Diarrhea in neonatal piglets caused by K99+ST+ enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli strains were isolated from the feces of 34.4% of 64 diarrheaic neonatal piglets on seven farms. Of a total of 518 isolates, 86 (16.6%) were enterotoxigenic and grouped into four phenotypes: K99+ST+ (K99 pilus antigen and heat-stable enterotoxin producing, 36 strains), ST+ (37 strains), K88+LT+ (K88 pilus antigen and heat-labile enterotoxin producing, 11 strains), and K88+ST+ (2 strains). K99+ST+ and ST+ isolates showed multiple drug resistance and most of those (58.3% and 56.8%, respectively) belonged to O serogroup 101. A K99+ST+ isolate proved to be capable of inducing diarrhea and death in hysterectomy-produced colostrum-deprived piglets when orally inoculated.     

Incidence and some characteristics of fimbriae FY and 31A of Escherichia coli isolates from calves with diarrhea in Japan

Escherichia coli isolates from calves with diarrhea (1 day to 8 weeks old, 140 individuals) were surveyed for the three immunologically distinct fimbrial adhesins FY, 31A, and K99. Of a total of 1,370 strains isolated, 96 (7.0%), 34 (2.5%), 75 (5.5%), and 13 (0.9%) were identified as FY+, 31A+, FY+.31A+, and K99+, respectively. The K99+ strains also manifested heat-stable enterotoxin production (ST+), while FY+, 31A+, and FY+.31A+ strains were ST-. Expression of FY and 31A was repressed at lower temperatures or poor aeration. The FY+ and 31A+ E. coli showed mannose-resistant hemagglutinating activity with bovine erythrocytes. Electron microscopy revealed that FY is a gently curled fimbria with a mean diameter of 4.2 nm, and 31A is a fimbria with a mean diameter of 5.1 nm. The molecular mass of protein subunits was found to be approximately 20 kilodaltons (Kd) and 19 Kd for FY and 31A, respectively. Lethal diarrhea of neonatal calves was induced by challenge with the combination of a K99+.ST+ E. coli strain and either a 31A+ E. coli strain or a 31A+ E. coli strain plus an FY+ E. coli strain under the experimental conditions in which lethal diarrhea was not induced by challenge with a K99+.ST+ E. coli strain alone.     

猪源大肠杆菌(ETEC、STEC、AEEC)毒力基因及其与O抗原型的关系

[目的]揭示从我国部分地区仔猪腹泻或水肿病病猪体内分离到的300个大肠杆菌分离株所属病原型(pathotype)、毒力基因及其与O血清型的关系.[方法]O血清型采用常规的凝集试验进行测定,毒力基因采用PCR方法检测.[结果]通过对这300个分离株的O血清型及其毒素、紧密素和黏附素基因进行鉴定,结果显示除50株未定型、17株自凝外,测定出233个分离株的血清型,这些分离株覆盖了45个血清型,其中以0149、0107、0139、093和091为主,共133株,占定型菌株的57.1%;拥有est Ⅰ、estⅡ、elt、stx2e和eae A基因的菌株分别为102(34.0%)、190(63.3%)、81(27.0%)、57(19.0%)和54(18.0%)株;分离株中有51株K88基因阳性(其中菌毛表达率为100%),75株F18基因阳性(其中菌毛表达率为50.7%),在K88菌株中,0149血清型与est Ⅰ或estⅡ elt密切相关,在F18菌株中,0107血清型与est Ⅰ或estⅡ、0139血清型与stx2e紧密相关.依其毒力特征可将这些分离株分为以下6种类型:ETEC、STEC、AEEC、ETEC/STEC、AEEC/ETEC和AEEC/ETEC/STEC,分别拥有190、24、36、32、17和1个菌株,占分离株的63.3%、8.0%、12.0%、10.7%、5.7%和0.3%.通过分析这些分离株的O血清型、毒素类型和黏附素型之间的相关性:猪源ETEC以0149、0107、093和098等血清型为主,0149:K88菌株主要与estⅡ或estⅡ elt肠毒素相关,0107:F18菌株主要与estⅡ相关,093和098血清型菌株主要与estⅡ肠毒素相关;STEC菌株以0139:F18血清型为主,拥有stx2e;AEEC菌株拥有紧密素,无明显优势血清型;ETEC/STEC菌株以0107:F18和0116:F18血清型为主,主要与est Ⅰ stx2e或estⅡ stx2e密切相关,ETEC/AEEC菌株以091和0107血清型为主,全部拥有肠毒素est Ⅰ和紧密素基因.[结论]我国至少存在6种病原型的猪肠道致病性大肠杆菌,其中ETEC为我国部分地区猪大肠杆菌病的主要病原,同时其病原型日益复杂.     

Biological properties of enterotoxigenic Escherichia isolated from patients with acute intestinal diseases of unknown etiology

As the result of the comparative examination of adult patients with acute enteric diseases and normal adults, 173 E. coli enterotoxigenic strains were isolated (161 strains from the patients and 12 strains from normal persons). 83% of the isolated enterotoxigenic E. coli (ETEC) produced two enterotoxins: thermolabile (LT) and thermostable (ST). Enterotoxigenicity was most pronounced in the strains of ETEC belonging to the prevaling variant ST + LT +. The enterotoxigenic properties of ETEC were highly stable: the production of ST and LT in the strains remained unchanged after their storage for up to 4 years. The isolated ETEC comprised 48 serogroups and 61 strains. The strains belonging to the same seroval had a similar degree of toxigenicity. The strains belonging to different serovars considerably differed in the activity of their enterotoxins. The production of two kinds of enterotoxins in the isolated E. coli strains was inter-related: the strains with a high activity of ST were, as a rule, good producers of LT.     

Fimbriae in Escherichia Coli Isolated from the Small Intestine of Piglets

Ninety E. coli strains, isolated from piglets which had died from neonatal diarrhea, were tested for the presence of K88, K99, 987P and type 1 fimbriae. Two or more types of fimbriae were demonstrated in 14 of the strains, a single fimbria! type in 44 strains while in 32 strains no fimbriae were detected. Of the 14 E. coli strains with more than 1 type of fimbriae, 12;, 10, 8 and 4 strains showed K88, K99, 987P and type 1, respectively. Twelve E. coli strains were isolated from piglets which had died in the neonatal period without showing signs of neonatal diarrhea at necropsy. One strain showed 987P and 3 strains showed type 1 fimbriae, while the remaining 8 strains were unfimbriated. Sixteen fimbriated E. coli strains were subcultured in order to examine the stability of fimbrial expression in the strains. The K88 and the type 1 fimbriae were regularly expressed, while the K99 and 987P were inconsistently demonstrated.     

山东省某地区奶牛源大肠埃希菌的血清型、耐药特性及分子特性

【目的】旨在对从山东省某地区4个健康奶牛养殖场分离到的大肠埃希菌进行优势血清型、耐药特性、Ⅰ类整合子基因盒携带情况以及系统进化群分析。【方法】采集194份来自山东省某地区4个规模化奶牛场奶牛新鲜粪便样品,进行大肠埃希菌分离和鉴定,利用常用大肠埃希菌诊断血清进行血清型鉴定;利用10%的绵羊血平板检测溶血性;利用K-B法检测对14种常规抗菌药物的敏感性;利用聚合酶链式反应(PCR)检测革兰阴性菌常见的6大类24种耐药基因、Ⅰ类整合子基因盒结构并对目的条带测序分析;利用细菌多位点序列分型(Multilocus sequence typing,MLST)技术分析大肠埃希菌的ST型并使用eBURST v3软件分析菌株之间的克隆关系。【结果】从194份新鲜粪便样品中分离到171株大肠埃希菌,其中主要为致病性(19.9%)和侵袭性大肠埃希菌(17.0%),优势血清型分别为O128:K67(12/171)和O143:K7(12/171)。另外,具有溶血性的大肠埃希菌阳性率为9.4%(16/171);药敏试验结果显示多重耐药菌株的比率为22.2%,其中对氨苄西林耐药率最高为33.9%,四环素次之,为24.0%;PCR检测耐药基因和整合子结果显示,59.1%的菌株携带β-内酰胺类耐药基因blaTEM,59.1%的菌株携带氨基糖苷类耐药基因ant(2′),未检测到四环素耐药基因tetA和tetB;Ⅰ类整合子的阳性率为4.1%(7/171),dfrA12-aadA2-sul1为优势基因盒结构(4/171);MLST将大肠埃希菌分为8种ST型,其中,ST155(10/171)和ST58(45/171)形成一个克隆复合物且没有发现新的ST型。【结论】本研究证实,从该地区规模化健康奶牛场新鲜粪便中分离到的大肠埃希菌优势血清型为O128:K67和O143:K7;少部分大肠埃希菌具有溶血性;仅对氨苄西林、四环素等具有较高的耐药率;优势基因盒结构为dfrA12-aadA2-sul1;MLST分型显示不同奶牛场分离出亲缘关系较近的菌株,其分布具有多态性,血清型与ST型之间无相关性。本研究表明源自表观健康的奶牛的大肠埃希菌存在多重耐药现象,具有食品公共卫生安全隐患,该研究对于提升规模化奶牛场奶制品的安全生产与质量评估具有一定的理论指导意义。     

Acquisition and maintenance of enterotoxin plasmids in wild-type strains of Escherichia coli

Enterotoxigenic strains of Escherichia coli (ETEC) may produce a heat-labile enterotoxin (LT), a heat-stable enterotoxin (ST) or both enterotoxins. Certain serogroups are represented more frequently than others in ETEC isolated from humans. The transfer of three plasmids encoding enterotoxin production (Ent) to 22 non-toxigenic E. coli strains of many different O:H serotypes was studied. The Ent plasmids encoded ST (TP276), or LT (TP277), or ST + LT (TP214), and all carried antibiotic-resistance determinants. Twenty-one recipient strains acquired TP214, 18 acquired TP277 and 14 acquired TP276. Strains of those serotypes to which ETEC in diarrhoeal studies commonly belong neither acquired nor maintained Ent plasmids with a higher frequency than strains of those serotypes to which ETEC rarely belong. The recipient strains, with one exception, all expressed ST, or LT, or ST and LT, when they had acquired the appropriate plasmid; a non-motile strain belonging to O serogroup 88 expressed LT but failed to express ST when it acquired TP214 or TP277.     

Response of nursery pigs to a synbiotic preparation of starch and an anti-Escherichia coli K88 probiotic

Postweaning diarrhea in pigs is frequently caused by enterotoxigenic Escherichia coli K88 (ETEC). The aim of this study was to test the efficacy of E. coli probiotics (PRO) in young pigs challenged with E. coli K88. We also tested the synbiotic interaction with raw potato starch (RPS), which can be used as a prebiotic. Forty 17-day-old weaned piglets were randomly assigned to four treatments: treatment 1, positive-control diet (C), no probiotics or RPS but containing in-feed antibiotics; treatment 2, probiotic (PRO), no feed antibiotics plus a 50:50 mixture of probiotic E. coli strains UM-2 and UM-7; treatment 3, 14% RPS, no antibiotics (RPS); treatment 4, 14% RPS plus a 50:50 mixture of probiotic E. coli strains UM-2 and UM-7, no antibiotics (PRO-RPS). The pigs were challenged with pathogenic E. coli K88 strains on day 7 of the experiment (24-day-old pigs) and euthanized on day 10 of the experiment (35-day-old pigs). Probiotic and pathogenic E. coli strains were enumerated by selective enrichment on antibiotics, and microbial community analysis was conducted using terminal restriction length polymorphism analysis (T-RFLP) of 16S rRNA genes. The combination of raw potato starch and the probiotic had a beneficial effect on piglet growth performance and resulted in a reduction of diarrhea and increased microbial diversity in the gut. We conclude that the use of E. coli probiotic strains against E. coli K88 in the presence of raw potato starch is effective in reducing the negative effects of ETEC in a piglet challenge model.     

Cloning of chromosomal DNA encoding the F41 adhesin of enterotoxigenic Escherichia coli and genetic homology between adhesins F41 and K88.

The genetic determinant for production of the adhesive antigen F41 was isolated from a porcine enterotoxigenic Escherichia coli strain by cosmid cloning. The cloned DNA included sequences homologous to those of hybridization probes prepared from the K88 adhesive antigen operon. Transposon insertions which inactivated F41 production mapped to the same region of DNA showing homology with the K88 genes, demonstrating the genetic relatedness of F41 and K88. Hybridization of a K88 gene probe to plasmid and total DNA from the porcine E. coli isolate from which the F41 gene was cloned indicated that F41 is chromosomally encoded by this strain. This observation was extended to other F41-producing animal isolates. A large number of animal E. coli isolates were examined with K88, F41, and K99 gene probes and for mannose-resistant hemagglutination of human group O erythrocytes and K88 and F41 antigen production. All K88 and F41 antigen producers possessed genetic homology with the K88 and F41 gene probes. Most, but not all, F41-producing strains possessed homology to the K99 gene probe, reflecting the previously observed association of F41 and K99 antigen production. In the strains examined, homology with the K99 gene probe was plasmid associated, whereas homology with the F41 gene probe was chromosomal. The K88 antigen-producing strains showed no homology with the K99 probe. A number of strains possessed homology with the K88 and F41 gene probes and were mannose-resistant hemagglutination positive, but did not produce K88 or F41 antigens. This suggests that there are adhesins among animal isolates of E. coli which are genetically related to but antigenically distinct from K88 and F41.     

Genome sequences and phylogenetic analysis of K88- and F18-positive porcine enterotoxigenic Escherichia coli

Porcine enterotoxigenic Escherichia coli (ETEC) continues to result in major morbidity and mortality in the swine industry via postweaning diarrhea. The key virulence factors of ETEC strains, their serotypes, and their fimbrial components have been well studied. However, most studies to date have focused on plasmid-encoded traits related to colonization and toxin production, and the chromosomal backgrounds of these strains have been largely understudied. Here, we generated the genomic sequences of K88-positive and F18-positive porcine ETEC strains and examined the phylogenetic distribution of clinical porcine ETEC strains and their plasmid-associated genetic content. The genomes of porcine ETEC strains UMNK88 and UMNF18 were both found to contain remarkable plasmid complements containing known virulence factors, potential novel virulence factors, and antimicrobial resistance-associated elements. The chromosomes of these strains also possessed several unique genomic islands containing hypothetical genes with similarity to classical virulence factors, although phage-associated genomic islands dominated the accessory genomes of these strains. Phylogenetic analysis of 78 clinical isolates associated with neonatal and porcine diarrhea revealed that a limited subset of porcine ETEC lineages exist that generally contain common toxin and fimbrial profiles, with many of the isolates belonging to the ST10, ST23, and ST169 multilocus sequencing types. These lineages were generally distinct from existing human ETEC database isolates. Overall, most porcine ETEC strains appear to have emerged from a limited subset of E. coli lineages that either have an increased propensity to carry plasmid-encoded virulence factors or have the appropriate ETEC core genome required for virulence.     

The prevalence and molecular typing of enterotoxigenic Escherichia coli strains isolated from diarrheic stools in Malatya, Turkey

This study was performed from June 2002 to November 2003 year in Malatya, eastern Turkey. Stools of 172 diarrheic patients and 90 healthy controls were analysed for enterotoxigenic Escherichia coli (ETEC). Heat-labile (LT) and heat-stable (ST) toxins were investigated by passive latex agglutination and enzyme immunoassay, respectively. Nine ETEC strains were isolated from 172 diarrheic stools (5.2%). Seven of the ETEC strains (10.1%) were isolated from 69 children in the 0-5 year age group. Two of these pediatric isolates were ST positive (2.9%) and five were LT positive (7.2%). ETEC was not isolated in the 6-18 year age group. Two ST producing E. coli strains were detected in diarrheic adult patients (> 18 years). In the 90 controls, two ETEC strains were detected (2.2%). One of them was a LT producer (1.1%) and the other was a ST producer (1.1%). E. coli strains producing both toxins simultaneously were not observed. ETEC positivity was higher in the diarrheic group than in the control group but statistically not significant (p > 0.05). The rate of resistance among ETEC strains to cefuroxime axetil, ampicillin, piperacillin, and trimethoprim-sulfamethoxazole was 72.7%, 54.5%, 45.5%, and 36.4%, respectively whereas the resistance rate to the same antibiotics in non-ETEC strains was 14%, 62%, 54%, and 66%, respectively. All ETEC isolates were intermediately resistant to cephalothin and fully susceptible to other antibiotics tested. Typing of the ETEC strains was done by arbitrary primed polymerase chain reaction (AP-PCR). Only two LT strains of the 11 typed strains had a unique profile. The remaining nine were mixed LT and ST strains and divided into two groups. The first group had three strains having a similarity coefficient ranging from 70-90%. The other one had six strains, five of them were similar and one was subtype isolate. It can be concluded that ETEC strains might be considerably important enteropathogens especially in pediatric patients in the 0-5 year age group. High clonal relation indicated that ETEC strains were epidemiologically related.     

Production of enterotoxins by strains of Escherichia coli isolated from pigs

The production of enterotoxins by 237 hemolytic strains of Escherichia coli isolated from pigs was determined with the use of CTE in CHO. Vero and Hela cells and ILT. More frequent (p less than 0.01) production of enterotoxins, determined by ILT, was found for the serotypes being pathogenic for the animals (63.8% of the strains). No correlation between intensity of ILT and particular serotype was observed. Both the serotypes pathogenic for pigs and other serotypes produced LT enterotoxins and ST toxin. The frequency of LT enterotoxin production was statistically insignificant compared to the frequency of ST enterotoxin production by strains with serotypes pathogenic for the pigs. Strains of E. coli producing only enterotoxin ST belonged both to the pathogenic serotypes as well as to other hemolytic serotypes. The cytotoxic activity of supernatants of E. coli strains with different serotypes isolated from pigs in Vero and Hela cells and simultaneous CTE in CHO cells was observed. This suggests the production by the strains of enterotoxin LT and cytotoxin VT. Seven out of the 96 isolates showing CTE in CHO cells gave no reaction in the ILT in pigs. This suggests the production by these isolates of a toxin (toxins) differing from the E. coli enterotoxins.     

Combinations of pathogenicity factors in Escherichia coli isolated from children and domestic animals

The study has shown that in E. coli strains isolated from children aged up to 1 year with acute intestinal diseases of unknown etiology different combinations of pathogenicity factors (Ent. K88, K99, Vir, CFAI, CFAII, HIy, ColV, ColI) can be detected in 47.1 +/- 3.8% of cases, while in E. coli strains isolated from practically healthy children of the same age combined carriership of these factors does not exceed 5.8 +/- 2.5%. In E. coli strains isolated from swine and cattle with diarrhea the combined carriership of pathogenicity factors is 68.5 +/- 2.8% and 58.4 +/- 4.9% respectively.     

Frequent production of toxins by Escherichia coli strains isolated from human urinary tract infections: relation with haemagglutination

Abstract We have investigated the production of heat-labile enterotoxin (LT), Verotoxin (VT), cytotoxic necrotizing factor (CNF), haemolysis (Hly) and lethal activity for mice in 48 Escherichia coli strains isolated from patients with urinary tract infections. Mannose-resistant and mannose-sensitive haemagglutination in these strains were also studied. Among the total strains investigated, 50% were haemolytic, 50% synthesized CNF and 58% were lethal for mice. A total of 33 (69%) strains were toxigenic, showing positive results at least in one of the tests employed for toxin detection. No strain was positive for LT and VT production. We conclude that, in addition to haemolytic and haemagglutinating activities, the production of CNF was closely associated to virulence in E. coli strains isolated from urinary tract infections.     

Serotypes, virulence genes, and PFGE patterns of enteropathogenic Escherichia coli isolated from Cuban pigs with diarrhea.

Thirty-six enteropathogenic Escherichia coli strains isolated from Cuban pigs with diarrhea were serotyped and screened by PCR for the presence of virulence genes. The 36 isolates belonged to 11 O serogroups and 14 O:H serotypes, with 53% of the isolates belonging to only two serotypes: O141:H- (13 isolates) and O157:H19 (6 isolates). Genes coding for STb, STa, VT2e, and LT toxins were identified in 69, 61, 53, and 6% of the isolates, respectively. The most prevalent fimbrial adhesin was F18, detected in 22 (61%) isolates. The gene encoding F6 (P987) colonization factor was identified in three (8%) isolates. None of the 36 isolates assayed contained genes encoding F4 (K88), F5 (K99), or F41. The seropathotype O141:H-:STa/STb/VT2e/F18 (13 isolates) was the most frequently detected, followed by O157:H19:VT2e/F18 (5 isolates). A genetic diversity study, carried out by pulsed-field gel electrophoresis (PFGE) of 24 representative isolates, revealed 21 distinct restriction patterns clustered in 18 groups (I-XVIII). Isolates of the same serotype were placed together in a dendrogram, but isolates of serotype O157:H19 showed a high degree of polymorphism. The results of this study demonstrate the presence in Cuba of different clusters among one of the most prevalent serotypes isolated from pigs with diarrhea. Further experiments are needed to determine whether some of these clusters have appeared recently; if so, their evolution, as well as their possible association with pathogenicity in farms should be studied.     

Detection of heat-stable and heat-labile enterotoxin genes of Escherichia coli in diarrhoeic faecal samples of mithun (Bos frontalis) calves by polymerase chain reaction

Aims:  To find out the prevalence of different serogroups of Escherichia coli ( E. coli ) and to detect heat-stable (ST) and heat-labile (LT) enterotoxin genes of enterotoxigenic E. coli (ETEC) from the faeces of mithun calves with diarrhoea.
Methods and Results:  Faecal samples obtained from 65 diarrhoeic mithun calves of under 2 months of age were examined for E. coli using polymerase chain reaction (PCR). Fifty-four E. coli isolates were obtained from those samples, which belonged to 38 different serogroups. Out of 54 isolates tested by PCR, two isolates (3·70%) belonging to serogroups O26 and O55 were found to possess gene that code for ST enterotoxin and one isolate (1·85%) belonging to serogroup O125 was found to carry LT enterotoxin gene.
Conclusions:  Escherichia coli isolates from diarrhoeic mithun calves were found to possess ST and LT enterotoxin genes, which are designated as ETEC, and these isolates can be detected through PCR using specific primers.
Significance and Impact of the Study:  This study reports the isolation of ETEC possessing ST and LT enterotoxin genes for the first time and ETEC could be a cause of diarrhoea in mithun calves leading to calf mortality.     

表达大肠杆菌K88ac-ST1-LTB融合蛋白基因工程菌株的构建

利用PCR技术,从大肠杆菌C83902质粒中扩增出K88ac基因、ST1突变基因和LTB基因,通过分离、纯化、内切酶酶切、连接和转化,构建了含K88ac-ST1-LTB融合基因表达载体的重组菌株BL21（DE3）（pXKST3LT5)。经酶切鉴定和DNA序列分析证实,构建的重组质粒pXKST3LT5中含有K88ac-ST1-LTB融合基因,且基因序列和阅读框架均正确。经ELISA检测,重组菌株表达的K88ac-ST1-LTB融合蛋白能够被ST1单抗、LTB和K88ac抗体识别。经乳鼠灌胃试验证实,表达的融合蛋白已丧失天然ST1肠毒素的活性。免疫实验结果表明,K88ac-ST1-LTB融合蛋白能够诱发小白鼠产生抗体,该抗体具有中和天然ST1肠毒素的毒性作用,表明构建的重组菌株可以作为预防仔猪黄、白痢基因工程菌苗的候选菌株。     

肠毒素大肠杆菌定居因子CS3和肠毒素融合抗原基因在减毒鼠伤寒沙门氏菌中的共表达

不耐热肠毒素（LT）和耐热肠毒素（ST）是产肠毒素大肠杆菌的主要致病因素,CS3为该菌的优势定居因子,是定居因子CFA/Ⅱ菌毛抗原的共有抗原组分。采用基因操作技术将编码CS3和融合肠毒素蛋白基因转化到减毒鼠伤寒沙门氏菌疫苗侯选株X4072中进行表达。用重组菌株口服免疫小鼠后,免疫动物能产生抗CS3、LT和ST的血清抗体。特别有意义的是,所产生的抗ST抗体能中和天然ST的生物活性。这一结果为研究载体疫苗防治肠毒素大肠杆菌腹泻疾病奠定了基础。     

Enterotoxigenic Bacteria in Food and Water from an Ethiopian Community

Food and water samples from an Ethiopian community were screened for the presence of enterotoxin-producing bacteria. Using the Chinese hamster ovary cell assay, 40 of 213 isolates (18.8%) produced heat-labile (LT) enterotoxin. These LT-producing isolates comprised 33 of 177 (18.6%) strains from 24 of 68 food samples (35.3%) and 7 of 36 (19.4%) isolates of 4 of 17 water samples (23.5%). One LT-producing strain each of Salmonella emek and of Shigella dysenteriae was found. Three pseudomonads, all LT producers, produced heat-stable enterotoxin as gauged by the suckling mouse test. Two strains of LT-enterotoxigenic Escherichia coli O68 were found in water samples. No enterotoxigenic E. coli were isolated from food samples, but 13 of the LT-producing strains were Enterobacter, Klebsiella, Serratia, and Proteus species, and 7 food samples yielded more than one species of enterotoxigenic bacterium. Of the enterotoxigenic isolates from food, 15 were oxidase-positive strains of the genera Aeromonas, Pseudomonas, Achromobacter, Flavobacterium, and Vibrio. LT-enterotoxigenic Enterobacter, Acinetobacter, Klebsiella, Proteus, Providencia, and Serratia species represented 20 of the food and water isolates. Culture supernatant fluids of representative strains of oxidase-positive and oxidase-negative species giving positive reactions in Chinese hamster ovary cell tests induced fluid accumulation in rabbit ileal loops. Eight of the food samples and two of the water samples contained more than one isolate or species of enterotoxigenic bacterium. The stability of the LT production by oxidase-positive bacteria and non-E. coli strains was assessed by the rabbit skin and adrenal cell tests after 9 months and 1 year of storage, respectively, in Trypticase soy broth with glycerol at −70°C. Only 33% of the oxidase-positive strains were still LT enterotoxigenic. Of the oxidase-negative strains, 50 and 33% were LT producing at 9 months and 1 year, respectively. None of the E. coli isolates, both enterotoxigenic and nonenterotoxigenic, possessed K88, K99, or colonization factor antigen. The survey demonstrates the presence in food and water of enterotoxigenic bacteria of the same species as those isolated from cases of infantile diarrhea in the same community, although a correlation between these sources and infantile diarrhea remains to be established.     

Phenotypic and molecular characteristics of Escherichia coli isolated from aquatic environment of Bangladesh

Pathogenic Escherichia coli remains important etiological agent of infantile diarrhea in Bangladesh. Previous studies have focused mostly on clinical strains, but very little is known about their presence in aquatic environments. The present study was designed to characterize potentially pathogenic E. coli isolated between November 2001 and December 2003 from aquatic environments of 13 districts of Bangladesh. Serotyping of 96 randomly selected strains revealed O161 to be the predominant serotype (19%), followed by O55 and O44 (12% each), and 11% untypable. Serotype-based pathotyping of the E. coli strains revealed 47%, 30%, and 6% to belong to EPEC, ETEC, and EHEC pathotypes, respectively. The majority of the 160 strains tested were resistant to commonly used antimicrobial agents. Plasmid pro-filing showed a total of 17 different bands ranging from 1.3 to 40 kb. However, 35% of the strains did not contain any detectable plasmid, implying no correlation between plasmid and drug resistance. Although virulence gene profiling revealed 97 (61%) of the strains to harbor the gene encoding heat-stable enterotoxin (ST), 2 for the gene encoding Shiga toxin (Stx), and none for the gene for heat-labile enterotoxin (LT), serotype-based pathotyping of E. coli was not fully supported by this gene profiling. A dendrogram derived from the PFGE patterns of 22 strains of three predominant serogroups indicated two major clusters, one containing mainly serogroup O55 and the other O8. Three strains of identical PFGE profiles belonging to serogroup O55 were isolated from three distinct areas, which may be of epidemiological significance. Finally, it may be concluded that serotype-based pathotyping may be useful for E. coli strains of clinical origin; however, it is not precise enough for reliably identifying environmental strains as diarrheagenic.