The Effect of ß-Lactam Antibiotics on the Evolution of Ceftazidime/Avibactam and Cefiderocol Resistance in KPC-Producing Klebsiella pneumoniae.

In this study, we aimed to clarify the evolutionary trajectory of a Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp) population during ß-lactam antibiotic therapy. Five KPC-Kp isolates were collected from a single patient. Whole-genome sequencing and a comparative genomics analysis were performed on the isolates and all blaKPC-2-containing plasmids to predict the population evolution process. Growth competition and experimental evolution assays were conducted to reconstruct the evolutionary trajectory of the KPC-Kp population in vitro. Five KPC-Kp isolates (KPJCL-1 to KPJCL-5) were highly homologous, and all harbor an IncFII blaKPC-containing plasmid (pJCL-1 to pJCL-5). Although the genetic structures of these plasmids were almost identical, distinct copy numbers of the blaKPC-2 gene were detected. A single copy of blaKPC-2 was presented in pJCL-1, pJCL-2, and pJCL-5, two copies of blaKPC (blaKPC-2 and blaKPC-33) were presented in pJCL-3, and three copies of blaKPC-2 were presented in pJCL-4. The blaKPC-33-harboring KPJCL-3 isolate presented resistance to ceftazidime-avibactam and cefiderocol. The blaKPC-2 multicopy strain KPJCL-4 had an elevated ceftazidime-avibactam MIC. The patient had been exposed to ceftazidime, meropenem, and moxalactam, after which KPJCL-3 and KPJCL-4 were isolated, which both showed a significant competitive advantage under antimicrobial pressure in vitro. Experimental evolution assays revealed that blaKPC-2 multicopy-containing cells were increased in the original single-copy blaKPC-2-harboring KPJCL-2 population under selection with ceftazidime, meropenem, or moxalactam, generating a low-level ceftazidime-avibactam resistance phenotype. Moreover, blaKPC-2 mutants with a G532T substitution, G820 to C825 duplication, G532A substitution, G721 to G726 deletion, and A802 to C816 duplication increased in the blaKPC-2 multicopy-containing KPJCL-4 population, generating high-level ceftazidime-avibactam resistance and reduced cefiderocol susceptibility. Ceftazidime-avibactam and cefiderocol resistance can be selected by ß-lactam antibiotics other than ceftazidime-avibactam. Notably, blaKPC-2 gene amplification and mutation are important in KPC-Kp evolution under antibiotic selection.

Disk Diffusion Testing for Detection of Methicillin-Resistant Staphylococci: Does Moxalactam Improve upon Cefoxitin?

Disk diffusion testing is widely used to detect methicillin resistance in staphylococci, and cefoxitin is currently considered the best marker for mecA-mediated methicillin resistance. In low-inoculum diffusion testing (colony suspension at 106 CFU/ml), the addition of moxalactam in combination with cefoxitin has been reported to improve on cefoxitin alone for the detection of methicillin-heteroresistant staphylococci. However, moxalactam is absent from EUCAST and CLSI guidelines, which use high-inoculum diffusion testing (colony suspension at 108 CFU/ml), calling into question the potential interest of including moxalactam in their recommendations. The inhibition zone diameters of cefoxitin and moxalactam, alone and in combination, were evaluated for concordance with mecA and mecC positivity in a large collection of clinical Staphylococcus isolates (611 Staphylococcus aureus, Staphylococcus lugdunensis, and Staphylococcus saprophyticus isolates and 307 coagulase-negative staphylococci other than S. lugdunensis and S. saprophyticus isolates, of which 22% and 53% were mecA-positive, respectively) and in 25 mecC-positive S. aureus isolates using high-inoculum diffusion testing. Receiver operating characteristic, sensitivity, and specificity analyses indicated that the detection of mecA- and mecC-positive and negative isolates did not improve with moxalactam, either alone or in combination with cefoxitin, compared to cefoxitin alone. These findings were similar in both the S. aureus/S. lugdunensis/S. saprophyticus group and in the coagulase-negative staphylococci group. Our results do not support the use of moxalactam as an additional marker of methicillin resistance when testing with high-inoculum disk diffusion.

[The activity of moxalactam against Enterobacteriaceae and anaerobia in vitro].

OBJECTIVE: To observe the antibacterial activity of moxalactam against Enterobacteriaceae bacteria and anaerobic bacteria in vitro, and to compare with other antibacterial drugs, for providing experimental basis for the clinical application of moxalactam. METHODS: Minimum inhibitory concentrations (MICs) of moxalactam and other antibacterial agents against 491 Enterobacteriaceae spp. and anaerobic spp.collecting from clinical settings were determined by agar dilution methods and E-test strips according to the Clinical and Laboratory Standards Institute (CLSI)(2014). RESULTS: Moxalactam showed great antibacterial activity to Enterobacteriaceae spp., including ESBLs-producing Escherichia coli, Klebsiella pneumoniae, and Proteus spp., with the MIC(50), MIC(90), and susceptibility rates of 0.25-4 mg/L, 0.5-8 mg/L, and >90%, respectively. The susceptibility rates of Enterobacteriaceae with ESBLs-producing or non-ESBLs-producing to imipenem and meropenem were both higher than 90%. The susceptibility rates of ESBLs-producing Escherichia coli, Klebsiella pneumoniae, and Proteus spp.to piperacillin/tazobactam and cefoperazone/sulbactam were 90%, 68%, 53% and 76%, 66%, 76.6%, respectively, while the susceptibility rates of non-ESBLs-producing Escherichia coli, Klebsiella pneumoniae, and Proteus spp.were all more than 95%. The susceptibility rates of Enterobacter spp. and other Enterobacter to piperacillin/tazobactam were 80%, 80%and that to cefoperazone/sulbactam were 80%, 76.7%, respectively.The MICs range of moxalactam on anaerobic spp.was from ≤0.064 to >256 mg/L, while MIC(50) was 2 mg/L and MIC(90) was 64 mg/L. CONCLUSIONS: Moxalactam showed well activity against ESBLs-producing and non-ESBLs-producing Enterobacteriaceae and anaerobia.

IMP-51, a novel IMP-type metallo-ß-lactamase with increased doripenem- and meropenem-hydrolyzing activities, in a carbapenem-resistant Pseudomonas aeruginosa clinical isolate.

A meropenem-resistant Pseudomonas aeruginosa isolate was obtained from a patient in a medical setting in Hanoi, Vietnam. The isolate was found to have a novel IMP-type metallo-ß-lactamase, IMP-51, which differed from IMP-7 by an amino acid substitution (Ser262Gly). Escherichia coli expressing blaIMP-51 showed greater resistance to cefoxitin, meropenem, and moxalactam than E. coli expressing blaIMP-7. The amino acid residue at position 262 was located near the active site, proximal to the H263 Zn(II) ligand.

Conformational Change Observed in the Active Site of Class C ß-Lactamase MOX-1 upon Binding to Aztreonam.

We solved the crystal structure of the class C ß-lactamase MOX-1 complexed with the inhibitor aztreonam at 1.9Å resolution. The main-chain oxygen of Ser315 interacts with the amide nitrogen of aztreonam. Surprisingly, compared to that in the structure of free MOX-1, this main-chain carboxyl changes its position significantly upon binding to aztreonam. This result indicates that the interaction between MOX-1 and ß-lactams can be accompanied by conformational changes in the B3 ß-strand main chain.

Potent antibacterial activities of latamoxef (moxalactam) against ESBL producing Enterobacteriaceae analyzed by Monte Carlo simulation.

Latamoxef (LMOX, Moxalactam) is one of the beta-lactam antibiotics which is stable against beta-lactamase. In this study, the antibacterial activity of LMOX was investigated, and Monte Carlo simulation was conducted to determine the appropriate dosing regimens of LMOX against extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae. The probability of target attainment (PTA) was analyzed at 40% and 70% of time above minimum inhibitory concentration (MIC) (time above MIC, T(>MIC)) for bacteriostatic and bactericidal effect respectively. All the tested regimens achieved 85% of PTA at 40% of T(>MIC) against ESBL producing Escherichia coli, and all the tested regimens except 1g q12h with 1 hour infusion achieved 85% of PTA at 40% of T(>MIC) against ESBL producing Klebsiella pneumoniae. The effective regimens to achieve 85% of PTA at 70% of T(>MIC )against E. coli were lg ql2h with 4 hours infusion, lg q8h with 1-4 hours infusion, 2g ql2h with 2-4 hours infusion, and lg q6h with 1-4 hours infusion. The effective regimens to achieve 85% of PTA at 70% of T(>MIC) against K. pneumoniae were 1g q8h with 3-4 hours infusion and 1g q6h with 1-4 hours infusion. These results of pharmacokinetics/pharmacodynamics (PK/PD) modeling showed the potent efficacy of LMOX against bacterial infections caused by ESBL producing Enterobacteriaceae.

A potential substrate binding conformation of ß-lactams and insight into the broad spectrum of NDM-1 activity.

New Delhi metallo-ß-lactamase 1 (NDM-1) is a key enzyme that the pathogen Klebsiella pneumonia uses to hydrolyze almost all ß-lactam antibiotics. It is currently unclear why NDM-1 has a broad spectrum of activity. Docking of the representatives of the ß-lactam families into the active site of NDM-1 is reported here. All the ß-lactams naturally fit the NDM-1 pocket, implying that NDM-1 can accommodate the substrates without dramatic conformation changes. The docking reveals two major binding modes of the ß-lactams, which we tentatively name the S (substrate) and I (inhibitor) conformers. In the S conformers of all the ß-lactams, the amide oxygen and the carboxylic group conservatively interact with two zinc ions, while the substitutions on the fused rings show dramatic differences in their conformations and positions. Since the bridging hydroxide ion/water in the S conformer is at the position for the nucleophilic attack, the S conformation may simulate the true binding of a substrate to NDM-1. The I conformer either blocks or displaces the bridging hydroxide ion/water, such as in the case of aztreonam, and is thus inhibitory. The docking also suggests that substitutions on the ß-lactam ring are required for ß-lactams to bind in the S conformation, and therefore, small ß-lactams such as clavulanic acid would be inhibitors of NDM-1. Finally, our docking shows that moxalactam uses its tyrosyl-carboxylic group to compete with the S conformer and would thus be a poor substrate of NDM-1.

[Comparison of oxacillin, cefoxitin, ceftizoxime, and moxalactam disk diffusion methods for detection of methicillin susceptibility in staphylococci]. / Stafilokoklarda Metisilin Duyarliliginin Belirlenmesinde Oksasilin, Sefoksitin, Seftizoksim ve Moksalaktam Disk Difüzyon Yöntemlerinin Karsilastirilmasi

Penicillin binding protein 2a/2' (PBP2a/PBP2') which is encoded by the mecA gene, is responsible for the methicillin resistance in staphylococci. Detection of methicillin resistance with phenotypic methods is still a problem especially because of heterogenous expression of mecA gene. Although mecA gene determination by polymerase chain reaction is considered as the gold standard method, molecular tests are not easily applied in all routine laboratories. Thus, for the rapid and accurate diagnosis of MRSA strains, easy and practical phenotypic tests are still required. This study was conducted to compare the oxacillin (OX), cefoxitin (CFX), ceftizoxime (CZX), and moxolactam (MOX) susceptibility testing by disk diffusion method for the detection of methicillin resistance in staphylococci. A total of 247 staphylococci (125 Staphylococcus aureus and 122 coagulase-negative staphylococci; CNS) isolated from various clinical specimens (114 wound and soft tissue materials, 51 urine, 48 blood, 30 respiratory tract, and four other samples) of inpatients and outpatients, were included in this study. PBP2a latex agglutination test was used as the reference method for the recognition of methicillin resistance; four antibiotic disks tested and sensitivity, specificity, positive and negative predictive values (PPV and NPV) were determined for each of them. According to PBP2a latex agglutination test 66 (54.1%) of CNS and 53 (42.4%) of S.aureus isolates were found methicillin- resistant. OX and MOX disks detected 113 (63 CNS and 50 S.aureus) methicillin-resistant strain out of 119 PBP2a positive isolates, where CFX and CZX disks detected 110 (60 CNS and 50 S.aureus) of them. Among 128 PBP2a negative isolates, 123 (52 CNS and 71 S.aureus) were detected as susceptible with OX, 127 (55 CNS and 72 S.aureus) with CFX and CZX, 126 (54 CNS and 72 S.aureus) with MOX. According to these results, the sensitivities and specificities of OX, CFX, CZX, and MOX disks were; 95.4% and 92.8%, 90.9% and 98.2%, 90.9% and 98.2%, 95.4% and 96.4%, respectively for CNS and 94.3% and 98.6%, 94.3% and 100%, 94.3% and 100%, 94.3% and 100%, respectively for S.aureus. The difference between sensitivities and specificities of tested antibiotic disks were not found statistically significant. In conclusion, due to the problems in detection of methicillin resistance with phenotypic methods, the use of different mecA gene-inducing antibiotic disks at the same time, and utilization of molecular methods as reference method might be suggested, when a discordance is observed between the antibiotic disks.

Sensitive and specific phenotypic assay for metallo-beta-lactamase detection in Enterobacteria by use of a moxalactam disk supplemented with EDTA.

Moxalactam is highly hydrolyzed by plasmid-mediated metallo-ß-lactamases (MBLs), whereas it is poorly inactivated by serine-active carbapenemases. This study demonstrated that moxalactam resistance constituted an effective screen for MBL expression in enterobacteria, which could be confirmed, even in low-MBL-producing isolates, by a disk potentiation test using moxalactam and EDTA.

Evaluation of new Vitek 2 card and disk diffusion method for determining susceptibility of Staphylococcus aureus to oxacillin.

Detection of methicillin resistance in Staphylococcus aureus is a challenge, especially low-level resistance, which is often misdiagnosed. The aim of this study was to compare the diagnostic accuracies of the automated Vitek 2 system and disk diffusion tests, using cefoxitin and moxalactam, for the detection of methicillin resistance in S. aureus strains. Four sets of genotypically diverse isolates were selected from a national reference collection, including mecA-negative S. aureus isolates (n = 56), hospital-acquired (n = 88) and community-acquired (n = 40) S. aureus isolates, and heterogeneous methicillin-resistant S. aureus isolates (n = 29). Oxacillin susceptibility was tested by the Vitek 2 system with the AST P549 card and by disk diffusion methods using 10, 30, and 60 microg cefoxitin and 30 microg moxalactam. Oxacillin resistance was confirmed by PCR for the mecA gene. The overall sensitivities for oxacillin resistance detection were 97.5% for the Vitek 2 automated system, 98.7% for 60-microg cefoxitin and moxalactam disk diffusion, and 99.6% for 10- and 30-microg cefoxitin disks, respectively. Methicillin-susceptible S. aureus isolates were correctly reported as susceptible by all methods. The median times for methicillin testing were 7 h for the Vitek 2 system versus 24 h for disk diffusion methods. In conclusion, the cefoxitin and moxalactam disk diffusion methods and the Vitek 2 automated system are highly accurate methods for methicillin resistance detection, including a range of representative Belgian methicillin-resistant S. aureus strains and unusual strains exhibiting cryptic or low-level oxacillin resistance.

Evaluation of moxalactam with the BD phoenix system for detection of methicillin resistance in coagulase-negative staphylococci.

The performance of moxalactam with the BD Phoenix system for the detection of methicillin resistance in coagulase-negative staphylococci was evaluated by use of a collection of 186 strains. Moxalactam was a better drug as an indicator of methicillin resistance for mecA-positive strains than oxacillin and cefoxitin were. For strains other than Staphylococcus saprophyticus, a moxalactam MIC >16 microg/ml was indicative of methicillin resistance.

Comparison of cefoxitin and moxalactam 30 microg disc diffusion methods for detection of methicillin resistance in coagulase-negative staphylococci.

OBJECTIVES: To compare cefoxitin and/or moxalactam 30 microg disc diffusion (DD) methods to detect methicillin resistance in coagulase-negative staphylococci (CoNS) using both high- and low-density (HD/LD) inoculum techniques. METHODS: A challenge set of 192 CoNS was tested. DD test results were compared with PBP2a detection. RESULTS: With the LD inoculum, the sensitivity/specificity of cefoxitin and moxalactam were 94.4%/100% and 100%/92.4%, respectively, using the DD breakpoints of the Comité de l'Antibiogramme de la Société Française de Microbiologie. With the HD inoculum, the sensitivity/specificity of cefoxitin and moxalactam were 93.7%/100% and 100%/96.9%, using the cefoxitin DD breakpoints of the CLSI and a resistant/susceptible breakpoint of < 20 mm/>or=20 mm for moxalactam. Comparison of receiver operating characteristic AUCs did not show significant difference between studied assays, but the overlapping zone where both PBP2a-positive and PBP2a-negative isolates were observed concerned a lower number of strains with moxalactam than with cefoxitin (P < 0.001). Combination of cefoxitin and moxalactam DD methods demonstrated that all isolates with a concordant cefoxitin/moxalactam phenotype were correctly classified. Interestingly, all isolates misclassified by each DD method used alone were cefoxitin-susceptible and moxalactam-resistant. CONCLUSIONS: Although all DD methods studied here performed well for detecting methicillin resistance in CoNS, moxalactam had a higher accuracy than cefoxitin to differentiate heteroresistant isolates from PBP2a-negative strains. Identification of isolates that should be submitted to a confirmatory test to conclude on methicillin resistance can be easily obtained by combining cefoxitin and moxalactam DD methods.

Antibiotic recognition by binuclear metallo-beta-lactamases revealed by X-ray crystallography.

Metallo-beta-lactamases are zinc-dependent enzymes responsible for resistance to beta-lactam antibiotics in a variety of host bacteria, usually Gram-negative species that act as opportunist pathogens. They hydrolyze all classes of beta-lactam antibiotics, including carbapenems, and escape the action of available beta-lactamase inhibitors. Efforts to develop effective inhibitors have been hampered by the lack of structural information regarding how these enzymes recognize and turn over beta-lactam substrates. We report here the crystal structure of the Stenotrophomonas maltophilia L1 enzyme in complex with the hydrolysis product of the 7alpha-methoxyoxacephem, moxalactam. The on-enzyme complex is a 3'-exo-methylene species generated by elimination of the 1-methyltetrazolyl-5-thiolate anion from the 3'-methyl group. Moxalactam binding to L1 involves direct interaction of the two active site zinc ions with the beta-lactam amide and C4 carboxylate, groups that are common to all beta-lactam substrates. The 7beta-[(4-hydroxyphenyl)malonyl]-amino substituent makes limited hydrophobic and hydrogen bonding contacts with the active site groove. The mode of binding provides strong evidence that a water molecule situated between the two metal ions is the most likely nucleophile in the hydrolytic reaction. These data suggest a reaction mechanism for metallo-beta-lactamases in which both metal ions contribute to catalysis by activating the bridging water/hydroxide nucleophile, polarizing the substrate amide bond for attack and stabilizing anionic nitrogen intermediates. The structure illustrates how a binuclear zinc site confers upon metallo-beta-lactamases the ability both to recognize and efficiently hydrolyze a wide variety of beta-lactam substrates.

Sourcing faecal pollution from onsite wastewater treatment systems in surface waters using antibiotic resistance analysis.

AIMS: To identify the sources of faecal contamination in investigated surface waters and to determine the significance of onsite wastewater treatment systems (OWTS) as a major contributor to faecal contamination. METHODS AND RESULTS: Antibiotic resistance patterns (ARP) were established for a library of 717 known Escherichia coli source isolates obtained from human, domesticated animals, livestock and wild sources. Eight commonly used antibiotics, including amoxicillin, cephalothin, erythromycin, gentamicin, ofloxacin, chlortetracycline, tetracycline and moxalactam, at four different concentrations were used to obtain ARPs for E. coli isolates. Discriminant analysis (DA) was used to differentiate between the ARP of sources isolates. The developed ARP library was found to be adequate for discriminating human from nonhuman isolates, and was used to classify 256 enumerated E. coli isolates collected from monitored surface water locations. CONCLUSIONS: The resulting ARP DA indicated that a majority of the faecal contamination in more rural areas was nonhuman; however, the percentage of human isolates increased significantly in urbanized areas using OWTS for wastewater treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study signifies the feasibility of using ARP for source tracking faecal contamination in surface waters, and linking faecal contamination to OWTS. The information will enable regulatory authorities to implement appropriate management practices to reduce the contamination of water resources caused by high densities and failing OWTS.

Evaluation of three techniques for detection of low-level methicillin-resistant Staphylococcus aureus (MRSA): a disk diffusion method with cefoxitin and moxalactam, the Vitek 2 system, and the MRSA-screen latex agglutination test.

Very-low-level methicillin-resistant Staphylococcus aureus (MRSA), or class 1 MRSA, is often misdiagnosed as methicillin-susceptible S. aureus (MSSA). We evaluated the performances of three methods for detection of low-level methicillin resistance: the disk diffusion method using the cephamycin antibiotics cefoxitin and moxalactam, the Vitek 2 system (bioMérieux), and the MRSA-screen test (Denka). Detection of the mecA gene by PCR was considered to be the "gold standard." We also determined the sensitivity of the oxacillin disk diffusion method with 5- and 1-microg disks and that of the Oxascreen agar assay with 6 mg of oxacillin liter(-1) for detection of MRSA. We compared the distributions of MICs of oxacillin and cefoxitin by the E-test (AB Biodisk), and those of moxalactam by dilutions in agar, for MRSA and MSSA isolates. The 152 clinical isolates of S. aureus studied were divided into 69 MSSA (mecA-negative) and 83 MRSA (mecA-positive) isolates, including 63 heterogeneous isolates and 26 class 1 isolates (low-level resistance). The cefoxitin and moxalactam disk diffusion tests detected 100% of all the MRSA classes: cefoxitin inhibition zone diameters were <27 mm, and moxalactam inhibition zone diameters were <24 mm. The Vitek 2 system and the MRSA-screen test detected 94 and 97.6% of all MRSA isolates, respectively. The sensitivities of the 5- and 1-microg oxacillin disks were 95.2 and 96.4%, respectively, whereas that of the Oxascreen agar screen assay was 94%. All of the tests except the 1-microg oxacillin disk test were 100% specific. For the class 1 MRSA isolates, the sensitivity of the Vitek 2 test was 92.3%, whereas those of the MRSA-screen test and the disk diffusion method with cefoxitin and moxalactam were 100%. Therefore, the cefoxitin and moxalactam disk diffusion methods were the best-performing tests for routine detection of all classes of MRSA.

Phosphate deprivation is associated with high resistance to latamoxef of gel-entrapped, sessile-like Escherichia coli cells.

Viable Escherichia coli cells were entrapped in agar gel layers and incubated in a phosphate-limited glucose medium. Immobilized bacteria displayed enhanced alkaline phosphatase activity and overexpressed the outer membrane protein PhoE as compared with free-floating organisms. These observations highlighted the existence of high phosphate deprivation within biofilm-like structures. In addition, the antimicrobial efficacy of latamoxef against immobilized bacteria was partly recovered in the presence of a high phosphate concentration. From these data, a possible role of phosphate deprivation in the high resistance of sessile-like organisms to antibiotics may be considered.

Inhibition of AmpC beta-lactamase through a destabilizing interaction in the active site.

Beta-lactamases hydrolyze beta-lactam antibiotics, including penicillins and cephalosporins; these enzymes are the most widespread resistance mechanism to these drugs and pose a growing threat to public health. beta-Lactams that contain a bulky 6(7)alpha substituent, such as imipenem and moxalactam, actually inhibit serine beta-lactamases and are widely used for this reason. Although mutant serine beta-lactamases have arisen that hydrolyze beta-lactamase resistant beta-lactams (e.g., ceftazidime) or avoid mechanism-based inhibitors (e.g., clavulanate), mutant serine beta-lactamases have not yet arisen in the clinic with imipenemase or moxalactamase activity. Structural and thermodynamic studies suggest that the 6(7)alpha substituents of these inhibitors form destabilizing contacts within the covalent adduct with the conserved Asn152 in class C beta-lactamases (Asn132 in class A beta-lactamases). This unfavorable interaction may be crucial to inhibition. To test this destabilization hypothesis, we replaced Asn152 with Ala in the class C beta-lactamase AmpC from Escherichia coli and examined the mutant enzyme's thermodynamic stability in complex with imipenem and moxalactam. Consistent with the hypothesis, the Asn152 --> Ala substitution relieved 0.44 and 1.10 kcal/mol of strain introduced by imipenem and moxalactam, respectively, relative to the wild-type complexes. However, the kinetic efficiency of AmpC N152A was reduced by 6300-fold relative to that of the wild-type enzyme. To further investigate the inhibitor's interaction with the mutant enzyme, the X-ray crystal structure of moxalactam in complex with N152A was determined to a resolution of 1.83 A. Moxalactam in the mutant complex is significantly displaced from its orientation in the wild-type complex; however, moxalactam does not adopt an orientation that would restore competence for hydrolysis. Although Asn152 forces beta-lactams with 6(7)alpha substituents out of a catalytically competent configuration, making them inhibitors, the residue is essential for orienting beta-lactam substrates and cannot simply be replaced with a much smaller residue to restore catalytic activity. Designing beta-lactam inhibitors that interact unfavorably with this conserved residue when in the covalent adduct merits further investigation.

Inactivation of Aeromonas hydrophila metallo-beta-lactamase by cephamycins and moxalactam.

Incubation of moxalactam and cefoxitin with the Aeromonas hydrophila metallo-beta-lactamase CphA leads to enzyme-catalyzed hydrolysis of both compounds and to irreversible inactivation of the enzyme by the reaction products. As shown by electrospray mass spectrometry, the inactivation of CphA by cefoxitin and moxalactam is accompanied by the formation of stable adducts with mass increases of 445 and 111 Da, respectively. The single thiol group of the inactivated enzyme is no longer titrable, and dithiothreitol treatment of the complexes partially restores the catalytic activity. The mechanism of inactivation by moxalactam was studied in detail. Hydrolysis of moxalactam is followed by elimination of the 3' leaving group (5-mercapto-1-methyltetrazole), which forms a disulfide bond with the cysteine residue of CphA located in the active site. Interestingly, this reaction is catalyzed by cacodylate.