Sixteen Escherichia coli clinical isolates which were resistant to ampicillin and amoxicillin-clavulanate but susceptible to cephalothin were studied. Eight strains showed the presence of a ,I-lactamase which comigrates with reference OXA-1 enzyme. The eight other strains produced different TEM-1 derivatives which had in common a higher Km for penicillins and a higher 50%o inhibitory concentration for the P-lactamase inhibitors.By oligotyping and sequencing of PCR products, it was shown that Ser (AGC) (TEM-30; also called TRI-1) in three strains and Cys (TGC) (TEM-31; also called TRI-2) in one strain were substituted for , that Leu (CTG) (TEM-33) and Val (GTG) (TEM-34) in one strain each were substituted for , and that in other mutants the two latter substitutions occurred together with the substitution of Asp (GAT) for Asn-272 (AAT). Therefore, different sets of amino acid substitutions of TEM-1 can be found in clinical isolates and lead to resistance to P-lactamase inhibitors.Resistance to aminopenicillins has become very frequent in Escherichia coli and is observed in France in up to 30 to 50% of the E. coli strains isolated in the hospital as well as in the community (25). This resistance is generally due to the presence of plasmid-mediated ,-lactamases, among which TEM P-lactamases are the most frequently found (22,25). This is one reason that combinations of ,B-lactam antibiotics with inhibitors of P-lactamases are now extensively used. Therefore, it is not surprising that many investigators have reported the emergence of clinical isolates of E. coli resistant to some of these combinations (3,4,26,28,31,32,(34)(35)(36). At least two major mechanisms are evoked to explain this resistance: either overproduction of the TEM P-lactamase or a modification of the kinetic properties of the TEM 1-lactamase due to some amino acid substitution (4,20,32,36). In this work, we have studied the mechanism of resistance in 16 clinical isolates of E. coli resistant to the combination of amoxicillin and clavulanate but susceptible to cephalothin. MATERIALS AND METHODSBacterial strains, medium, and antibiotics. Sixteen clinical isolates of amoxicillin-clavulanate-resistant and cephalothinsusceptible E. coli were collected during a 1-year period (1991 to 1992) from different units and different patients in two hospitals (Hopital Broussais and Hopital Saint-Joseph Identification of the TEM 13-lactamase. After separation on a sodium dodecyl sulfate-15% polyacrylamide gel of the proteins present in the crude P-lactamase preparations of the different strains, polyclonal anti-TEM antibodies (kindly provided by J. Davies) and immunoblotting were used to identify the 1-lactamase as previously described (1).Oligotyping and sequencing of DNA amplified by PCR. Colony hybridizations were performed as follows. Nylon filters (0.45-,um pore size) (Hybond-N; Amersham) were placed on Mueller-Hinton agar and inoculated with 10 ,ul of 18-h broth cultures. After 6 h of incubation, colonies were lysed and DNA was fixed as described previously (1...
We have previously described a clinical isolate of Escherichia coli (Q2) that is highly resistant to fluoroquinolones (MIC of ciprofloxacin, 16 p,g/ml) but susceptible to nalidixic acid (MIC of nalidixic acid, 4 ,ug/ml) (N. Moniot-Ville, J. Guibert, N. Moreau, J. F. Acar, E. Collatz, and L. Gutmann, Antimicrob. Agents Chemother. 35:519-523, 1991). Transformation of strain Q2 with a plasmid carrying the wild-type gyrA gene from E. coli K-12(pAFF801) resulted in a 32-fold decrease in the MIC of ciprofloxacin, suggesting that at least one mutation in gyrA was involved in the resistance of Q2. Intragenic gyrA fragments of 668 and 2,500 bp from strain Q2 were amplified by the polymerase chain reaction. We sequenced the 668-bp fragment and identified a single novel point mutation (transition from G to A at position 242), leading to an amino acid substitution (Gly-81 to Asp) in the gyrase A subunit. We constructed hybrid plasmids by substituting either the 668-bp fragment or the 2,500-bp fragment from Q2 DNA, both of which contained the gyrA point mutation, for the corresponding fragments in wild-type gyrA (2,625 bp) of E. coli K-12. When introduced into E. coli KNK453 (gyrA temperature sensitive), both plasmids conferred an eightfold increase in the MIC of ciprofloxacin, but only a twofold increase in the MIC of nalidixic acid. When introduced into E. coli Q2, neither plasmid conferred any change in the MICs of ciprofloxacin or nalidixic acid, suggesting that only the point mutation found in gyrA was involved in the resistance that we observed.Quinolones are among the most potent antibacterial agents (1, 2) developed so far for use in humans. On the basis of their structures and antimicrobial spectra, quinolones have been separated into two groups; the original agents include nalidixic acid, flumequine, and oxolinic acid, while the newer fluoroquinolones include all the recently developed molecules, such as norfloxacin, ofloxacin, pefloxacin, and ciprofloxacin. Besides the antimicrobial activities of the older quinolones, which are essentially directed against enterobacteria, the newer quinolones have broader spectra of activity, including activities against nearly all gramnegative species (bacilli and cocci), some gram-positive species (staphylococci), and intracellular bacterial species, such as rickettsii and mycobacteria (2). The chemical structures of the two groups differ by a piperazine ring at position C-7 and a fluorine atom at position C-6 in the second group (5). Quinolones have been shown to inhibit DNA gyrase activities such as DNA supercoiling and DNA replication (8). DNA gyrase, an A2B2 tetrameric enzyme which is the target of the quinolones (11), is a type II topoisomerase that is essential for replication and gene expression and that is also involved in recombination and conjugation (16,32,33). Until now, chromosomal mutations are the only observed genetic changes which can lead to quinolone resistance (9). The mechanisms involved in quinolone resistance include a decrease in membrane permeability,...
Three clinical isolates, Enterobacter cloacae EC1562 and EC1563 and Citrobacter freundii CFr564, displayed an aminoglycoside resistance profile evocative of low-level 6′-N acetyltransferase type II [AAC(6′)-II] production, which conferred reduced susceptibility to gentamicin but not to amikacin or isepamicin. Aminoglycoside acetyltransferase assays suggested the synthesis in the three strains of an AAC(6′) which acetylated amikacin practically as well as it acetylated gentamicin in vitro. Both compounds, however, as well as isepamicin, retained good bactericidal activity against the three strains. The aacgenes were borne by conjugative plasmids (pLMM562 and pLMM564 of ca. 100 kb and pLMM563 of ca. 20 kb). By PCR mapping and nucleotide sequence analysis, an aac(6′)-Ib gene was found in each strain upstream of an ant(3")-I gene in asulI-type integron. The size of the AAC(6′)-Ib variant encoded by pLMM562 and pLMM564, AAC(6′)-Ib7, was deduced to be 184 (or 177) amino acids long, whereas in pLMM563 a 21-bp duplication allowing the recruitment of a start codon resulted in the translation of a variant, AAC(6′)-Ib8, of 196 amino acids, in agreement with size estimates obtained by Western blot analysis. Both variants had at position 119 a serine instead of the leucine typical for the AAC(6′)-Ib variants conferring resistance to amikacin. By using methods that predict the secondary structure, these two amino acids appear to condition an α-helical structure within a putative aminoglycoside binding domain of AAC(6′)-Ib variants.
High‐level carbapenem‐resistant (CpmR) mutants, with MICs for imipenem and carbapenem of > 128 μg/ml, were selected in vitro from four carbapenem‐susceptible (CpmS) clinical isolates of Bacteroides fragilis. The CpmS strains produced very low levels of β‐lactamase activity, which was increased approx. 50‐ to 100‐fold in the CpmR mutants. Isoelectric focussing and enzyme kinetic analysis (Km and Vrel) of the ‘carbapenemases’ from the CpmR mutants and similarly resistant clinical isolates suggested a close relatedness of the enzymes. A probe covering most of the cfiA gene encoding such an enzyme (Thompson, J.S. and Malamy, M.H. (1990) J. Bacteriol. 172, 2584–2593) hybridized with DNA from the CpmR mutants, their CpmS parental strains as well as clinical CpmR isolates, but not from randomly chosen carbapenem‐susceptible strains. The possibility is considered that mutations leading to expression of the silent carbapenemase gene, and thereby to clinically relevant carbapenem resistance, may also occur in the clinical setting.
Seventy amikacin-resistant clinical isolates of gram-negative bacteria belonging to nine genera were examined by immunoblotting and by DNA-DNA hybridisation for the presence of ACC(6')1b enzyme, previously called AAC(6')-4, or its encoding gene aacA1b. The organisms mostly had resistance profiles compatible with AAC(6') production and were from South and North America, the Far East and Europe. Polyclonal (rabbit) anti-AAC(6')-1b antisera and an intragenic aacA1b (aacA4) probe derived from the multiresistance plasmid pAZ007 were used. The aacA1b gene was found to be widespread. Positive hybridisation, and immunologically cross-reactive proteins, were observed in 44% of the isolates examined. They were present most frequently (greater than or equal to 70%) in isolates of Klebsiella, Escherichia and Enterobacter spp., but less often (less than or equal to 25%) in Serratia, Citrobacter, Acinetobacter and Pseudomonas spp. The strains that reacted with the probe produced enzymes that varied in their apparent mol. wts between c. 24,000 and 26,000. The existence of multiple electrophoretic forms of amikacin-acetylating enzymes of the ACC(6')-1b type may be useful in epidemiological surveys of AAC(6')-mediated amikacin resistance.
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