Class 1 integrons contributes to antibiotic resistance among clinical isolates of Escherichia coli producing extended-spectrum beta-lactamases

Objectives: The objective of this study is to determine the prevalence of antibiotic resistance factors, including the production of extended-spectrum beta-lactamases (ESBLs) and the presence of class 1 integrons among Escherichia coli isolated from clinical specimens. Materials and Methods: Bacterial species identification was performed using a VITEK-2 system (VITEK2 GN-card; bioMérieux, France). Antimicrobial susceptibility testing was determined using the disk diffusion method according to the 2010 Clinical and Laboratory Standards Institute guidelines. Polymerase chain reaction (PCR) was used to detect integrons and amplify variable regions of the bla_TEM, bla_SHV and blaCTX-M genes. Gene cassettes were detected by deoxyribonucleic acid sequencing. Results: In this study, 58% (100/172) of clinical E. coli isolates were identified as ESBL producers. We found that 90% of the ESBL-producing E. coli isolates harbored the bla_CTX-M gene, whereas only 59% and 32% possessed the bla_TEM and bla_SHV genes respectively. The presence of class 1 integrons was based on the detection of the integrase gene by PCR. A total of 69% of the ESBL-producing isolates were integron-positive. Resistance to 10 antibiotics, including quinolones, sulfonamides and β-lactam/enzyme inhibitors, was significantly higher in the class 1 integron-positive isolates (P < 0.05). The occurrence of class 1 integrons in bla_TEM, bla_SHV and bla_CTX-M gene carriers was 72.9%, 84.4% and 68.9%, respectively. Class 1 integrons were detected in 61.5% of the isolates with only one ESBL genotype, but in 69.0% and 92.3% of the isolates with two or three different ESBL genotypes, respectively. Conclusions: Our findings indicate that clinical strains of bacteria with multiple ESBL genotypes may have greater opportunities to carry class 1 integrons.     

Long-term study of the frequency of Escherichia coli and Klebsiella pneumoniae isolates producing extended-spectrum β-lactamases

In total, 438 (1.7%) Escherichia coli and 125 (3.98%) Klebsiella pneumoniae isolates were found to be producers of extended-spectrum beta-lactamase (ESBL) during 1995-2003 in southern Spain. There was a significant increase in the frequency of ESBL-producing E. coli isolates, from < 0.36% before 1999 to 4.8% in 2003, while the frequency of ESBL-producing K. pneumoniae isolates decreased during the same period. The most common ESBLs detected in K. pneumoniae were SHV type, whereas both CTX-M and SHV types were detected in E. coli. In addition, E. coli isolates showed greater clonal diversity (84 distinct REP-PCR patterns, compared with five in K. pneumoniae), fewer enzymes per isolate, and a higher number of isolates recovered from outpatients. These differences may have implications for the control measures that should be used for these two microorganisms.     

SHV-5 extended-spectrum beta-lactamases in clinical isolates of Escherichia coli in Malaysia

Escherichia coli isolates resistant to ceftazidime isolated in the University Malaya Medical Center (UMMC) Kuala Lumpur, Malaysia, between the years 1998 and 2000 were studied for extended-spectrum beta-lactamase (ESBL) production. All strains were analysed phenotypically and genotypically and found to be ESBL-producing organisms harbouring SHV-5 beta-lactamase. This was confirmed by PCR-SSCP and nucleotide sequencing of the blaSHV amplified gene. As there was no evidence of ESBL activity in E. coli prior to this, coupled with the fact that there was a predominance of SHV-5 beta-lactamases in Klebsiella pneumoniae isolates in UMMC, we postulate that the E. coli obtained the SHV-5 beta-lactamase genes by plasmid transfer from the ESBL-producing K. pneumoniae.     

Prevalence and antimicrobial susceptibility of extended-spectrum beta lactamases-producing Escherichia coli and Klebsiella pneumoniae isolated in selected hospitals of Anyigba,Nigeria

BackgroundEscherichia coli and Klebsiella pneumoniae are commonly implicated in urinary tract infections accounting for majority of the antimicrobial resistance encountered in hospitals.ObjectivesTo determine the prevalence and antimicrobial susceptibility of extended-spectrum beta-lactamases (ESBLs) producing E. coli and K. pneumoniae among patients in Anyigba, Nigeria.MethodsThis hospital-based cross-sectional study was conducted using urine samples from 200 patients of Grimmard Catholic hospital and Maria Goretti hospital. Urine samples were processed to identify ESBL-producing E. coli and K. pneumoniae using standard microbiological techniques. Isolates were then tested against antimicrobial agents.ResultsA total of 156 bacterial isolates were recovered consisting 128 of E. coli and 28 of K. pneumoniae. Extended spectrum beta-lactamases production was observed in 69% of E. coli and 31% of K. pneumoniae. These pathogens were resistant to 3 or more antibiotics. Of the antimicrobials tested, cefotaxime demonstrated the highest rates of resistance (100%) for both ESBL-producing E. coli and K. pneumoniae. Fifty-four isolates of ESBL-producing E. coli showed a high level of resistance to amoxicillin clavulanic acid (83.3%), ciprofloxacin (83.3%), and ceftazidime (79.6%). ESBL-positive K. pneumoniae isolates were highly resistant to ciprofloxacin (75%), and amoxicillin clavulanic acid (83.3%). Cefoxitin (62.5%) and gentamicin (66.7%) showed substantially higher rates of resistance against these isolates while all 24 strains were resistant to imipenem.ConclusionThis study indicated the prevalence of ESBL-positive Gram-negative pathogens in these study sites and also demonstrated their resistance to a few antibiotics. This highlights the need for new antimicrobials that are potent and improved policy on use of antibiotics.     

头孢西丁诱导产ACT-1型AmpC酶大肠埃希菌的比较蛋白质组学研究

目的研究产AmpC酶大肠埃希菌在头孢西丁诱导前后的蛋白质谱差异,筛选有价值的靶位。方法提取产ACT-1AmpC酶大肠埃希菌在头孢西丁诱导前后的全菌蛋白,应用双向凝胶电泳技术分析和Blue silver法染色。凝胶图像分析后,对差异蛋白质进行MALDI-TOF质谱分析。结果在所鉴定的8个差异蛋白当中,AmpC酶、外膜蛋白II和转座酶表达显著上调,而磷酸转移酶系统的葡萄糖特异性组分IIA蛋白、抗碲酸盐蛋白、丙三醇磷酰基二酯酶、硫醇过氧化物酶及细菌分化蛋白FtsZ表达下调。结论所鉴定的这些差异蛋白将为阐明产AmpC酶大肠埃希菌耐药产生的机制提供线索,也为筛选具有潜在价值的靶位提供理论依据。     

产AmpC酶大肠埃希菌和肺炎克雷伯菌中整合子与多重耐药的研究

目的探讨整合子介导的耐药机制在产AmpC酶大肠埃希菌和肺炎克雷伯菌多重耐药中的作用.方法5株产AmpC酶大肠埃希菌和肺炎克雷伯菌分离自2002年1月-2004年5月间我院呼吸科住院的患者,采用E-test试验条进行药敏试验、电转化试验,筛选、分离耐药质粒.PCR扩增Ⅰ型整合子基因盒插入序列,分子克隆和序列分析.结果所有产酶菌株通过电转化试验可将头孢西丁耐药性传递给受体菌,5个产AmpC酶耐药质粒中,有4个检出整合酶序列,其中3个携带2种抗药性基因盒,包括氨基糖苷乙酰转移酶基因aacA4;氨基糖苷腺苷转移酶基因aadA5;二氢叶酸还原酶基因dfrA17;氯霉素外排蛋白编码基因cmL44.结论整合子介导的抗药性基因盒参与了产质粒AmpC酶大肠埃希菌和肺炎克雷伯菌多重耐药的形成,应引起高度重视.     

E.coli临床分离株的基因组结构分型

目的：通过基因组结构分析对临床分离的Escherichia coli(E.coli)进行分型，并探讨分型与临床疾病的关系。方法：取临床不同疾病病人的痰、尿、血、分泌物等标本，分离E.coli。用I-Ceu Ⅰ酶切全基因组DNA，用脉冲场凝胶电泳分离DNA片段后，根据酶切图谱的异同进行分型。结果：从临床上分离的64株E.coli中，62株有7个I-Ceu Ⅰ酶切位点，2株有8个I-Ceu Ⅰ酶切位点。菌株间I-Ceu Ⅰ酶切图谱差异明显。这些菌株根据基因组结构的差异分为32个型，分型与疾病之间的对应关系分散。结论：临床分离的E.coli基因组结构存在多样性，其与临床疾病之间的关系有待进一步探讨。     

First identification of an Escherichia coli clinical isolate producing both metallo-β-lactamase VIM-2 and extended-spectrum β-lactamase IBC-1

An Escherichia coli strain with decreased susceptibility to carbapenems was isolated from a hospitalised patient in Athens, Greece. The strain was resistant to all beta-lactams, including aztreonam, whereas the MIC of imipenem and meropenem was 0.5 mg/L. A positive EDTA-disk synergy test suggested the production of a metallo-beta-lactamase. PCR experiments revealed the presence of the bla(VIM-2), bla(IBC-1), and bla(TEM-1) genes. Resistance to beta-lactams was not transferable by conjugation. This is the first report of a clinical isolate of E. coli producing VIM-2, and the first report of the coexistence of bla(VIM-2) and bla(IBC-1) in a single clinical isolate.     

Pathogenicity island markers in commensal and uropathogenic Escherichia coli isolates

Uropathogenic isolates of Escherichia coli (UPEC) contain blocks of DNA, termed pathogenicity islands (PAIs), that contribute to their virulence. Two multiplex PCR assays were developed to detect eight PAI markers among 50 commensal E. coli and 100 UPEC isolates. In total, 40% of commensal isolates and 93% of UPEC carried PAIs. Despite this difference, the distribution of various PAIs showed the same pattern in both groups, with the most prevalent being PAI IV(536) (38% commensal vs. 89% UPEC), followed by PAI I(CFT073) (26% vs. 73%), PAI II(CFT073) (14% vs. 46%), PAI II(J96) (8% vs. 34%), PAI I(536) (8% vs. 33%) and PAI II(536) (4% vs. 20%). PAI III(536) was detected only in UPEC (2%), while PAI I(J96) was not detected in any isolate. Although the mean number of PAIs per isolate was higher among UPEC (2.97) than in commensal (0.98) isolates, there were no statistical differences among group B2 E. coli from the two origins; however, commensal isolates from groups D and B1 appeared to be less virulent than pathogenic isolates. Regardless of their phylogenetic group, nearly all the commensal and UPEC isolates with the same number of PAIs had the same PAI combinations. Although group B2 E. coli are uncommon among commensal intestinal flora, they are highly virulent when present, suggesting that the intestinal flora may act as a reservoir for bacteria that can cause urinary tract infection.     

Prevalence and pathogenesis of extended-spectrum beta-lactamase producing Escherichia coli causing urinary tract infection in hospitalized patients

A total of 296 E. coli strains isolated from hospitalized patients with urinary tract infection were included in this study. These strains were tested for their resistance to 22 antimicrobial drugs and the presence of ESBLs genes coding for TEM, SHV, OXA, and CTX-M. We further characterized them for their interaction with a renal cell line (A-498) and a gastrointestinal cell line (Caco-2). Strains were also typed using a combination of RAPD-PCR, PhP-typing and phylogenetic grouping. Only eight strains (2.7?%) were confirmed as ESBLs producers. The most common clonal type contained 35 isolates and only two of them were ESBLs producers and both showed a high degree of adhesion to both cell lines but only one was able to translocate in Caco-2 cells. These strains belonged to phylogenetic group B2, were resistant to nine antibiotics and carried CTX-M-type of ESBL. The remaining six strains belonged to single clones with different phylogenetic groups and ESBL genotypes and were resistant to between 12 and 15 antibiotics. They also showed a high rate of adhesion to A-498 cells (19?±?2 to 35?±?3?CFU/cell) and all translocated in this cell line. The rate of adhesion of ESBL-producing strains to Caco-2 cells (11?±?3.4?CFU/cell) was significantly lower than A-498 cells (26?±?8?CFU/cell) (p?=?0.0002) and only four of them translocated in Caco-2 cells. Our results suggest that the ESBL-producing clones of E. coli have a potential to translocate and cause septicemia in hospitalized patients with UTI.     

Attaching and effacing Escherichia coli isolates from Danish children: clinical significance and microbiological characteristics

This study describes the prevalence, clinical manifestations and microbiological characteristics of attaching and effacing Escherichia coli isolates, i.e., enteropathogenic E. coli (EPEC) belonging to the classical EPEC serotypes, non-EPEC attaching and effacing E. coli (A/EEC) and verocytotoxin-producing E. coli (VTEC), isolated in a case-control study of Danish children aged <5 years. Among 424 children with diarrhoea and 866 healthy controls, EPEC and VTEC were more prevalent in cases (2.4% and 2.6%, respectively) than in controls (0.7% and 0.7%, respectively). There was a high frequency of A/EEC isolates (n = 121), but these were equally prevalent in cases (11.3%) and controls (12.5%), and comprised a heterogeneous distribution of O:H serotypes. The intimin (eae) subtypes in A/EEC isolates showed an even distribution; the eae-gamma subtype predominated in classical EPEC cases. The virulence genes encoding the bundle-forming pilus (bfpA) and enteroaggregative heat-stable enterotoxin (astA) were rare among all isolates, and seemed to be of limited pathogenic importance in this population. Virulence characterisation of A/EEC isolates did not reveal any significant differences between cases and controls. Colonisation of children with A/EEC was associated with contact with sheep or goats (OR 2.2). The role of A/EEC, not being VTEC or belonging to the classical EPEC serotypes, requires further clarification, but serotyping is useful in discriminating between EPEC and A/EEC strains.     

膀胱上皮细胞对大肠杆菌抗菌作用观察

目的：了解膀胱上皮细胞和大肠杆菌的相互作用,观察其对大肠杆菌杀菌作用的动态变化。方法：采用人膀胱上皮细胞株T24和大肠杆菌标准株K12共同孵育,分别与作用后的15分钟、30分钟、60分钟、90分钟、120分钟,用Triton X-100释放粘附在细胞上及细胞内的活菌,用平板菌落计数法计数活菌数。结果：T24对大肠杆菌K12有杀菌作用,在相互作用15分钟时即表现出来,且在30分钟时杀菌作用最强,与对照组相比细菌减少53％。结论：膀胱上皮细胞对大肠杆菌有杀菌作用,这可能是泌尿道抵抗外来细菌感染的一种天然防御机制。     

Molecular diversity of Proteus mirabilis isolates producing extended-spectrum β-lactamases in a French university hospital

Between February 1997 and December 2002, 3340 hospitalised patients yielded samples positive for Proteus mirabilis, of whom 45 (1.3%) were colonised/infected by P. mirabilis producing extended-spectrum beta-lactamases (ESBLs). The gross incidence of patients colonised/infected by ESBL-producing P. mirabilis was 1.61/10(5) days of hospitalisation, with 20% of isolates being collected from patients in urology wards, most frequently (53.3%) from urine samples. Seventeen (37.7%) of the 43 isolates were obtained from samples collected within 48 h of hospitalisation, indicating that they were community-acquired. Isoelectric focusing assays and sequencing identified the TEM-24, TEM-92 and TEM-52 ESBLs. Pulsed-field gel electrophoresis revealed eight pulsotypes (I-VIII), with the two most common pulsotypes, IV and VI, comprising ten (23.3%) and 12 (26.6%) isolates, respectively. These pulsotypes were considered to represent epidemic strains and spread in various wards of the hospital.     

Identification of CTX-M-14 extended-spectrum beta-lactamase in clinical isolates of Shigella sonnei, Escherichia coli, and Klebsiella pneumoniae in Korea

CTX-M-14 beta-lactamase was identified in a stool isolate of Shigella sonnei and in blood isolates of Escherichia coli (one isolate) and Klebsiella pneumoniae (two isolates) from different parts of Korea. The amino acid sequence differed by one amino acid from CTX-M-9 (Ala-231--> Val) and was identical to that of beta-lactamases recently found in China and Japan.     

聚类分析在大肠埃希菌耐药性研究中的应用

目的 探讨聚类分析在大肠埃希菌临床分离株同类系株的分离及在耐药性监测中的实用价值。方法 用WHONETSE软件对收集的大肠埃希菌的监测数据进行处理，用SPSSl0．0对收集到的药敏试验数据进行聚类分析。结果( 1)聚类分析可以将同类系菌株与非同类系菌株分成不同的类别，以树状图的形式可以直观地反映菌株间的相似性。(2)变量的数量和种类的选择对聚类分析的结果会产生明显的影响。结论 利用大肠埃希菌的药物敏感试验数据进行聚类分析可以将产超广谱β-内酰胺酶(ESBLs)的大肠埃希菌与普通株分开，对推断抗生素的临床疗效具有一定意义；同时可以辨别菌株间的相关性，为进行细菌基因同源性研究提供参考数据。     

致泻性大肠杆菌中发现小肠结肠炎耶尔森菌毒力岛

目的　研究肠产志贺样毒素且具侵袭力的大肠杆菌 (ESIEC)菌株是否含有耶尔森菌的HPI(毒力岛 )基因。方法　采用PCR扩增和Southern杂交的方法。结果　从 35 %的ESIEC菌的染色体上同时扩出irp1、irp2和fyuA 3个片段 ,片段大小分别与小肠结肠炎耶尔森菌WA菌株的相应片段一致 ,鼠疫耶尔森菌HPI上的 6对引物均未扩出目的片段。 6 5 %的ESIEC使用以上 9对引物未扩出目的片段。ESIEC菌的染色体EcoRI酶切产物电泳后与小肠结肠炎耶尔森菌WA菌株的fyuA探针杂交出大小一致的条带 ,与irp1和irp2探针杂交出不同的带型。结论　ESIEC菌含有小肠结肠炎耶尔森菌的HPI,且有变异现象。ESIEC和EAEC的关系还有待于进一步的研究     

Risk-factors for acquisition of extended-spectrum β-lactamase-producing Escherichia coli among hospitalised patients

Between 1996 and 2002, 103 hospitalised patients yielding one or more clinical isolates of extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-EC) were identified. A significant increase was observed in the incidence of ESBL-EC colonisation or infection during the study period (1.65 episodes/100 000 patient-days in 1996 to 12.6 episodes/100 000 patient-days in 2002; p 0.01). Infection developed in 70 (68%) patients (75 episodes), with surgical site (44%) and urinary tract (17%) infections being the most frequent. Pulsed-field gel electrophoresis showed extensive clonal diversity among the isolates. A case-control study and multivariate analysis identified female gender (OR 2.1; p 0.01), use of a nasogastric tube (OR 3.5; p 0.001) and previous antibiotic therapy (OR 3.9; p < 0.001) as independent variables associated with acquisition of ESBL-EC. The study demonstrated a progressive increase in the number of ESBL-EC isolates in a non-epidemic setting. Most cases of ESBL-EC colonisation or infection occurred in hospitalised patients exposed to invasive procedures and antibiotic pressure.     

Flooding adds pathogenic Escherichia coli strains to the water sources in southern Khyber Pakhtunkhwa,Pakistan

Purpose: Seasonal rains in Pakistan result in heavy floods across the country, whereby faecal contaminants will be added to the water bodies and cause numerous food-borne outbreaks. The present study was aimed to determine the prevalence of diarrheagenic Escherichia coli (DEC) strains in the water sources. Materials and Methods: Two hundred water samples collected during (2011–2012) were processed for the isolation of E. coli (EC) strains. EC strains were further analysed for antibiotic susceptibility patterns, and pathogroups-specific virulence factors stx1, stx2, stx2c, eae, tir, hlyA, bfpA, estA and eltA were detected using multiplex polymerase chain reaction. Results: Thirty-three percent of the water samples were contaminated with EC pathotypes. Fifty percent (33/66) of the DEC pathotypes were identified as enterotoxigenic EC (ETEC). Seventy-two percent (13/18) of the enteropathogenic EC (EPEC) strains were identified as typical EPEC and 28% (5/18) as atypical EPEC. Eleven percent (7/66) of the Shiga toxin EC (STEC) isolates carried a combination of stx1 and stx2 genes. Summer was found as a peak season with 47% (31/66) for EC pathogroups’ activities. Eighty-nine percent of the strains showed resistance against tetracycline. Conclusion: ETEC and EPEC are the primary causes of water contamination in southern regions of Khyber Pakhtunkhwa province, Pakistan. Firm adherence to the prescribed drugs can decrease trends in antibiotic resistance.     

Risk-factors for emerging bloodstream infections caused by extended-spectrum β-lactamase-producing Escherichia coli

Risk-factors for bloodstream infections caused by extended-spectrum β-lactamase (ESBL)-producing Escherichia coli were investigated using an exploratory case-double control study in which 43 cases (70% producing CTX-M enzymes) were compared with: (i) 86 patients with bacteraemia caused by non-ESBL-producing E. coli ; and (ii) 86 hospitalised patients. Previous follow-up as an outpatient, urinary catheterisation and use of oxyimino-β-lactams or fluoroquinolones were independent risk-factors for ESBL-producing E. coli among patients with E. coli bacteraemia, and previous use of oxyimino-β-lactams or fluoroquinolones were also independent risk-factors among hospitalised patients. These findings may help in identifying patients at greater risk for bloodstream infection caused by ESBL-producing E. coli in endemic areas.