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PubMed2004

Mechanism of the drug interaction between valproic acid and carbapenem antibiotics in monkeys and rats.

The Ministry of Health and Welfare, Japan banned coadministration of carbapenems, such as panipenem/betamipron (PAPM), meropenem (MEPM), and valproic acid (VPA) because clinical reports have indicated that the coadministration caused seizures in epileptic patients due to lowered plasma levels of VPA. In this study, we have clarified the mechanism of the drug-drug interaction using PAPM, MEPM, and doripenem [S-4661; (+)-(4R,5S,6S)-6-[(1R)-1-hydroxyethyl]-4-methyl-7-oxo-3-[[(3S,5S)-5-[(sulfamoylamino)methyl]-3-pyrrolidinyl]thio]-1-azabicyclo[3.2.0]hept-2-ene-2-caboxylic acid monohydrate], a newly synthesized carbapenem. In vitro experiments using monkey liver slices suggested that the apparent synthetic rate of VPA glucuronide (VPA-G) increased in the presence of carbapenems. However, no such increase was observed in the experiment using monkey liver microsomes. Although no increase of uridine 5'-diphosphate D-glucuronic acid was found in monkey liver slices in the presence of carbapenems, potent inhibitory activity of carbapenems for the hydrolysis of VPA-G was found in monkey and rat liver homogenate. In vivo hydrolysis of VPA-G was clearly shown by the existence of VPA in plasma after dosing of VPA-G to rats, and its inhibition by carbapenems was also clearly shown by the negligible levels of VPA in rat plasma after coadministration of carbapenems and VPA-G. These results clearly indicate one of the important causes of drug interaction as follows: carbapenems would inhibit the hydrolytic enzyme, which is involved in the hydrolysis of VPA-G to VPA, resulting in a decrease of plasma concentration of VPA.

Animals↗

PubMed2006

Antibiotic coresistance in extended-spectrum-beta-lactamase-producing Enterobacteriaceae and in vitro activity of tigecycline.

The spread of extended-spectrum-beta-lactamase (ESBL)-producing organisms, particularly those harboring the CTX-M-type enzymes, both in the hospital and in the community, is difficult to discontinue due to the successful mobilization and evolution of the genetic elements harboring ESBL genes and coresistance rates in these isolates. The activities of tigecycline against 285 non-clonally related isolates (172 from Escherichia coli, 84 from Klebsiella spp., 20 from Enterobacter spp., 5 from Salmonella spp., and 4 from Citrobacter spp.) expressing well-characterized ESBLs and recovered in our hospital and its community area of influence were comparatively assessed (CLSI microdilution). Susceptibility rates for meropenem, imipenem, tigecycline, amikacin, and piperacillin-tazobactam were 100%, 100%, 97.5%, 93.3%, and 93%, respectively. Tigecycline (mode MIC, 0.5 microg/ml; MIC(90), 1 microg/ml) was 4- to 256-fold more active than doxycycline and minocycline (mode MIC range, 2 to 128 microg/ml). CTX-Ms were the most frequent ESBLs (61.4%), 65.8% in community and 58.6% in nosocomial isolates. CTX-M-9 (22%), CTX-M-14 (15.8%), and CTX-M-10 (14%) were the most represented derivatives. SHV and TEM variants constituted 22.8% and 15.8% of the ESBLs, respectively. Overall coresistance rates were as follows: gentamicin, 27.4%; tobramycin, 27.4%; amikacin, 6.7%; and chloramphenicol, 29.1%. Sulfonamide (61.7%), trimethoprim (52.3%), streptomycin (50.5%), and ciprofloxacin (37.2%) resistance levels were significantly (P < 0.001) associated with CTX-M-9 producers. No tigecycline resistance was observed, although seven Klebsiella pneumoniae isolates exhibited intermediate MICs (4 mug/ml). Tigecycline, lacking cross-resistance with other compounds, could represent an opportunity to reduce the intensity of selection for ESBL-producing organisms derived from the use of other antimicrobial agents. However, this in vitro promise requires support from clinical studies.

Anti-Bacterial Agents↗

PubMed2006

High-level carbapenem resistance in a Klebsiella pneumoniae clinical isolate is due to the combination of bla(ACT-1) beta-lactamase production, porin OmpK35/36 insertional inactivation, and down-regulation of the phosphate transport porin phoe.

Clinical isolates of Klebsiella pneumoniae resistant to carbapenems and essentially all other antibiotics (multidrug resistant) are being isolated from some hospitals in New York City with increasing frequency. A highly related pair of K. pneumoniae strains isolated on the same day from one patient in a hospital in New York City were studied for antibiotic resistance. One (KP-2) was resistant to imipenem, meropenem, and sulopenem (MICs of 16 to 32 microg/ml) while the other (KP-1) was susceptible (MIC of 0.5 microg/ml); both contained the bla(ACT-1), bla(SHV-1), and bla(TEM-1) beta-lactamases. bla(ACT-1) in both strains was encoded on a large approximately 150-kb plasmid. Both isolates contained an identical class 1 integron encoding resistance to aminoglycosides and chloramphenicol. They each had identical insertions in ompK35 and ompK36, resulting in disruption of these key porin genes. The carbapenem-resistant and -susceptible isolates were extensively studied for differences in the structural and regulatory genes for the operons acrRAB, marORAB, romA-ramA, soxRS, micF, micC, phoE, phoBR, rpoS, and hfq. No changes were detected between the isolates except for a significant down-regulation of ompK37, phoB, and phoE in KP-2 as deduced from reverse transcription-PCR analysis of mRNA and polyacrylamide gel electrophoresis separation of outer membrane proteins. Backcross analysis was conducted using the wild-type phoE gene cloned into the vector pGEM under regulation of its native promoter as well as the lacZ promoter following transformation into the resistant KP-2 isolate. The wild-type gene reversed carbapenem resistance only when under control of the heterologous lacZ promoter. In the background of ompK35-ompK36 gene disruption, the up-regulation of phoE in KP-1 apparently compensated for porin loss and conferred carbapenem susceptibility. Down-regulation of phoE in KP-2 may represent the normal state of this gene, or it may have been selected from KP-1 in vivo under antibiotic pressure, generating the carbapenem-resistant clone. This is the first study in the Enterobacteriaceae where expression of the phosphate-regulated PhoE porin has been associated with resistance to antimicrobials. Our results with this pair of Klebsiella clinical isolates highlight the complex and evolving nature of multiple drug resistance in this species.

Bacterial Proteins↗

PubMed2006

In vitro susceptibility of Clostridium difficile clinical isolates from a multi-institutional outbreak in Southern Québec, Canada.

Clostridium difficile isolates from a 2004 outbreak in Québec, Canada, were all found to be susceptible to metronidazole, vancomycin, rifampin, and meropenem but resistant to bacitracin, cefotaxime, ciprofloxacin, and levofloxacin, and most (>80%) were resistant to ceftriaxone, clarithromycin, gatifloxacin, and moxifloxacin. The predominant NAP1 isolates were susceptible to clindamycin, while the NAP2 isolates were resistant.

Anti-Bacterial Agents↗

PubMed2006

Antimicrobial susceptibility and synergy studies of Burkholderia cepacia complex isolated from patients with cystic fibrosis.

Susceptibility (18 antimicrobial agents including high-dose tobramycin) and checkerboard synergy (23 combinations) studies were performed for 2,621 strains of Burkholderia cepacia complex isolated from 1,257 cystic fibrosis patients. Minocycline, meropenem, and ceftazidime were the most active, inhibiting 38%, 26%, and 23% of strains, respectively. Synergy was rarely noted (range, 1% to 15% of strains per antibiotic combination).

Anti-Bacterial Agents↗

PubMed2006

In vitro activities of tigecycline and eight other antimicrobials against different Nocardia species identified by molecular methods.

The in vitro activities of tigecycline and other antimicrobials against 51 isolates of Nocardia spp. were evaluated. MIC(90)s and MIC ranges were as follows: tigecycline, 4 and < or =0.06 to 8 mg/liter, respectively; minocycline, 2 and < or =0.06 to 2 mg/liter, respectively; linezolid, 1 and < or =0.06 to 2 mg/liter, respectively; moxifloxacin, 2 and < or =0.06 to >64 mg/liter, respectively; ertapenem, 32 and < or =0.06->64 mg/liter, respectively; imipenem, 2 and < or =0.06 to >64 mg/liter, respectively; meropenem, 8 and < or =0.06 to >64 mg/liter, respectively; amikacin, 1 and < or =0.06 to 32 mg/liter, respectively; and trimethoprim-sulfamethoxazole, 1/19 and < or =0.5/9.5 to >2/38 mg/liter, respectively.

Anti-Bacterial Agents↗

PubMed2006

Postgenomic scan of metallo-beta-lactamase homologues in rhizobacteria: identification and characterization of BJP-1, a subclass B3 ortholog from Bradyrhizobium japonicum.

The diffusion of metallo-beta-lactamases (MBLs) among clinically important human pathogens represents a therapeutic issue of increasing importance. However, the origin of these resistance determinants is largely unknown, although an important number of proteins belonging to the MBL superfamily have been identified in microbial genomes. In this work, we analyzed the distribution and function of genes encoding MBL-like proteins in the class Rhizobiales. Among 12 released complete genomes of members of the class Rhizobiales, a total of 57 open reading frames (ORFs) were found to have the MBL conserved motif and identity scores with MBLs ranging from 8 to 40%. On the basis of the best identity scores with known MBLs, four ORFs were cloned into Escherichia coli for heterologous expression. Among their products, one (blr6230) encoded by the Bradyrhizobium japonicum USDA110 genome, named BJP-1, hydrolyzed beta-lactams when expressed in E. coli. BJP-1 enzyme is most closely related to the CAU-1 enzyme from Caulobacter vibrioides (40% amino acid sequence identity), a member of subclass B3 MBLs. A kinetic analysis revealed that BJP-1 efficiently hydrolyzed most beta-lactam substrates, except aztreonam, ticarcillin, and temocillin, with the highest catalytic efficiency measured with meropenem. Compared to other MBLs, BJP-1 was less sensitive to inactivation by chelating agents.

Agrobacterium tumefaciens↗

PubMed1990

In vitro susceptibilities of Pseudomonas pseudomallei to 27 antimicrobial agents.

Clinical isolates of Pseudomonas pseudomallei isolated in Thailand from 1981 to 1989 were tested for their in vitro susceptibilities to 27 antimicrobial agents, including older and newer quinolones, broad-spectrum cephems, carbapenems, monobactams, penicillins, tetracyclines, chloramphenicol, rifamycin, sulfamethoxazole, trimethoprim, and fosfomycin. Tosufloxacin, meropenem, CS-533, and minocycline were active against P. pseudomallei at levels comparable to or even greater than those of antimicrobial agents tested in previous studies, such as ciprofloxacin, ceftazidime, imipenem, carumonam, and piperacillin. Drug-resistant P. pseudomallei was found in only 1% of the isolates. The drug-resistant P. pseudomallei isolates displayed a unique pattern of susceptibility to the above-listed drugs.

Anti-Bacterial Agents↗

PubMed1990

Characterization of beta-lactamases from non-Bacteroides fragilis group Bacteroides spp. belonging to seven species and their role in beta-lactam resistance.

Twelve beta-lactamase-positive non-Bacteroides fragilis group Bacteroides spp. belonging to seven different species were examined by MIC determination and enzyme characterization. MICs of most beta-lactams except cefoxitin, cefotetan, imipenem, and meropenem were relatively high or very high. All enzymes hydrolyzed cephaloridine (Vmax, 100%; Km, 12 to 70 microM), cephalothin (Vmax, 25 to 826%; Km, 8 to 143 microM), cefamandole (Vmax, 13 to 158%; Km, 17 to 170 microM), and cefuroxime (hydrolysis rate, 19 to 98%), and 11 of 12 hydrolyzed cefotaxime (Vmax, 26 to 145%; Km, 13 to 127 microM); no hydrolysis of cefoxitin or moxalactam was observed. Penicillins were hydrolyzed at lower rates, with Vmax values less than or equal to 20% of that obtained with cephaloridine. Addition of clavulanate, sulbactam, or tazobactam led to a 4- to 2,048-fold lowering of MICs of penicillins as well as cephalosporins. All enzymes were inhibited by clavulanate (50% inhibitory concentration [IC50], 0.01 to 1.8 microM), sulbactam (IC50, 0.02 to 1.9 microM), tazobactam (IC50, 0.001 to 0.9 microM), cefoxitin (IC50, 0.002 to 0.35 microM), and moxalactam (IC50, 0.03 to 6.6 microM). No enzymes were inhibited by 100 microM EDTA or p-chloromercuribenzoic acid; an enzyme of one strain of B. loescheii was inhibited by 100 microM cloxacillin (IC50, 2.35 microM). Ten enzymes had optimal activity at pH 5.0 to 6.0, and two had optimal activity at pH 8.0. Isoelectric focusing revealed pIs between 4.2 and 5.6. These enzymes seem to belong to a previously unclassified group of beta-lactamases, related (but not identical) to beta-lactamases of the B. fragilis group.

Anti-Bacterial Agents↗

PubMed1990

Diffusion of beta-lactam antibiotics through liposome membranes reconstituted from purified porins of the outer membrane of Pseudomonas aeruginosa.

Determination of the rates of diffusion of beta-lactam antibiotics through purified Pseudomonas aeruginosa porins C, D2, and E in liposomes yielded the following results. (i) The rates of carbapenem (imipenem and meropenem) diffusion through the protein D2 pore were roughly 2 to 70 times higher than those through other porin pores. It is not clear why the protein D2 pore allowed rapid diffusion of carbapenems. The rates of diffusion of glucosamine and triglycine through the protein D2 pore were about 14 and 4 times higher, respectively, than that of an uncharged test solute with a similar Mr, glucose. (ii) The rates of diffusion of antipseudomonal anionic beta-lactams such as piperacillin, ceftazidime, cefsulodin, and aztreonam through the protein C pore were higher than those through other porin pores. This was probably due to the slightly larger pore size and the slight anion selectivity of protein C, since the apparent exclusion limit of the protein C pore for uncharged saccharides is higher than that of other porins and the rate of diffusion of gluconic acid through the protein C pore is about double that for glucose. (iii) The rates of diffusion of cefoperazone through all three species of porin were relatively high. These results indicate that the antipseudomonal beta-lactams permeate the P. aeruginosa outer membrane via newly identified porins.

Anti-Bacterial Agents↗

PubMed1991

In vitro activity of a catechol-substituted cephalosporin, GR69153.

The in vitro activity of GR69153, a new catechol-substituted cephalosporin, was compared with those of ceftazidime, imipenem, meropenem, and ceftriaxone against 604 recent clinical isolates and other strains with known mechanisms of resistance. The MICs of GR69153 for 90% of the members of the family Enterobacteriaceae tested were less than 0.5 micrograms/ml, with the exceptions of those for Serratia spp. (4 micrograms/ml), Citrobacter spp. (2 micrograms/ml), and Enterobacter spp. (8 micrograms/ml). Ninety percent of Pseudomonas aeruginosa isolates were susceptible to less than or equal to 1 microgram of GR69153 per ml. With the exception of methicillin-resistant strains, 90% of Staphylococcus aureus isolates were susceptible to less than or equal to 2 micrograms/ml, and GR69153 was four- to eightfold more active than ceftazidime and ceftriaxone against these strains. Isolates of Haemophilus influenzae, Branhamella catarrhalis, Neisseria spp., and Streptococcus pneumoniae (penicillin susceptible) were highly susceptible (MIC for 90% of the strains, less than or equal to 0.12 micrograms/ml). GR69153 was stable to hydrolysis by the TEM-1 and TEM-5, SHV-1 and SHV-2, and K1 beta-lactamases, but some susceptibility to hydrolysis by the TEM-3, TEM-9, and P99 enzymes was observed. The protein-binding activity of GR69153 was 74.5 to 66.8%, depending on the concentration, and serum had little effect upon activity.

Bacteria↗

PubMed1991

Susceptibilities of 394 Bacteroides fragilis, non-B. fragilis group Bacteroides species, and Fusobacterium species to newer antimicrobial agents.

The susceptibilities of 374 selected beta-lactamase-producing gram-negative anaerobes (including 22 cefoxitin-resistant strains and 36 strains refractory to the enhancing effect of beta-lactamase inhibitors) and 20 beta-lactamase-negative strains were tested by agar dilution against selected new agents. The organisms included 217 Bacteroides fragilis group strains, 137 non-B. fragilis group Bacteroides spp., and 40 fusobacteria. All strains were susceptible to piperacillin-tazobactam, imipenem, and meropenem. For the B. fragilis group, 96% were susceptible to ampicillin-sulbactam, 95% were susceptible to amoxicillin-clavulanate and cefoperazone-sulbactam, 94% were susceptible to tosufloxacin, 91% were susceptible to cefoxitin, 88% were susceptible to trospectomycin, and 73% were susceptible to cefotetan. For the beta-lactamase-positive non-B. fragilis group Bacteroides spp., greater than or equal to 94% were susceptible to cefoxitin, amoxicillin-clavulanate, ampicillin-sulbactam, cefoperazone-sulbactam, and trospectomycin, 90% were susceptible to cefotetan, and 85% were susceptible to tosufloxacin (the most resistant strains were B. bivius and B. disiens). For the beta-lactamase-positive fusobacteria, greater than or equal to 97% were susceptible to amoxicillin-clavulanate, ampicillin-sulbactam, cefoperazone-sulbactam, trospectomycin, and cefoxitin, 90% were susceptible to cefotetan, and 89% were susceptible to tosufloxacin. All agents showed excellent activity against beta-lactamase-negative strains (for trospectomycin, 95% were susceptible; for all other drugs, 100% were susceptible). Overall, both carbapenems and piperacillin-tazobactam were most active. Amoxicillin-clavulanate, ampicillin-sulbactam, and cefoperazone-sulbactam lacked activity against some cefoxitin-resistant B. fragilis group strains but had excellent activity against other organisms. Tosufloxacin, a new quinolone, had very good activity against B. fragilis group strains (94% susceptible), good activity against other beta-lactamase-positive strains (less than or equal 85% susceptible), and excellent activity against beta-lactamase-negative strains (100% susceptible; MIC for 90% of strains, 0.5 microgram/ml). Trospectomycin was active against >90% of all strains except for B. fragilis group strains (88% susceptible; MIC for 90% of strains, 32 microgram/ml). Clinical studies are required to delineate the role of newer agents in the therapy of anaerobic infections.

Anti-Bacterial Agents↗

PubMed1991

Bactericidal effects of antibiotics on slowly growing and nongrowing bacteria.

Antimicrobial agents are most often tested against bacteria in the log phase of multiplication to produce the maximum bactericidal effect. In an infection, bacteria may multiply less optimally. We examined the effects of several classes of antimicrobial agents to determine their actions on gram-positive and gram-negative bacteria during nongrowing and slowly growing phases. Only ciprofloxacin and ofloxacin exhibited bactericidal activity against nongrowing gram-negative bacteria, and no antibiotics were bactericidal (3-order-of-magnitude killing) against Staphylococcus aureus. For the very slowly growing gram-negative bacteria studied, gentamicin (an aminoglycoside), imipenem (a carbapenem), meropenem (a carbapenem), ciprofloxacin (a fluoroquinolone), and ofloxacin (a fluoroquinolone) exhibited up to 5.7 orders of magnitude more killing than piperacillin or cefotaxime. This is in contrast to optimally growing bacteria, in which a wide variety of antibiotic classes produced 99.9% killing. For the gram-positive and gram-negative bacteria we examined, antibiotic killing was greatly dependent on the growth rate. The clinical implications of slow killing by chemotherapeutic agents for established bacterial infections and infections involving foreign bodies are unknown.

4-Quinolones↗

PubMed1992

Interplay of impermeability and chromosomal beta-lactamase activity in imipenem-resistant Pseudomonas aeruginosa.

Mutational loss of the D2 porin causes imipenem resistance in Pseudomonas aeruginosa. It was found that this mechanism could function only when the chromosomal beta-lactamase was expressed. Mutants lacking both the beta-lactamase and the D2 porin were almost as susceptible as those that lacked the beta-lactamase but retained the porin. Thus, imipenem resistance reflected an interplay of the enzyme and impermeability, not either factor alone. These findings suggest that the activity of a carbapenem more beta-lactamase stable than imipenem should be less affected by the porin loss. Meropenem approached this behavior.

Bacterial Outer Membrane Proteins↗

PubMed1993

Biochemical properties of a carbapenem-hydrolyzing beta-lactamase from Enterobacter cloacae and cloning of the gene into Escherichia coli.

A clinical isolate of Enterobacter cloacae, strain NOR-1, exhibited resistance to imipenem and remained susceptible to extended-spectrum cephalosporins. Clavulanic acid partially restored the susceptibility of the strain to imipenem. Two beta-lactamases with isoelectric points (pI) of 6.9 and > 9.2 were detected in strain E. cloacae NOR-1; the higher pI corresponded to AmpC cephalosporinase. Plasmid DNA was not detected in E. cloacae NOR-1 and imipenem resistance could not be transferred into Escherichia coli JM109. The carbapenem-hydrolyzing beta-lactamase gene was cloned into plasmid pACYC184. One recombinant plasmid, pPTN1, harbored a 5.3-kb Sau3A fragment from E. cloacae NOR-1 expressing the carbapenem-hydrolyzing beta-lactamase. This enzyme (pI 6.9) hydrolyzed ampicillin, cephalothin, and imipenem more rapidly than it did meropenem and aztreonam, but it hydrolyzed extended-spectrum cephalosporins only weakly and did not hydrolyze cefoxitin. Hydrolytic activity was partially inhibited by clavulanic acid, sulbactam, and tazobactam, was nonsusceptible to chelating agents such as EDTA and 1,10-o-phenanthroline, and was independent of the presence of ZnCl2. Its relative molecular mass was 30,000 Da. Induction experiments concluded that the carbapenem-hydrolyzing beta-lactamase biosynthesis was inducible by cefoxitin and imipenem. Subcloning experiments with HindIII partial digests of pPTN1 resulted in a recombinant plasmid, designated pPTN2, which contained a 1.3-kb insert from pPTN1 and which conferred resistance to beta-lactam antibiotics. Hybridization studies performed with a 1.2-kb HindIII fragment from pPtN2 failed to determine any homology with ampC of E. cloacae, with other known beta-lactamase genes commonly found in members of the family Enterobacteriaceae (bla(TEM-1)) and bla(SHV-3) derivatives), and with previously described carbapenemase genes such as those from Xanthomonas maltophilia, Bacillus cereus, Bacteroides fragilis (cfiA), and Aeromonas hydrophila (cphA). This work describing the biochemical properties of a novel chromosome-encoded beta-lactamase from E. cloacae indicates that this enzyme differs from all the previously described carbapenemases. This is the first reported cloning of a carbapenem-hydrolyzing gene from a member of the family Enterobacteriaceae.

Carbapenems↗

PubMed1993

High specificity of cphA-encoded metallo-beta-lactamase from Aeromonas hydrophila AE036 for carbapenems and its contribution to beta-lactam resistance.

The Aeromonas hydrophila AE036 chromosome contains a cphA gene encoding a metallo-beta-lactamase highly active against carbapenem antibiotics. This enzyme was induced in strain AE036 to the same extent by both benzylpenicillin and imipenem. When the cphA gene was inserted into plasmid pACYC184, used to transform Escherichia coli DH5 alpha, the MICs of imipenem, meropenem, and penem HRE664 for recombinant clone DH5 alpha(pAA20R), expressing the Aeromonas metallo-beta-lactamase, were significantly increased, but those of penicillins and cephalosporins were not. When the metallo-beta-lactamase purified from E. coli DH5 alpha(pAA20R) was assayed with several beta-lactam substrates, it hydrolyzed carbapenems but not penicillins or cephalosporins efficiently. These results demonstrate that this metallo-beta-lactamase possesses an unusual spectrum of activity compared with all the other class B enzymes identified so far, being active on penems and carbapenems only. This enzyme may thus contribute to the development of resistance to penems and carbapenems but not other beta-lactams.

Aeromonas hydrophila↗

PubMed1993

OXA-11, an extended-spectrum variant of OXA-10 (PSE-2) beta-lactamase from Pseudomonas aeruginosa.

Pseudomonas aeruginosa ABD, which was isolated in October 1991 from blood cultures of a burn patient in Turkey, was resistant to cephalosporins, particularly ceftazidime (MIC, 512 micrograms/ml), penicillins, aztreonam, and meropenem, but not to imipenem. Cephalosporin and penicillin resistance transferred to P. aeruginosa PU21 and was associated with a beta-lactamase with a pI of 6.4 encoded by a 100-MDa plasmid designated pMLH52. Like extended-spectrum TEM and SHV beta-lactamases, this enzyme hydrolyzed penicillins and newer cephalosporins but did not hydrolyze cefoxitin or carbapenems. However, it differed from TEM and SHV derivatives in being a potent oxacillinase, and its encoding gene did not hybridize with probes to TEM and SHV genes. To characterize the enzyme, libraries of total DNA were cloned into plasmid pUC19 and were transformed into Escherichia coli DH5 alpha. Recombinant plasmids that gave ceftazidime resistance all contained a 3.65-kb BamHI fragment. Deletions from this fragment allowed the beta-lactamase gene to be located on a 1.4-kb section of DNA, which contained an open reading frame of 798 bases. This encoded a protein that was deduced to differ from PSE-2 beta-lactamase only in having serine instead of asparagine at position 143 and aspartate instead of glycine at position 157. It is concluded that the resistance of isolate ABD dependent on an extended-spectrum variant of the PSE-2 enzyme. The ability of this enzyme to cause ceftazidime resistance dependent primarily on a low Km for the compound; Vmax remained low. It is proposed that PSE-2 should be transferred to the OXA group as OXA-10 and that the new enzyme be designated OXA-11.

Amino Acid Sequence↗

PubMed1994

Rapid identification of metallo- and serine beta-lactamases.

Simple methods to detect, identify, and differentiate metallo- and serine beta-lactamases were developed and used to differentiate enzymes produced by 17 clinical isolates of Xanthomonas maltophilia. All isolates exhibited beta-lactamase activity, and in 16 strains this was induced by imipenem. All but one isolate hydrolyzed imipenem (and meropenem), and in all cases this activity was inhibited by 1 mM EDTA. The metallo- and serine beta-lactamases in the cell extracts were distinguished on isoelectric focusing (IEF) gels by using the following procedures. (i) Cell lysates were preincubated with 83 mM EDTA prior to IEF and subsequent visualization with nitrocefin, and (ii) after IEF, the gels were overlaid with either 1 mM zinc sulfate or 100 microM BRL 42715 before staining with nitrocefin. Bands of beta-lactamase activity which were removed by BRL 42715 but unaffected by EDTA or zinc sulfate were categorized as serine beta-lactamases. Bands which were unaffected by BRL 42715 but inhibited by EDTA or enhanced by zinc sulfate were classified as metallo-beta-lactamases. By using this approach, seven metallo-beta-lactamases were differentiated with pI values of 4.8 (two strains), 5.5 (four strains), 5.7 (one strain), 6.0 (one strain), 6.4 (four strains), 6.6 (one strain), and 6.8 (three strains). The metallo-beta-lactamase band with a pI of 6.4 aligned with the recently characterized metallo-beta-lactamase from X. maltophilia 511. Heterogeneity was also observed for the serine beta-lactamases: 14 isolates elaborated serine beta-lactamase activity which focused with major bands with at least eight different pIs. The remaining three strains produced serine beta-lactamases which focused with five distinct bands with pIs of 6.4, 6.2, 5.7, 5.5, and 5.2. We conclude that X. maltophilia produces many types of metallo- and serine beta-lactamases distinguishable by these new methods and that the previously reported L-1 and L-2 enzymes are not solely representative of the beta-lactamases produced by this species.

Anti-Bacterial Agents↗