cefoxitin and nitrocefin

Limited treatment options available for Mycobacterium abscessus infections include the parenteral β-lactam antibiotics cefoxitin and imipenem, which show moderate in vitro activity. Other β-lactam antibiotics (except meropenem) have no considerable in vitro activity, due to their rapid hydrolysis by a broad-spectrum β-lactamase (Bla_Mab). We here addressed the impact of β-lactamase production and β-lactam in vitro stability on M. abscessus MIC results and determined the epidemiological cut-off (ECOFF) values of cefoxitin, imipenem and meropenem.. By LC high-resolution MS (LC-HRMS), we assessed the in vitro stability of cefoxitin, imipenem and meropenem. M. abscessus ATCC 19977 strain and its isogenic blaMab deletion mutant were used for MIC testing. Based on MIC distributions for M. abscessus clinical strains, we determined ECOFFs of cefoxitin, imipenem and meropenem.. A functional Bla_Mab increased MICs of penicillins, ceftriaxone and meropenem. LC-HRMS data showed significant degradation of cefoxitin, imipenem and meropenem during standard antibiotic susceptibility testing procedures. MIC, MIC50 and ECOFF values of cefoxitin, imipenem and meropenem are influenced by incubation time.. The results of our study support administration of imipenem, meropenem and cefoxitin, for treatment of patients infected with M. abscessus. Our findings on in vitro instability of imipenem, meropenem and cefoxitin explain the problematic correlation between in vitro susceptibility and in vivo activity of these antibiotics and question the clinical utility of susceptibility testing of these chemotherapeutic agents.

Topics: Anti-Bacterial Agents; beta-Lactamases; beta-Lactams; Cefoxitin; Cephalosporins; Drug Stability; Humans; Imipenem; Meropenem; Microbial Sensitivity Tests; Mutation; Mycobacterium abscessus; Mycobacterium Infections, Nontuberculous; Thienamycins

The constitutively expressed, chromosomally encoded β-lactamase (BlaC) is the enzyme responsible for the intrinsic resistance to β-lactam antibiotics in Mycobacterium tuberculosis. Previous studies from this laboratory have shown that the enzyme exhibits an extended-spectrum phenotype, with very high levels of penicillinase and cephalosporinase activity, as well as weak carbapenemase activity [Tremblay, L. W., et al. (2008) Biochemistry 47, 5312-5316]. In this report, we have determined the pH dependence of the kinetic parameters, revealing that the maximal velocity depends on the ionization state of two groups: a general base exhibiting a pK value of 4.5 and a general acid exhibiting a pK value of 7.8. Having defined a region where the kinetic parameters are pH-independent (pH 6.5), we determined solvent kinetic isotope effects (SKIEs) for three substrates whose kcat values differ by 5.5 orders of magnitude. Nitrocefin is a highly activated, chromogenic cephalosporin derivative that exhibits steady-state solvent kinetic isotope effects of 1.4 on both V and V/K. Cefoxitin is a slower cephalosporin derivative that exhibits a large SKIE on V of 3.9 but a small SKIE of 1.8 on V/K in steady-state experiments. Pre-steady-state, stopped-flow experiments with cefoxitin revealed a burst of β-lactam ring opening with associated SKIE values of 1.6 on the acylation step and 3.4 on the deacylation step. Meropenem is an extremely slow substrate for BlaC and exhibits burst kinetics in the steady-state experiments. SKIE determinations with meropenem revealed large SKIEs on both the acylation and deacylation steps of 3.8 and 4.0, respectively. Proton inventories in all cases were linear, indicating the participation of a single solvent-derived proton in the chemical step responsible for the SKIE. The rate-limiting steps for β-lactam hydrolysis of these substrates are analyzed, and the chemical steps responsible for the observed SKIE are discussed.

Topics: Acylation; Bacterial Proteins; beta-Lactam Resistance; beta-Lactamases; Cefoxitin; Cephalosporins; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Meropenem; Mycobacterium tuberculosis; Solvents; Thienamycins

Samples were taken under strictly anaerobic conditions from the root canals of 22 patients having a tooth in which necrotic pulp was associated with an inflammatory peri-apical lesion an optical periodontitis. In the majority of cases (85%), these lesions were chronic and the pulp chamber was closed. One hundred and two strains were isolated, 71.7% of the bacteria being obligate anaerobes and 49.8% being Gram-negative bacilli. Their ability to produce beta-lactamases was tested and 8.8% gave a positive reaction in a cefinase test. These cefinase positive strains were nevertheless susceptible to 3rd-generation cephalosporins (cefoxitin) and to amoxicillin-clavulanate, with the exception of M. Morganii.

Topics: Actinomyces; Amoxicillin-Potassium Clavulanate Combination; Anti-Bacterial Agents; Bacteriological Techniques; Bacteroides; beta-Lactam Resistance; beta-Lactamases; Cefoxitin; Cephalosporins; Dental Pulp Cavity; Dental Pulp Necrosis; Gram-Negative Bacteria; Gram-Positive Bacteria; Humans; Indicators and Reagents; Peptostreptococcus; Periapical Periodontitis; Prevotella; Streptococcus

The widespread use of beta-lactam antibiotics has lead to the development of resistance to this group of antibiotics in bacterial pathogens due to beta-lactamase production. Information on such pathogens is not available from eastern region of India. This study was undertaken to determine the AmpC beta-lactamase production in pathogens isolated from hospitalized patients in Kolkata.. Non-repeat clinical isolates (284) from pus, urine, sputum and other clinical specimens of hospitalized patients were taken. Disk agar diffusion (DAD) and minimum inhibitory concentration (MIC) with different beta-lactam antibiotics, and double disc synergy test (DDST) with clavulanic acid and sulbactam were done. Disk antagonism test (DAT) and three-dimensional extract test (TDET) were conducted for phenotypic confirmation of AmpC and inducible AmpC beta-lactamase production. Nitrocefin spot test and microiodometric assay of beta-lactamase were also performed.. Twenty seven isolates were found to be resistant to cefoxitin, a alpha-methoxy-beta-lactam. Of these, 19 were observed to be AmpC beta-lactamase producers and 4 were inducible AmpC beta- lactamase producers by DDST, DAT and TDET. Remaining 4 were non AmpC beta-lactamase producers. Of the 23 AmpC beta-lactamase producers, the distribution of different species was as follows: Escherichia coli 11 (47.8%), Pseudomonas aeruginosa 4 (17.3%) Klebsiella pneumoniae 3 (13%), and Klebsiella aeruginosa 1 (4.3%).. Our finding showed 6.7 per cent AmpC beta-lactamase and 1.4 per cent inducible AmpC beta-lactamase producing clinical isolates from Kolkata. AmpC beta-lactamase producing bacterial pathogens may cause a major therapeutic failure if not detected and reported in time.

Topics: Anti-Bacterial Agents; Bacteria; Bacterial Proteins; beta-Lactamases; Cefoxitin; Cephalosporins; Clavulanic Acid; Drug Resistance, Bacterial; Enzyme Induction; Hospitals; Humans; India; Isoelectric Focusing; Microbial Sensitivity Tests; Species Specificity

Isolates previously identified as Citrobacter diversus are now known as Citrobacter koseri. We measured sequence variation at the beta-lactamase structural gene among a group of clinical isolates originally identified as C. diversus by API 20E profiling.. beta-Lactamase and 16S rRNA genes were amplified by PCR and sequenced by standard methods. beta-Lactamase induction was attempted in liquid-grown cultures using cefoxitin. Nitrocefin hydrolysis assays were performed using a spectrophotometer.. Analysis of 16S rRNA gene sequences showed that Citrobacter spp. isolates with an inducible beta-lactamase gene, cdiA, closely related to 'C. koseri ' NF85 and ULA27 are actually Citrobacter amalonaticus. C. koseri isolates, whose identities were confirmed by 16S rRNA sequencing, produce a class A beta-lactamase, Cko, constitutively at low levels. The cko and cdiA beta-lactamase genes share <45% identity.. We have confirmed that cko is a beta-lactamase gene carried by C. koseri, and that isolates previously identified as 'C. koseri ', but carrying the cdiA beta-lactamase gene are C. amalonaticus. Thus, beta-lactamase-gene-specific PCR may provide a valuable tool to differentiate these biochemically homogeneous Citrobacter species.

Topics: beta-Lactamases; Cefoxitin; Cephalosporins; Cephamycins; Citrobacter; Citrobacter koseri; Gene Expression Regulation, Bacterial; Microbial Sensitivity Tests; Phylogeny; Reverse Transcriptase Polymerase Chain Reaction; RNA, Ribosomal, 16S; Sequence Homology, Nucleic Acid

Susceptibilities to beta-lactam antibiotics and beta-lactamase content of two groups of Bacteroides strains were compared. Type cultures produced low levels of beta-lactamase and were susceptible to cefoxitin, latamoxef, imipenem and the combination of benzylpenicillin and clavulanic acid. Other Bacteroides strains that produced higher levels of beta-lactamase were generally less susceptible to these antibiotics; this resistance was more closely related to enzyme type than to the amount of enzyme present. The beta-lactamases produced by the test strains fell into three broad groups on the basis of antibiotic degradation and inhibitor profiles: those that inactivated benzylpenicillin, but not cefoxitin, latamoxef or imipenem, and were susceptible to inhibition by beta-lactamase inhibitors; those that hydrolysed benzylpenicillin, cefoxitin and latamoxef, but not imipenem, and which were less susceptible to inhibition by beta-lactamase inhibitors; an enzyme that inactivated all the antibiotics and was not inhibited by beta-lactamase inhibitors.

Topics: Bacteroides fragilis; beta-Lactamase Inhibitors; beta-Lactamases; Cefoxitin; Cephalosporins; Drug Resistance, Microbial; Imipenem; Isoelectric Point; Kinetics; Microbial Sensitivity Tests; Moxalactam; Nephelometry and Turbidimetry; Thienamycins

Using a nitrocefin competition assay, I determined the relative substrate affinity index (RSAI) values of nine clinically significant beta-lactamase enzymes against a range of beta-lactams. Using selected beta-lactam substrates, I observed large differences in the RSAI values of the nine enzymes that were sufficient in many cases to positively identify specific enzymes. I made use of the unique RSAI values of SHV-1, TEM-1, and TEM-2 beta lactamases with cefoxitin to screen for the presence of these enzymes in Klebsiella aerogenes clinical isolates. The RSAI values also allow for the prediction of the outcome of beta-lactam therapy against specific beta-lactamase-producing isolates.

Topics: Anti-Bacterial Agents; beta-Lactamases; Cefoxitin; Cephalosporins; Enterobacter; Escherichia coli; Klebsiella pneumoniae; Microbial Sensitivity Tests; Substrate Specificity