Emergence of resistance to beta-lactam agents in enterobacteriaceae species with group I beta-lactamases in Spain.

The contribution of induction and stable derepression of chromosomal group I beta-lactamases to beta-lactam antibiotics resistance was studied in clinical isolates of Enterobacteriaceae, collected from patients treated with these antibiotics. Multiple isolates of the same species from the same patient were characterized by different typing methods. Sonicated extracts of cells were assayed for chromosomal and plasmid-mediated beta-lactamases by isoelectric focusing and cloxacillin inhibition studies. The specific beta-lactamase activity, basal and induced with cefoxitin, was determined to differentiate strains with inducible or derepressed production of the enzyme. Induction of beta-lactamases was performed in each strain against the beta-lactams used in the therapy of each patient. Older penicillins resulted in a moderate to strong increase in beta-lactamase activity, whereas the results obtained with first-generation cephalosporins were species dependent. Expanded-spectrum cephalosporins were weak inducers of beta-lactamases. Indeed, the use of cefotaxime for treatment preceded the appearance of strains that produced chromosomal group I beta-lactamases constitutively. These strains showed a remarkable reduction in sensitivity to ureidopenicillins, carboxipenicillins, expanded-spectrum cephalosporins, and monobactams, but not to carbapenems.

Five-year survey of cefotaxime resistance in Spain.

During 1991-1995 a Spain collaborative study group surveyed the resistance to cefotaxime both in community as well as in hospital isolates of bacteria. The isolates tested during the study period of 5 years were 813, 875, 3631, 3184, and 3050 strains, respectively. Antimicrobial activity of cefotaxime was assayed by broth or agar microdilution, in accordance with criteria of the National Committee of Clinical Laboratory Standards (NCCLS). Cefotaxime resistance included 2.5% of all isolates: 2.6% Enterobacteriaceae, 1.7% Streptococcus pneumoniae, 0.5% Haemophilus influenzae, 0.0% Haemophilus spp., and 0.0% Moraxella catarrhalis. The overall incidence of resistance to cefotaxime decreased fro member of Enterobacteriaceae from 3.6% in 1991 to 2.5% in 1995. The incidence of resistance varied with the species and was highest in Enterobacter and in Citrobacter freundii.

Non-radioactive PCR-SSCP with a single PCR step for detection of inhibitor resistant beta-lactamases in Escherichia coli.

A method based on PCR-SSCP has been developed to detect presumptive Inhibitor-Resistant TEM (IRT) beta-lactamases in Escherichia coli. The capacity of this technique to differentiate genes from 11 control strains encoding IRT beta-lactamases was evaluated with PCR products digested with RsaI. All the bla(TEM) genes studied could be distinguished by their electrophoretic mobilities. Applied to 29 epidemiologically unrelated clinical isolates of E. coli resistant to amoxicillin-clavulanate (MIC, > or =32 microg/ml), the electrophoretic mobilities of the digested bla(TEM) PCR products were identical to those of the reference bla(TEM-1A) (6 strains) and bla(TEM-1B) (18 strains) genes. The remaining five bla(TEM) PCR products displayed SSCP profiles different from those of the reference bla(TEM) genes and their nucleotide sequence identified them as bla(TEM-1C) in one strain, bla(TEM-30/IRT-2) in two strains, bla(TEM-37/IRT-8) in one strain, and bla(TEM-40/IRT-11) in one isolate. Overexpression of the wild-type bla(TEM-1) gene, as detected by high-level resistance to beta-lactams and enzyme assay, accounted for resistance in the 24 E. coli containing bla(TEM-1). We report a simple one PCR step SSCP that can be used in epidemiological studies for rapid preliminary detection of IRT beta-lactamases; identification should be confirmed by sequence data.

[Profile of bacterial isolates and antimicrobial susceptibility: Multicenter study using a one-day cut-off]. / Perfil de aislamientos bacterianos y sensibilidad antimicrobiana. Estudio multicéntrico mediante corte de un día.

The frequency and antimicrobial susceptibility of bacterial isolates from the largest clinical samples collected in 19 Spanish hospitals were studied. A total of 523 strains were identified and grouped by sample. Blood stream: Staphylococcus coagulase-negative (41%) and Escherichia coli (19.7%); oxacillin resistance occurred in 44% of coagulase-negative strains, strains which were also resistant to nonbetalactam agents. All antimicrobial agents tested had good activity against E. coli, with the exception of penicillins (25 to 33% susceptible). Urine: E. coli (59.1%) and Enterococcus faecalis (15%); aminoglycosides and third generation cephalosporins were the most active compounds against E. coli, whereas penicillins and cotrimoxazole were the least active. E. faecalis isolates showed low rates of resistance to the antibiotics tested and no glycopeptide-resistant strains were detected. Skin and soft tissues: Staphylococcus aureus (24.1%), Pseudomonas aeruginosa (17.7.%); oxacillin resistance occurred in 15.8% of S. aureus strains and co-resistance to nonbetalactam agents was frequently observed among these strains. Ceftazidime susceptibility was elevated among P. aeruginosa (76.9%) and the most active agents were aminoglycosides (100% susceptibility). Lower respiratory tract: P. aeruginosa (21.4%) and Haemophilus influenzae (15.5%). Aminoglycosides (88.8 to 94.4%) and ceftazidime (72%) presented the highest susceptibility rates in P. aeruginosa. All the agents tested were very active against H. influenzae (89% susceptibility). Among Gram-positive cocci, no vancomycin and/or teicoplanin-resistant strains were detected.

[Molecular study of ampicillin resistance in clinical isolates of Salmonella]. / Estudio molecular de la resistencia a ampicilina en aislamientos clínicos de Salmonella.

BACKGROUND: The purpose of this work was to study the molecular basis of beta-lactamase production in ampicillin-resistant strains of Salmonella spp. METHODS: It was performed analytical isoelectric focusing of beta-lactamases produced by a group of 33 strains selected in basis of their resistance phenotype. Plasmid profile analysis and assays of transferable drug resistance were developed. The study was completed by hybridization experiments with an intragenic TEM probe which allowed the location of the bla-TEM gene. RESULTS: By analytical isoelectrofocusing we found that 26 out of the 27 ampicillin-resistant strains produced beta-lactamases with pl 5.4 and/or 5.6 corresponding to TEM-1 and/or TEM-2 type. Analysis of plasmid DNA revealed in almost all strains plasmids ranging in size from 1.1 to 125 Mdal. This plasmids were responsible of the resistance and, moreover, were able to transfer the resistance by conjugation mechanisms. Southern blot analysis detected the gene that code the TEM beta-lactamase at the 125, 8 and 5.8 Mdal plasmids. CONCLUSIONS: Resistance to ampicillin in the strains of Salmonella studied was due to the presence of TEM type beta-lactamases coded by conjugative plasmids. These plasmids coded also resistance to other antimicrobial agents. Our results showed that the use of a DNA probe to the detect TEM-type beta-lactamases using a non radioactive probe, could be a suitable alternative to isoelectric focusing.

In vitro activity of biapenem against beta-lactamase producing Enterobacteriaceae.

The activity of biapenem was compared with that of imipenem and cefotaxime against 108 strains of beta-lactamase producing Enterobacteriaceae. Biapenem and imipenem were very active, inhibiting 90% of the strains at a concentration of 0.5 microgram/ml. Both carbapenems were very active against plasmidic beta-lactamase producers, with MIC90s below 1 microgram/ml. However, the MIC90 of biapenem for cephalosporinase producers was 1 microgram/ml. Against strains producing extended-spectrum beta-lactamases, biapenem exhibited better activity against TEM-type producers (MIC90 0.25 microgram/ml) than against SHV-type producers (MIC90 0.5 microgram/ml). Overall, the in vitro antibacterial activity of biapenem is similar to that of imipenem.