Interaction energies between beta-lactam antibiotics and E. coli penicillin-binding protein 5 by reversible thermal denaturation.
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Penicillin-binding proteins (PBPs) catalyze the final stages of bacterial cell wall biosynthesis. PBPs form stable covalent complexes with beta-lactam antibiotics, leading to PBP inactivation and ultimately cell death. To understand more clearly how PBPs recognize beta-lactam antibiotics, it is important to know their energies of interaction. Because beta-lactam antibiotics bind covalently to PBPs, these energies are difficult to measure through binding equilibria. However, the noncovalent interaction energies between beta-lactam antibiotics and a PBP can be determined through reversible denaturation of enzyme-antibiotic complexes. Escherichia coli PBP 5, a D-alanine carboxypeptidase, was reversibly denatured by temperature in an apparently two-state manner with a temperature of melting (T(m)) of 48.5 degrees C and a van't Hoff enthalpy of unfolding (H(VH)) of 193 kcal/mole. The binding of the beta-lactam antibiotics cefoxitin, cloxacillin, moxalactam, and imipenem all stabilized the enzyme significantly, with T(m) values as high as +4.6 degrees C (a noncovalent interaction energy of +2.7 kcal/mole). Interestingly, the noncovalent interaction energies of these ligands did not correlate with their second-order acylation rate constants (k(2)/K'). These rate constants indicate the potency of a covalent inhibitor, but they appear to have little to do with interactions within covalent complexes, which is the state of the enzyme often used for structure-based inhibitor design. (+info)
Inactivation of Aeromonas hydrophila metallo-beta-lactamase by cephamycins and moxalactam.
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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. (+info)
Phosphate deprivation is associated with high resistance to latamoxef of gel-entrapped, sessile-like Escherichia coli cells.
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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. (+info)
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.
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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 (bioMerieux), 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. (+info)
Effect of beta-lactams on peptidoglycan metabolism of Haemophilus influenzae grown in animals.
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We have examined bacterial determinants that influence beta-lactam activity in Haemophilus influenzae cells cultivated in a system that reproduces in vivo growth conditions. Bacteria grown in diffusion chambers were recovered from the peritoneal cavities of rats, and their cell properties were compared with those of bacteria grown in broth cultures by various tests performed in vitro. The rate of peptidoglycan synthesis was measured as the incorporation of [14C]alanine into cell wall material in the presence of chloramphenicol. The total incorporation of [14C]alanine into peptidoglycan was markedly increased in cells grown in rats prior to the assay but was efficiently reduced by the beta-lactams. The extent of cross-linking was lower in the peptidoglycan of in vivo-grown bacteria, as estimated by sodium dodecyl sulfate- to trichloroacetic acid-insoluble radioactive cell wall material ratios. A whole-cell labeling assay with 125I-penicillin was used to characterize the penicillin-binding proteins (PBPs). Four PBPs showed a striking reduction in the binding of the labeled penicillin in cells grown in rats. Such changes resembled the PBP alterations seen in beta-lactamase-negative clinical strains that were resistant to the beta-lactams. Although ampicillin and moxalactam showed delayed inhibitory activities in vitro for cells collected from rats, cells recovered from beta-lactam-treated rats showed evidence of antibiotic effectiveness (binding of the beta-lactams to PBPs in vivo and altered morphology), and the killing of cells exposed to antibiotics in broth or in peritoneal fluid was equally good. Finally, the frequencies of spontaneous resistance or tolerance to ampicillin or moxalactam were estimated, and there was no significant difference for in vitro- or in vivo-grown cells. These data demonstrated that the cultivation of H. influenzae in animals created changes in PBPs and the overall peptidoglycan metabolism. Such alterations did not impair the bactericidal activities of the beta-lactams, although they resulted in delayed bacterial inhibition, a phenomenon that may have important consequences in antibiotherapy. (+info)
Comparison of cefoxitin and moxalactam 30 microg disc diffusion methods for detection of methicillin resistance in coagulase-negative staphylococci.
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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 Comite de l'Antibiogramme de la Societe Francaise 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. (+info)
Evaluation of moxalactam with the BD phoenix system for detection of methicillin resistance in coagulase-negative staphylococci.
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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. (+info)
Evaluation of new Vitek 2 card and disk diffusion method for determining susceptibility of Staphylococcus aureus to oxacillin.
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