikacin and ciprofloxacin. The results obtained by the radiometric method seem to indicate that, as for mycobacteria, this method may also give a more accurate evaluation of the antimicrobial susceptibility of Nocardiae.
Abstract: Sixteen different compounds usually considered beta-lactamase stable or representing potential beta-lactam inhibitors and inactivators were tested against the beta-lactamase produced by Mycobacterium fortuitum. The compounds exhibiting the most interesting properties were BRL42715, which was by far the best inactivator, and CGP31608 and ceftazidime, which were not recognized by the enzyme. These compounds thus exhibited adequate properties for fighting mycobacterial infections. Although cloxacillin, dicloxacillin, cefoxitin, and CP65207-2 exhibited poor inhibitory efficiency against the enzyme, they were also rather poor substrates and might be considered potential antimycobacterial agents. By contrast, CGP31523A and ceftamet were good substrates.
Abstract: Cefodizime (formerly HR221) was tested either for in vitro microbiological activity or for its stability to beta-lactamases in the presence of two beta-lactamase inhibitors (clavulanic acid, tazobactam). Cefodizime was a poor substrate of class C enzymes but hyperproducer strains were generally resistant with or without a beta-lactamase inhibitor used in combination. On the contrary, class A enzymes were able to hydrolyze cefodizime. However, strains expressing class A beta-lactamase were susceptible to cefodizime in combination with clavulanic acid.
Abstract: A beta-lactamase produced by Pseudomonas stutzeri was purified to protein homogeneity, and its physicochemical and catalytic properties were determined. Its profile was unusual since, in addition to penicillins, the enzyme hydrolysed second- and third-generation 'beta-lactamase-stable' cephalosporins and monobactams with similar efficiencies. On the basis of the characteristics of the interaction with beta-iodopenicillanic acid, the enzyme could be classified as a class-A beta-lactamase. However, when compared with most class-A beta-lactamases, it exhibited significantly lower kcat./Km values for the compounds usually considered to be the best substrates of these enzymes.
Abstract: S-adenosylhomocysteine hydrolase (SAHase) was purified to homogeneity from the Gram negative strain Acinetobacter calcoaceticus 501. The molecular weight of the native enzyme, estimated by gel permeation, was about 288 KDa, while sodium dodecyl sulfate polyacrylamide gel electrophoresis yielded a relative molecular mass of 48 KDa. The determination of the coenzyme content gave 4 mol of NAD+ and 2 mol of NADH per mol of enzyme. The isoelectric point of native SAHase was at pH 5.1. When assayed in the hydrolytic direction, the Km for S-adenosylhomocysteine and the Vmax of the enzyme for this substrate were 84 microM and 357 mumol/min/mg, respectively; in the synthetic direction, instead, the Km for adenosine and the corresponding Vmax value were 1.6 microM and 37 mumol/min/mg. Substrate analogs were tested for their ability to act as inhibitors and inactivators of the enzyme. Among these compounds, 9-beta-arabinofuranosyl adenine (Ara A) appeared as the most powerful competitive inhibitor (Ki = 18 microM) as well as the strongest time-dependent inactivator. The common feature of all the assayed analogs was the presence of the adenine ring in their molecular structure. It can thus concluded that the presence of the adenine moiety is an essential element in substrate and/or inhibitor interaction with this bacterial enzyme.
Abstract: It is widely assumed that the high level of intrinsic resistance to beta-lactam antibiotics exhibited by mycobacteria results from the combination of factors including permeability to the drugs, beta-lactamase production, and affinity for penicillin-binding proteins (PBPs). We conducted an evaluation of the second and third factors by isolating nitrosoguanidine-induced mutants from the beta-lactamase-producing strain Mycobacterium fortuitum ATCC 19542 that displayed either elevated or reduced resistance to various beta-lactam antibiotics. The mutants studied included D1 (a beta-lactamase producer with high penicillin resistance), gamma 27 (a low-level beta-lactamase producer with low penicillin resistance), and D316 (a high-level beta-lactamase producer with high penicillin resistance). In all strains examined, four major PBPs, named 1, 2a, 2b, and 3, with apparent molecular weights of 102,000, 90,000, 87,000, and 50,000, respectively, were found. The MICs of various beta-lactams toward ATCC 19542 and its mutants were considered in the context of beta-lactamase production, the quantity of PBPs synthesized, and their affinities for beta-lactam antibiotics. The data obtained show that beta-lactamase production is likely to be an important factor in the expression of resistance by clinical isolates and that PBP alterations can contribute to resistance at least in laboratory-derived mutants.
Abstract: Citrobacter diversus ULA-27, a clinical isolate showing a broad resistance pattern towards both penicillins and cephalosporins, produces chromosome encoded beta-lactamases. However, the strain remains susceptible to some cephamycins, imipenem, ceftazidime and tetracyclines. Crude bacterial extracts analyzed by isoelectric focusing on polyacrylamide gels, revealed the presence of two main isoforms and some "satellite" bands focusing in the pH range 5.7-7.2. The isoform showing the pIs 6.8 and 6.2 were characterized as class "A" beta-lactamases (according to Ambler's classification) based on the rate of interaction of beta-iodopenicillanate and the amino acid sequence around the active site serine. The substrate specificity of the Citrobacter diversus beta-lactamases explains the resistance phenomenon of this bacterium to penicillins and cephalosporins.
Abstract: In this paper we report some structural features of human seminal transferrin (HSmT) in comparison with the homologous protein purified from human serum (HSrT). In particular, the sequence of the first 13 N-terminal amino acids of HSmT shows 12/13 of identity with the first 13 N-terminal amino acids of HSrT, the ninth residue of the former protein being not definitely determined. Moreover, HSrT and HSmT analysed under the same conditions, by means of reversed phase HPLC, thiol groups determination and second derivative spectroscopy, show a different content of amino acids. In particular, HSmT exhibits mainly: i) a lower Asx/Glx ratio; ii) a reduction of about 50% in Cys residues; iii) a decrease of Tyr and Trp residues. Eventually oligosaccharide parallel analyses of HSmT and HSrT show the same glycosidic bond and almost the same sugar content (around 5.5% w/w); conversely, HSmT lacks of sialic acid residues and probably it contains fucose. These results, taken all together, could be sound of interest to a better understanding of the possible physiological roles of HSmT.
Abstract: The N-terminal sequences of the two major beta-lactamases produced by Citrobacter diversus differed only by the absence of the first residue in form II and the loss of five amino acid residues at the C-terminal end. Limited proteolysis of the homogeneous form I protein yielded a variety of enzymatically active products. In the major product obtained after the action of papain, the first three N-terminal residues of form I had been cleaved, whereas at the C-terminal end the treated enzyme lacked five residues. However, this cannot explain the different behaviours of form I, form II and papain digestion product upon chromatofocusing. Form I, which was sequenced up to position 56, exhibited a very high degree of similarity with a Klebsiella oxytoca beta-lactamase. The determined sequence, which contained the active serine residue, demonstrated that the chromosome-encoded beta-lactamase of Citrobacter diversus belong to class A.
Abstract: The gene coding for the class A beta-lactamase of Citrobacter diversus has been cloned and sequenced. It contains the information for a 294-amino-acid precursor protein, including a 27-residue N-terminal signal peptide. The deduced sequence of the N-terminal portion of the mature protein is in excellent agreement with that determined by microsequencing of the protein and readily explains the pI differences observed between the naturally occurring forms I and II of the enzyme. The sequence of the mature protein exhibits a very high degree of similarity with that of the Klebsiella oxytoca class A beta-lactamase.
Abstract: The kinetics of both intracellular and extracellular beta-lactamase production and the relationship between extracellular enzyme and in vitro susceptibility of Mycobacterium fortuitum to beta-lactam antibiotics have been studied. To this end we used a panel of stable nitrosoguanidine-induced mutants of M. fortuitum derived from the parental strain ATCC 19542 and differing in beta-lactamase production from 0.0001 to 278 U/liter in Mueller-Hinton broth. For overproducers of beta-lactamase (mutants A188, B180, C207, D316, and E31), MICs of benzylpenicillin, amoxicillin, ampicillin, and cephaloridine progressively increased with the amount of enzyme released into the medium, whereas MICs of imipenem and cefoxitin did not. The resistance of the mutants to amoxicillin was reduced up to 32-fold by clavulanic acid, whereas that to ampicillin was reduced 8-fold by sulbactam. These data suggest that the enzyme participated in the mechanisms of resistance to the beta-lactam antibiotics. However, for a mutant of M. fortuitum (gamma 27) with virtually nonexistent beta-lactamase production, the antibiotics still had relatively high MICs (for instance, benzylpenicillin and cephaloridine had MICs of 64 and 32 micrograms/ml, respectively). This suggests that, aside from beta-lactamase production, other mechanisms such as cell wall permeability and/or affinity for penicillin-binding proteins could coexist in M. fortuitum and explain its natural resistance to beta-lactam antibiotics.
Abstract: Inducible beta-lactamases were obtained after exposure to several beta-lactams in clinical isolates of Enterobacter cloacae and Citrobacter diversus. Enzyme production was related to the inducer and medium composition. beta-lactamase is able to inactivate only labile compounds, thus generating minimum inhibitory concentrations higher than in the absence of the inducer; imipenem susceptibilities usually were not changed.
Abstract: In this study the kinetic features of cefotaxime (CTX) and desacetyl-cefotaxime towards several representative beta-lactamases were investigated. Desacetyl-CTX was more stable to hydrolysis in comparison with cefotaxime for all the investigated enzymes. However, a cephalosporinase produced in Acinetobacter was progressively inactivated by both CTX and des-CTX. After prolonged incubation, dialysis partially restored the enzyme activity. Finally, both compounds were tested against selected resistant strains. It is concluded that des-CTX, because of either poor hydrolysis or prolonged half-life in body fluids, could contribute in vivo to the good antimicrobial properties of cefotaxime.
Abstract: Citrobacter diversus NF85 produced a chromosomal beta-lactamase that was induced by a variety of beta-lactam antibiotics. Two major forms of the enzyme, with isoelectric points (pI's) of 5.7 and 6.2, were found in crude cell extracts. Derepressed mutants of NF85, generated by nitrosoguanidine treatment, displayed different levels of beta-lactamase expression to the parent strain and had different patterns of resistance to a range of beta-lactam antibiotics. Those mutants of NF85 that were totally derepressed, expressing high, constitutive levels of enzyme, were found to have an additional beta-lactamase activity with a pI of 6.8.
Abstract: A beta-lactamase from Mycobacterium fortuitum D316 was purified and some physico-chemical properties and substrate profile determined. On the basis of its N-terminal sequence and of its sensitivity to beta-iodopenicillanate inactivation, the enzyme appeared to be a class A beta-lactamase, but its substrate profile was quite unexpected, since nine cephalosporins were among the eleven best substrates. The enzyme also hydrolysed ureidopenicillins and some so-called 'beta-lactamase-stable' cephalosporins.
Abstract: In order to elucidate the role of beta-lactamase in the resistance of M. fortuitum to beta-lactams, M. fortuitum ATCC 19542 and three mutants, strains D 316, D 319 and D 170 obtained from it by nitrosoguanidine treatment, were studied. Furthermore the kinetics of the beta-lactamase production during the bacterial growth and many biochemical and enzymatic characteristics of parent and mutant strains were investigated. Amoxicillin MICs well correlated with the beta-lactamase production in the high producer strains D 316 and D 319; on the contrary in strain D 170 a high MIC was joined with a moderate production of the enzyme showing that not only beta-lactamase but also other mechanisms can be effective in the resistance of M. fortuitum to beta-lactams. Clavulanic acid, an inhibitor of beta-lactamase, reduced MICs to amoxicillin in high and in low producer strains. The production of extracellular beta-lactamase occurred in a M. fortuitum mutant strain mainly during the stationary phase, indicating that a cell wall damage or initial autolysis could be responsible for the release of enzyme. Enzymatic and biochemical characteristics were not affected by nitrosoguanidine treatment except for nitrate test which showed only a weak positivity in high producer strains.
Abstract: Boric and boronic acids were used as inhibitors of beta-lactamases produced by two Citrobacter diversus strains and by one strain of Pseudomonas aeruginosa; all strains were clinical isolates. The beta-lactamases produced by the two Citrobacter diversus strains were inhibited by both borates and boronates, using cephazolin as substrate. The enzyme from Pseudomonas aeruginosa was inhibited only by boronates, using benzylpenicillin as substrate. These inhibitors were also used in combination with selected beta-lactams so as to determine if a synergism of antimicrobial activity occurred. All data reported in the present paper indicate that the minimum inhibitory concentration (MIC) values were lowered in the presence of these inhibitors for the two Citrobacter diversus strains. In the Pseudomonas aeruginosa strains the MIC values were not significantly altered, thus indicating the presence of a permeability barrier for 3-aminophenylboronic acid.
Abstract: The inhibiting or inactivating effects of some beta-lactam antibiotics on beta-lactamase from Mycobacterium fortuitum were studied. Among all substrates tested, clavulanic acid and sulbactam were the strongest competitive inhibitors of the enzyme although the latter was slightly hydrolyzed. Imipenem and cefoxitin scarcely inhibited the beta-lactamase yet expressed good activity against the microorganism in vitro, suggesting that the effectiveness of these drugs on M. fortuitum might be due to high permeation through the cell wall. All the isoxazolylpenicillins tested and methicillin inactivated the enzyme of M. fortuitum by a first rapid phase of acylation followed by a steady-state process of enzyme reactivation (deacylation). Clavulanic acid and sulbactam showed Ki values for the enzyme inactivation closely corresponding to hematic concentrations achievable in vivo during antibiotic treatment.
Abstract: We measured the kinetics of hydrolysis of various cephalosporins by the chromosomally-encoded beta-lactamases of Citrobacter diversus ULA-27. Cefonicid, cefamandole, cefatrizine and cefoperazone were all hydrolyzed but these antibiotics showed a different feature in their kinetic parameters. Moreover, cefoperazone was a non-competitive inhibitor of this type of enzyme. Cefotetan was stable to hydrolysis and behaved like a progressive inactivator. The ability of these enzymes to inactivate the reported antibiotics contributes largely to the resistance of the studied strain. We conclude that hydrolysis is the main mechanism of resistance of this strain to the new cephalosporins.
Abstract: The activity of beta-lactamases from Citrobacter diversus ULA-27 on ceftriaxone, a widely recognized third-generation cephalosporin, has been examined and compared to the activity of various other beta-lactamases from different sources. Ceftriaxone (Roche S.p.A. Milan) was found to be resistant to hydrolysis by beta-lactamases from Enterobacter cloacae and Bacillus cereus, but susceptible to beta-lactamases from Mycobacterium fortuitum strain Cow 18 and, mostly, to beta-lactamases from various strains of Citrobacter diversus. Derivatives with substituents in the 3-position of ceftriaxone, namely cefotaxime (Roussel Maestretti S.p.A., Milan) and ceftizoxime (Farmitalia Carlo Erba, Milan), were much less susceptible to hydrolysis by C. diversus ULA-27 enzymes (22 and 6% of ceftriaxone hydrolysis, respectively), the hydrolysis rate being paralleled by differences in MIC values. Ceftriaxone inhibited the activity of E. cloacae beta-lactamases toward cefazolin as substrate, but the inhibition was totally abolished by preincubation of ceftriaxone with the enzyme before addition of the substrate. Overall, the data point to a relevance of C. diversus ULA-27 beta-lactamases in the mechanism of resistance of this strain to the various third-generation cephalosporins.
Abstract: Two chromosome-encoded beta-lactamases have been purified from Citrobacter diversus ULA-27. They exhibited slightly different isoelectric points (6.8 and 6.2) and very similar Mr values (congruent to 29,000). Their specificity spectrum was rather wide, since they hydrolysed some cephalosporins with kcat: values similar to those observed with the best penicillin substrates. Cloxacillin, methicillin and imipenem were hydrolysed very slowly. Hydrolysis of azthreonam could not be detected.
