Molecular characterization of cefoxitin-resistant Escherichia coli from Canadian hospitals.
(33/339)
A study designed to gain baseline information on strains of Escherichia coli displaying resistance to cefoxitin in Canada is described. A total of 29,323 E. coli isolates were screened at 12 participating hospital sites as part of an extended-spectrum beta-lactamase surveillance initiative. A total of 411 clinically significant, nonrepeat isolates displaying reduced susceptibilities to the NCCLS-recommended beta-lactams were submitted to a central laboratory over a 1-year period ending on 30 September 2000. Two hundred thirty-two isolates were identified as resistant to cefoxitin. All cefoxitin-resistant strains were subtyped by pulsed-field gel electrophoresis, and of these, 182 strains revealed a unique fingerprint and 1 strain was untypeable. PCR and sequence analysis of the ampC promoter region revealed 51 different promoter or attenuator variants and 14 wild-type promoters. Three promoter regions were interrupted by insertion elements, two contained IS10 elements, and one contained an IS911 variant. PCR and sequence analysis for the detection of acquired AmpC resistance (by the acquisition of ACT-1/MIR-1, CMY-2, or FOX) revealed that 25 strains contained CMY-2, including 7 of the strains found to have wild-type promoters. The considerable genetic variability in both the strain fingerprint and the promoter region suggests that AmpC-type resistance may emerge spontaneously by mutation of sensitive strains rather than by the spread of strains or plasmids in the hospital setting. (+info)
Evaluation of cefoxitin 5 and 10 microg discs for the detection of methicillin resistance in staphylococci.
(34/339)
OBJECTIVE: To evaluate cefoxitin 5 and 10 microg discs for detection of methicillin resistance in staphylococci. METHODS: Six hundred and forty-one Staphylococcus aureus (261 mecA-negative and 380 mecA-positive) and 344 coagulase-negative staphylococci (CoNS) (132 mecA-negative and 212 mecA-positive) were investigated. The CoNS represented nine species, Staphylococcus epidermidis being the most frequent (n = 231). All isolates were tested using semi-confluent growth on Iso-Sensitest agar (ISA), and Mueller-Hinton agar (MH) using a 5 and a 10 microg cefoxitin disc and overnight incubation in ambient air at 35-37 degrees C. RESULTS: For S. aureus, both cefoxitin discs performed with high accuracy on both media. The sensitivity and specificity for the following proposed interpretive zone diameters were: ISA 5 microg, R < 14 mm (99.5% and 98.1%); ISA 10 microg, R < 22 mm (99.5% and 98.1%); MH 5 microg, R < 12 mm (99.7% and 98.1%); and MH 10 microg, R < 18 mm (99.5% and 98.9%), respectively. All four variants were superior to oxacillin using the former SRGA methodology. In CoNS, a substantial overlap was seen for all variants. However, by avoiding primary interpretation in the overlapping interval, highly accurate results could be obtained for 81%, 80%, 91% and 97% of the isolates, respectively. CONCLUSION: For S. aureus, cefoxitin 5 and 10 microg discs performed with high accuracy on both ISA and MH using semi-confluent growth and standard incubation conditions. With the introduction of a defined interval in which primary interpretation should be avoided, the method could also be used for CoNS. (+info)
Cefoxitin resistance as a surrogate marker for the detection of methicillin-resistant Staphylococcus aureus.
(35/339)
OBJECTIVES: To evaluate the usefulness of cefoxitin when used as a surrogate marker for the detection of methicillin resistance. PATIENTS AND METHODS: Eight hundred and seventy-one strains of Staphylococcus aureus, collected from eight tertiary referral centres serving diverse socio-economic populations, were included in the study using NCCLS disc diffusion and the agar dilution methods. RESULTS: Using cefoxitin and NCCLS criteria for disc diffusion, the sensitivity and specificity for recognizing methicillin resistance were both 100%. Similar results were obtained when the strains were tested by the agar dilution method. The cefoxitin MICs for methicillin-susceptible strains were < or = 4 mg/L. CONCLUSIONS: Testing with cefoxitin as a surrogate marker for the detection of methicillin resistance was very accurate with both disc diffusion and agar dilution methods. Such testing clearly distinguished methicillin-resistant strains of S. aureus from methicillin-susceptible strains. (+info)
Increase in ampC promoter strength due to mutations and deletion of the attenuator in a clinical isolate of cefoxitin-resistant Escherichia coli as determined by RT-PCR.
(36/339)
OBJECTIVES: To characterize the mechanism of cefoxitin resistance in clinical isolate Escherichia coli N99-0001. METHODS: Plasmid analysis, PCR for beta-lactamases, and sequencing of the ampC genes was carried out. An RT-PCR method was developed to determine relative ampC expression. RESULTS: Analysis of the ampC promoter region of E. coli N99-0001 revealed a T-->A mutation at -32, a C-->A mutation at -11, an insertion of a T between -20 and -21, and a 28 bp deletion including the entire attenuator. RT-PCR showed that ampC was expressed 140-fold higher in E. coli N99-0001 than in E. coli ATCC 25922. CONCLUSIONS: Cefoxitin resistance in E. coli N99-0001 was due to overexpression of ampC caused by an increase in promoter strength. (+info)
Laboratory tests in the detection of extended spectrum beta-lactamase production: National Committee for Clinical Laboratory Standards (NCCLS) screening test, the E-test, the double disk confirmatory test, and cefoxitin susceptibility testing.
(37/339)
Extended spectrum beta-lactamase (ESBL) production by Klebsiella sp. and E. coli is an emerging problem. In this study, 107 clinical isolates (53 E. coli, 47 K. pneumoniae and 7 K. oxytoca) screened as ESBL producers by the NCCLS disk diffusion procedure were submitted to a double disk confirmatory test (DDT) and to the E-test double strip for confirmation of ESBL production by demonstration of clavulanic acid inhibition effect (CAIE). Only 72/107 (67%) of the isolates were confirmed as ESBL producers by DDT, with diverse results among species. By the E-test, 58/107 (54%) isolates were confirmed as ESBL producers, and 18/107 (17%) were not determinable. Susceptibility to cefoxitin was found in 57/68 (83%) of strains that did not show CAIE. ESBL detection remains a controversial issue and clinical laboratories are in need of a simple and effective way to recognize strains with this kind of resistance. (+info)
Structure, function, and inhibition along the reaction coordinate of CTX-M beta-lactamases.
(38/339)
CTX-M enzymes are an emerging group of extended spectrum beta-lactamases (ESBLs) that hydrolyze not only the penicillins but also the first-, second-, and third-generation cephalosporins. Although they have become the most frequently observed ESBLs in certain areas, there are few effective inhibitors and relatively little is known about their detailed mechanism. Here we describe the X-ray crystal structures of CTX-M enzymes in complex with different transition-state analogues and beta-lactam inhibitors, representing the enzyme as it progresses from its acylation transition state to its acyl enzyme complex to the deacylation transition state. As the enzyme moves along this reaction coordinate, two key catalytic residues, Lys73 and Glu166, change conformations, tracking the state of the reaction. Unexpectedly, the acyl enzyme complex with the beta-lactam inhibitor cefoxitin still has the catalytic water bound; this water had been predicted to be displaced by the unusual 7alpha-methoxy of the inhibitor. Instead, the 7alpha-group appears to inhibit by preventing the formation of the deacylation transition state through steric hindrance. From an inhibitor design standpoint, we note that the best of the reversible inhibitors, a ceftazidime-like boronic acid compound, binds to CTX-M-16 with a K(i) value of 4 nM. When used together in cell culture, this inhibitor reversed cefotaxime resistance in CTX-M-producing bacteria. The structure of its complex with CTX-M enzyme and the structural view of the reaction coordinate described here provide templates for inhibitor design and intervention to combat this family of antibiotic resistance enzymes. (+info)
Cefoxitin, a semisynthetic cephamycin antibiotic: antibacterial spectrum and resistance to hydrolysis by gram-negative beta-lactamases.
(39/339)
The in vitro activity of cefoxitin, 3-carbamolyloxymethyl-7-alpha-methoxy-7[2-(2-thienyl)acetamido]-3-cephem-4-carboy xlic acid, was investigated. Activity against gram-positive organisms was less than that of cephalothin and cephloridine. It was highly active against gram-negative bacilli, with activity against Escherichia coli, Proteus mirabilis, and Klebsiella pneumoniae equal to that of currently available cephalosporins. In addition, it was active against certain Enterobacter strains, Serratia marcescens, indole-positive Proteae and Herellea. The strains of these latter bacteria were strains susceptible to carbenicillin and ticarcillin. Pseudomonas aeruginosa and other Pseudomonas species were resistant. Changes in pH, inoculum size, and type of growth medium had no significant effect on the activity of the antibiotic. Cefoxitin was highly resistant to hydrolysis by various types of gram-negative beta-lactamases. The precise role of resistance to beta-lactamase hydrolysis varied from strain to strain. Bacterial resistance to cefoxitin was not necessarily related to hydrolysis of the antibiotic. However, the resistance of cefoxitin to hydrolysis did contribute to its activity. Cefoxitin could function as an inducer of beta-lactamase activity and effectively bound to purified beta-lactamases. (+info)
Cefoxitin and cephalothin: antimicrobial activity, human pharmacokinetics, and toxicology.
(40/339)
Cefoxitin, a semisynthetic cephamycin, has been compared with the widely used parenteral cephalosporin, cephalothin, in terms of antibacterial activity, human pharmacokinetics, and toxicity. For both compounds, minimal inhibitory concentrations were within the therapeutic range against the 156 gram-positive cocci tested (except group D streptococci), but cephalothin was 8 to 20 times more active. Regarding the 313 gram-negative organisms tested, both antibiotics were of approximately equal activity against cephalothin-susceptible strains, but cefoxitin was outstandingly superior against Providencia spp. and indole-producing Proteus spp., and markedly better against Serratia marcescens and Bacteroides fragilis. Against these organisms, cefoxitin but not cephalothin would be expected to be therapeutically valuable. Antibiotic activity levels in the serum and urine of 18 human volunteers after parenteral administration were higher and more prolonged in the case of cefoxitin, which had an average terminal serum half-life of about 45 min and a urinary recovery of about 90%. Cefoxitin was entirely nontoxic and, given intramuscularly, slightly less painful then cephalothin. These preliminary results suggest that cephamycins may prove to be a significant chemotherapeutic advance. (+info)