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.     

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.     

Construction by polymerase chain reaction and intragenic DNA probes for three main types of transferable β-lactamases (TEM, SHV, CARB)

Intragenic DNA probes were synthesized by polymerase chain reaction using fragments of the genes of three major types of beta-lactamases (TEM, SHV, CARB) as templates. The TEM probe hybridized with the genes encoding TEM-1, TEM-2 and six extended-spectrum related enzymes (TEM-3 to TEM-7, TEM-2O) in colony hybridizations and Southern-blot analysis. The SHV probe hybridized with the genes for SHV-1, OHIO-1 and four derived extended-spectrum beta-lactamases (SHV-2, SHV-3, SHV-4 and SHV-5). The CARB probe hybridized with the genes for PSE-1 (CARB-2), PSE-4 (CARB-1), CARB-3 and CARB-4. None of the probes hybridized with genes for any of eight oxacillin-hydrolysing enzymes, PSE-2, OXA-1 to OXA-7, ROB-1 and chromosomal beta-lactamases of various Enterobacteriaceae (except Klebsiella pneumoniae) and Pseudomonas aeruginosa. Investigations of Escherichia coli clinical isolates using these probes indicate the presence of a novel type of extended-spectrum, transferable beta-lactamase.     

Impact of three ampicillin dosage regimens on selection of ampicillin resistance in Enterobacteriaceae and excretion of blaTEM genes in swine feces

The aim of this study was to assess the impact of three ampicillin dosage regimens on ampicillin resistance among Enterobacteriaceae recovered from swine feces by use of phenotypic and genotypic approaches. Phenotypically, ampicillin resistance was determined from the percentage of resistant Enterobacteriaceae and MICs of Escherichia coli isolates. The pool of ampicillin resistance genes was also monitored by quantification of bla(TEM) genes, which code for the most frequently produced beta-lactamases in gram-negative bacteria, using a newly developed real-time PCR assay. Ampicillin was administered intramuscularly and orally to fed or fasted pigs for 7 days at 20 mg/kg of body weight. The average percentage of resistant Enterobacteriaceae before treatment was between 2.5% and 12%, and bla(TEM) gene quantities were below 10(7) copies/g of feces. By days 4 and 7, the percentage of resistant Enterobacteriaceae exceeded 50% in all treated groups, with some highly resistant strains (MIC of >256 microg/ml). In the control group, bla(TEM) gene quantities fluctuated between 10(4) and 10(6) copies/g of feces, whereas they fluctuated between 10(6) to 10(8) and 10(7) to 10(9) copies/g of feces for the intramuscular and oral routes, respectively. Whereas phenotypic evaluations did not discriminate among the three ampicillin dosage regimens, bla(TEM) gene quantification was able to differentiate between the effects of two routes of ampicillin administration. Our results suggest that fecal bla(TEM) gene quantification provides a sensitive tool to evaluate the impact of ampicillin administration on the selection of ampicillin resistance in the digestive microflora and its dissemination in the environment.     

Investigation of extended-spectrum beta-lactamases produced by clinical isolates of Klebsiella pneumoniae and Escherichia coli in Korea

AIMS: Isolates obtained from various regions in Korea in 2002 were identified and their susceptibility to extended-spectrum cephalosporins, monobactams and/or cephamycins was studied along with any production of extended-spectrum beta-lactamases (ESBLs). METHODS AND RESULTS: Bacteria identified by the conventional techniques and Vitek GNI card were Klebsiella pneumoniae and Escherichia coli. Using disk diffusion and double-disk synergy tests, we found that 39.2% of strains produced ESBLs. About 52% of isolates transferred resistance to ceftazidime by conjugation. Banding patterns of PCR amplification with the designed primers showed that 837- and 259-bp fragments specific to bla(TEM) genes were amplified in 63.3% of strains. 929- and 231-bp fragments (bla(SHV)), 847- and 520-bp fragments (bla(CMY)), 597- and 858-bp fragments (bla(CTX-M)) were amplified in 61.5, 17.3 and 7.7% of strains respectively. About 51.9% of strains contained more than two types of beta-lactamase genes. Especially, one strain contained bla(TEM), bla(CMY) and bla(CTX-M) genes. SIGNIFICANCE: Resistance mechanisms to beta-lactams, comprising mostly ESBL production, lead to the resistance against even recently developed beta-lactams in enterobacteria, which is now a serious threat to antibiotic therapy. The high prevalence of bla(CMY) genes and multidrug-resistant genes may also make therapeutic failure and lack of eradiation of these strains by extended-spectrum cephalosporins or cephamycins.     

Genetic determinants of antibacterial resistance among nosocomial Escherichia coli, Klebsiella spp., and Enterobacter spp. isolates collected in Russia within 2003-2007

Nosocomial bacterial isolates collected within 2003-2004 (n=411) and 2005-2007 (n=422) were highly resistant to cephalosporins III-IV and antibacterials of other groups (aminoglycosides, fluoroquinolons, chloramphenicol, and co-trimoxazole). Genes encoding TEM, SHV, CTX-M, OXA-2, and AmpC types of beta-lactamases (BLs) in the E. coli, Klebsiella spp., and Enterobacter spp. isolates were detected using polymerase chain reaction (PCR). Prevalent CTX-M-type BLs were detected in 85% of the E. coli, 87% of the Klebsiella spp., and 38% of the Enterobacter spp. isolates of the first strain collection and in 94% of the E. coli, 91% of the Klebsiella spp., and 38% of the Enterobacter spp. isolates of the second one. Genes belonging to three subtypes of blacTx-M genes were identified: bla(CTX-M-1) (228 bla(CTX-M-15) and six bla(CTX-M-3) of the first strain collection; 275 bla(CTX-M-15), three bla(CTX-M-3), and one bla(CTX-M-22) of the second one), bla(CTX-M-2) (one bla(CTX-M-5) of the first strain collection and one bla(CTX-M-2) of the second one), bla(CTX-M-9) (17 bla(CTX-M-14) and one bla(CTX-M-9) of the first strain collection; seven bla(CTX-M-14) and one bla(CTX-M-9) of the second one). Three isolates of the first strain collection and one isolate of the second one carried two genes belonging to two different subtypes, i.e., bla(CTX-M-15) and bla(CTX-M-14) simultaneously. The bacterial isolates had high levels of associative resistance to ciprofloxacin, co-trimoxazole, gentamicin, amikacin, and chloramphenicol associated with the resistance gene cassettes aadA1, aadA2, aadA5, aadB, aacA4, aac(6')Ib; dfrA1, dfrA5, dfrA12, dfrA17, cmlA1, ereA2, and catB8 in the class 1 integrons and the resistance gene cassettes dfrA1, sat1, and aadA1 in the class 2 integrons.     

Detection and characterization of beta-lactamase genes in subgingival bacteria from patients with refractory periodontitis

Fifty-three beta-lactamase-producing strains of oral bacteria isolated from patients with refractory periodontitis in Norway and USA were screened for the presence of the bla(TEM), bla(SHV), bla(OXA), bla(ampC), bla(cfxA), and bla(cepA/cblA) genes by the polymerase chain reaction (PCR). The PCR products were characterized by direct sequencing of the amplified DNA. Thirty-four of the 53 enzyme-producing strains (64%) were positive in one of the PCR assays. All beta-lactamase-producing Prevotella and Capnocytophaga spp. were CfxA positive. TEM-type beta-lactamases were identified in one strain each of Escherichia coli and Neisseria sp., and one strain of Citrobacter freundii possessed an AmpC-type beta-lactamase. Screening for gene cassettes and genes known to be associated with integrons did not reveal the presence of integrons in these oral bacteria. Sequence analyses showed that most CfxA positive Prevotella and Capnocytophaga isolates from patients with refractory periodontitis harboured variants of the CfxA2 and CfxA3 enzyme. The present study also showed that many different genetic determinants of beta-lactamase production are found in bacteria isolated from refractory periodontitis, many of which remain to be characterized.     

Distribution of blaOXA genes among carbapenem-resistant Acinetobacter baumannii nosocomial strains in Poland

Acinetobacter baumannii is an important nosocomial pathogen occurring particularly in intensive care (ICU) as well as burn therapy units (BTU). A. baumannii strains have emerged as resistant to almost all antimicrobial agents, including carbapenems. b-lactamase-mediated resistance is the most common mechanism for carbapenem resistance in this species. Carbapenem-hydrolysing class D b-lactamases - OXA are widespread among A. baumannii strains. It is suggested that ISAba1 plays an important role in drug resistance. The aims of the study were detection of OXA encoding genes and presence of ISAba1. The study included the total of 104 isolates of carbapenem-resistant A. baumannii, obtained from patients hospitalized in ICU and BTU of Specialized Hospital in Krakow. Multiplex PCR was applied for detection of selected OXA carbapenemases encoding genes. PCR analysis showed the presence of bla OXA-51-like gene and ISAba1 in all isolates. 46 strains carried bla OXA-51-like and bla OXA-23-like genes while 48 bla OXA-51-like and bla OXA-40-like genes. 3 isolates carried: bla OXA-51-like , bla OXA-23-like and bla OXA-40-like genes. 7 strains encoded an OXA-51-like carbapenemase but were negative for enzymes belonging to the other families tested. Comparative analysis of ICU and BTU isolates revealed the dominance of: bla OXA-51-like and bla OXA-40-like among ICU while bla OXA-51-like and bla OXA-23-like in BTU.     

Analysing diversity among beta-lactamase encoding genes in aquatic environments

The most common mechanism of resistance to beta-lactam antibiotics is the production of beta-lactamases. These enzymes are encoded by genes that evolve rapidly, thus constituting a group characterized by high levels of molecular diversity. Most of the genetic determinants of resistance to beta-lactam antibiotics characterized until now were obtained from clinical isolates. This study was designed in order to exploit the presence of beta-lactamase gene sequences in an aquatic environment, and to get information on the distinctive features of those sequences when compared to others available on databases. DNA sequences potentially encoding proteins of three different families of clinically relevant beta-lactamases were assessed: TEM, IMP and OXA-2 derivatives. The presence of bla sequences in DNA extracted from water samples from the lagoon Ria de Aveiro was checked by PCR and hybridization. Sequences representing the three families of beta-lactamases studied were detected. The molecular diversity of the amplicons was assessed by cloning and sequence analysis, and denaturing gradient gel electrophoresis (PCR-DGGE) separation. Most of the retrieved sequences (particularly sequences representing bla(TEM)and bla(OXA-2)) were identical or very similar to beta-lactamase gene sequences previously characterized from clinical isolates. Phylogenetic analysis suggests that this aquatic ecosystem is a reservoir of molecular diverse putative bla sequences. The patterns of molecular diversity found within the beta-lactamase gene families studied do not correspond to those reported in studies focussing on clinical isolates.     

The role of bla(OXA-like carbapenemase) and their insertion sequences (ISS) in the induction of resistance against carbapenem antibiotics among Acinetobacter baumannii isolates in Tehran hospitals

This study aimed to evaluate the occurrence and dissemination of bla(OXA-like) carbapenemase genes and their insertion sequences among Acinetobacter baumannii isolates, taken from different hospitals in Tehran city and also their roles in the induction of resistance to carbapenem drugs. A total number of 100 non duplicate Acinetobacter baumannii with different origins, were isolated from patients with proved nosocomial infections at eight university hospital in Tehran city. Antimicrobial susceptibility of these strains was done by E-test against 7 antimicrobial agents according to CLSI guideline. PCR of bla(OXA-51-like), bla(OXA-23-like), bla(OXA-24-like), bla(OXA-58-like), IS(ABA-1), IS(1133) was carried out by specialized primers and then these strains were typed by REP-fingerprinting. Colistin, imipenem and meropenem were the most sensitive antibiotics against Acinetobacter baumannii isolates with 96%, 51% and 51% sensitivity respectively. All the isolates had a bla(OXA-51-like) intrinsic to these species. The rates of bla(OXA-23), 23 and 58-like were 38%, 32% and 1% respectively. Coexistence of bla(OXA-51/23/24-like) was observed among 16% of these isolates. All bla(OXA-23-like) carbapenemase genes had only one IS(ABA1). REP fingerprinting showed 5 genotypes among carbapenem resistant isolates, 16 of them being genotype A. This study emphasized on the major role of bla(OXA-like) carbapenemase, particularly bla(OXA-23-like) carbapenemase and their IS(ABA1), in the dissemination of carbapenem resistant Acinetobacter baumannii. This study confirmed a presumptive role of IS element neighboring the carbapenemase gene in the elevation of resistance to carbapenem drug among Acinetobacter baumannii isolates for the first time in Iran.     

A novel sequence framework (bla(TEM-1G)) encoding the parental TEM-1 beta-lactamase

A novel parental bla(TEM) gene (bla(TEM-1G)), encoding a TEM-1 beta-lactamase (pI of 5.4) produced by the uropathogenic Escherichia coli strain FMV194 was isolated from a dog. We report PCR-restriction fragment length polymorphism analysis and nucleotide sequencing of this gene. The bla(TEM-1G) sequence was identical to the bla(TEM-1C) gene framework in the coding and promoter (P3) regions, except for a silent G(604)-->T mutation in the coding region. Molecular phylogenetic analysis of parental bla(TEM) genes indicated two distinct groups, one comprising bla(TEM-1F) and bla(TEM-2). The other group comprises bla(TEM-1C) which is the probable ancestor of bla(TEM-1A), bla(TEM-1D) and bla(TEM-1G). The bla(TEM-1G) gene has the same framework as a gene encoding an inhibitor-resistant TEM beta-lactamase produced by an E. coli strain of human origin. Thus, parental bla(TEM) genes encoding beta-lactamases in E. coli strains isolated from different host species, in this case human and canine, may be phylogenetically very close.     

Characterisation of extended-spectrum beta-lactamases of the SHV family using a combination of PCR-single strand conformational polymorphism (PCR-SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP)

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) has been developed to extend the identification of SHV beta-lactamases previously characterised by PCR-single strand conformational polymorphism (PCR-SSCP) analysis alone. Eight bacteria, each producing a different SHV beta-lactamase, were used in this study. These bacteria harbour bla(SHV-1), bla(SHV-2a), bla(SHV-3), bla(SHV-4), bla(SHV-5) (two strains), bla(SHV-11) and bla(SHV-12). All isolates were characterised by PCR-SSCP and PCR-RFLP with DdeI and NheI digestion. By a combination of these techniques, the genes encoding these beta-lactamases could be differentiated from each other. In addition, the PCR-RFLP technique theoretically can be applied to distinguish the genes encoding SHV-7, SHV-9, SHV-10, SHV-15, SHV-17 and SHV-24 from those encoding other SHV variants. We report a simple PCR-RFLP technique that can be used in epidemiological studies to enable the rapid characterisation of known SHV beta-lactamases in a combination with the previously published PCR-SSCP analysis.     

Antibiotic susceptibility of bacterial strains isolated from patients with various infections

AIMS: Isolates from various samples obtained during 1998 and 1999 were identified and their susceptibility to third-generation cephalosporins, monobactams and/or cephamycins studied along with any production of ESBLs. METHODS AND RESULTS: Of these samples, bacteria most frequently isolated by the conventional techniques and Vitek GNI card were Escherichia coli (37%), Klebsiella pneumoniae (27%) and Enterobacter cloacae (16%). Using disk diffusion and double-disk synergy tests, we found that 71% strains produced ESBLs and 18% strains produced ESBLs and cephamycinases. Banding patterns of PCR amplification with the designed primers showed that 57% strains were capable of harbouring bla(SHV) genes. The bla(TEM), bla(CMY) and bla(AmpC) genes were harboured by 55%, 31% and 12% strains, respectively. Forty-five percent of strains contained more than two types of beta-lactamase genes. In particular, one strain contained bla(TEM), bla(SHV), bla(CMY) and bla(AmpC) genes. CONCLUSIONS: The percentage of ESBL-producing strains was high. The most prevalent beta-lactamase gene was bla(SHV) gene. The bla(CMY) genes have been prevalent in cephamycin-resistant strains. The multidrug-resistant strains resistant to third-generation cephalosporins and cephamycins were detected in high percentage. SIGNIFICANCE AND IMPACT OF THE STUDY: Resistance mechanisms to beta-lactams, comprising mostly extended-spectrum beta-lactamase (ESBL) production, lead to the resistance against even recently developed beta-lactams in enterobacteria, which is now a serious threat to antibiotic therapy. The high prevalence of bla(CMY) genes and multidrug-resistant genes may also cause therapeutic failure and lack of eradication of these strains by third-generation cephalosporins or cephamycins.     

Coastal Seawater Bacteria Harbor a Large Reservoir of Plasmid-Mediated Quinolone Resistance Determinants in Jiaozhou Bay, China

Diversity and prevalence of plasmid-mediated quinolone resistance determinants were investigated in environmental bacteria isolated from surface seawater of Jiaozhou Bay, China. Five qnr gene alleles were identified in 34 isolates by PCR amplification, including qnrA3 gene in a Shewanella algae isolate, qnrB9 gene in a Citrobacter freundii isolate, qnrD gene in 22 Proteus vulgaris isolates, qnrS1 gene in 1 Enterobacter sp. and 4 Klebsiella spp. isolates, and qnrS2 gene in 1 Pseudomonas sp. and 4 Pseudoalteromonas sp. isolates. The qnrC, aac(6')-Ib-cr, and qepA genes could not be detected in this study. The 22 qnrD-positive Proteus vulgaris isolates could be differentiated into four genotypes based on ERIC-PCR assay. The qnrS1 and qnrD genes could be transferred to Escherichia coli J53 Azi(R) or E. coli TOP10 recipient strains using conjugation or transformation methods. Among the 34 qnr-positive isolates, 30 had a single point mutation in the QRDRs of GyrA protein (Ala67Ser, Ser83Ile, or Ser83Thr), indicating that cooperation of chromosome- and plasmid-mediated resistance contributed to the spread and evolution of quinolone resistance in this coastal bay. Eighty-five percent of the isolates were also found to be resistant to ampicillin, and bla(CMY), bla(OXY), bla(SHV), and bla(TEM) genes were detected in five isolates that also harbored the qnrB9 or qnrS1 gene. Our current study is the first identification of qnrS2 gene in Pseudoalteromonas and Pseudomonas strains, and qnrD gene in Proteus vulgaris strains. High prevalence of diverse qnr genes in Jiaozhou Bay indicates that coastal seawater may serve as an important reservoir, natural source, and dissemination vehicle of quinolone resistance determinants.     

Susceptibility and molecular mechanisms of resistance to cephalosporins of Escherichia coli strains isolated from patients with nosocomial infections

Resistance to wide spectrum of antibiotics was studied and most widespread genetic determinants of resistance to beta-lactam antibiotics were revealed. Susceptibility testing was performed using serial broth microdilution method. Detection of class A expanded spectrum beta-lactamases genes (TEM, SHV, CTX) by polymerase-chain reaction method was performed in 90 strains. Carbapenems remained the most active antibacterial agents with respect to studied E. coli strains. Among the 3rd generation cephalosporins the lowest minimal inhibitory concentrations were observed for inhibition-protected combined agents (ceftazidime/clavulanic acid and cefoperazone/ sulbactam). Alone or in various combinations TEM, SHV, and CTX types of beta-lactamases were found in 58.9%, 14.4%, and 77.8% of strains. Combinations of 2 determinants were detected in 55.6% of the isolates, and all 3 determinants--in 5.6%. Most often E. coli was isolated in patients with urinary tract infections. Carbapenems and inhibition-protected combined 3rd generation cephalosporins are the most active agents against E. coli.     

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.     

Molecular characterization of multidrug-resistant Escherichia coli isolates from Irish cattle farms

This study describes the genotypic characteristics of a collection of 100 multidrug-resistant (MDR) Escherichia coli strains recovered from cattle and the farm environment in Ireland in 2007. The most prevalent antimicrobial resistance identified was to streptomycin (100%), followed by tetracycline (99%), sulfonamides (98%), ampicillin (82%), and neomycin (62%). Resistance was mediated predominantly by strA-strB (92%), tetA (67%), sul2 (90%), bla(TEM) (79%), and aphA1 (63%) gene markers, respectively. Twenty-seven isolates harbored a class 1 integrase (intI1), while qacEΔ1 and sul1 markers were identified in 25 and 26 isolates, respectively. The variable regions of these integrons contained aminoglycoside, trimethoprim, and β-lactam resistance determinants (aadA12, aadB-aadA1, bla(OXA-30)-aadA1, dfrA1-aadA1, dfrA7). Class 2 integrons were identified less frequently (4%) and contained the gene cassette array dfrA1-sat1-aadA1. Resistance to ampicillin, neomycin, streptomycin, sulfonamide, and tetracycline was associated with transferable high-molecular-weight plasmids, as demonstrated by conjugation assays. A panel of virulence markers was screened for by PCR, and genes identified included vt1, K5 in 2 isolates, papC in 10 isolates, and PAI IV(536) in 37 isolates. MDR commensal E. coli isolates from Irish cattle displayed considerable diversity with respect to the genes identified. Our findings highlight the importance of the commensal microflora of food-producing animals as a reservoir of transferable MDR.     

Occurrence of qnr-positive clinical isolates in Klebsiella pneumoniae producing ESBL or AmpC-type beta-lactamase from five pediatric hospitals in China.

The plasmid-mediated quinolone resistance qnr genes in clinical isolates in adults have been described in different countries; however, the frequency of their occurrence has not been detected in pediatric patients. A total of 410 clinical isolates of Klebsiella pneumoniae, identified as producers of an extended-spectrum beta-lactamase (ESBL), or AmpC beta-lactamase, were collected from five children's hospitals in China during 2005-2006. The isolates were screened for the presence of the qnrA, qnrB, and qnrS genes, and then the harboring qnr gene isolates were detected for a bla gene coding for the TEM, SHV, CTX-M, and plasmid-mediated ampC gene by a PCR experiment. Ninety-two isolates (22.7%) were positive for the qnr gene, including 10 of qnrA (2.4%), 25 of qnrB (6.1%), and 62 of qnrS (15.1%). Eighty-one of the 92 (88.0%) qnr-positive isolates carried at least one bla gene for TEM, SHV, CTX-M, or DHA-1. The ciprofloxacin resistance increased 16-256-fold and oflaxacin resistance increased 2-32-fold in transconjugants, respectively. These results indicated that the plasmid-mediated qnr quinolone resistance gene was qnrS, followed by qnrB and qnrA. Most of the isolates also carried a bla gene coding ESBL or ampC gene coding DHA-1 among Klebsiella pneumoniae isolated from Chinese pediatric patients.     

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.     

Antibiotic-resistant Escherichia coli bacteria, including strains with genes encoding the extended-spectrum beta-lactamase and QnrS, in waterbirds on the Baltic Sea Coast of Poland

Individual cloacal swabs of mallards (Anas platyrhynchos) and of herring gulls (Larus argentatus), as well as samples of waterbird feces obtained in 2008 and 2009, were cultivated for Escherichia coli. Isolates of E. coli were tested for susceptibilities to 12 antimicrobial agents by the disk diffusion method. Moreover, the samples were subcultivated on MacConkey agar (MCA) containing cefotaxime (2 mg liter(-1)) to detect E. coli with extended-spectrum beta-lactamase (ESBL) and subsequently on MCA supplemented with ciprofloxacin (0.05 mg liter(-1)) and MCA with nalidixic acid (20 mg liter(-1)) to isolate fluoroquinolone-resistant E. coli. PCR was used to detect specific antibiotic resistance genes. We found 9 E. coli isolates producing ESBL with bla genes: bla(CTX-M-1) (6 isolates), bla(CTX-M-9) plus bla(TEM-1b) (1 isolate), bla(CTX-M-15) plus bla(OXA-1) (1 isolate), and bla(SHV-12) (1 isolate). In the isolate with bla(CTX-M-15), the gene aac(6)-Ib-cr was also detected. The bla genes were harbored by transferable plasmids of the IncN and IncI1 groups. Nine quinolone-resistant E. coli isolates with qnrS genes were found and characterized. The gene qnrS was associated with a Tn3-like transposon on the IncX1 plasmid together with bla(TEM-1) in two isolates. The gene qnrS was also harbored by conjugative plasmids of the IncN and IncX2 groups. Even if populations of wild birds are not directly influenced by antibiotic practice, we have demonstrated that antibiotic-resistant E. coli strains, including strains with various ESBL and qnrS genes, are found in the feces of wild birds on the coast of the Baltic Sea in Poland.