Organisms producing extended-spectrum -lactamases (ESBLs) have been reported in many countries, but there is no information on the prevalence of ESBL-producing members of the family Enterobacteriaceae in Cameroon. A total of 259 Enterobacteriaceae strains were isolated between 1995 and 1998 from patients at the Yaounde Central Hospital in Cameroon. Enterobacterial isolates resistant to extended-spectrum cephalosporin and monobactam were screened for ESBL production by the double-disk (DD) synergy test. Thirty-one (12%) of these Enterobacteriaceae strains were shown to be positive by the DD synergy test, suggesting the presence of ESBLs. Resistance to oxyimino-cephalosporins and monobactams of 12 (38.7%) of the 31 strainsi.e., 6 Klebsiella pneumoniae, 4 Escherichia coli, 1 Citrobacter freundii, and 1 Enterobacter cloacae strain-was transferred to E. coli HK-225 by conjugation. Resistance to gentamicin, gentamicin plus trimethoprimsulfamethoxazole, or trimethoprim-sulfamethoxazole was cotransferred into 6, 2, and 1 of these transconjugants, respectively. All 12 transconjugants were resistant to amoxicillin, piperacillin, all of the cephalosporins, and aztreonam but remained susceptible to cefoxitin and imipenem. Crude extracts of -lactamase-producing transconjugants were able to reduce the diameters of inhibition zones around disks containing penicillins, narrow-to expanded-spectrum cephalosporins or monobactams when tested against a fully susceptible E. coli strain but had no effect on such zones around cefoxitin, imipenem, and amoxicillin-clavulanate disks. The -lactamases produced by the 12 tranconjugants turned out to be SHV-12 by DNA sequencing. Therefore, the ESBL SHV-12 is described for the first time in Cameroon.Many extended-spectrum -lactamases (ESBLs) are plasmid-mediated derivatives from TEM-and SHV-type enzymes and cause resistance to expanded-spectrum cephalosporins. They belong to Bush group 2be (6). Since their initial description in Germany in 1983 (13), ESBLs have diversified and spread worldwide. Several ESBLs appear to be particularly widely disseminated, being found in many countries, whereas others seem to occur more commonly in one or few countries (4). The various national patterns of antibiotic consumption in hospitals probably account for the differences in distribution of these enzymes. In an attempt to detect and study the dissemination of ESBLs in a central African country (Cameroon), we collected and characterized producers of such enzymes among clinical isolates of Enterobacteriaceae at Yaounde Central Hospital between 1995 and 1998. The ESBL SHV-12 was found in several species of Enterobacteriaceae for the first time in Cameroon. MATERIALS AND METHODSBacterial strains. A total of 259 isolates, members of the Enterobacteriaceae family were collected from patients in Yaounde Central Hospital (Table 1). Isolates were collected over 3-year period (April 1995 to March 1998) from urine, pus, and blood. The isolates were identified by conventional techniques (9) and were confirmed by t...
Extended-spectrum -lactamases (ESBLs), e.g., ESBLs of the TEM or SHV type, compromise the efficacies of expanded-spectrum cephalosporins. An SHV non-ESBL that hydrolyzes only narrow-spectrum cephalosporins can be converted into an SHV ESBL through substitutions at three amino acid positions, 179, 238, or 238-240. In order to improve detection of SHV ESBLs, a novel method, based on real-time PCR monitored with fluorescently labeled hybridization probes and followed by melting curve analysis, was developed. It is able to (i) detect bla SHV genes with high degrees of sensitivity and specificity, (ii) discriminate between bla SHV non-ESBL and bla SHV ESBL , and (iii) categorize the SHV ESBL producers into three phenotypically relevant subgroups. This method, termed the SHV melting curve mutation detection method, represents a powerful tool for epidemiological studies with SHV ESBLs. It even has the potential to be used in the diagnostic microbiology laboratory, because up to 32 clinical isolates can be processed in less than 1 h by starting with just a few bacterial colonies.The production of extended-spectrum -lactamases (ESBLs) of the TEM or SHV type by bacterial pathogens is a major threat to the use of the clinically important expanded-spectrum cephalosporins. Since 1983 (19, 20), clinical isolates producing SHV-2 or related ESBLs have increasingly been reported. SHV ESBLs are derived through single amino acid substitutions from a narrow-spectrum cephalosporin-hydrolyzing enzyme, SHV-1. Over 20 SHV ESBLs, designated SHV-2 through SHV-26, have been described in the literature (6, 14, 32) and on an Internet site (http://www.lahey.org/studies/webt .htm). Since the responsible genes are often easily transferable due to their localization on plasmids (34), the situation has recently been called a "plague of plasmids" (12). SHV enzymes have frequently been found in the widespread pathogens Klebsiella, Escherichia, and Salmonella (11), rarely in other members of the family Enterobacteriaceae, and once, recently, in Pseudomonas aeruginosa (26). Phenotypic differences due to various substitutions within the ESBLs were noted early and were found to be responsible for failure of treatment with expanded-spectrum cephalosporins (16,21).In order to improve the phenotypic detection of ESBL production, standard susceptibility tests have been refined (3,13,15,17,36,37), including two commercially available tests (7, 10). Other investigators developed molecular biology-based methods, such as oligotyping (23) and PCR-restriction fragment length polymorphism analysis (2), for differentiation of different TEM ESBLs, a family of enzymes analogous to the SHV ESBLs. For detection of SHV ESBLs, methods based on single-strand conformation polymorphism analysis (8, 25) and PCR-NheI restriction analysis (30) and two tests based on the ligase chain reaction were elaborated (18,28 We describe a PCR that uses special fluorescently labeled oligonucleotide hybridization probes on a LightCycler instrument. We demonstrate a rapid, sensitive, an...
SHV extended-spectrum -lactamases (ESBLs) arise through single amino acid substitutions in the parental enzyme, SHV-1. In order to evaluate the effect of genetic dissimilarities around the structural gene on MICs, we had previously devised an isogenic system of strains. Here, we present an extended version of the system that now allows assessment of all major types of SHV -lactamases as well as of two types of promoters of various strengths. Moreover, we devised a novel vector, pCCR9, to eliminate interference of the selection marker. A substitution within the signal sequence, I8F found in SHV-7, slightly increased MICs, suggesting more efficient transfer of enzyme precursor into the periplasmic space. We also noted that combination of G238S and E240K yielded higher resistance than G238S alone. However, the influence of the additional E240K change was more pronounced with ceftazidime and aztreonam than with cefotaxime and ceftriaxone. The SHV enzymes characterized by the single change, D179N, such as SHV-8, turned out to be the weakest SHV ESBLs. Only resistance to ceftazidime was moderately increased compared to SHV-1. Since 1983 (15, 16), clinical isolates resistant to expandedspectrum cephalosporins have increasingly been reported. They were derived through single amino acid substitutions from one of three parental enzymes, TEM-1, TEM-2, or SHV-1. The resulting structures were designated extendedspectrum -lactamases (ESBLs) (13, 29), and they were classified in a new subgroup, 2be (4). Since the responsible genes are easily transferable due to frequent localization on plasmids (34) the situation has recently been called a "plague of plasmids" (6).Phenotypic differences due to the variably mutated -lactamases were noted early in vitro, and, although ESBL production appears to frequently lead to treatment failure, MICs for ESBL producers may be barely significantly increased compared to those for fully susceptible variants (18). Therefore, it is crucial to be able (i) to detect ESBLs or bla ESBL genes easily and reliably (19) and (ii) to judge the clinical significance of given ESBLs by studying the structure-function relationships of the various ESBLs. The present study is a contribution to the second aim.In order to examine the influence of amino acid substitutions in known SHV -lactamases as well as of various promoter strengths on the level of resistance, we exploited a previously developed system of strains (26) which allows direct phenotypic comparison of such derivatives under isogenic conditions. To some extent, the effect of the plasmid copy number could also be estimated through introduction of a novel lowcopy-number vector system. MATERIALS AND METHODSBacterial strains and plasmids. Escherichia coli DH5␣ (10) was used as a recipient for transformation with cloned bla SHV genes based on a novel plasmid vector, pCCR9 (this study). Relevant information on recombinant strains and plasmids are given in Tables 1 and 2.Antibiotics. Ampicillin was obtained from SmithKline Beecham Pharmaceuticals (Surrey...
Five different methods for detection of different types of SHV extended-spectrum beta-lactamases (ESBL) were compared: minimum inhibitory concentration (MIC) determination of beta-lactam with and without clavulanic acid, double-disk synergy test (DDST), inhibitor potentiated disk diffusion test (IPDDT), three-dimensional test (TDT) and PCR/Nhe I test. MIC determination of beta-lactam with and without clavulanic acid was the most sensitive method regardless of the type of beta-lactamase. However the specificity of this method was a little above 90%. IPDDT turned out to be a very sensitive method too but it lacks specificity because 26.9% of ceftazidime sensitive strains (putative ESBL negative), gave a positive result. It is important to put all four disks on the plate because ceftazidime and aztreonam were more sensitive indicators for SHV-5 and SHV-12 beta-lactamase producers while cefotaxime and ceftriaxone were more reliable in detecting SHV-2 beta-lactamase producers. The DDST detected all SHV-5 and SHV-12 beta-lactamase producers and 95.2% of SHV-2, so it was less sensitive than MIC determination but was highly specific, since there were no false negative results observed. The sensitivity of DDST can be improved by using all four disks and placing them at the smaller distance from the central disk (2.5 cm). The TDT was the least sensitive method, particularly for SHV-5 and SHV-12 beta-lactamase producers. The PCR/Nhe I test for detection of ESBL blaSHV genes is a highly sensitive and specific method but it is rather laborious and thus not very practical for use in routine clinical laboratories. Nevertheless it has potential to serve as the gold standard in epidemiological investigations on ESBLs. According to the results of this investigation MIC determination of beta-lactam with and without clavulanic acid, even if only one antibiotic is used and the PCR/Nhe I tests are the most reliable methods for detection of SHV ESBLs.
In order to assess the molecular epidemiology of 40 previously identified extended-spectrum beta-lactamase (ESBL)-producing clinical isolates of Klebsiella pneumoniae, gene sequencing was performed. While the previous examination of these isolates revealed one TEM producer, the sequencing procedure performed in this study identified 13 additional TEM producers, and all of the sequenced genes reflected production of nonESBL TEM-1. All 38 suspected SHV producers were confirmed to be carriers of blaSHV-ESBL genes using the PCR/Nhel test and sequencing. Among them, types SHV-2, SHV-5, and SHV-12 were found in 20, 10, and 7 isolates, respectively, and SHV-2a was identified in 1. SHV-5 and SHV-12 conferred higher resistance to ceftazidime and cefepime, while SHV-2 and SHV-2a raised the minimum inhibitory concentrations of cefotaxime and cefpirome. Fourth-generation cephalosporins were found to be more active against the isolates than third-generation cephalosporins.
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