Characterization of extended-spectrum β-lactamase genes found among Escherichia coli isolates from duck and environmental samples obtained on a duck farm

In this study, we focused on evaluating the occurrence of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in fecal samples of healthy ducks and environmental samples from a duck farm in South China. Duck cloacal swabs and pond water samples were cultivated on MacConkey agar plates supplemented with ceftiofur. Individual colonies were examined for ESBL production. Bacteria identified as E. coli were screened for the presence of ESBL and plasmid-borne AmpC genes. The genetic relatedness, plasmid replicon type, and genetic background were determined. Of 245 samples analyzed, 123 had E. coli isolates with ceftiofur MICs higher than 8 μg/ml (116 [50.4%] from 230 duck samples and 7 [46.7%] from 15 water samples). bla(CTX-M), bla(SHV-12), bla(CMY-2), and bla(DHA-1) were identified in 108, 5, 9, and 1 isolates, respectively. The most common bla(CTX-M) genes were bla(CTX-M-27) (n = 34), bla(CTX-M-55) (n = 27), bla(CTX-M-24e) (n = 22), and bla(CTX-M-105) (n = 20), followed by bla(CTX-M-14a), bla(CTX-M-14b), bla(CTX-M-24a), and bla(CTX-M-24b). Although most of the CTX-M producers had distinct pulsotypes, clonal transmission between duck and water isolates was observed. bla(CTX-M) genes were carried by transferable IncN, IncF, and untypeable plasmids. The novel CTX-M gene bla(CTX-M-105) was flanked by two hypothetical protein sequences, partial ISEcp1 upstream and truncated IS903D, iroN, orf1, and a Tn1721-like element downstream. It is suggested that the horizontal transfer of bla(CTX-M) genes mediated by mobile elements and the clonal spread of CTX-M-producing E. coli isolates contributed to the dissemination of bla(CTX-M) in the duck farm. Our findings highlight the importance of ducks for the dissemination of transferable antibiotic resistance genes into the environment.     

Mechanisms of resistance to cephalosporin and emergence of O25b-ST131 clone harboring CTX-M-27 β-lactamase in extraintestinal pathogenic Escherichia coli from dogs and cats in Japan

Thirty-three cefazolin-resistant extraintestinal pathogenic Escherichia coli strains from companion animals were screened for bla(CMY-1) , bla(CMY-2) , bla(SHV) , bla(TEM) , and bla(CTX-M) genes. Extended-spectrum β-lactamase-producing strains were further characterized by O serotyping and multilocus sequence typing. It was found that 20 and 17 isolates harbored TEM-1 and CMY-2 β-lactamases, respectively, and 13 isolates harbored both β-lactamases. One isolate harbored DHA-1 β-lactamase. Eleven isolates were found to possess CTX-M β-lactamases (CTX-M-27 [n= 6], CTX-M-14 [n= 3], CTX-M-15 [n= 1], and CTX-M-55 [n= 1]). Of 11 CTX-M-positive strains, four strains were O25b-ST131 clones harboring CTX-M-27, and the remaining seven strains belonged to O6-ST127, ONT-ST354, O159-ST539, O1-ST648, O8-ST1642, O25b-ST2042, and ONT-ST2178.     

呼吸道产超广谱β-内酰胺酶分离株耐药基因初步分型

目的了解产超广谱β-内酰胺酶 (ESBLs)呼吸道分离株的主要基因型分布特点.方法用表型确证试验确定临床呼吸道标本中产ESBLs的大肠埃希菌和肺炎克雷伯菌.应用聚合酶链反应(PCR)方法扩增产ESBLs株的bla(TEM)、bla(SHV)和bla(CTX-M)基因.结果 PCR结果显示bla(TEM)、bla(SHV)和bla(CTX-M)基因的总阳性率分别为40 .7%、45.7%和75.3%,其中大肠埃希菌分别为:64.9%、2.7%和91.9%,肺炎克雷伯菌分别为:20.5%、81.8%和61.4%.67.6%的大肠埃希菌和95.5%的肺炎克雷伯菌同时携带多个基因.结论深圳市人民医院呼吸道分离的产ESBLs大肠埃希菌的主要基因型为CTX-M,肺炎克雷伯菌主要基因型为SHV.大多数菌株同时携带多个基因.     

Impact of feed supplementation with antimicrobial agents on growth performance of broiler chickens, Clostridium perfringens and enterococcus counts, and antibiotic resistance phenotypes and distribution of antimicrobial resistance determinants in Escherichia coli isolates

The effects of feed supplementation with the approved antimicrobial agents bambermycin, penicillin, salinomycin, and bacitracin or a combination of salinomycin plus bacitracin were evaluated for the incidence and distribution of antibiotic resistance in 197 commensal Escherichia coli isolates from broiler chickens over 35 days. All isolates showed some degree of multiple antibiotic resistance. Resistance to tetracycline (68.5%), amoxicillin (61.4%), ceftiofur (51.3%), spectinomycin (47.2%), and sulfonamides (42%) was most frequent. The levels of resistance to streptomycin, chloramphenicol, and gentamicin were 33.5, 35.5, and 25.3%, respectively. The overall resistance levels decreased from day 7 to day 35 (P < 0.001). Comparing treatments, the levels of resistance to ceftiofur, spectinomycin, and gentamicin (except for resistance to bacitracin treatment) were significantly higher in isolates from chickens receiving feed supplemented with salinomycin than from the other feeds (P < 0.001). Using a DNA microarray analysis capable of detecting commonly found antimicrobial resistance genes, we characterized 104 tetracycline-resistant E. coli isolates from 7- to 28-day-old chickens fed different growth promoters. Results showed a decrease in the incidence of isolates harboring tet(B), bla(TEM), sulI, and aadA and class 1 integron from days 7 to 35 (P < 0.01). Of the 84 tetracycline-ceftiofur-resistant E. coli isolates, 76 (90.5%) were positive for bla(CMY-2). The proportions of isolates positive for sulI, aadA, and integron class 1 were significantly higher in salinomycin-treated chickens than in the control or other treatment groups (P < 0.05). These data demonstrate that multiantibiotic-resistant E. coli isolates can be found in broiler chickens regardless of the antimicrobial growth promoters used. However, the phenotype and the distribution of resistance determinants in E. coli can be modulated by feed supplementation with some of the antimicrobial agents used in broiler chicken production.     

High prevalence of extended-spectrum beta lactamases among Salmonella enterica Typhimurium isolates from pediatric patients with diarrhea in China

We investigated the extended-spectrum beta lactamases among 62 Salmonella enterica Typhimurium isolates recovered from children with diarrhea in a Chinese pediatric hospital. A large proportion of S. enterica Typhimurium isolates were resistant to multiple antimicrobial agents, including ampicillin (90.3%), tetracycline (80.6%), trimethoprim/sulfamethoxazole (74.2%), chloramphenicol (66.1%), cefotaxime (27.4%). Forty-nine (79.0%) of S. enterica Typhimurium isolates were positive for bla(TEM-1b) and resistant to ampicillin. Thirteen S. enterica Typhimurium isolates (21.0%) were positive for bla(CTX-M-1-group) and bla(CTX-M-9-group), and all isolates harboring bla(CTX-M) genes were positive for ISEcp1. Two main clones (PFGE type A and D) accounted for nearly 70% of S. enterica Typhimurium isolates, and 7 CTX-M-producing isolates belonged to PFGE type D. Collectively, our data reveal multi-drug resistance and a high prevalence of extended spectrum beta lactamases among S. enterica Typhimurium isolates from children in China. In addition, we report the first identification of bla(CTX-M-55) within Salmonella spp. Our data also suggest that clonal spread is responsible for the dissemination of S. enterica Typhimurium isolates.     

产超广谱β-内酰胺酶大肠埃希菌和肺炎克雷伯菌blaCTX-M基因的检测

目的了解产CTX-M型超广谱β-内酰胺酶 (ESBLs) 大肠埃希菌和肺炎克雷伯菌在深圳市人民医院的分布情况.方法应用美国国家临床实验室标准化委员会(NCCLS)推荐的表型确证试验,从2000年6月～2003年8月该院临床标本分离株中筛选出不重复的产ESBLs菌株215株,其中大肠埃希菌151株,肺炎克雷伯菌64株,应用聚合酶链反应(PCR)检测所有产ESBLs株的bla(CTX-M)基因.结果 PCR结果显示,大肠埃希菌bla(CTX-M)基因阳性率为92.1%(139/151),肺炎克雷伯菌的阳性率为65.6%(42/64).bla(CTX-M)基因阳性菌株主要来源于临床送检尿和痰标本,并广泛分布于20多个临床科室.结论该院临床分离的大肠埃希菌和肺炎克雷伯菌产生的ESBL大多数为CTX-M型,该类酶广泛分布于各临床科室,需引起重视.     

Acquired AmpC type beta-lactamases: an emerging problem in Italian long-term care and rehabilitation facilities

We report the multiple detection of Proteus mirabilis isolates, from 4 different long-term care and rehabilitation facilities (LTCRFs) of Northern Italy, resistant to expanded-spectrum cephalosporins and cephamycins and producing an acquired ampC-like beta-lactamase, named CMY-16. Genotyping by PFGE showed that isolates were clonally related to each other, although not identical. In all isolates the bla(CMY16) gene was not transferable by conjugation and was found to be carried on the chromosome. These results revealed multifocal spreading of a CMY-16 producing P. mirabilis clone in Northern Italy and emphasize the emergence of similar acquired resistance determinants in the LTCRFs setting.     

Genetic environments of bla(CTX-M) genes located on conjugative plasmids of Enterobacteriaceae nosocomial isolates collected in Russia within 2003-2007

The study showed that bla(CTX-M) genes were present in the genomes of 71% of cephalosporin resistant Enterobacteriaceae nosocomial isolates (n=833) collected in Russian hospitals within 2003-2007, including 91% of E.coli, 90% of Klebsiella spp., 38% of Enterobacter spp., 31% of Citrobacter spp. (n=9), and 36% of the other Enterobacteriaceae species. The genes belonging to the following subtypes (clusters) were identified: bla(CTX-M-1) (529 bla(CTX-M-15) genes; 25 bla(CTX-M-3) genes; 1 bla(CTX-M-22) gene, 1 bla(CTX-M-23) gene, and 1 bla(CTX-M-34) gene); bla(CTX-M-2) (1 bla(CTX-M-2) gene, and 4 bla(CTX-M-5) genes), and bla(CTX-M-9) (2 bla(CTX-M-9) genes, and 28 bla(CTX-M-14) genes). It was shown that bla(CTX-M) genes were located on high-molecular weight (60-160 bp) conjugative plasmids belonging mainly to the incompatibility groups IncF, IncL/M and IncA/C (bla(CTX-M-15) gene); IncL/M(bla(CTX-M-3) gene); and IncF, IncL/Mand IncI1-ly (CTX-M-14 gene). The gene environments of bla(CTX-M) genes were shown specific for the subtype of the genes. A mobile genetic element ISEcp1 (in some cases deleted or inserted by IS26, IS1, IS10, resTn2, or resTn3 sequences, in direct or reverse position) were detected upstream of bla(CTX-M-3), bla(CTX-M-14), and bla(CTX-M-15) genes. A special characteristic was the sequence between ISEcp1 and bla(CTX-M) gene: 48 bp for bla(CTX-M-15) (except 1 E.coli isolate having such a sequence deleted by 3 bp); 127 bp for bla(CTX-M-3); 42 bp for bla(CTX-M-14). Downstream of bla(CTX-M) and bla(CTX-M-15) genes in the major bacterial isolates orf477 mucA and Delta orf477-Delta mucA sequences were detected respectively. Two isolates had additional Delta orf3 insertion inside of Delta orf477-Delta mucA sequence. Insertion sequence IS903 (intact or deleted) was detected downstream of bla(CTX-M-14) gene. Unlike the others, bla(CTX-M-2) and bla(CTX-M-9) genes were located inside of ISCR1 mobile element, downstream of class 1 integron and orf513 sequence.     

Molecular epidemiology of ESbetaL producing P. mirabilis strains from a long-term care and rehabilitation facility in Italy

We report the detection of multidrug resistant ESbetaL producing Proteus mirabilis isolates from a long-term care and rehabilitation facility (LTCRF) in Northern Italy. 53% of the collected P. mirabilis strains were ESbetaL producers. PCR and sequencing techniques confirmed the presence of the bla(TEM-92) and bla(CMY-16) resistance genes in 23/26 (88.5%) and 3/26 (11.5%) of the ESbetaL producers respectively. PFGE showed that the TEM-92 beta-lactamase producing isolates were not clonally related, indicating the presence of at least four different clonal lineages (A, B, C, D), whereas all the CMY-16 enzyme producers belonged in the same lineage. The bla(TEM-92) and bla(CYY-16) determinants were distributed in seven different wards, but in three of them they coexisted. Our results show that the most patients are co-colonized by ESbetaLs producing P. mirabilis strains at the time of admission to an LTCRF. An effective strategy to curtail the spread of ESbetaLs mediated resistance in LTCRFs could be to activate sourveillance programs to monitor routinely the entry of resistant bacteria.     

Role of Ceftiofur in Selection and Dissemination of blaCMY-2-Mediated Cephalosporin Resistance in Salmonella enterica and Commensal Escherichia coli Isolates from Cattle

Third-generation cephalosporin resistance of Salmonella and commensal Escherichia coli isolates from cattle in the United States is predominantly conferred by the cephamycinase CMY-2, which inactivates β-lactam antimicrobial drugs used to treat a wide variety of infections, including pediatric salmonellosis. The emergence and dissemination of bla_CMY-2--bearing plasmids followed and may in part be the result of selection pressure imposed by the widespread utilization of ceftiofur, a third-generation veterinary cephalosporin. This study assessed the potential effects of ceftiofur on bla_CMY-2 transfer and dissemination by (i) an in vivo experimental study in which calves were inoculated with competent bla_CMY-2-bearing plasmid donors and susceptible recipients and then subjected to ceftiofur selection and (ii) an observational study to determine whether ceftiofur use in dairy herds is associated with the occurrence and frequency of cephalosporin resistance in Salmonella and commensal E. coli. The first study revealed bla_CMY-2 plasmid transfer in both ceftiofur-treated and untreated calves but detected no enhancement of plasmid transfer associated with ceftiofur treatment. The second study detected no association (P = 0.22) between ceftiofur use and either the occurrence of ceftiofur-resistant salmonellosis or the frequency of cephalosporin resistance in commensal E. coli. However, herds with a history of salmonellosis (including both ceftiofur-resistant and ceftiofur-susceptible Salmonella isolates) used more ceftiofur than herds with no history of salmonellosis (P = 0.03) These findings fail to support a major role for ceftiofur use in the maintenance and dissemination of bla_CMY-2-bearing plasmid mediated cephalosporin resistance in commensal E. coli and in pathogenic Salmonella in these dairy cattle populations.The major mechanism of third-generation cephalosporin resistance among U.S. human and veterinary clinical isolates of Salmonella enterica subsp. enterica is the beta-lactamase CMY-2 (12, 17, 43, 44, 46). bla_CMY-2, which likely originated from the chromosomal AmpC locus of Citrobacter freundii, is disseminated among a group of similar plasmids harbored by diverse Enterobacteriaceae species (1, 2, 20, 26, 30, 31, 42, 45). In Salmonella, bla_CMY-2-bearing plasmids have been observed in more than 30 serovars, notably including serovar Newport, which has gained specific attention from public health officials as a rapidly emerging threat (2, 6, 31).Commensal Escherichia coli frequently harbors bla_CMY-2-bearing plasmids (15, 33, 44), and these plasmids may be transferable to pathogens, since bla_CMY-2 plasmids isolated from E. coli and S. enterica share extensive sequence similarity in addition to the bla_CMY-2 open reading frame (5, 12, 42, 44). This transfer may occur in the gastrointestinal tracts of cattle, where these bacterial species periodically coexist and where transconjugants may be subjected to specific antimicrobial selection pressure. In fact, in vivo transfer of bla_CMY-2 in the gastrointestinal tract has been reported between a Klebsiella pneumoniae bla_CMY-2 plasmid donor and a Salmonella enterica serovar Typhimurium isolate in cattle and goats (29).Ceftiofur is the only third-generation cephalosporin antimicrobial drug that is used in cattle production systems and is labeled for the treatment of pneumonia, postpartum metritis, necrotizing pododermatitis, and mastitis. Two ceftiofur preparations, ceftiofur sodium (Naxcel) and ceftiofur hydrochloride (Excenel) (Pfizer Animal Health, New York, NY), are unique in the veterinary pharmacopeia because they require no withholding and discard of milk collected from treated cows, making them frequent therapeutic choices in lactating animals (19, 35). Ceftiofur was licensed in 1988 (41) and its resistance in Salmonella spp. isolated from U.S. cattle, presumably conferred by bla_CMY-2, was first documented in 1998 (6).The effects of ceftiofur use on selection of bla_CMY-2-bearing commensal E. coli has been examined for cattle both epidemiologically and experimentally. Tragesser et al. studied 18 Ohio dairy herds and determined that the 11 herds that used ceftiofur in any capacity (labeled indications and/or extralabel use) were 25 times more likely to have E. coli with reduced susceptibility to ceftriaxone (an expected bla_CMY-2 phenotype) than the seven herds that reported no ceftiofur use (40). Interestingly, however, within eight herds that had detailed treatment records, no association was detected between the prevalence of E. coli with reduced susceptibility to ceftriaxone and use of ceftiofur on an individual-animal basis (40). In an experimental study by Jiang et al., ceftiofur administered to dairy calves was correlated with a 14% increase in ceftriaxone-resistant fecal E. coli compared to untreated controls (21). Together, these studies show a correlation between selection pressure within the gastrointestinal tracts at the individual-animal level and show that ceftiofur use may promote the dissemination of resistance in commensal E. coli at the whole-herd level.Whether or not ceftiofur treatment directly affects in vivo horizontal transfer of bla_CMY-2-bearing elements among E. coli and Salmonella has yet to be addressed. The diversity of bla_CMY-2 plasmid-bearing bacterial hosts is consistent with wide dissemination of this genetic element. One hypothesis that could explain this wide dissemination is that ceftiofur may itself promote the in vivo horizontal transfer of bla_CMY-2-bearing plasmids. Specifically, due to the relatively slow bactericidal activity of aminothiazolyl cephalosporins such as ceftiofur, it has been suggested that exposure to these compounds promotes filament formation in gram-negative bacteria prior to cell death that may increase the surface area and increase receptiveness of the cells for resistance plasmids (11).Because bla_CMY-2 may be disseminated by horizontal transfer of R plasmids and/or clonal expansion of individual strains, we examined the effect of ceftiofur use on these processes with two approaches; the first approach specifically considered the issue of horizontal transfer in an experimental in vivo calf model, while the second approach, a field study, assessed the overall relationship between ceftiofur use and bla_CMY-2 prevalence in the primary agricultural animal niche where it is used.     

Potential pathogenicity and host range of extended-spectrum beta-lactamase-producing Escherichia coli isolates from healthy poultry

Thirty of 33 epidemiologically unrelated extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates from healthy poultry lacked the virulence genes commonly associated with human-pathogenic strains. The main zoonotic risk is associated with the broad host range of avian E. coli belonging to sequence type complex 10 and of IncN and IncI1 plasmids carrying bla(CTX-M) or bla(SHV).     

Human-associated extended-spectrum β-lactamase in the Antarctic

Escherichia coli bacteria with extended-spectrum β-lactamase (ESBL) type CTX-M resistance were isolated from water samples collected close to research stations in Antarctica. The isolates had bla(CTX-M-1) and bla(CTX-M-15) genotypes and sequence types (ST) indicative of a human-associated origin. This is the first record of ESBL-producing enterobacteria from Antarctica.     

Molecular characteristics of extended-spectrum beta-lactamases and qnr determinants in Enterobacter species from Japan

The incidence of extended-spectrum β-lactamases (ESBLs) has been increasing worldwide, but screening criteria for detection of ESBLs are not standardized for AmpC-producing Enterobacteriaceae such as Enterobacter species. In this study, we investigated the prevalence of ESBLs and/or AmpC β-lactamases in Japanese clinical isolates of Enterobacter spp. and the association of plasmid-mediated quinolone resistance (PMQR) determinants with ESBL producers. A total of 364 clinical isolates of Enterobacter spp. collected throughout Japan between November 2009 and January 2010 were studied. ESBL-producing strains were assessed by the CLSI confirmatory test and the boronic acid disk test. PCR and sequencing were performed to detect CTX-M, TEM, and SHV type ESBLs and PMQR determinants. For ESBL-producing Enterobacter spp., pulsed-field gel electrophoresis (PFGE) was performed using XbaI restriction enzyme. Of the 364 isolates, 22 (6.0%) were ESBL producers. Seven isolates of Enterobacter cloacae produced CTX-M-3, followed by two isolates producing SHV-12. Two isolates of Enterobacter aerogenes produced CTX-M-2. Of the 22 ESBL producers, 21 had the AmpC enzyme, and six met the criteria for ESBL production in the boronic acid test. We found a significant association of qnrS with CTX-M-3-producing E. cloacae. The 11 ESBL-producing Enterobacter spp. possessing bla(CTX-M), bla(SHV), or bla(TEM) were divided into six unique PFGE types. This is the first report about the prevalence of qnr determinants among ESBL-producing Enterobacter spp. from Japan. Our results suggest that ESBL-producing Enterobacter spp. with qnr determinants are spreading in Japan.     

Analysis of bla(CTX-M)-carrying plasmids from Escherichia coli isolates collected in the BfT-GermVet study

In this study, 417 Escherichia coli isolates from defined disease conditions of companion and farm animals collected in the BfT-GermVet study were investigated for the presence of extended-spectrum β-lactamase (ESBL) genes. Three ESBL-producing E. coli isolates were identified among the 100 ampicillin-resistant isolates. The E. coli isolates 168 and 246, of canine and porcine origins, respectively, harbored bla(CTX-M-1), and the canine isolate 913 harbored bla(CTX-M-15), as confirmed by PCR and sequence analysis. The isolates 168 and 246 belonged to the novel multilocus sequence typing (MLST) types ST1576 and ST1153, respectively, while isolate 913 had the MLST type ST410. The ESBL genes were located on structurally related IncN plasmids in isolates 168 and 246 and on an IncF plasmid in isolate 913. The bla(CTX-M-1) upstream regions of plasmids pCTX168 and pCTX246 were similar, whereas the downstream regions showed structural differences. The genetic environment of the bla(CTX-M-15) gene on plasmid pCTX913 differed distinctly from that of both bla(CTX-M-1) genes. Detailed sequence analysis showed that the integration of insertion sequences, as well as interplasmid recombination events, accounted for the structural variability in the bla(CTX-M) gene regions.     

Molecular epidemiology of ceftiofur-resistant Escherichia coli isolates from dairy calves

Healthy calves (n = 96, 1 to 9 weeks old) from a dairy herd in central Pennsylvania were examined each month over a five-month period for fecal shedding of ceftiofur-resistant gram-negative bacteria. Ceftiofur-resistant Escherichia coli isolates (n = 122) were characterized by antimicrobial resistance (disk diffusion and MIC), serotype, pulsed-field gel electrophoresis subtypes, beta-lactamase genes, and virulence genes. Antibiotic disk diffusion assays showed that the isolates were resistant to ampicillin (100%), ceftiofur (100%), chloramphenicol (94%), florfenicol (93%), gentamicin (89%), spectinomycin (72%), tetracycline (98%), ticarcillin (99%), and ticarcillin-clavulanic acid (99%). All isolates were multidrug resistant and displayed elevated MICs. The E. coli isolates belonged to 42 serotypes, of which O8:H25 was the predominant serotype (49.2%). Pulsed-field gel electrophoresis classified the E. coli isolates into 27 profiles. Cluster analysis showed that 77 isolates (63.1%) belonged to one unique group. The prevalence of pathogenic E. coli was low (8%). A total of 117 ceftiofur-resistant E. coli isolates (96%) possessed the bla(CMY2) gene. Based on phenotypic and genotypic characterization, the ceftiofur-resistant E. coli isolates belonged to 59 clonal types. There was no significant relationship between calf age and clonal type. The findings of this study revealed that healthy dairy calves were rapidly colonized by antibiotic-resistant strains of E. coli shortly after birth. The high prevalence of multidrug-resistant nonpathogenic E. coli in calves could be a significant source of resistance genes to other bacteria that share the same environment.     

An algorithm for identification of CTX-M-type beta-lactamase genes using restriction fragment length polymorphism analysis of PCR-product

The algorithm of the identification of the bla(CTX-M) genes coding CTX-M-type beta-lactamases providing resistance to cephalosporins III-IV was developed. This algorithm provides identification of 49 genes of 96 genes presented in the GenBank database so far. Remaining 47 genes can be identified as consisting of small sub-groups composed of 2-6 genes with the exception of sub-group of the bla(CTX-M-14)-like genes composed of 13 genes. The identification of the bla(CTX-M) genes is based on two-step restriction fragment length polymorphism analysis of 544 bp PCR-product (PCR-RFLP). In the first step, determination of subtype (cluster) of the bla(CTX-M) gene occurred using the restriction nuclease Alu I: cluster 1, -2, -8, -9 or -25. Moreover, four genes can be identified just at this step: bla(CTX-M)-59, (cluster 2); bla(CTX-M-63) (cluster 8), bla(CTX-M-45) (cluster 9), and bla(CTX-M-78) (hybrid gene between cluster 2 and cluster 25). At the second step gene identification goes on inside of each cluster separately using a set of 26 restriction nucleases. As a result of the PCR-RFLP-analysis, 23 bla(CTX-M) genes can be identified at the cluster 1, 11 genes--at the cluster 2, 4 genes--at the cluster 8, 9 genes--at the cluster 9, 1 gene--at the cluster 25, and 2 hybrid genes: bla(CTX-M-78) (between clusters 2 and 25), and bla(CTX-M-64) (between clusters 1 and 9). The described algorithm was used for identification of the blac(CTX-M) genes (n = 585) detected in Enterobacteriaceae nosocomial isolates (n = 877), collected from Russial hospitals in 2003-2007. It was shown that major genes belonged to cluster 1 (n = 543), namely--bla(CTX-M-15) gene (n = 515), bla(CTX-M-3) (n = 25), bla(CTX-M-22) (n = 1), bla(CTX-M-23) (n = 1), and bla(CTM-34) (n = 1). Moreover, the genes atributed to cluster 2 were identified: bla(CTX-M-2) (n = 1), and bla(CTX-M-5) (n = 4); and genes belonged to cluster 9: bla(CTX-M-9) (n = 2), and bla(CTX-M-14) (n = 35).     

Resistance to broad-spectrum antibiotics in aquatic systems: anthropogenic activities modulate the dissemination of bla(CTX-M)-like genes

We compared the resistomes within polluted and unpolluted rivers, focusing on extended-spectrum beta-lactamase (ESBL) genes, in particular bla(CTX-M). Twelve rivers from a Portuguese hydrographic basin were sampled. Physicochemical and microbiological parameters of water quality were determined, and the results showed that 9 rivers were classified as unpolluted (UP) and that 3 were classified as polluted (P). Of the 225 cefotaxime-resistant strains isolated, 39 were identified as ESBL-producing strains, with 18 carrying a bla(CTX-M) gene (15 from P and 3 from UP rivers). Analysis of CTX-M nucleotide sequences showed that 17 isolates produced CTX-M from group 1 (CTX-M-1, -3, -15, and -32) and 1 CTX-M that belonged to group 9 (CTX-M-14). A genetic environment study revealed the presence of different genetic elements previously described for clinical strains. ISEcp1 was found in the upstream regions of all isolates examined. Culture-independent bla(CTX-M)-like libraries were comprised of 16 CTX-M gene variants, with 14 types in the P library and 4 types in UP library, varying from 68% to 99% similarity between them. Besides the much lower level of diversity among CTX-M-like genes from UP sites, the majority were similar to chromosomal ESBLs such as bla(RAHN-1). The results demonstrate that the occurrence and diversity of bla(CTX-M) genes are clearly different between polluted and unpolluted lotic ecosystems; these findings favor the hypothesis that natural environments are reservoirs of resistant bacteria and resistance genes, where anthropogenic-driven selective pressures may be contributing to the persistence and dissemination of genes usually relevant in clinical environments.     

Clonal populations of thermotolerant Enterobacteriaceae in recreational water and their potential interference with fecal Escherichia coli counts

Bacterial strains were isolated from beach water samples using the original Environmental Protection Agency method for Escherichia coli enumeration and analyzed by pulsed-field gel electrophoresis (PFGE). Identical PFGE patterns were found for numerous isolates from 4 of the 9 days sampled, suggesting environmental replication. 16S rRNA gene sequencing, API 20E biochemical testing, and the absence of beta-glucuronidase activity revealed that these clonal isolates were Klebsiella, Citrobacter, and Enterobacter spp. In contrast, 82% of the nonclonal isolates from water samples were confirmed to be E. coli, and 16% were identified as other fecal coliforms. These nonclonal isolates produced a diverse range of PFGE patterns similar to those of isolates obtained directly from untreated sewage and gull droppings. beta-Glucuronidase activity was critical in distinguishing E. coli from other fecal coliforms, particularly for the clonal isolates. These findings demonstrate that E. coli is a better indicator of fecal pollution than fecal coliforms, which may replicate in the environment and falsely elevate indicator organism levels.     

Molecular characterization of β-lactam-resistant Escherichia coli isolated from Fu River, China

The present study aims to demonstrate the β-lactam resistance phenotypes and genotypes of Escherichia coli isolates from the Fu River in Chengdu, southwestern China. We obtained 108 E. coli isolates from nine sampling sites during May and December 2010. The total bacterial count varied from 79 colony-forming units (CFU)/ml to 14,550 CFU/ml, and coliform group number from 13 to 1,435 MPN/ml. Among the 108 isolates, 0-31.48% were resistant to β-lactams and β-lactam inhibitors, 1.85-7.40% to aminoglycoside, 1-20% to fluoroquinolone, and 50% to trimethoprim- sulfamethoxazole. The total bacterial count and antimicrobial resistance of different sites were significantly correlated (P < 0.05). Among the 34 ampicillin-resistant isolates, polymerase chain reaction (PCR) amplification and DNA sequencing showed that bla (TEM), bla (SHV), and bla (CTX-M) were detected in 85.29% (n = 29), 41.18% (n = 14), and 5.88% (n = 2) of the isolates, respectively, whereas bla (KPC) and bla (GES) were not observed in any of the isolates. Enterobacterial repetitive intergenic consensus-PCR patterns revealed that the 34 ampicillin-resistant E. coli isolates belonged to three distinct groups. Plasmid DNAs from the 14 SHV producer isolates yielded one to five bands of ca. 0.15-40 kb. To our knowledge, the current study is the first to describe the phenotypic and genetic characterizations of β-lactam resistance in E. coli isolates of river water origin from the Fu River, Chengdu, southwestern China. Results of the present study suggest that the river water may be considered as a reservoir for antibiotic resistance genes.     

Interaction of CTX-M-15 enzyme with cefotaxime: a molecular modelling and docking study

Extended-spectrum β-lactamases (ESBLs) are the bacterial enzymes that make them resistant to advanced-generation cephalosporins. CTXM enzymes (the most prevalent ESBL-type) target cefotaxime. Aims of the study were: Modelling of CTX-M enzyme from bla(CTX-M) sequences of clinical Escherichia coli isolatesDocking of cefotaxime with modelled CTX-M enzymes to identify amino acid residues crucial to their interaction To hypothesize a possible relationship between 'interaction energy of the docked enzyme-antibiotic complex' and 'minimum inhibitory concentration (MIC) of the antibiotic against the bacteria producing that enzyme'. Seven E. coli strains of clinical origin which were confirmed as PCR-positive for bla(CTX-M) were selected for the study. C600 cells harboring cloned bla(CTX-M) were tested for ESBL-production by double-disk-synergy test. BLAST analysis confirmed all the bla(CTX-M) genes as blaCTX-M-15. Four of the 7 strains were found to be clonally related. Modelling was performed using Swiss Model Server. Discovery Studio 2.0 (Accelrys) was used to prepare Ramachandran plots for the modelled structures. Ramachandran Z-scores for modelled CTX-M enzymes from E. coli strains D8, D183, D253, D281, D282, D295 and D296 were found to be -0.449, 0.096, 0.027, 0.043, 0.032, -1.249 and -1.107, respectively. Docking was performed using Hex 5.1 and the results were further confirmed by Autodock 4.0. The amino acid residues Asn 104, Asn132, Gly 227, Thr 235, Gly 236, and Ser237 were found to be responsible for positioning cefotaxime into the active site of the CTX-M-15 enzyme. It was found that cefotaxime MICs for the CTX-M-15-producers increased with the increasing negative interaction energy of the enzyme-antibiotic complex.