Enteroaggregative Escherichia coli Heat-Stable Enterotoxin 1 (EAST1): A New Toxin with an Old Twist

Enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) is a small protein that was first detected more than a decade ago in an enteroaggregative E. coli (EAEC) strain isolated from the stools of a diarrheic child. The EAST1 gene, astA, is not solely present in EAEC, but also in other categories of diarrheagenic E. coli. Strains expressing EAST1 have been shown to induce diarrhea principally in humans, although they have also been associated with piglets and calves. EAST1 toxin has been proposed as a virulence factor implicated in the mechanism of pathogenesis of EAEC and could play a role in the pathogenicity of other enteropathogens as well. This toxin is often compared to E. coli STa enterotoxin because they share some physical and mechanistic similarities. This review summarizes the various observations on EAST1 since its discovery.     

Improved detection of Shigella using Escherichia coli medium enrichment: Polymerase chain reaction from stool samples

Laboratory diagnosis of shigellosis using conventional culture technique is limited by lower sensitivity and higher turnaround time. Here, we have evaluated the role of polymerase chain reaction from stool samples after enrichment in Escherichia coli medium for detection of Shigellae. The technique not only increased the sensitivity but also decreased the turnaround time.     

Diagnostic value of quantitative PCR for adenovirus detection in stool samples as compared with antigen detection and cell culture in haematopoietic stem cell transplant recipients

Adenovirus (AdV) infections constitute a significant cause of morbidity and mortality during haematopoietic stem cell transplantation. Recent guidelines recommend repeated screening for AdV in whole blood (WB), with quantitative PCR (qPCR) as the reference standard. Despite pre-emptive antiviral treatment based on qPCR in WB, the mortality rate after disseminated AdV infection remains very high. The aim of our study was to advance early screening for AdV, using a standardized method, so as to enable the earlier initiation of antiviral treatment or adoptive immunotherapy. The diagnostic value of AdV DNA quantification in stool samples was investigated retrospectively and compared with antigen detection and cell culture in 21 patients with AdV infection, from 182 patients followed in the Transplant Unit of Nancy University Hospital Centre, including 18 patients with systemic infection. In 16/18 patients with positive AdV viraemia, AdV DNA was present in stool samples earlier than in WB (median, 42 days; range, 3–199 days), whereas both antigen detection and cell culture were still negative for 11/18 patients with systemic AdV infection. The course of AdV viral loads in stool samples was predictive of adenoviraemia (sensitivity, 89%). Very late and lethal AdV infections were observed in cord blood transplant recipients, and would have been detected much earlier with the use of qPCR on stool samples. This study confirmed that quantification of AdV in stool samples by qPCR is beneficial for the management of transplant recipients, with or without antigen detection.     

福田区致泻性大肠埃希菌感染的流行病学分析

目的了解深圳市福田区门诊腹泻病例中致泻性大肠埃希菌感染状况及相关因素,为临床治疗和预防提供科学依据。方法采集门诊腹泻监测病例的大便样本、肛拭子或呕吐物标本,进行致病菌的分离培养,对阳性菌株进行分子生物学特征分析。结果福田区门诊腹泻病例监测中致泻性大肠埃希菌感染率为10.31%（114/1106）,病例以EPEC、ETEC、EIEC感染为主。EPEC、EIEC、ETEC在不同年龄组的感染情况不同,差异有统计学意义（χ2=2.680,P=0.001;χ2=18.278,P=0.006;χ2=13.948,P=0.030）,且以青壮年居多;但在季节性分布、男女性别比例和户籍分布中差异均无统计学意义（P〉0.05）。结论福田区致泻性大肠埃希菌阳性病例以EPEC、ETEC、EIEC感染为主。应重视辖区内腹泻病例的致泻性大肠埃希菌感染,继续做好腹泻监测并积极防治含致泻性大肠埃希菌感染的腹泻病例,避免疫情爆发。     

Diarrhoeagenic Escherichia coli isolated from children with acute diarrhoea at Rakai hospital,Southern Uganda

BackgroundDiarrhoeagenic Escherichia coli (DEC) is a leading cause of childhood diarrhoea. This study estimated the prevalence of DEC and DEC pathotypes among children with acute diarrhoea in Southern Uganda.MethodsA cross-sectional study was conducted on 267 children less than 5 years with acute diarrhoea, admitted to Rakai General Hospital in Southern Uganda. Faecal samples were collected from the children and processed for isolation of E. coli. The presence of DEC and the distribution of DEC pathotypes were determined by polymerase chain reaction.ResultsA total of 102 (38.2%, 102/267) children had DEC of various pathotypes – enteroaggregative E. coli (EAEC) (14.2%); enteropathogenic E. coli (EPEC) (6.7%); enterotoxigenic E. coli (ETEC) (6%); enteroinvasive E. coli (EIEC) (7.5%); enterohemorrhagic E. coli (EHEC) (3%); and cell-detaching E. coli (CDEC) (0.75%). The difference in the overall prevalence of DEC was not significant regarding HIV but individually, EAEC and CDEC were associated with HIV-positive status while ETEC was associated with HIV-negative status.ConclusionsDEC is prevalent in children with acute diarrhoea in Southern Uganda and its identification in children should be considered among strategies for combatting childhood diarrhoea in Africa.     

Changes in Escherichia coli resistance patterns during and after antibiotic therapy: a longitudinal study among outpatients in Germany.

There is worldwide concern about the appearance and rise of bacterial resistance to commonly used antibiotics. Although the gut is an important reservoir for resistant Escherichia coli, data from large-scale epidemiological studies concerning the colonisation dynamics of the normal gut flora with resistant E. coli during and after antibiotic therapy are sparse. Accordingly, a large community-based study was conducted to ascertain changes in the prevalence of resistant E. coli during and after antibiotic treatment. Stool samples before, during and after antibiotic therapy were obtained from 541 patients (aged >/=40 years) with a febrile infection who attended a general practitioner in southern Germany. The MICs of commonly prescribed antibiotics for E. coli isolates from the stools were determined. The prevalence of resistance to the corresponding antibiotics rose from 18% to 38%, from 29% to 58% and from 33% to 67% during treatment with beta-lactam antibiotics, doxycycline and co-trimoxazole, respectively. Prevalences of resistance in the E. coli isolates also rose for other antibiotic classes. With the exception of co-trimoxazole resistance, prevalences of resistance returned to baseline levels in <2 weeks after the cessation of antibiotic therapy. Thus, there was a substantial, but rapidly reversible, increase in the prevalence of resistant E. coli isolates during antibiotic treatment.     

Prevalence of diarrheagenic Escherichia coli strains detected by PCR in patients with travelers' diarrhea

Objective: To determine the prevalence of the different categories of diarrheagenic Escherichia coli , enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), verotoxin-producing E. coli (VTEC), enteroaggregative E. coli (EAggEC), diffusely adherent E. coli (DAEC), and enteropathogenic E. coli (EPEC), associated with travelers' diarrhea.
Methods: Stool specimens from 350 patients with travelers' diarrhea were collected between 1994 and 1996. The virulence factors of the diarrheagenic E. coli isolated were detected by PCR technique, in subcultures of single colonies of all morphotypes of E. coli observed in culture on MacConkey agar.
Results: ETEC (15.7%), EAggEC (13.4%) and DAEC (9.14%) are significantly more prevalent than EIEC (3.4%), EPEC (2.86%) and VTEC (0.86%) (p < 0.05; z -test). The prevalence of ETEC and EAggEC was similar in all geographic areas visited.
Conclusions: PCR is a rapid and specific technique to use in the identification of the different categories of diarrheagenic E. coli and greatly increases the yield of potential enteropathogens from cases of travelers' diarrhea. Not only ETEC but also EAggEC and DAEC strains play a major role in the etiology of travelers' diarrhea, whereas EIEC, EPEC, and VTEC strains play a minor role, leading to the question of whether it is necessary to search routinely for these three types of E. coli in diagnostic laboratories.     

Rapid and sensitive detection of fluoroquinolone-resistant Escherichia coli from urine samples using a genotyping DNA microarray

Urinary tract infections (UTI) are among the most common bacterial infections in humans, with Escherichia coli being the major cause of infection. Fluoroquinolone resistance of uropathogenic E. coli has increased significantly over the last decade. In this study a microarray-based assay was developed and applied, which provides a rapid, sensitive and specific detection of fluoroquinolone-resistant E. coli in urine. The capture probes were designed against previously identified and described hotspots for quinolone resistance (codons 83 and 87 of gyrA). The key goals of this development were to reduce assay time while increasing the sensitivity and specificity as compared with a pilot version of a gyrA genotyping DNA microarray. The performance of the assay was demonstrated with pure cultures of 30 E. coli isolates as well as with urine samples spiked with 6 E. coli isolates. The microarray results were confirmed by standard DNA sequencing and were in full agreement with the phenotypic antimicrobial susceptibility testing using standard methods. The DNA microarray test displayed an assay time of 3.5h, a sensitivity of 100CFU/ml, and the ability to detect fluoroquinolone-resistant E. coli in the presence of a 10-fold excess of fluoroquinolone-susceptible E. coli cells. As a consequence, we believe that this microarray-based determination of antibiotics resistance has a true potential for the application in clinical routine laboratories in the future.     

How to evaluate PCR assays for the detection of low‐level DNA

High sensitivity of PCR‐based detection of very low copy number DNA targets is crucial. Much focus has been on design of PCR primers and optimization of the amplification conditions. Very important are also the criteria used for determining the outcome of a PCR assay, e.g. how many replicates are needed and how many of these should be positive or what amount of template should be used? We developed a mathematical model to obtain a simple tool for quick PCR assay evaluation before laboratory optimization and validation procedures . The model was based on the Poisson distribution and the Binomial distribution describing parameters for singleplex real‐time PCR‐based detection of low‐level DNA. The model was tested against experimental data of diluted cell‐free foetal DNA. Also, the model was compared with a simplified formula to enable easy predictions. The model predicted outcomes that were not significantly different from experimental data generated by testing of cell‐free foetal DNA. Also, the simplified formula was applicable for fast and accurate assay evaluation. In conclusion, the model can be applied for evaluation of sensitivity of real‐time PCR‐based detection of low‐level DNA, and may also assist in design of new assays before standard laboratory optimization and validation is initiated.     

Screening for Escherichia coli O157:H7 in Illinois

Objective: To determine the incidence of infection with Escherichia coli O157:H7 in a tertiary referral center in Chicago, where a similar study had been performed in 1984, to evaluate cases of disease reported to the Illinois Department of Public Health (IDPH) in 1993, and to determine laboratory practices used to detect this infection throughout the state.
Methods: During a 6-month period in 1993, all stool specimens at Rush-Presbyterian-St Luke's Medical Center (RPSLMC) were tested for E. coli O157:H7. Reports of diagnosed E. coli O157:H7 cases investigated by IDPH were also reviewed. A survey of 73 hospitals in the Chicago area was performed to determine routine culturing practices, specifically, the selection of stool specimens for evaluation for this pathogen.
Results: In the RPSLMC survey, two cases were identified among 1985 samples (incidence 0.1%), similar to the 0.08% incidence detected in a similar study conducted at the same institution in 1984. Through passive surveillance, the IDPH received 44 reports of E. coli O157:H7 in 1993. The hospital survey revealed that, in the seven labs testing all stool specimens for E. coli O157:H7, an incidence of 16/8137 specimens (0.2%) was determined.
Conclusions: These data suggest that sporadic E. coli O157:H7 remains uncommon in Illinois and that the incidence may not have changed over a 9-year period. The low yield and substantial cost of culturing all stools suggest that only specimens from patients with bloody diarrhea should be evaluated routinely in areas of low endemicity.     

Detection of enterotoxigenic Escherichia coli in stool samples by using nonradioactively labeled oligonucleotide DNA probes and PCR.

The detection of heat-labile enterotoxin LT-A and heat-stable enterotoxin ST Ia and ST Ib genes from enterotoxigenic Escherichia coli (ETEC) by using oligonucleotide DNA probes and the PCR was evaluated in reconstruction experiments and by testing stool specimens from 29 healthy subjects and from 50 patients with diarrhea who had returned from the (sub)tropics. ETEC strains were detected in concentrations ranging from 10(6) to 10(8) CFU/g of feces when oligonucleotide probes were applied to colony blots from five randomly picked E. coli-like colonies from CLED (cystine lactose electrolyte deficient) agar plates inoculated with the feces. When these probes were applied to blots from whole stool cultures collected from the agar plates (sweep blot), the detection limit was 10(6) CFU/g of feces. PCR of the sweep material could detect toxin genes when the concentration of ETEC strains was 10(2) CFU/g of feces. Results obtained with stool specimens from 29 healthy control subjects were negative. Testing stool specimens from 50 patients confirmed the observation that the number of samples containing ETEC enterotoxin genes was higher when PCR of sweeps was used than when oligonucleotide DNA probe hybridization of either sweep blots or colony blots was used. Furthermore, PCR of sweeps is an easy and rapid method which does not require DNA extraction and purification from fecal specimens.     

Identification of serotypes and virulence markers of Escherichia coli isolated from human stool and urine samples in Egypt

Purpose: Haemorrhagic colitis and haemolytic-uremic syndrome are associated with Shiga-toxin producing Escherichia coli (STEC). There are others DEC (Diarrhoeagenic E. coli) pathotypes responsible for outbreaks and others toxins associated to these. Most clinical signs of disease arise as a consequence of the production of Shiga toxin 1 (Stx1), Stx2 or combinations of these toxins. Other major virulence factors include E. coli haemolysin (hlyA), and intimin, the product of the eaeA gene that is involved in the attaching and effacing adherence phenotype. Materials and Methods: In this study, the PCR assay was used to detect 12 E. coli genes associated with virulence (stx1, stx2, hylA, Flic h7 , stb, F41, K99, sta, F17, LT-I, LT-II and eaeA). Results: A total of 108 E. coli strains were serotyped into 64 typable strains. The investigated strains from the stool, 8/80 (10%) strains were O 164:K, while the 56/110 strains isolated from the urine were O126:K71 (44/110, 40%) and O 86:K 61 (12/110, 11%). The distribution pattern of the detected virulence genes was observed to be in the following order: F17 (10% from the stool and 44% from the urine), Sta (10% from the stool), hylA (10% from the stool and 44% from the urine), Stb (44% from the urine) and stx1 (27% from the urine). The 8 faecal strains encoded a combination of the F17, Sta and hylA genes, while the 56 urine strains encoded a combination of the F17 0+ Stb + hylA (44/110, 40%) and Stx1 only (12/60, 20%). Conclusion: This is the first report on the molecular characterization of E. coli diarrhoeagenic strains in Egypt and the first report on the potential role of E. coli in diarrhoea and urinary tract infections in a localized geographic area where the people engage in various occupational activities.     

Characterization of CTX‐M‐producing Escherichia coli by repetitive sequence‐based PCR and real‐time PCR‐based replicon typing of CTX‐M‐15 plasmids

The emergence of extended‐spectrum β‐lactamase (ESBL)‐producing Enterobacteriaceae is a major global concern. CTX‐M is the dominating ESBL type worldwide, and CTX‐M‐15 is the most widespread CTX‐M type. The dissemination of CTX‐M appears to be in part due to global spread of the Escherichia coli clone O25b‐ST131. However, the gene‐encoding CTX‐M is mainly located on mobile genetic elements, such as plasmids, that also promote the horizontal dissemination of the CTX‐M genes. In this study, 152 CTX‐M‐producing E. coli isolated in 1999–2008 in Örebro County, Sweden, were typed using a commercial repetitive sequence‐based PCR (the DiversiLab system), and the prevalence of ST131 was investigated by pabB PCR. Real‐time PCR‐based plasmid replicon typing was performed on 82 CTX‐M‐15‐producing E. coli isolates. In general, the CTX‐M‐producing E. coli population was genetically diverse; however, ST131 was highly prevalent (27%), and the dominating clone in our area. The bla_CTX‐M‐15 gene was mainly located on IncF plasmids (69%), but a relatively high proportion of IncI1 plasmids (29%) were also detected among E. coli with diverse rep‐PCR patterns, indicating that horizontal transmission of IncI1 plasmids carrying bla_CTX‐M‐15 may have occurred between different E. coli strains.     

Occurrence and molecular characterization of enteropathogenic Escherichia coli serotypes isolated from children with diarrhoea in Najaf, Iraq

Purpose: Enteropathogenic Escherichia coli (EPEC) are among the most important pathogens infecting children worldwide and are one of the main causes of diarrhoea. The study was carried out to investigate the occurrence of EPEC as a cause of infectious diarrhoea in children younger than 2 years of age and characterize their virulence genes. Materials and Methods: During the study period, a total of 656 faecal specimens from children with diarrhoea and 54 from healthy children were analyzed. E. coli isolates were serotypically identified with EPEC polyvalent and monovalent antisera. The isolated EPEC were examined for the presence of the attaching and effacing (eaeA), bundle-forming pilus (bfpA), Shiga like toxins (stx₁ and stx₂), enterohaemorrhagic E. coli enterohaemolysin (EHEC hlyA) and EPEC adherence factor (EAF) genes by the PCR assay. Results: The study has shown that 22 (3.4%) had diarrhoea due to EPEC, while no EPEC isolates were detected in asymptomatic children. The highest number of the EPEC isolated belonging to polyvalent 2. The primers encoding virulence genes were subjected to all the EPEC isolates. Only 9.1%, 27.3%, and 9.1% isolates gave positive re sults with intimin (eaeA), bfbA and (EAF) genes, respectively. None of the isolates were positive for stx₁, stx₂, and hlyA genes. Typical EPEC (eaeA⁺, bfpA⁺) was diagnosed in two isolates, while, atypical EPEC was manifested in four isolates. Conclusions: According to the results, the frequency of EPEC isolates in Najaf was lower than what has been suspected and the investigation including the use of molecular technique and serotyping, are necessary to allow precise identification and epidemiological study of these pathogens.     

A high‐throughput assay for the measurement of uropathogenic Escherichia coli attachment to urinary bladder cells

Strains of uropathogenic Escherichia coli (UPEC) are the major causative agent of urinary tract infections (UTI), the most common infectious diseases in the world. Their ability to attach and enter into cells in the urinary tract is a limiting step for their pathogenicity. Many studies are thus focussing on these key mechanisms to propose new therapeutic strategies. To facilitate such studies, we developed a fast and high‐throughput assay which makes it possible to monitor the interaction of UPEC with cultured human uroepithelial cells. This assay allows measurement of the in vitro association of fluorescently labelled clinical isolates with bladder epithelial cells using flow cytometry in a microplate format. The assay was sensitive enough to detect variations between isolates expressing different adhesins and virulence factors and the inhibitory effect of proanthocyanidins. Thus we have developed a fast and robust assay which allows us to measure variations in the adhesion properties of UPEC to human bladder cells. This novel assay will be valuable for the study of initial steps of pathogenesis in UTI and for the screening or validation of inhibitory molecules.     

Characterization of verotoxigenic Escherichia coli (VTEC) isolates from faeces of small ruminants and environmental samples in southern Jordan

This study was conducted to determine the prevalence rate of VTEC in slaughtered sheep and goats and to evaluate the contamination rate of VTEC in slaughterhouses and butchers' shops in southern Jordan. 201 E. coli isolates from animals' faecal samples and 33 E. coli isolates from slaughterhouse/butcher shop samples were characterized by multiplex PCR (mPCR) reaction for detection of stx1, stx2, eae A and E‐hly A virulent genes. Twenty‐six virulent E. coli isolates were characterized by mPCR to seven different virulent patterns: stx1, stx1+stx2, stx1+eae A, stx1+E‐hly A, stx1+eae A+E‐hly A, eae A and E‐hly A. It was found that VTEC comprised 6.4% and 21% of the total E. coli isolates from slaughtered small ruminants and slaughterhouses/ butchers' shops, respectively. The VTEC comprised 76.2% of the virulent isolates. The proportion of stx1:stx1+stx2 patterns was 19:1. It was found that the characterized complex VTEC (containing eae A and/or E‐hly A) possessed three virulence patterns, including (VTEC) stx1 +eae A, (VTEC/EHEC) stx1 +E‐hly A and (VTEC/EHEC) stx1 +eae A +E‐hly A in percentages of 30%, 25% and 10%, respectively, in relation to the total VTEC isolates. Only two VTEC isolates were characterized as E. coli O157 and O26 serotypes, as highly pathogenic strains. Each of the O157 and O26 VTEC isolates was in a percentage of 0.4% in relation to the total E. coli isolates with virulent patterns stx1, eae A and E‐hly A. The rest of the VTEC isolates were non‐O157 VTEC. The antibiotic sensitivity test showed that the isolated VTEC was highly sensitive to gentamicin and co‐trimoxazole and highly resistant to tetracycline and ampicillin. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)