An adhesive factor found in strains ofEscherichia coli belonging to the traditional infantile enteropathogenic serotypes

Escherichia coli strains isolated from outbreaks of diarrheal disease were tested for the presence of adhesive factors. Fifty-one of these strains belonged to traditional infantile entero-pathogenic serotypes (EPEC) and 17 belonged to other serotypes. None of these strains were enterotoxigenic and none possessed colonization factors CFA/I or CFA/II, which have been described among strains of enterotoxigenicE. coli (ETEC). EnterotoxigenicE. coli strains from patients with diarrhea and strains which were neither EPEC nor ETEC from subjects without diarrhea were also examined. By means of a tissue culture technique using HEp-2 cells, a new adhesive factor was found to occur with greater frequency in EPEC strains. The adhesive factor was found less frequently in the other groups ofE. coli studied. It was distinct from type 1 pili and was not inhibited by the presence ofD-mannose.     

First isolation and further characterization of enteropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> (EPEC) O157:H45 strains from cattle

Background  

Enteropathogenic Escherichia coli (EPEC), mainly causing infantile diarrhoea, represents one of at least six different categories of diarrheagenic E. coli with corresponding distinct pathogenic schemes. The mechanism of EPEC pathogenesis is based on the ability to introduce the attaching-and-effacing (A/E) lesions and intimate adherence of bacteria to the intestinal epithelium. The role and the epidemiology of non-traditional enteropathogenic E. coli serogroup strains are not well established. E. coli O157:H45 EPEC strains, however, are described in association with enterocolitis and sporadic diarrhea in human. Moreover, a large outbreak associated with E. coli O157:H45 EPEC was reported in Japan in 1998. During a previous study on the prevalence of E. coli O157 in healthy cattle in Switzerland, E. coli O157:H45 strains originating from 6 fattening cattle and 5 cows were isolated. In this study, phenotypic and genotypic characteristics of these strains are described. Various virulence factors (stx, eae, ehxA, astA, EAF plasmid, bfp) of different categories of pathogenic E. coli were screened by different PCR systems. Moreover, the capability of the strains to adhere to cells was tested on tissue culture cells.     

Cloning and Sequencing of Two New Verotoxin 2 Variant Genes of Escherichia coli Isolated from Cases of Human and Bovine Diarrhea

We cloned and sequenced two new Verotoxin 2 (VT2) variant genes: one from an Escherichia coli strain from a case of bovine diarrhea and the other from an E. coli strain from a patient with diarrhea. The nucleotide and amino acid sequences of these two genes were highly homologous with, but distinct from those of the VT2, VT2vha, VT2vhb, SLT-IIv (VT2vp1) and SLT-IIva (VT2vp2) genes. Their nucleotide sequences were much more closely homologous to that of VT2vh than to that of VT2vp. Search for these two new genes in other Verocytotoxin-producing E. coli strains resulted in the isolation of 2 strains carrying one of the new VT2 variant genes, one strain from Tokyo and the other from Canada.     

Involvement of Ca2+ -Calmodulin-Dependent Protein Kinase II in the Intestinal Secretory Action of Escherichia coli Heat-Stable Enterotoxin II

Treating the mouse intestine with the calmodulin antagonist W-7 and KN-93, an inhibitor of Ca²⁺ -calmodulin-dependent protein kinase II (CaMK II), reduced the sensitivity of the host to the action of Escherichia coli heat-stable enterotoxin II (STII). CaMK II activity in mouse intestinal cells increased after exposure to STII. These results indicate that CaMK II is involved in the mechanism of action of STII.     

Occurrence of Genes Associated with Enterotoxigenic and Enterohemorrhagic Escherichia coli in Agricultural Waste Lagoons

The prevalence among all Escherichia coli bacteria of the LTIIa toxin gene and STII toxin gene, both associated with enterotoxigenic E. coli, and of three genes (stxI, stxII, and eaeA) associated with enterohemorrhagic E. coli was determined in farm waste disposal systems seasonally for 1 year. Single- and nested-PCR results for the number of E. coli isolates carrying each toxin gene trait were compared with a five-replicate most-probable-number (MPN) method. The STII and LTIIa toxin genes were present continuously at all farms and downstream waters that were tested. Nested-MPN-PCR manifested sensitivity increased over that of single-MPN-PCR by a factor of 32 for LTIIa, 10 for STII, and 2 for the stxI, stxII, and eaeA genes. The geometric mean prevalence of each toxin gene within the E. coli community in waste disposal site waters after nested MPN-PCR was 1:8.5 E. coli isolates (1:8.5 E. coli) for the LTIIa toxin gene and 1:4 E. coli for the STII toxin gene. The geometric mean prevalence for the simultaneous occurrence of toxin genes stxI, stxII, and eaeA, was 1:182 E. coli. These findings based on total population analysis suggest that prevalence rates for these genes are higher than previously reported in studies based on surveys of single isolates. With a population-based approach, the frequency of each toxin gene at the corresponding disposal sites and the endemic nature of diseases on farms can be easily assessed, allowing farmers and public health officials to evaluate the risk of infection to animals or humans.     

Application of polymerase chain reaction for determination of toxins in Escherichia coli strains isolated from pigs with diarrhea.

The PCR method was used for the determination of LTI, STI, STII enterotoxins and Stx2e toxin genes in E. coli strains isolated from pigs with diarrhea. It was shown that most of the strains (77.3%) were enterotoxigenic, producing mainly LTI and STII (30.8%) or STI and STII (21.3%) toxins. It was found that 10.6% of isolates possessed the stx2e genes responsible for the elaboration of shiga toxin 2e.     

A biomarker for the identification of swine fecal pollution in water,using the STII toxin gene from enterotoxigenic<Emphasis Type="Italic"> Escherichia coli</Emphasis>

This research developed a PCR method to identify swine fecal pollution in water, using a portion of the STII toxin gene from enterotoxigenic Escherichia coli as the target sequence. This method showed the gene to have a wide-spread geographical distribution and temporal stability; and the primers demonstrated high specificity, sensitivity, and reliability. A total of 110 DNA extracts from different animal fecal and human sewage samples were screened using the primers and no positives resulted. Centrifugation and filtration methods for concentrating E. coli seeded into stream, ocean, secondary effluent, and dairy lagoon waters resulted in detection limits at the femtogram and attogram levels. E. coli with the biomarker seeded into stream, ocean, and secondary effluent waters remained stable for approximately 2 weeks for all water types. Of the farm lagoon and waste samples tested, 94% were positive for the STII trait, regardless of the number of E. coli screened and 100% were positive when 35 E. coli isolates were screened. As the PCR product of the target sequence yielded a single band, the method is applicable to dot blot detection methodology, yielding great accuracy in determining the presence of swine fecal sources.     

Bovine attaching and effacing Escherichia coli possess a pathogenesis island related to the LEE of the human enteropathogenic Escherichia coli strain E2348/69

Attaching and effacing Escherichia coli (AEEC) has been described as a cause of diarrhea in calves. The molecular pathogenesis of AEEC was mainly studied in human enteropathogenic E. coli strain E2348/69 in which the virulence correlated with the presence of a 35.4 kb pathogenesis island called LEE. We showed that several strains isolated from calves with diarrhea were able to produce attaching and effacing lesions in a rabbit ileal loop model and that they possess a pathogenesis island related to the LEE. Moreover, we showed that the LEE from bovine strains was inserted mainly at a different position in the chromosome compared to the human enteropathogenic E. coli strain E2348/69.     

Occurrence and antimicrobial drug susceptibility patterns of commensal and diarrheagenic <Emphasis Type="Italic">Escherichia coli</Emphasis> in fecal microbiota from children with and without acute diarrhea

Acute diarrhea is a public health problem and an important cause of morbidity and mortality, especially in developing countries. The etiology is varied, and the diarrheagenic Escherichia coli pathotypes are among the most important. Our objectives were to determine the occurrence of commensal and diarrheagenic E. coli strains in fecal samples from children under five years old and their drug susceptibility patterns. E. coli were isolated from 141 fresh fecal samples; 84 were obtained from clinically injured donors with acute diarrhea (AD) and 57 from clinically healthy donors without diarrhea (WD). Presumptive phenotypic species identification was carried out and confirmed by amplification of specific 16S ribosomal RNA encoding DNA. Multiplex PCR was performed to characterize the diarrheagenic E. coli strains. Drug susceptibility patterns were determined by the disc-diffusion method. In total, 220 strains were recovered from the fecal specimens (61.8% from AD and 38.2% from WD). Diarrheagenic E. coli was identified at a rate of 36.8% (n=50) in diarrheic feces and 29.8% (n=25) in non-diarrheic feces. Enteroaggregative E. coli was the most frequently identified pathotype in the AD group (16.2%) and the only pathotype identified in the WD group (30.9%). Enteropathogenic E. coli was the second most isolated pathotype (10.3%), followed by Shiga toxin-producing E. coli (7.4%) and enterotoxigenic E. coli (2.9%). No enteroinvasive E. coli strains were recovered. The isolates showed high resistance rates against ampicillin, tetracycline, and sulfamethoxazole-trimethoprim. The most effective drugs were ceftazidime, ceftriaxone, imipenem and piperacillin-tazobactam, for which no resistance was observed. Differentiation between the diarrheagenic E. coli pathotypes is of great importance since they are involved in acute diarrheal diseases and may require specific antimicrobial chemotherapy. The high antimicrobial resistance observed in our study raises a broad discussion on the indiscriminate or improper use of antimicrobials, besides the risks of self-medication.     

Enteroaggregative and cell-detaching<Emphasis Type="Italic">Escherichia coli</Emphasis> strains among polish children with and without diarrhea

To determine the association of enteroaggregative (EAEC) and cell-detaching (CDEC)Escherichia coli with diarrhea of unknown origin among children from Wroc?aw (Poland),E. coli strains isolated from stool specimens of children with diarrhea were examined for mannose-resistant adherence to HEp-2 cells. EAEC were isolated from 10 of 39 (26%) children examined with diarrhea and 4 of 20 (20%) age-matched controls. CDEC were present in 14 (36%) cases of diarrhea and 7 (35%) healthy subjects. Cell-detaching activity was distinctly associated with hemolysin production. Among hemolytic CDEC strains cytotoxic necrotizing factor 1 (CNF1) synthesis prevailed among isolates obtained from cases of diarrhea (57%) in comparison with isolates obtained from healthy controls (14.3%). Although neither EAEC nor CDECE. coli strains were associated with diarrhea of children in this setting, there were differences among EAEC and CDEC strains isolated from children with and without diarrhea.     

通辽地区犊牛腹泻大肠杆菌耐药性检测及一株多重耐药菌全基因组测序分析

【背景】大肠杆菌(Escherichia coli)是引起犊牛腹泻的最主要病原菌,其耐药性菌株的不断出现引起广泛关注。【目的】了解内蒙古自治区通辽市犊牛腹泻大肠杆菌耐药性及耐药基因流行情况。【方法】从通辽市多个旗县采集犊牛腹泻样品40份,经细菌分离纯化及16S rRNA基因测序,最终鉴定出20株大肠杆菌。采用药敏试验和PCR方法对分离菌进行耐药性及耐药基因检测分析,并对其中1株多重耐药菌株进行全基因组测序。【结果】20株分离菌均具有多重耐药性,对链霉素、环丙沙星、恩诺沙星和复方新诺明的耐药率达80%以上。所检耐药基因中,aphA1、strB、TEM-1和qnrS检出率达100%。通过对代表性菌株TL-13全基因组测序发现,其基因组大小为4897185bp,GC含量为50.68%,同时携带2个质粒,大小分别为108288bp(pTL13-1)和64018bp(pTL13-2)。质粒中共携带18个可移动耐药基因。【结论】通辽地区犊牛腹泻大肠杆菌多重耐药性普遍存在,4种常见耐药基因普遍流行。     

Comparison between Enterotoxigenic Escherichia coli Strains Expressing “F42,” F41 and K99 Colonization Factors

Two enterotoxigenic Escherichia coli (ETEC) strains (coded 567/7 and 103) isolated from piglets with neonatal diarrhea were described as producers of a new adhesin (F42). With the use of molecular biology and immunology techniques such as DNA hybridization with probes for F41 and K99 genes and Western-blotting of the superficial proteins of these strains and standard E. coli strains carrying genes for F41 and K99 adhesins, it was demonstrated that this new adhesin either shares extensive genetic and immunological determinants with F41 adhesin or they are the same fimbriae.     

Detection of Shiga toxins,intimin and enterohemolysin in Escherichia coli strains isolated from children in eastern Slovakia

FiftyEscherichia coli strains isolated from stool samples of 51 healthy children, 143 strains isolated from stool samples of 327 children with diarrhea and 24 strains isolated from stool samples of 21 children with suspected hemolytic uremic syndrome were examined for the presence of Shiga toxin-producingE. coli virulence factors (shiga toxin 1 and 2, intimin and enterohemolysin) and their genes. Vero-cell assay and latex agglutination were used for detection of Shiga toxin 1 and 2, TSB agar with washed erythrocytes was used for detection of enterohemolysin; genes encoding shiga toxin 1 and 2, intimin and enterohemolysin were detected using multiplex PCR. The presence ofE. coli strains harboring genes encoding shiga toxin 1 and 2 (12 strains), intimin (34 strains) and enterohemolysin (12 strains) was demonstrated.     

Production of enterotoxin,verotoxin, hemolysin and cytotoxic necrotizing factor byEscherichia coli of intestinal and extraintestinal origin

Seventy-sixEscherichia coli strains were examined for heat-labile enterotoxin (LT), verotoxin (VT), hemolysin (HLy) and cytotoxic necrotizing factor (CNF). Thirty-six strains were isolated from patients suffering from diarrhea and forty from different extraintestinal infections. The number of LT-producing strains was low (2.6%) (one of intestinal and one of extraintestinal origin). Verotoxin was produced only by one extraintestinal strain. Four intestinal strains were hemolytic (11.2%) and also positive for CNF. From 24 hemolytic strains of extraintestinal origin (60%), 17 produced also CNF. Most of the hemolytic (30%) as well as CNF-producing strains (22.5%) were isolated from urine. Our results are similar to those of other studies confirming the close association between hemolysin and CNF production as well as a possible role of these toxic factors in pathogenesis of extraintestinal, infections caused byE. coli.     

Isolation and characterization ofEscherichia coli O157:H7 strains in China

Five strains ofEscherichia coli O157:H7 were isolated from 486 stool specimens collected in 1986, 1987, and 1988 from patients with diarrhea in Xuzhou City, Jiangsu Province, China; 21 of the specimens were from patients with bloody diarrhea. The biochemical reactions of all five strains were almost identical with those of the well-knownE. coli O157:H7 strain 933. All of the strains were found to carry a 60 Md plasmid and two small plasmids. The plasmid DNA Hind 111 restriction patterns were identical. The strains were lysed byE. coli typing phage E1, E2, and E3, but not by E4 or E5. Data suggested that it might belong to a single phage or plasmid group. All strains produced vero toxin and caused diarrhea and death in infant rabbits and mice.     

Isolation and characterisation of dog uropathogenic Escherichia coli strains and their fimbriae

A number of Escherichia coli strains have been isolated from dogs with urinary tract infections. These strains have been characterised with respect to their O, K, H, and fimbrial antigens, colicin production, antibiotic resistance, plasmid content and their ability to haemagglutinate erythrocytes from various species. Crossed immunoelectrophoresis of fimbrial extracts, as well as the reaction of partly purified fimbriae of a number of these strains with monoclonal antibodies revealed homology or a strong crossereaction with an F12 fimbrial subunit protein of human uropathogenic E. coli strains. Unlike human F12 fimbriae producing strains, the dog isolates did agglutinate dog erythrocytes in the presence of D-mannose but not human erythrocytes, indicating that the adhesin carried by these strains is different from the adhesin on fimbriae of human uropathogenic E. coli. Similar indications were obtained from experiments with latex beads coated with the receptor for P-fimbriae. These beads were agglutinated by Escherichia coli strains from human urinary tract infections, but not by the dog isolates described here. Preliminary adhesion experiments of human and dog Escherichia coli to human bladder epithelial and canine kidney epithelial cells also showed differences in adhesion depending on the origin of the strain tested.     

Fimbriation ofEscherichia coli in urinary tract infections. Comparisons between bacteria in the urine and subcultured bacterial isolates

Midstream urine samples from 37 patients with urinary tract infections were studied by electron microscopy, hemagglutination, and the salt aggregation test (SAT) to measure the hydrophobicity of the bacterial surface.Escherichia coli subcultured from these urine samples were tested in the same way. Fimbriae were visualized onE. coli in the urine of 31 specimens, and all these urines containedE. coli that expressed pronounced surface hydrophobicity and aggregated in ammonium sulfate of 0.1–1.6 M final concentration. Hemagglutination of human and/or guinea pig erythrocytes was expressed by 21E. coli in the urine. TheE. coli strains subcultured from these 31 urine samples were also fimbriated, but the number of fimbriae per bacterium as well as the percentage of fimbriated bacteria varied compared with the directly collected strains. The surface hydrophobicity and hemagglutination were similar to the results with the directly collected bacteria. However, after serial transfer in CFA-broth under static conditions, all non-hemagglutinating strains expressed mannose-sensitive hemagglutination of guinea pig erythrocytes, and three strains also expressed weak mannose-resistant hemagglutination of human erythrocytes. Following serial transfer, fimbriae were also visualized on the sixE. coli strains that appeared non-fimbriate in the urine. It is thus concluded thatE. coli causing urinary tract infection are often fimbriated and express surface hydrophobicity in the urine. Based on these findings, a rapid method to isolate hydrophobic, possibly fimbriated bacteria was tried in which the urine was mixed with a hydrophobic gel. Hydrophobic bacteria bound to the gel and could be eluted from the sedimented gel.     

Diarrheagenic <Emphasis Type="Italic">Escherichia coli</Emphasis> Strains Recovered from Urban Pigeons (<Emphasis Type="Italic">Columba livia</Emphasis>) in Brazil and Their Antimicrobial Susceptibility Patterns

Urban pigeons (Columba livia) come into close contact with humans and animals, and may contribute to the spread of infectious agents. These may include human pathogens such as diarrheagenic Escherichia coli strains, which are able to survive in pigeon feces, thus creating potential for human exposure and infection. Our objectives were to determine the occurrence of diarrheagenic E. coli strains in fresh feces from urban pigeons and their drug susceptibility patterns. E. coli strains were isolated from 100 fresh feces samples and presumptive phenotypic species identification was carried out, confirmed by amplification of specific 16S ribosomal RNA encoding DNA. Multiplex PCR was performed to characterize pathogenic strains. Drug susceptibility patterns were determined by the agar dilution method. Enteroinvasive E. coli, Shiga toxin-producing E. coli, enteropathogenic E. coli, and enterotoxigenic E. coli were detected at an overall rate of 12.1%. Among the isolated E. coli strains, 62.1% were susceptible to all tested drugs, whereas 37.9% were resistant to at least one of the antimicrobials tested. Amikacin was the less effective drug (36.8% resistance), followed by ampicillin (7.8%). No resistance was detected to gentamicin, ceftriaxone, and ceftazidime and almost all the isolates were susceptible to ampicillin-sulbactam (98.4%), levofloxacin (97.8%), and trimethoprim-sulfametoxazole (96.1%). Since these pigeons may harbor multidrug-resistant pathogens, their presence in an urban environment could be an important component of infection spread, with impact on public health.     

Relationship between Phylogenetic Groups, Genotypic Clusters, and Virulence Gene Profiles of Escherichia coli Strains from Diverse Human and Animal Sources

Escherichia coli strains in water may originate from various sources, including humans, farm and wild animals, waterfowl, and pets. However, potential human health hazards associated with E. coli strains present in various animal hosts are not well known. In this study, E. coli strains from diverse human and animal sources in Minnesota and western Wisconsin were analyzed for the presence of genes coding for virulence factors by using multiplex PCR and biochemical reactions. Of the 1,531 isolates examined, 31 (2%) were found to be Shiga toxin-producing E. coli (STEC) strains. The majority of these strains, which were initially isolated from the ruminants sheep, goats, and deer, carried the stx_1c and/or stx_2d, ehxA, and saa genes and belonged to E. coli phylogenetic group B1, indicating that they most likely do not cause severe human diseases. All the STEC strains, however, lacked eae. In contrast, 26 (1.7%) of the E. coli isolates examined were found to be potential enteropathogenic E. coli (EPEC) strains and consisted of several intimin subtypes that were distributed among various human and animal hosts. The EPEC strains belonged to all four phylogenetic groups examined, suggesting that EPEC strains were relatively widespread in terms of host animals and genetic background. Atypical EPEC strains, which carried an EPEC adherence factor plasmid, were identified among E. coli strains from humans and deer. DNA fingerprint analyses, done using the horizontal, fluorophore-enhanced repetitive-element, palindromic PCR technique, indicated that the STEC, potential EPEC, and non-STEC ehxA-positive E. coli strains were genotypically distinct and clustered independently. However, some of the potential EPEC isolates were genotypically indistinguishable from nonpathogenic E. coli strains. Our results revealed that potential human health hazards associated with pathogenic E. coli strains varied among the animal hosts that we examined and that some animal species may harbor a greater number of potential pathogenic strains than other animal species.     

Shiga Toxin 2e-Producing Escherichia coli Isolates from Humans and Pigs Differ in Their Virulence Profiles and Interactions with Intestinal Epithelial Cells

Thirteen Escherichia coli strains harboring stx_2e were isolated from 11,056 human stools. This frequency corresponded to the presence of the stx_2e allele in 1.7% of all Shiga toxin-producing E. coli (STEC) strains. The strains harboring stx_2e were associated with mild diarrhea (n = 9) or asymptomatic infections (n = 4). Because STEC isolates possessing stx_2e are porcine pathogens, we compared the human STEC isolates with stx_2e-harboring E. coli isolated from piglets with edema disease and postweaning diarrhea. All pig isolates possessed the gene encoding the F18 adhesin, and the majority possessed adhesin involved in diffuse adherence; these adhesins were absent from all the human STEC isolates. In contrast, the high-pathogenicity island encoding an iron uptake system was found only in human isolates. Host-specific patterns of interaction with intestinal epithelial cells were observed. All human isolates adhered to human intestinal epithelial cell lines T84 and HCT-8 but not to pig intestinal epithelial cell line IPEC-J2. In contrast, the pig isolates completely lysed human epithelial cells but not IPEC-J2 cells, to which most of them adhered. Our data demonstrate that E. coli isolates producing Shiga toxin 2e have imported specific virulence and fitness determinants which allow them to adapt to the specific hosts in which they cause various forms of disease.