Occurrence of antibiotic resistance and class 1, 2 and 3 integrons in Escherichia coli isolated from a densely populated estuary (Seine, France)

Over 6 years, Escherichia coli were isolated from water samples from seven Seine estuary stations, characterized by a densely populated watershed (654 isolates). Resistances of these E. coli to 16 antibiotics were determined and compared with the same resistances in E. coli isolated from a small stream (120 isolates) and from the treated effluent of the largest estuary wastewater treatment plant (123 isolates). Between 30.2% and 56.6% of the estuary isolates were resistant, whatever the station or time of sampling; of these, 60.5–80% were resistant to at least two and up to 12 antibiotics. In the three contrasting sites, resistances to tetracycline, amoxicillin and ticarcillin were the commonest. DNA was extracted from 279 estuary isolates (January 2006) and class 1, 2 and 3 integrons were detected by multiplex real-time PCR and confirmed by classic PCR. IntI1 and intI2 genes were found in 11% of isolates. No intI3 gene was detected. The variable regions of the class 1 and 2 integrons sequenced contained predominantly gene cassettes aadA and dfr . However, for slightly over half of the E. coli isolates exhibiting the class 1 integron, the variable region could not be amplified, because part of the 3' conserved sequence was missing.     

The diversity of Escherichia coli serotypes and biotypes in cattle faeces

AIM: To study the diversity of commensal Escherichia coli populations shed in faeces of cattle fed on different diets. METHODS AND RESULTS: Thirty Brahman-cross steers were initially fed a high grain (80%) diet and then randomly allocated into three dietary treatment groups, fed 80% grain, roughage, or roughage + 50% molasses. Up to eight different E. coli isolates were selected from primary isolation plates of faecal samples from each animal. Fifty-two distinct serotypes, including nine different VTEC strains, were identified from a total of 474 E. coli isolates. Cattle fed a roughage + molasses diet had greater serotype diversity (30 serotypes identified) than cattle fed roughage or grain (21 and 17 serotypes identified respectively). Cluster analysis showed that serotypes isolated from cattle fed roughage and roughage + molasses diets were more closely associated than serotypes isolated from cattle fed grain. Resistance to one or more of 11 antimicrobial agents was detected among isolates from 20 different serotypes. Whilst only 2.3% of E. coli isolates produced enterohaemolysin, 25% were found to produce alpha-haemolysin. CONCLUSIONS: Diverse non-VTEC populations of E. coli serotypes are shed in cattle faeces and diet may affect population diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new information on the serotype diversity and phenotypic traits of predominant E. coli populations in cattle faeces, which could be sources of environmental contamination.     

Analysis of integrons in clinical isolates of Escherichia coli in China during the last six years

A multiple PCR for the detection of the integrase genes of the three classes of integrons was carried out, and their gene cassettes were characterized in 111 clinical strains of Escherichia coli isolated in Guangzhou City, China during the last 6 years. IntI1 and intI2 genes were detected in 95 isolates (85.6%) and four isolates (3.6%), respectively. No intI3 gene was detected. Six different gene cassettes were found in these strains, and a high prevalence of dfr and aad genes was observed. The E. coli isolates that contained a 1664-bp amplicon of dfrA17-aadA5 in class 1 integron were found to be phylogenetically unrelated to each other by using the enterobacterial repetitive intergenic consensus PCR, as the cassette could be transferred to recipient strains, indicating that the gene cassettes might be disseminated in the clinical strains by a horizontal gene transfer. Therefore, it is important that guidelines for the prudent use of antimicrobial agents are adopted and surveillance programs are established.     

High prevalence of antimicrobial-resistant genes and integrons in Escherichia coli isolates from Black-headed Gulls in the Czech Republic

AIMS: To carry out an assessment of the occurrence of resistance to antimicrobials in Escherichia coli that has been isolated from young Black-headed Gulls in three nesting colonies. METHODS AND RESULTS: A total of 257 isolates were tested for sensitivity to eight antibacterial substances by disk diffusion method. The polymerase chain reaction was used for detecting specific genes of antibacterial resistance and class 1 integrons in resistant E. coli isolates. A total 75 (29.9%) of 257 isolates were resistant to one or more antimicrobial agents. The dominant type of resistance was to tetracycline, detected in 49 (19.1%) isolates. Resistance to ampicillin was detected in 30 (11.7%), cephalothin in 11 (4.3%), streptomycin in 24 (9.3%), sulphonamides in 20 (7.8%) and chloramphenicol in 5 (1.9%) isolates. Nine isolates carrying integrons were detected. CONCLUSIONS: The study suggests that young Black-headed Gulls are an important host reservoir of resistant E. coli strains, probably reflecting the presence of such strains in their sources of food and/or water. SIGNIFICANCE AND IMPACT OF THE STUDY: Although Black-headed Gulls do not naturally come into contact with antibiotics, these birds can be infected with resistant E. coli and potentially serve as their reservoirs, vectors and bioindicators in the environment.     

Genetic diversity analyses of class 1 integrons and their associated antimicrobial resistance genes in Enterobacteriaceae strains recovered from aquatic habitats in China

Aims: To characterize the molecular diversity of class 1 integrons and antibiotic resistance (AR) genes of Enterobacteriaceae strains recovered from aquatic habitats in Jinan, Shandong Province, China. Methods and Results: Six hundred and thirty‐eight antimicrobial‐resistant Enterobacteriaceae isolated from wastewater were examined for class 1 integron. Of these, 293 were positive for the class 1 integrase gene intI1; among these, 34 gene cassettes and 29 AR genes were detected. Twenty‐nine distinct gene cassette arrays were identified by restriction fragment length polymorphism (RFLP). Seven strains harboring novel gene cassette arrays were subjected to further study, in which antimicrobial susceptibility profiles were determined, and the presence of other AR genes outside of the integrons was assayed. Several of the resistance determinants were found to be transferable by conjugation or transformation. Conclusions: This study established the assessment of class 1 integron and antimicrobial resistance gene patterns among environmental Enterobacteriaceae. Also, a restriction enzyme EcoRII was employed to develop a rapid and simple method for characterizing gene cassette arrays by RFLP analysis, which facilitated further study of novel gene cassette arrays. Significance and Impact of Study: These data not only illustrated the diversity of class 1 integron gene cassettes but also provided direct evidence that integrons mobilized gene cassettes, generating new linkages of resistance genes, and they could be integrated in gene transfer units such as conjugative plasmids to contribute to the dissemination of AR genes by horizontal gene transfer (HGT) in aquatic environments.     

Distribution and molecular profiling of class 1 integrons in MDR Acinetobacter baumannii isolates and whole genome-based analysis of antibiotic resistance mechanisms in a representative strain

The class 1 integron is an important driver of the nosocomial dissemination of multidrug-resistant (MDR) bacteria, such as Acinetobacters. In this study, we characterized the gene cassette arrays of class 1 integrons in Acinetobacter baumannii, where the detailed structure of these integrons for 38 clinical strains was analyzed. The results showed that there are three types of gene cassette arrays that are carried by different class 1 integrons, among them the aac(6′)-IId-catB8-aadA1 array was the most prevalent. For detailed analysis of the integron structure, whole genome sequencing was carried out on strain AB16, and it was found that a single integron on its chromosome has a partial Tn21 transposon in its 5′ flanking region and two complete copies of the insertion element IS26 in both the 5′ and 3′ flanking regions, indicating that the integron could be acquired by horizontal gene transfer. Furthermore, there is one resistance island AbaR22, one bla gene containing a transposon, four intrinsic resistant genes and one efflux pump that together confer six types of antibiotic resistance.     

A high prevalence of antimicrobial resistant Escherichia coli isolated from pigs and a low prevalence of antimicrobial resistant E. coli from cattle and sheep in Great Britain at slaughter

The incidence of antimicrobial resistance and expressed and unexpressed resistance genes among commensal Escherichia coli isolated from healthy farm animals at slaughter in Great Britain was investigated. The prevalence of antimicrobial resistance among the isolates varied according to the animal species; of 836 isolates from cattle tested only 5.7% were resistant to one or more antimicrobials, while only 3.0% of 836 isolates from sheep were resistant to one or more agents. However, 92.1% of 2480 isolates from pigs were resistant to at least one antimicrobial. Among isolates from pigs, resistance to some antimicrobials such as tetracycline (78.7%), sulphonamide (66.9%) and streptomycin (37.5%) was found to be common, but relatively rare to other agents such as amikacin (0.1%), ceftazidime (0.1%) and coamoxiclav (0.2%). The isolates had a diverse range of resistance gene profiles, with tet(B), sul2 and strAB identified most frequently. Seven out of 615 isolates investigated carried unexpressed resistance genes. One trimethoprim-susceptible isolate carried a complete dfrA17 gene but lacked a promoter for it. However, in the remaining six streptomycin-susceptible isolates, one of which carried strAB while the others carried aadA, no mutations or deletions in gene or promoter sequences were identified to account for susceptibility. The data indicate that antimicrobial resistance in E. coli of animal origin is due to a broad range of acquired genes.     

Distribution of integron-associated trimethoprim–sulfamethoxazole resistance determinants among Escherichia coli from humans and food-producing animals

Aims: To compare the distribution of integrons and trimethoprim–sulfamethoxazole resistance genes among Escherichia coli isolates from humans and food‐producing animals. Methods and Results: A collection of 174 multidrug‐resistant E. coli isolates obtained from faecal samples of food‐producing animals (n = 64) and humans (n = 59), and patients with urinary tract infections (n = 51) in Hong Kong during 2002–2004 were studied. The strains were analysed for their phylogenetic groups, the presence of sul genes (sul1 and sul2), integrons (intl1 and intl2) and class 1 integron‐associated dfr cassette genes by PCR, restriction enzyme analysis and sequencing. Integrons were identified in 110 (63·2%) isolates. The prevalence of integrons was significantly different according to the specimen sources (animal faecal 84·4%, human faecal 67·8% and human urinary 31·4%) and phylogenetic groups (B1 80·8%, A 77·6%, D 54·1% and B2 11·5%). Faecal isolates (both human and animal) are more likely to belong to group A and B1. In contrast, most urinary isolates were either groups B2 and D. Among dfr containing isolates, dfrA1 and dfrA12 were almost exclusively found in strains of phylogenetic groups A and B1; and were present in animal and human faecal isolates. In contrast, dfrA17 was found in both faecal and urinary isolates and comprised strains from all phylogenetic groups. The sul1 and sul2 genes were equally prevalent among the isolates irrespective of the specimen source and phylogenetic group status. Pulsed‐field gel electrophoresis analysis of isolates with identical cassette genes showed that they were genetically diverse. Conclusions: More animal faecal isolates carry class 1 integrons than human faecal and human urinary isolates, and the distribution of phylogenetic groups is common across animal and human faecal isolates but different from human urinary isolates. Significance and Impact of the Study: Commensal isolates from food‐producing animals are an important reservoir for integrons carrying antibiotic resistance genes.     

Genetic diversity of Escherichia coli recovered from the oral cavity of beef cattle and their relatedness to faecal E. coli

AIMS: To determine the genetic diversity of generic Escherichia coli recovered from the oral cavities of beef cattle and their relatedness to E. coli isolated from the faeces of cattle during pasture grazing and feedlot finishing. METHODS AND RESULTS: A total of 484 E. coli (248 oral and 236 faecal isolates) were obtained from eight beef cattle after 1 and 5 months of grazing on pasture and after 1 and 5 months in a feedlot. The random amplification of polymorphic DNA (RAPD) method was used to genetically characterize these isolates. The RAPD patterns showed that ca 60% of E. coli recovered from the oral cavities and faeces during pasture and feedlot shared a close genetic relatedness. A number of E. coli with unique RAPD types were also found either in the oral cavities or faeces. Most of the E. coli RAPD types recovered from the oral cavities were shared among animals, but there were also RAPD types which were unique to individual animals. The E. coli populations of the oral cavities were genetically diverse and changed over time. CONCLUSIONS: This study indicates that there are large numbers of E. coli carried in the oral cavities of beef cattle and those E. coli are closely related to strains found in the faeces. The oral cavities of cattle harbour a genetically diverse E. coli population. SIGNIFICANCE AND IMPACT OF THE STUDY: The oral cavity may be an important reservoir of enteric pathogens which may transfer to meat during carcass dressing. A better understanding of the molecular ecology of E. coli in cattle would assist the design of approaches to control pathogenic strains during beef production and processing.     

Occurrence, virulence characteristics and antimicrobial resistance of Escherichia coli O157 in slaughtered cattle and diarrhoeic calves in West Bengal, India

AIMS: (i) To study the occurrence of Escherichia coli serotype O157 in cattle stool in West Bengal, India, and (ii) the virulence properties and antimicrobial resistance of the E. coli isolates. METHODS AND RESULTS: Following enrichment in modified EC broth and plating onto HiCrome MS.O157 agar, a total of 14 strains of E. coli serotype O157 was isolated from faecal samples from two (2.04%) slaughtered cattle and six (7.59%) diarrhoeic calves. By multiplex PCR, Shiga toxin genes were detected in all the isolates. The enterohaemolysin phenotype was found in all, but one strain. Among 14 strains, ten were resistant to at least one of the antimicrobial agents tested. Multiple antibiotic resistance was frequent. CONCLUSIONS: The study showed that occurrence of Shiga toxin-producing and multiple antibiotic-resistant E. coli O157 among cattle population in this region of India is significant. SIGNIFICANCE AND IMPACT OF THE STUDY: Considering routine human contacts with cattle, a large human population in this region may be at risk for exposure to Shiga toxin-producing E. coli O157.     

环境中抗生素抗性基因与I型整合子的研究进展

抗生素抗性基因(Antibiotic resistance genes,ARGs)作为一种新型污染物在不同环境中广泛分布、来源复杂,对生态环境和人类健康造成了很大的潜在风险。同时,Ⅰ型整合子(Int Ⅰ)介导的ARGs水平转移是环境中微生物产生耐药性的重要途径,Ⅰ型整合子整合酶基因(intI1)与ARGs丰度在环境中表现出了较高的正相关性,Int Ⅰ可以作为标记物在一定程度上反映ARGs在环境中的迁移转化规律和人类活动影响程度。本文介绍ARGs与Int Ⅰ在环境中的来源与分布,总结Int Ⅰ介导的ARGs迁移转化机制以及相关研究方法,并展望未来的研究发展趋势。     

水产养殖环境耐药细菌中复合1型整合子的流行特征

【目的】了解复合1型整合子在水产养殖环境中的分布和流行特征。【方法】对108株分离自福建水产养殖场的耐药细菌,通过PCR和序列分析,检测其复合1型整合子上下游保守区和可变区的携带情况。【结果】有86株(79.6%)和47株(43.5%)分别携带1型整合子和ISCR1元件,这两种上下游保守区均携带的耐药细菌则有26株(24.1%),其中16株(14.8%)耐药细菌成功地扩增出上下游可变区,分布于8属9种。进一步对ISCR1上下游序列的拼接和分析,表明这16株细菌携带两种类型的复合1型整合子:(1)int I1-aac(6')-Ib-cr-arr-3-dfr A27-aad A16-qac E 1-sul1-ISCR1-sdr-qnr B6-qac E 1-sul1(15株);(2)int I1-aac(6')-Ib-cr-arr-3-dfr A27-aad A16-qac E 1-sul1-ISCR1-sap A-like-qnr B2-qac E 1(truncated)-sul1(1株,肺炎克雷伯菌C12),该阵列为新发现的复合1型整合子结构。【结论】复合1型整合子在水产养殖环境中并不少见,且存在于多种细菌中,但其基因阵列结构缺乏多样性。     

Characterization of an extremely heat-resistant Escherichia coli obtained from a beef processing facility

Aims:  This study aimed to determine the survival of Escherichia coli strains during steam and lactic acid decontamination interventions currently used by the beef‐processing industry, and to determine their heat resistance. Methods and Results:  Strains were grouped into cocktails of five strains each differing in their RAPD patterns for subsequent identification. Steam and lactic acid treatments on meat reduced cell counts of E. coli strain cocktails by 90–99%. The 20 slaughter plant isolates exhibited only minor variation in their resistance to steam and lactic acid treatments but were more resistant than reference strains (three strains) or isolates from live cattle (seven strains). D₆₀ values of strains from live cattle, and reference strains ranged from 0·1 to 0·5 min, in keeping with literature data. However, D₆₀ values of current slaughter plant isolates ranged between 15 for E. coli DM18.3 and 71 min AW 1.7. Cell counts of E. coli AW 1.7 were reduced by <5 log₁₀ CFU g^?1 in ground beef patties cooked to an internal temperature of 71°C. Conclusions:  Strains of E. coli that survive cooking of ground beef to the recommended internal temperature of 71°C can be isolated from beef‐processing facilities. Significance and Impact of the Study:  Pathogen interventions in current commercial beef slaughter may select for extremely heat‐resistant strains of E. coli.     

Virulence markers and serotypes of Shiga toxin-producing Escherichia coli, isolated from cattle in Rio Grande do Sul, Brazil

AIMS: To determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) and serotypes and virulence markers of the STEC isolates from beef and dairy cattle in Rio Grande do Sul, Brazil. METHODS AND RESULTS: Faecal samples from beef cattle were collected at slaughterhouses. The isolates were submitted to colony hybridization assay with specific DNA probes for stx1, stx2 and eae genes, and serotyped for the identification of O and H antigens. Thirty-nine per cent of beef cattle surveyed harboured at least one STEC strain. Among the distinct serotypes identified, 10 were shared by both beef and dairy cattle. Most of the strains isolated harboured stx2. Genotypic and phenotypic profiles allowed the identification of 34 and 31 STEC strains, isolated from beef and dairy cattle, respectively. Serotypes O10:H14, O15:H21, O96:H21, O119:H4, O124:H11, O128:H21, O137:H-, O141:H19, O159:H42, O160:H2 and O177:H11, identified in this study, have not been previously reported as STEC isolated from cattle. CONCLUSIONS: Cattle are an important reservoir of STEC strains associated with human diseases in South America. SIGNIFICANCE AND IMPACT OF THE STUDY: Determining the prevalence, genotypic profile and serotypes of STEC strains isolated from cattle enables the prediction of possible risk for public health.     

产超广谱β-内酰胺酶大肠埃希菌Ⅰ类整合子检测与耐药性关系

目的了解I类整合子在产ESBLs和非产ESBLs大肠埃希菌中分布状况,分析I类整合子在细菌多重耐药中的作用。方法用PCR方法扩增I类整合酶基因,经电泳后检测扩增产物。用2χ检验进行统计学分析,P<0.05为差异有显著性。结果105株大肠埃希菌检出I类整合子46株,检出率为43.8%。I类整合子在产ESBLs菌的检出率为53.4%,明显高于非产ESBLs菌(31.9%),2χ检验,P<0.01。I类整合子阳性菌株多重耐药率为68.8%(33/48),明显高于阴性菌株(33.3%),P<0.05。I类整合子阳性菌株和产ESBLs菌均对青霉素类、喹诺酮类、磺胺类抗生素表现出较高的耐药率。所有菌株均对亚胺培南敏感。结论I类整合子携带与产ESBLs菌株耐药有关,I类整合子阳性菌株对多种抗生素的耐药率大于整合子阴性菌株。     

质粒介导的大肠埃希菌对喹诺酮类抗菌药物耐药机制的研究

目的了解临床分离的耐环丙沙星的大肠埃希菌质粒介导的喹诺酮类耐药基因的携带情况,并进行相关耐药机制的分析。方法采用VITEK-2全自动微生物检测系统鉴定细菌,用K-B法检测细菌对16种常用抗生素的敏感性,采用聚合酶链反应检测喹诺酮类耐药基因qnrS、qnrA、qnrB、qepA和aac（6′）-Ib,并对阳性的aac（6′）-Ib结果进行测序分析。结果 30株耐环丙沙星的大肠埃希菌中,2株（6.67%）检出qepA基因,8株（26.67%）检出aac（6′）-Ib基因,经测序证实其中6株为aac（6）′-Ib-cr（20.0%）。未检出qnrS、qnrA和qnrB基因。结论对环丙沙星耐药的大肠埃希菌携带aac（6′）-Ib-cr和qepA基因,引起质粒介导的对喹诺酮类抗菌药物的低水平耐药。     

Characterization of class 1 integrons-mediated antibiotic resistance among calf pathogenic Escherichia coli

Escherichia coli isolates from calf diarrhea cases (n=22) in the Beijing surrounding region in China were characterized for disease serotype, virulence factors, antimicrobial susceptibility pattern and class 1 integrons. 59% (n=13) of the isolates were positive for the int I1 gene. The presence and genetic content of class 1 integrons in 13 E. coli isolates were examined by PCR and sequencing. Sequencing analysis revealed six gene cassettes, which encoded resistance to trimethoprim (dfrA1, dfrA17), aminoglycosides (aadB, aadA1 and aadA5) and chloramphenicol (cmlA). The gene cassette arrays dfrA1-orf (45%) and aadB-orf-cmlA (32%) were most prevalent among these isolates. These data revealed the high prevalence of class 1 integrons among calf pathogenic E. coli isolates in the Beijing surrounding region in China, which may provide important and useful surveillance information reflecting specific antibiotic selective pressure.