Mixing of Shiga toxin-producing and enteropathogenic Escherichia coli in a wastewater treatment plant receiving city and slaughterhouse wastewater

Wastewater of human and animal may contain Shiga toxin-producing (STEC) and enteropathogenic (EPEC) Escherichia coli. We evaluated the prevalence of such strains in a wastewater treatment plant (WWTP) receiving both city and slaughterhouse wastewater. PCR screenings were performed on 12,248 E. coli isolates. The prevalence of STEC in city wastewater, slaughterhouse wastewater and treated effluent was 0.22%, 0.07% and 0.22%, respectively. The prevalence of EPEC at the same sampling sites was 0.63%, 0.90% and 0.55%. No significant difference was observed between the sampling points. Treatment had no impact on these prevalences. Enterohemorrhagic E. coli (EHEC) O157:H7 and O111:H8 were isolated from the treated effluent rejected into the river. The characteristics of STEC and EPEC differed according to their origin. City wastewater contained STEC with various stx subtypes associated with serious human disease, whereas slaughterhouse wastewater contained exclusively STEC with stx_2e subtype. All the EPEC strains were classified as atypical and were screened for the ε, γ1 and β1 subtypes, known to be associated with the EHEC mainly involved in human infections in France. In city wastewater, eae subtypes remained largely unidentified; whereas eae-β1 was the most frequent subtype in slaughterhouse wastewater. Moreover, the EPEC isolated from slaughterhouse wastewater were positive for other EHEC-associated virulence markers, including top five serotypes, the ehxA gene, putative adherence genes and OI-122 associated genes. The possibility that city wastewater could contain a pool of stx genes associated with human disease and that slaughterhouse wastewater could contain a pool of EPEC sharing similar virulence genes with EHEC, was highlighted. Mixing of such strains in WWTP could lead to the emergence of EHEC by horizontal gene transfer.     

上海市2012－2013年4种致泻性大肠埃希菌监测

目的 基于临床医院开展4种致泻性大肠埃希菌(DEC)人群监测,探讨公共卫生实验室对临床实验室需求的直接技术指导的实践模式。方法 设立哨点医院,以标准化方法筛选和鉴定DEC菌型;构建DEC流行特征基线;对疑似暴发病例开展基于实验室和流行病学调查。结果 2012－2013年选择上海地区4家哨点医院检测7 204份腹泻标本确认的712例DEC感染病例,阳性率为9.9%。其中肠致病性大肠埃希菌(EPEC)感染351例;肠产毒性大肠埃希菌(ETEC)感染292例;肠侵袭性大肠埃希菌(EIEC)感染32例;产志贺样毒素大肠埃希菌(STEC/EHEC)感染6例;DEC混合感染31例。EPEC感染以1～5岁儿童最多见,菌型均为aEPEC;ETEC流行峰值在8月,阳性率>20%,感染病例2012年聚集于1～28日龄和2013年的20～60岁人群(P< 0.05),菌型以耐热肠毒素(ST)型最多(59.6%),其次为不耐热肠毒素(LT)型(27.8%)和ST/LT型(12.6%);2013年儿童感染EIEC病例明显增加(P< 0.01);未监测到EHEC O157 : H7,但确认2例EHEC O26 : H11(eae-hlyA-stx1a)儿童病例;调查确认2012年上海地区15例新生儿ETEC聚集性感染病例与四川省自贡市新生儿病例属于同一克隆(STh-CS21-CFA/I-ClyA-EatA-ST2332- SHNL0005)。结论 上海地区DEC型谱特征已发生改变,ETEC对新生儿院内感染和食源性感染性腹泻构成潜在暴发风险,需加强实验室主动监测。     

Cytolethal distending toxin producing Escherichia coli O157:H43 strain T22 represents a novel evolutionary lineage within the O157 serogroup

Enterohemorrhagic Escherichia coli (EHEC) O157:H7/NM strains are significant foodborne pathogens intensively studied, while other sero- and pathotypes of the O157 serogroup only began to receive more attention. Here we report the first genome sequence of a cytolethal distending toxin (CDT-V) producing E. coli O157:H43 strain (T22) isolated from cattle. The genome consists of a 4.9 Mb chromosome assembled into three contigs and one plasmid of 82.4 kb. Comparative genomic investigations conducted with the core genomes of representative E. coli strains in GenBank (n = 62) confirmed the separation of T22 from the EHEC and enteropathogenic (EPEC) O157 lineages. Gene content based pangenome analysis revealed as many as 261 T22-specific coding sequences without orthologs in EDL933 EHEC O157 prototypic and two phylogenetically related commensal E. coli strains. The genome sequence revealed 10 prophage-like regions which harbor several virulence-associated genes including cdt and heat-labile enterotoxin (LT-II) encoding operons. Our results indicate that the evolutionary path of T22 is largely independent from that of EHEC and EPEC O157:H7/NM strains. Thus, the CDT-producing T22 E. coli O157:H43 strain represents a unique lineage of E. coli O157.     

Virulence genotypes and serotypes of verotoxigenic Escherichia coli isolated from cattle and foods in Argentina

Virulence factors of Verotoxin-producing Escherichia coli(VTEC) strains isolated from hamburgers and ground beef were studied in Argentina by PCR. Their virulence profiles were correlated with those corresponding to strains isolated from calves and adult cattle. Most virulent profiles (VTs⁺ eae ⁺Mp⁺) were present in E. colifrom healthy and diarrheic calves corresponding to O5:H-, O5:H27, O20:H?, O26:H11, O38:H?, O103:H-, O103:H2, O111:H-, O118:H16, O165:H-serotypes. The presence of the eaegene was significantly more frequent among VTEC strains isolated from calves (20/26; 76%) than from adult cattle (1/39; 2.5%) (p< 0.005). VT2⁺ eae ^? E. coliwas prevalent in foods and adult cattle at slaughterhouse. The prevalence of the eaegene was similar between VTEC strains isolated from meat (0/21) and adult cattle (1/39; 2.5%) which constitutes the main population processed at slaughterhouses in Argentina. Serotyping showed that VTEC strains were distributed among 31 serotypes, some of which (O20:H19, O91:H21, O113:H21, O116:H21, O117:H7, O171:H2, OX3:H21) were shared between bovine and food strains. These O serogroups have been isolated from cases of haemorrhagic colitis (HC) and haemolytic-uraemic syndrome (HUS) in humans in several continental European countries. This study confirms the role of cattle as a reservoir of many VTEC serotypes other than O157:H7 and represents a base for future diagnostic, prevention and control strategies of EHEC in this country. In addition, this study affirms the advantages of PCR-based screening of E. coliisolates given the finding of so many verotoxin-producing strains.     

太原地区携带OI一28毒力基因组岛致泻大肠埃希菌性小儿腹泻

目的探讨携带OI-28毒力基因组岛致泻大肠埃希菌在小儿腹泻中的病原学作用。方法在山西省儿童医院采集257例腹泻病患儿粪便,做肠道病原菌常规培养和致泻大肠埃希菌血清学分型鉴定,用PCR和DNA斑点杂交检测致泻大肠埃希菌EHEC OI-28毒力基因组岛中与RTX相关的5个毒力基因。结果257例腹泻病患儿检出病原菌206株(80.16%)。其中大肠埃希菌149株(57.98%),其他肠道致病菌57株(22.18%)。血清分型检出致泻大肠埃希菌EPEC 3株(2.01%)、ETEC 2株(1.34%)、EHEC 2株(1.34%),其余142株为"疑似致泻大肠埃希菌"(55.25%)。149株大肠埃希菌中OI-28 5个基因全阳性者21株(14.09%),1个基因阳性者8株(5.37%),2个基因阳性者2株(1.34%)。21例携带OI-28毒力基因组岛大肠埃希菌感染的腹泻患儿,以3岁以下小儿为主(80.95%)。结论携带OI-28毒力基因组岛大肠埃希菌是夏季小儿腹泻的重要病原菌之一。     

产志贺样毒素且具侵袭力的大肠杆菌的研究

我国临床腹泻病人粪便标本中，约有６０％检不出任何已知病原，而能分离出几乎纯培养的大肠杆菌。这些大肠杆菌过去被误认为是肠道正常菌样，我们对在北京西城区三个医院从腹泻病患者粪便本本中分离的１７２株所谓正肠杆菌，进行质粒ＤＮＡ，Ｈｅｐ－２细胞粘附试验，和１０种ＤＮＡ探针分析，结果表发现这些所谓的肠道正常大肠杆菌并不正常，其中４４％可为致泻性大肠杆菌，包括ＥＨＥＣ１６株，占所检查菌株的９．３％；ＥＰＥＣ８     

泰州市腹泻病人中致泻大肠埃希氏菌感染状况及 病原学特征分析

摘要:目的　了解泰州市腹泻病人中致泻大肠埃希氏菌感染状况、毒力基因、分子分型以及耐药性情况,为致泻大肠埃希氏菌防治提供依据。方法　对2013年泰州市215份腹泻病人粪便标本进行多重PCR鉴定和分离培养,并对分离的致泻大肠埃希氏菌菌株进行生化鉴定、脉冲场凝胶电泳（Pulsed Field Gel Electrophoresis,PFGE）分子分型以及药敏试验。结果　215份腹泻病人粪便标本共检测出致泻大肠埃希氏菌30株,其中EPEC 20株（占9.30%）,EAEC 5株（占2.33%）,ETEC 5株（占2.33%）;PFGE结果表明,30株致泻大肠埃希氏菌PFGE带型种类多,各亲缘关系较近,但无100%的同源菌,同时PFGE分析结果表明在同一个粪便中有2种不同致泻大肠埃希氏菌感染情况存在;药敏试验表明,30株致泻大肠埃希氏菌对阿莫西林、四环素耐药性最高,3耐以及以上占63.33%。结论　泰州市腹泻病人中致泻大肠埃希氏菌感染普遍存在,并以EPEC、EAEC、ETEC感染为主,菌株的耐药性较强,耐药谱广;基因型呈多态分布,且具有流行相关性。     

Phenotypic and Genotypic Antibiotic Resistant diarrheagenic Escherichia coli pathotypes isolated from Children with Diarrhea in Nairobi City,Kenya

BackgroundThe marked genome plasticity of diarrheagenic Escherichia coli promotes emergence of pathotypes displaying unique phenotypic and genotypic resistance. This study examined phenotypic and genotypic antibiotic resistant diarrheagenic Escherichia coli pathotypes among children in Nairobi City, Kenya.MethodsIn a cross-sectional study, diarrheagenic Escherichia coli pathotypes were isolated from stool samples and their phenotypic and genotypic resistance against eight antimicrobial agents assayed.ResultsDiarrheagenic Escherichia coli was detected in 136(36.4%) children. Most of diarrheagenic Escherichia coli that were resistant to ampicillin, ceftriaxone, streptomycin, gentamycin, ciprofloxacin, chloramphenicol, erythromycin and tetracycline, harbored citm, bla CMY, aadA1, aac(3)-IV, qnr, catA, ere(A) and tet(A) corresponding resistant genes.ConclusionAntimicrobial-resistant genes are highly prevalent among phenotypic resistant ETEC pathotypes indicating a possibility of horizontal gene transfer in spreading antibiotic resistant genes among E. coli pathotypes.     

2010-2012年河南省致泻大肠埃希菌监测

目的建立河南省致泻大肠埃希菌多重PCR分型的基线数据,分析致泻大肠埃希菌感染的流行病学特征。方法在河南省郑州、登封、周口、睢县4个地区的多家医疗机构设立监测哨点,收集可疑致泻大肠菌感染样本,采用多重PCR方法进行分型,并对致泻大肠菌分型结果和病例个人信息进行统计分析。结果 2010-2012年河南省共检测可疑致泻大肠埃希菌病例样本数1 934例,检出致泻大肠菌阳性样本为139例,检出率为7.19%。其中EPEC检出率最高为67例（3.46%）,之后依次为EAEC 56例（2.90%）、ETEC 9例（0.47%）、EHEC 7例（0.36%）、EIEC未检出。致泻大肠埃希菌感染高峰在4-5月,1-2岁组腹泻患者的检出率最高（10.81%）。结论 EAEH和EPEC是河南主要的致泻大肠菌型,应用多重PCR技术能快速鉴别粪便标本中的致泻大肠埃希菌。     

HEp-2-adherent Escherichia coli strains associated with acute infantile diarrhea,São Paulo,Brazil

In this paired case-control study of infants with diarrhea in São Paulo, we examined the association between HEp-2–adherent Escherichia coli strains and diarrhea. We tested isolates from stool specimens of infants with diarrhea and matched controls in an HEp-2 cell adherence assay; we then hybridized isolates with DNA probes and identified enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), and diffusely adherent E. coli (DAEC). From 100 patient-control pairs, we isolated 78 HEp-2–adherent strains; of these, 61 strains were single pathogens identified in stools of infants with diarrhea. While typical EPEC was significantly associated with diarrhea (p<0.001), EAEC was more frequently associated with diarrhea in clinical cases (20%) compared with healthy controls (3%) (p<0.001). Atypical EPEC, showing a localized adherence-like pattern, was also more common in patients than controls (p>0.1). DAEC was isolated with equal frequency from patients and controls (p>0.1).     

High prevalence of clade 8 Escherichia coli O157:H7 isolated from retail meat and butcher shop environment

Escherichia coli O157:H7 is an enteric pathogen associated with food safety threats and with significant morbidity and mortality worldwide. In Argentina, post-enteric hemolytic uremic syndrome (HUS) is endemic, with > 70% of cases associated with E. coli O157 infection. To date the biological basis behind the severity among E. coli O157 infections is unknown. However, single nucleotide polymorphism (SNP) typing has helped to define nine E. coli O157:H7 clades, of which clade 8 strains are associated with severe disease cases. The aim of this study was to characterize a collection of 20 STEC O157:H7 strains isolated between 2011 and 2013 from ground beef and different environmental samples from butcher shops of Argentina. All strains harbored the eae, ehxA, fliC_H7, efa, iha, and toxB genes, with stx_2a/stx_2c as the predominant genotype (75%). The XbaI-PFGE analysis showed that the E. coli O157 strains had high genetic diversity. Nine strains were grouped in four XbaI-PFGE clusters, whereas 11 strains showed unique XbaI-PFGE patterns. In contrast, the SNP analysis allowed us to separate the strains in two distinct lineages representing clade 8 (70%) and clade 6 (30%). Our results show the molecular characterization of E. coli O157 strains isolated from ground beef and environmental samples from Argentinean butcher shops.     

An outbreak of gastroenteritis in Osaka,Japan due to Escherichia coli serogroup O166:H15 that had a coding gene for enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1)

In an outbreak of gastroenteritis on 23 July 1996, in Osaka, Japan, 54 of 91 persons who had attended a meeting the previous day became ill. Escherichia coli O166:H15 was isolated from stool specimens of patients (29/33, 88%). Laboratory tests for other bacterial pathogens and viruses were negative. The E. coli 0166 organisms did not adhere to HEp-2 cells in a localized, diffuse, or enteroaggregative manner. The organisms did not express known enterotoxigenic E. coli (ETEC) colonization factors. In polymerase chain reaction tests, the bacteria did not have coding genes for shigatoxin of enterohemorrhagic E. coli (EHEC), heat-labile, or heat-stable enterotoxin of ETEC, attachment and effacement (eaeA) of EPEC, or invasion (invE) of enteroinvasive E. coli (EIEC). Consequently, they could not be assigned to any of the recognized diarrhoeagenic groups of E. coli: EPEC, ETEC, EHEC, EIEC, enteroaggregative E. coli (EAggEC), or diffusely adhering E. coli. However, the organisms possessed the EAggEC heat-stable enterotoxin (EAST1) gene. To our knowledge, this is the first report of an outbreak caused by E. coli that did not have well-characterized virulence genes other than EAST1. The isolates showed the same DNA banding pattern in pulsed-field gel electrophoresis after digestion with the restriction enzymes XbaI or NotI. Three O166:H15 strains isolated from two sporadic cases and another outbreak during 1997-8 were distinct, indicating that multiple clones have spread already. We propose that diarrhoeal specimens should be examined for E. coli possessing the EAST1 gene.     

Towards an attenuated enterohemorrhagic Escherichia coli O157:H7 vaccine characterized by a deleted ler gene and containing apathogenic Shiga toxins

The aim of this study was to develop a candidate vaccine against enterohemorrhagic Escherichia coli O157:H7. A ler deletion mutant derived from wild-type EHEC O157:H7 86-24 was constructed by use of suicide vector pCVD442. The bacteriophage encoding Shiga toxin (Stx) was excised by serial passage to produce a ler/stx deletion mutant, F25. Stx1 and Stx2 mutants were constructed by site-directed mutagenesis within the active center and membrane-spanning region of the toxin A subunit. Mutants stx1 and stx2 were then introduced into F25 to construct live attenuated candidate vaccine F105. The cytotoxicity of F25 was inactivated and that of F105 was significantly reduced in comparison with wild-type E. coli strain EDL933. Mice injected with candidate vaccine strains F25 and F105 gained weight and showed no clinical signs of disease. F25 and F105 reduced the colonization of wild-type O157:H7 in mouse intestine. Immunized pregnant mice were able to protect their suckling newborns from intragastric challenge with wild-type O157:H7. Immunized mice were protected against infection with wild-type O157:H7 and exhibited normal weight gain. Such attenuated vaccine strains may therefore have potential use as oral vaccines against O157:H7.     

Aetiology of Diarrhoea and Virulence Properties of Diarrhoeagenic Escherichia coli among Patients and Healthy Subjects in Southeast Nigeria

Diarrhoeal diseases are one of the most important causes of illness and death all over the world. In Nigeria, the aetiology of diarrhoeagenic bacteria and the virulence of various Escherichia coli pathotypes have not been well-studied because most currently-published data were from the southwestern axis of the country. In total, 520 stool samples were collected from infants, young children, and other age-groups with acute diarrhoeal diseases in Enugu and Onitsha, southeastern Nigeria. Stool samples were collected from 250 apparently-healthy individuals, with similar age distribution and locality, who were considered control subjects. The stool samples were screened for diarrhea-causing bacterial agents. E. coli strains were isolated from both the groups and were examined by polymerase chain reaction (PCR) for 16 virulence genes. Of the 520 stool samples in the diarrhoea group, 119 (44.74%) were E. coli. Fifty (49.02%) were enteropathogenic E. coli (EPEC), 22 (21.57%) were enterotoxigenic E. coli (ETEC) while 7.84% was enteroaggregative E. coli (EAEC). Sex had no effect on the distribution of diarrhoeagenic bacteria, except for EIEC. The E. coli strains isolated from the diarrhoea and healthy asymptomatic age-matched control groups examined by PCR for 16 virulence genes indicate that the detection of EAEC, ETEC, EPEC, and EIEC was significantly associated with diarrhoea (p=0.0002). The study confirmed that several bacterial pathogens, such as E. coli, play an important role in the aetiology of acute diarrhoea in southeastern Nigeria. A routine surveillance, especially for diarrhoeagenic E. coli, would be useful in identifying outbreaks and help identify the potential reservoirs and transmission routes.Key words: Diarrhoea, Escherichia coli, Virulence, Nigeria     

我国2012－2015年门诊腹泻患者中致泻性大肠埃希菌流行特征分析

目的 了解我国门诊腹泻患者中致泻性大肠埃希菌（DEC）流行特征。方法 2012－2015年在27省（直辖市、自治区）170家医院门、急诊中开展腹泻病监测,收集腹泻病例的临床和流行病学资料,同时采集粪便标本,送往92家网络实验室开展DEC检测。结果 在46 721例腹泻监测病例中,DEC的总检出率为7.7%,且各地区检出率存在较大差异。在2 982例（6.4%）报告了PCR菌型鉴定分类信息的DEC阳性病例中,肠集聚性黏附大肠埃希菌（EAEC）是最主要的类型（1 205例,40.4%）,其次是肠致病性大肠埃希菌（EPEC）（815例,27.3%）和肠产毒性大肠埃希菌（ETEC）（653例,21.9%）。整体分析,25～34岁青壮年、暖温带地区和黏液便的DEC检出率较高,检出率分别为10.1%、11.1%和9.4%。DEC的季节性发病特征明显,各菌型的流行高峰均在夏季。结论 DEC是我国腹泻病门诊重要病原体之一,流行菌型以EAEC、EPEC和ETEC为主,不同地区、不同年龄人群、不同季节的流行特征存在差异。     

Over-expression of major colonization factors of enterotoxigenic Escherichia coli, alone or together,on non-toxigenic E. coli bacteria

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease and deaths among children in developing countries and the major cause of traveller's diarrhea. Since surface protein colonization factors (CFs) of ETEC are important for pathogenicity and immune protection is mainly mediated by locally produced IgA antibodies in the gut, much effort has focused on the development of an oral CF-based vaccine. We have recently described the development of recombinant strains over-expressing CFA/I; the most prevalent CF among human clinical ETEC isolates. Here, non-toxigenic recombinant E. coli strains over-expressing Coli surface antigen 2 (CS2), CS4, CS5, and CS6, either alone, or each in combination with CFA/I were constructed by cloning the genes required for expression and assembly of each CF into expression vectors harboring a strong promoter. Immunological assays showed that recombinant strains expressing single CFs produced those in significantly larger amounts than did corresponding naturally high producing reference strains. Recombinant strains co-expressing CFA/I together with another CF also expressed significantly larger amounts of both CFs compared with the corresponding references strains. Further, when tested in mice, oral immunization with formalin-killed recombinant bacteria co-expressing one such double-expression CF pair, CFA/I + CS2, induced specific serum IgG + IgM and fecal IgA antibody responses against both CFs exceeding the responses induced by immunizations with natural reference strains expressing CFA/I and CS2, respectively. We conclude that the described type of recombinant bacteria over-expressing major CFs of ETEC, alone or in combination, may be useful as candidate strains for use in an oral whole-cell CF-ETEC vaccine.     

Serotypes and colonization factors of enterotoxigenic Escherichia coli isolated in various countries

One hundred and six enterotoxigenic E. coli (ETEC) isolated from many geographical areas were serotyped and investigated for the presence of colonization factor antigens CFA/I and CFA/II, the expression of mannose-resistant haemagglutination (MRHA) and the levels of surface hydrophobicity. CFA/I was found in 6 (17%) of 36 LT⁺STa⁺ strains and in 15 (54%) of 28 STa⁺ strains; CFA/II was found in 16 (44%) of 36 LT⁺STa⁺ strains. None of 42 LT⁺ strains showed CFA/I or CFA/II. CFA/I was found in ETEC of serotypes O63:K?:H?, O78:K80, O128:K67 and O153:K?:H45, whereas CFA/II was found in serotypes O6:H?, O6:K15:H16 and 06:K?:H40. Of the 69 CFA/I^? CFA/II^? ETEC strains, 9 (13%) showed MRHA with some of the seven erythrocyte species used and 21 (30%) were hydrophobic. Among the 21 hydrophobic strains CFA-negative we have detected: (i) 6 LT⁺ strains of serogroup O25 negative for MRHA, (ii) 5 strains O159 (4 LT⁺ and 1 LT⁺ STa⁺) also negative for MRHA, and (iii) 3 STa⁺ strains of serotype O27:K?:H7 that haemagglutinated calf and sheep erythrocytes when grown on Minca-Is. The 106 ETEC strains belonged to 20 different 0 serogroups. However, 77 (73%) were of one of nine serogroups (O6, O8, O25, O27, O78, O148, O153, O159 and O167). E. coli strains belonging to O6 and O153 groups predominated among ETEC isolated in Spain, O159 strains in the Central African Republic, O25 and O148 strains in Japan, and O15 and O78 strains in India.     

Prevalence of virulence factors in Escherichia coli isolated from healthy animals and water sources in Brazil

The aim of this work was to verify the presence of seven virulence factors (ST, LT, eae, stx₁, stx₂, INV and EAEC) among Escherichia coli strains isolated from healthy humans, bovines, chickens, sheep, pigs and goats, from two sewage treatment plants and from the Tietê River. We have found a high prevalence of eae, stx₁ and stx₂ in ruminants. The EAEC gene was only found in humans and sewage. No strains presented ST, LT or INV. BOX-PCR fingerprints revealed a high diversity among the strains analysed and a non-clonal origin of strains that presented the same virulence factors. Therefore, we concluded that ruminants may constitute an important reservoir of most diarrheagenic E. coli in Brazil, except for EAEC strains. These results emphasize the importance of the identification of the animal source of fecal contamination for the correct water risk assessment.     

Construction of a non-toxigenic Escherichia coli oral vaccine strain expressing large amounts of CS6 and inducing strong intestinal and serum anti-CS6 antibody responses in mice

Coli surface antigen 6 (CS6) is one of the most prevalent non-fimbrial colonization factors (CFs) of enterotoxigenic Escherichia coli (ETEC) bacteria, which are the most common cause of diarrhea among infants and children in developing countries. Since immune protection against ETEC is mainly mediated by locally produced IgA antibodies in the gut, much effort is focused on the development of an oral CF-based vaccine. Previous work has described the preparation of candidate E. coli vaccine strains expressing immunogenic amounts of fimbrial CF antigens such as CFA/I and CS2, which are retained after formalin treatment. However, attempts to generate E. coli expressing immunogenic amounts of CS6 and to preserve the immunological activity of the CS6 protein in a killed whole-cell vaccine have failed until now. Here we describe the construction of a recombinant non-toxigenic E. coli strain, with thyA as a non-antibiotic-based selection, which expresses large amounts of CS6 antigen on the bacterial surface, and show that phenol inactivation of the bacteria does not destroy the CS6 antigen properties. Oral immunization of mice with such phenol-killed CS6 over-expressing E. coli bacteria induced strong fecal and intestinal IgA and serum IgG + IgM antibody responses to CS6 that exceeded the responses induced by an ETEC reference strain naturally expressing CS6 and previously used as a vaccine strain. Our data indicate that the described phenol-inactivated non-toxigenic and CS6 over-expressing E. coli strain may be a useful component in an oral ETEC vaccine.