Resistance to antibiotics in clinical isolates of Pseudomonas aeruginosa.

OBJECTIVES: To analyse the global resistance to some antibiotics used to treat nosocomial infections by Pseudomonas aeruginosa, specially to carbapenems, and its relationship with the presence of carbapenemases, OXA, VIM and IMP. METHODS: The study included 229 P. aeruginosa isolates from a Hospital in Northern Spain (year 2002). Susceptibility to antimicrobial agents was determined by the analysis of the MIC. Genetic typing was carried out by RAPD-PCR fingerprinting with primer ERIC-2. Genetic experiments to detect class-1 integrons were performed by PCR with primers 5'CS and 3'CS. Detection of carbapenemases was done by phenotypic (Hodge test and DDST) and genotypic methods (PCR with primers for imp, vim1, vim2 and oxa40 genes). RESULTS: 23.9% of isolates were resistant to ceftazidime, 35.9% to cefotaxime, 5.3% to amikacin, 54.9% to gentamicin, 14.6% to imipenem and 6.6% to meropenem. Isolates resistant to imipenem (33) were furtherly tested. Genetic typing didn't show clonal relatedness among the most of the isolates. Class-1 integrons were present in most isolates (sizes 600-1700 bp). Phenotypic methods for carbapenemases showed 5 positive isolates. Genotypic methods showed the presence of two isolates with the oxa40 gene. CONCLUSIONS: Meropenem, amikacin and imipenem were the most active agents to treat infections caused by Pseudomonas aeruginosa. In our study, the presence of carbapenemase enzymes wasn't high. Phenotypic tests cannot be considered as accurate screening tool to detect carbapenemases. This is the fist report of the oxa40 gene in Pseudomonas aeruginosa isolates.

[Transfer of plasmid beta-lactamases in enterobacteria]. / Transferencia de betalactamasas plasmídicas en enterobacterias.

The aim of the present study was to determine which types of beta-lactamases codified by plasmids are transferred by conjugation from several species of enterobacteria. To this end, 352 strains of ampicillin-resistant enterobacteria from clinical samples from the Hospital Civil of Bilbao were evaluated. Their beta-lactamase activity and their capacity to transfer this capacity by conjugation were evaluated. The several types of plasmidic beta-lactamases in the strains that conjugated and in their respective transconjugants were characterized by analytic isoelectric approach, and also the sensitivity of these stains to 20 beta-lactamic antibiotics and the size of their plasmids. Twenty different types were detected, with a clear predominance of TEM 1. Type TEM 2 was found in 19% of the strains which conjugated, and much less commonly the types SHV 1, HMS 1 and a beta-lactamase of an approximate pl of 4.9 were found. The transfer of these beta-lactamases is mediated by a great variety of plasmids and is associated with variable levels of resistance to penicillins and unstable cephalosporins. The presence of betalactamases with activity on the more stable cephalosporins has not been detected.

Conjugal transfer of R plasmids to and from Enterobacteriaceae isolated from sewage.

The potential of the transfer of natural plasmids between sewage strains has been studied. In vitro transfer was conducted at 37 degrees C in tryptone soya broth and sterile raw sewage as mating media. In situ transfer was carried out in sterile raw sewage within membrane diffusion chambers at 10.6 degrees C. When the recipient was a laboratory strain of Escherichia coli K-12, the in situ frequency values were significantly lower (P less than 0.001) than those obtained in vitro for the same mating pair. When the laboratory recipient was replaced with recipients from the same sewage source, frequency values decreased progressively from the optimum conditions to the most adverse. However, in situ frequency values were higher than those for the same donors mated with a laboratory recipient.

[DNA probe for the detection of uropathogenic strains of P-fimbriated Escherichia coli]. / Sonda de ADN para la detección de cepas uropatógenas de Escherichia coli P fimbriadas.

BACKGROUND: P fimbria is one of the main factors of virulence of the uropathogenic Escherichia coli strains thus developing methods for its detection is of interest. P fimbriation may manifest through associated characteristic hemagglutination patterns. Another way of directly detecting its expression is by the PF test consisting in a specific agglutination test with latex particles which have the specific receptor of the fimbria incorporated. METHODS: The two phenotypic techniques (hemagglutination pattern using human, bovine, and sheep erythrocytes, and the PF test) were compared with colony hybridization with a specific DNA probe (pap1) in 35 strains of uropathogenic E. coli. RESULTS: Eight of the 35 strains studied were positive for the PF test with 7 strains presenting mannose-resistant agglutination to human erythrocytes without agglutinating the other erythrocytes tried. However, with hybridization with the DNA probe the number of positives was higher (25/35). CONCLUSIONS: The difference found in the number of positive strains may be due to the probe used corresponding to cluster pap, thus the use of a smaller more specific probe for fimbrial expression obtained from pap should be used given that the hybridization technique is easy to perform and is carried out in less time than phenotypic detection which requires long periods of culture prior to the test.

[Detection of virulence factors using DNA probes in uropathogenic strains of Escherichia coli]. / Detección de factores de virulencia mediante sondas de DNA en cepas de Escherichia coli uropatógenas.

BACKGROUND: There are several bacterial determinants that contribute to the onset of urinary tract infection by E. coli. The present study focuses on some of the virulence factors considered to be most important, as P fimbriae, the siderophore aerobactin and bacterial capsule, which were studied among 123 uropathogenic E. coli strains isolated from outpatients from the Basque Community. METHODS: Virulence factors were detected using Molecular Biology techniques, namely DNA hybridization to specific probes prepared in our laboratory. RESULTS: When probe pap2, specific for fimbrial adherence was used, 36.5% of the strains showed positive hybridization, and 66 and 73% of the strains hybridized to probes for aerobactin and common capsule region, respectively. CONCLUSIONS: We believe that this technology provides a very useful tool for rapid and easy screening of strains harbouring different virulence factors. Nevertheless, the fact that these methods detect genetic determinants that are not always being expressed must be borne in mind.

[Antimicrobial resistance in strains of Salmonella enterica isolated in the Hospital de Basurto (Bilbao) from 1987 to 1990]. / Resistencia antimicrobiana en cepas de Salmonella enterica aisladas en el Hospital de Basurto (Bilbao) desde 1987 hasta 1990.

BACKGROUND: Rate development of antimicrobial resistance of Salmonella strains in Hospital Basurto of Bilbao from 1987 to 1990 and to study their resistance mechanism. METHODS: The antimicrobial resistance for all strains isolated (1201 strains) was performed by means of a agar-diffusion test. We selected 32 multi-resistant strains for additional study (MIC, conjugation, IEF, and plasmid profile). RESULTS: The most frequent isolated serotypes were S. enteritidis (79.01%), S. typhimurium (8.5%) and Salmonella serogroup C1 (6.9%). The resistance to one or more of 17 antimicrobial rose significantly: 9.6% in 1987; 10.25% in 1988; 16.45% in 1989 and 13.73% in 1990. The percentage of resistant serotypes were: S. typhimurium (40.2%); Salmonella serogroup B (31.8%); Salmonella serogroup C1 (16.8%); Salmonella serogroup D1 (13.04%); Salmonella serogroup C2 (9.09%) and S. enteritidis (8.4%). In 27 multi-resistant strains and their transconjugants, beta-lactamase bands with a pl: 5.4 and/or 5.6 compatible with TEM-1 and/or TEM-2 were observed. Also, these strains carried a plasmid of high molecular weight (125 MD). CONCLUSIONS: Although, the resistance of Salmonella is not a serious problem in our environment this situation is raising progressively with a greater number of strains with plasmid mediated beta-lactamases. So, the antimicrobial policy will be more severe and righ in both hospital and extrahospital surrounding.

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.

[Modulation of P-fimbriation by ciprofloxacin in uropathogenic Escherichia coli]. / Modulación de la fimbriación P por ciprofloxacina en Escherichia coli uropatógena.

BACKGROUND: The aim of this study was to determine the effect of ciprofloxacin at subinhibitory concentrations on the expression of P fimbriae of uropathogenic Escherichia coli. Thirty-nine strains of Escherichia coli isolated from out patients with urinary tract infection were studied. Thirty-nine of these strains had been previously characterized as P-fimbriated and the remaining non fimbriated strain was used as a negative control. METHODS: Fimbriation was quantitatively studied by electron microscope observation of the strains before and after treatment. To determine possible qualitative variations in the fimbrial proteins and in the external membrane (OMPs), extraction and electrophoretic separation was performed in polyacrylamide gels. RESULTS: No qualitative differences were observed in the OMPs profile and fimbrial proteins induced by ciprofloxacin in any of the strains studied. However, electron microscopic observation generally showed a decrease in the percentage fimbriated bacterial cells by the antimicrobial effect. CONCLUSIONS: The mechanism of action of ciprofloxacin at subinhibitory doses may correspond to a process of fimbrial protein synthesis inhibition secondary to the initiation of general repair mechanism of the cell exposed to the antimicrobial and not to a process of specific mutations which qualitatively affect fimbrial protein composition.

[Virulence factors and 0 serogroups of Escherichia coli as a cause of community-acquired urinary infections]. / Factores de virulencia y serogrupos 0 de Escherichia coli causantes de infecciones urinarias comunitarias.

BACKGROUND: The aim of this study was to determine the virulence factors and 0 serogroups of E. coli strains that cause community acquired urinary tract infections (UTI). METHODS: We examined 103 E. coli strains isolated from the urine of patients with UTI. The following virulence factors were investigated using phenotypic techniques: the alpha-haemolysin (Hly), the cytotoxic necrotizing factor type 1 (CNF-1) and the mannose-resistant haemagglutination (MRHA) types III, IVa and IVb expressed by P fimbriated E. coli. Serotyping of 0 antigen was carried out by means of a microtechnique using 163 antisera. RESULTS: Fifty-five (53%) of the 103 E. coli strains examined showed some of the virulence factors investigated in this study; 41% of the strains were Hly+, 28% were CNF-1+ and 48% expressed MRHA types III, IVa or IVb. The uropathogenic strains characterized belonged to 27 different 0 serogroups. However, 68% were from one of 10 serogroups (01, 02, 04, 06, 09, 018, 027, 073, 075 and 077) and 36% were from one of only 3 serogroups (02, 04 and 06). Furthermore, the virulence factors were concentrated in strains belonging to the 3 serogroups most frequently detected. Thus, 36 (97%) of the 37 strains of these 3 serogroups showed virulence factors, versus only 19 (29%) of 66 belonging to other serogroups (p < 0.001). CONCLUSIONS: Our results support the special pathogenicity theory and suggest that many cases of community acquired UTI may be caused by a limited number of uropathogenic E. coli strains that produce toxins (Hly+ and/or CNF-1+) and possess P fimbriae or P-related adhesins (with MRHA types III, IVa or IVb), and that usually belong to 02, 04 and 06 serogroups.