Comparison of Virulence Gene Profiles of Escherichia coli Strains Isolated from Healthy and Diarrheic Swine

A combination of uni- and multiplex PCR assays targeting 58 virulence genes (VGs) associated with Escherichia coli strains causing intestinal and extraintestinal disease in humans and other mammals was used to analyze the VG repertoire of 23 commensal E. coli isolates from healthy pigs and 52 clinical isolates associated with porcine neonatal diarrhea (ND) and postweaning diarrhea (PWD). The relationship between the presence and absence of VGs was interrogated using three statistical methods. According to the generalized linear model, 17 of 58 VGs were found to be significant (P < 0.05) in distinguishing between commensal and clinical isolates. Nine of the 17 genes represented by iha, hlyA, aidA, east1, aah, fimH, iroN_{E. coli}, traT, and saa have not been previously identified as important VGs in clinical porcine isolates in Australia. The remaining eight VGs code for fimbriae (F4, F5, F18, and F41) and toxins (STa, STb, LT, and Stx2), normally associated with porcine enterotoxigenic E. coli. Agglomerative hierarchical algorithm analysis grouped E. coli strains into subclusters based primarily on their serogroup. Multivariate analyses of clonal relationships based on the 17 VGs were collapsed into two-dimensional space by principal coordinate analysis. PWD clones were distributed in two quadrants, separated from ND and commensal clones, which tended to cluster within one quadrant. Clonal subclusters within quadrants were highly correlated with serogroups. These methods of analysis provide different perspectives in our attempts to understand how commensal and clinical porcine enterotoxigenic E. coli strains have evolved and are engaged in the dynamic process of losing or acquiring VGs within the pig population.     

Comparative analysis of virulence genes, genetic diversity, and phylogeny of commensal and enterotoxigenic Escherichia coli isolates from weaned pigs

If the acquisition of virulence genes (VGs) for pathogenicity were not solely acquired through horizontal gene transfers of pathogenicity islands, transposons, and phages, then clonal clusters of enterotoxigenic Escherichia coli (ETEC) would contain few or even none of the VGs found in strains responsible for extraintestinal infections. To evaluate this possibility, 47 postweaning diarrhea (PWD) ETEC strains from different geographical origins and 158 commensal E. coli isolates from the gastrointestinal tracts of eight group-housed healthy pigs were screened for 36 extraintestinal and 18 enteric VGs using multiplex PCR assays. Of 36 extraintestinal VGs, only 8 were detected (fimH, traT, fyuA, hlyA, kpsMtII, k5, iha, and ompT) in the ETEC collection. Among these, hlyA (alpha-hemolysin) and iha (nonhemagglutinating adhesin) occurred significantly more frequently among the ETEC isolates than in the commensal isolates. Clustering analysis based on the VG profiles separated commensal and ETEC isolates and even differentiated serogroup O141 from O149. On the other hand, pulsed-field gel electrophoresis (PFGE) successfully clustered ETEC isolates according to both serotype and geographical origin. In contrast, the commensal isolates were heterogeneous with respect to both serotype and DNA fingerprint. This study has validated the use of VG profiling to examine pathogenic relationships between porcine ETEC isolates. The clonal relationships of these isolates can be further clarified by PFGE fingerprinting. The presence of extraintestinal VGs in porcine ETEC confirmed the hypothesis that individual virulence gene acquisitions can occur concurrently against a background of horizontal gene transfers of pathogenicity islands. Over time, this could enable specific clonotypes to respond to host selection pressure and to evolve into new strains with increased virulence.     

Comparative Analysis of Virulence Genes, Genetic Diversity, and Phylogeny of Commensal and Enterotoxigenic Escherichia coli Isolates from Weaned Pigs

If the acquisition of virulence genes (VGs) for pathogenicity were not solely acquired through horizontal gene transfers of pathogenicity islands, transposons, and phages, then clonal clusters of enterotoxigenic Escherichia coli (ETEC) would contain few or even none of the VGs found in strains responsible for extraintestinal infections. To evaluate this possibility, 47 postweaning diarrhea (PWD) ETEC strains from different geographical origins and 158 commensal E. coli isolates from the gastrointestinal tracts of eight group-housed healthy pigs were screened for 36 extraintestinal and 18 enteric VGs using multiplex PCR assays. Of 36 extraintestinal VGs, only 8 were detected (fimH, traT, fyuA, hlyA, kpsMtII, k5, iha, and ompT) in the ETEC collection. Among these, hlyA (α-hemolysin) and iha (nonhemagglutinating adhesin) occurred significantly more frequently among the ETEC isolates than in the commensal isolates. Clustering analysis based on the VG profiles separated commensal and ETEC isolates and even differentiated serogroup O141 from O149. On the other hand, pulsed-field gel electrophoresis (PFGE) successfully clustered ETEC isolates according to both serotype and geographical origin. In contrast, the commensal isolates were heterogeneous with respect to both serotype and DNA fingerprint. This study has validated the use of VG profiling to examine pathogenic relationships between porcine ETEC isolates. The clonal relationships of these isolates can be further clarified by PFGE fingerprinting. The presence of extraintestinal VGs in porcine ETEC confirmed the hypothesis that individual virulence gene acquisitions can occur concurrently against a background of horizontal gene transfers of pathogenicity islands. Over time, this could enable specific clonotypes to respond to host selection pressure and to evolve into new strains with increased virulence.     

DNA microarray-based identification of serogroups and virulence gene patterns of Escherichia coli isolates associated with porcine postweaning diarrhea and edema disease

Escherichia coli strains causing postweaning diarrhea (PWD) and edema disease (ED) in pigs are limited to a number of serogroups, with O8, O45, O138, O139, O141, O147, O149, and O157 being the most commonly reported worldwide. In this study, a DNA microarray based on the O-antigen-specific genes of all 8 E. coli serogroups, as well as 11 genes encoding adhesion factors and exotoxins associated with PWD and ED, was developed for the identification of related serogroups and virulence gene patterns. The microarray method was tested against 186 E. coli and Shigella O-serogroup reference strains, 13 E. coli reference strains for virulence markers, 43 E. coli clinical isolates, and 12 strains of other bacterial species and shown to be highly specific with reproducible results. The detection sensitivity was 0.1 ng of genomic DNA or 10(3) CFU per 0.3 g of porcine feces in mock samples. Seventeen porcine feces samples from local hoggeries were examined using the microarray, and the result for one sample was verified by the conventional serotyping methods. This microarray can be readily used to screen for the presence of PWD- and ED-associated E. coli in porcine feces samples.     

Virulence factor gene profiles of Escherichia coli isolates from clinically healthy pigs

Nonpathogenic, intestinal Escherichia coli (commensal E. coli) supports the physiological intestinal balance of the host, whereas pathogenic E. coli with typical virulence factor gene profiles can cause severe outbreaks of diarrhea. In many reports, E. coli isolates from diarrheic animals were classified as putative pathogens. Here we describe a broad variety of virulence gene-positive E. coli isolates from swine with no clinical signs of intestinal disease. The isolation of E. coli from 34 pigs from the same population and the testing of 331 isolates for genes encoding heat-stable enterotoxins I and II, heat-labile enterotoxin I, Shiga toxin 2e, and F4, F5, F6, F18, and F41 fimbriae revealed that 68.6% of the isolates were positive for at least one virulence gene, with a total of 24 different virulence factor gene profiles, implying high rates of horizontal gene transfer in this E. coli population. Additionally, we traced the occurrence of hemolytic E. coli over a period of 1 year in this same pig population. Hemolytic isolates were differentiated into seven clones; only three were found to harbor virulence genes. Hemolytic E. coli isolates without virulence genes or with only the fedA gene were found to be nontypeable by slide agglutination tests with OK antisera intended for screening live cultures against common pathogenic E. coli serogroups. The results appear to indicate that virulence gene-carrying E. coli strains are a normal part of intestinal bacterial populations and that high numbers of E. coli cells harboring virulence genes and/or with hemolytic activity do not necessarily correlate with disease.     

Occurrence of intestinal and extraintestinal virulence genes in Escherichia coli isolates from rainwater tanks in Southeast Queensland, Australia

In this study, 200 Escherichia coli isolates from 22 rainwater tank samples in Southeast Queensland, Australia, were tested for the presence of 20 virulence genes (VGs) associated with intestinal and extraintestinal pathotypes. In addition, E. coli isolates were also classified into phylogenetic groups based on the detection of the chuA, yjaA, and TSPE4.C2 genes. Of the 22 rainwater tanks, 8 (36%) and 5 (23%) were positive for the eaeA (belonging to enteropathogenic E. coli [EPEC] and Shiga-toxigenic E. coli [STEC]) and ST1 (belonging to enterotoxigenic E. coli [ETEC]) genes, respectively. VGs (cdtB, cvaC, ibeA, kpsMT allele III, PAI, papAH, and traT) belonging to extraintestinal pathogenic E. coli (ExPEC) were detected in 15 (68%) of the 22 rainwater tanks. Of the 22 samples, 17 (77%) and 11 (50%) contained E. coli belonging to phylogenetic groups A and B1, respectively. Similarly, 10 (45%) and 16 (72%) contained E. coli belonging to phylogenetic groups B2 and D, respectively. Of the 96 of the 200 strains from 22 tanks that were VG positive, 40 (42%) were carrying a single VG, 36 (37.5%) were carrying two VGs, 17 (18%) were carrying three VGs, and 3 (3%) had four or more VGs. This study reports the presence of multiple VGs in E. coli strains belonging to the STEC, EPEC, ETEC, and ExPEC pathotypes in rainwater tanks. The public health risks associated with potentially clinically significant E. coli in rainwater tanks should be assessed, as the water is used for drinking and other, nonpotable purposes. It is recommended that rainwater be disinfected using effective treatment procedures such as filtration, UV disinfection, or simply boiling prior to drinking.     

Pathogenic Escherichia coli found in sewage treatment plants and environmental waters

We previously demonstrated that some Escherichia coli strains with uropathogenic properties survived treatment stages of sewage treatment plants (STPs), suggesting that they may be released into the environment. We investigated the presence of such strains in the surrounding environmental waters of four STPs from which these persistent strains were isolated. In all, 264 E. coli isolates were collected from 129 receiving water sites in a 20-km radius surrounding STPs. We also included 93 E. coli strains collected from 18 animal species for comparison. Isolates were typed using a high-resolution biochemical fingerprinting method (the PhPlate system), and grouped into common (C) types. One hundred forty-seven (56%) environmental isolates were identical to strains found in STPs' final effluents. Of these, 140 (95%) carried virulence genes (VGs) associated with intestinal pathogenic E. coli (IPEC) or uropathogenic E. coli (UPEC) and were found in a variety of sites within areas sampled. Of the remaining 117 environmental strains not identical to STP strains, 105 belonged to 18 C types and 102 of them carried VGs found among IPEC or UPEC strains. These strains belonged mainly to phylogenetic groups A (A0 and A1) and B1 and to a lesser extent B2(2), B2(3), D1, and D2. Eight of 18 environmental C types, comprising 50 isolates, were also identical to bird strains. The presence of a high percentage of environmental E. coli in waters near STPs carrying VGs associated with IPEC and UPEC suggests that they may have derived from STP effluents and other nonpoint sources.     

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.     

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.     

DNA Microarray-Based Identification of Serogroups and Virulence Gene Patterns of Escherichia coli Isolates Associated with Porcine Postweaning Diarrhea and Edema Disease

Escherichia coli strains causing postweaning diarrhea (PWD) and edema disease (ED) in pigs are limited to a number of serogroups, with O8, O45, O138, O139, O141, O147, O149, and O157 being the most commonly reported worldwide. In this study, a DNA microarray based on the O-antigen-specific genes of all 8 E. coli serogroups, as well as 11 genes encoding adhesion factors and exotoxins associated with PWD and ED, was developed for the identification of related serogroups and virulence gene patterns. The microarray method was tested against 186 E. coli and Shigella O-serogroup reference strains, 13 E. coli reference strains for virulence markers, 43 E. coli clinical isolates, and 12 strains of other bacterial species and shown to be highly specific with reproducible results. The detection sensitivity was 0.1 ng of genomic DNA or 10³ CFU per 0.3 g of porcine feces in mock samples. Seventeen porcine feces samples from local hoggeries were examined using the microarray, and the result for one sample was verified by the conventional serotyping methods. This microarray can be readily used to screen for the presence of PWD- and ED-associated E. coli in porcine feces samples.     

Antimicrobial resistance and virulence genes of Escherichia coli isolates from swine in Ontario

A total of 318 Escherichia coli isolates obtained from diarrheic and healthy pigs in Ontario from 2001 to 2003 were examined for their susceptibility to 19 antimicrobial agents. They were tested by PCR for the presence of resistance genes for tetracycline, streptomycin, sulfonamides, and apramycin and of 12 common virulence genes of porcine E. coli. Antimicrobial resistance frequency among E. coli isolates from swine in Ontario was moderate in comparison with other countries and was higher in isolates from pigs with diarrhea than in isolates from healthy finisher pigs. Resistance profiles suggest that cephamycinases may be produced by > or = 8% of enterotoxigenic E. coli (ETEC). Resistance to quinolones was detected only in enterotoxigenic E. coli (< or = 3%). The presence of sul3 was demonstrated for the first time in Canada in porcine E. coli isolates. Associations were observed among tetA, sul1, aadA, and aac(3)IV and among tetB, sul2, and strA/strB, with a strong negative association between tetA and tetB. The paa and sepA genes were detected in 92% of porcine ETEC, and strong statistical associations due to colocation on a large plasmid were observed between tetA, estA, paa, and sepA. Due at least in part to gene linkages, the distribution of resistance genes was very different between ETEC isolates and other porcine E. coli isolates. This demonstrates that antimicrobial resistance epidemiology differs significantly between pathogenic and commensal E. coli isolates. These results may have important implications with regards to the spread and persistence of resistance and virulence genes in bacterial populations and to the prudent use of antimicrobial agents.     

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 stx2e were isolated from 11,056 human stools. This frequency corresponded to the presence of the stx2e allele in 1.7% of all Shiga toxin-producing E. coli (STEC) strains. The strains harboring stx2e were associated with mild diarrhea (n = 9) or asymptomatic infections (n = 4). Because STEC isolates possessing stx2e are porcine pathogens, we compared the human STEC isolates with stx2e-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.     

Autotransporter-encoding sequences are phylogenetically distributed among Escherichia coli clinical isolates and reference strains

Autotransporters are secreted bacterial proteins exhibiting diverse virulence functions. Various autotransporters have been identified among Escherichia coli associated with intestinal or extraintestinal infections; however, the specific distribution of autotransporter sequences among a diversity of E. coli strains has not been investigated. We have validated the use of a multiplex PCR assay to screen for the presence of autotransporter sequences. Herein, we determined the presence of 13 autotransporter sequences and five allelic variants of antigen 43 (Ag43) among 491 E. coli isolates from human urinary tract infections, diarrheagenic E. coli, and avian pathogenic E. coli (APEC) and E. coli reference strains belonging to the ECOR collection. Clinical isolates were also classified into established phylogenetic groups. The results indicated that Ag43 alleles were significantly associated with clinical isolates (93%) compared to commensal isolates (56%) and that agn43K12 was the most common and widely distributed allele. agn43 allelic variants were also phylogenetically distributed. Sequences encoding espC, espP, and sepA and agn43 alleles EDL933 and RS218 were significantly associated with diarrheagenic E. coli strains compared to other groups. tsh was highly associated with APEC strains, whereas sat was absent from APEC. vat, sat, and pic were associated with urinary tract isolates and were identified predominantly in isolates belonging to either group B2 or D of the phylogenetic groups based on the ECOR strain collection. Overall, the results indicate that specific autotransporter sequences are associated with the source and/or phylogenetic background of strains and suggest that, in some cases, autotransporter gene profiles may be useful for comparative analysis of E. coli strains from clinical, food, and environmental sources.     

PicU, a second serine protease autotransporter of uropathogenic Escherichia coli

Escherichia coli is the major aetiological agent of urinary tract infections (UTI). Like diarrhoeagenic strains of E. coli, uropathogenic isolates possess virulence determinants that distinguish them from commensal strains and allow them to produce the clinical manifestations associated with UTI. Several autotransporter proteins have been associated with the ability of E. coli, and other Gram-negative bacteria, to cause disease. Recently, we described the existence within uropathogenic E. coli (UPEC) strains of Sat, a toxin of the serine protease autotransporter of Enterobacteriaceae (SPATE) subfamily. Using features common to proteins secreted via the autotransporter pathway we have identified nine additional autotransporter proteins from the genomic sequence data of UPEC CFT073. Surprisingly, two additional members of the SPATE subfamily were identified. One protein, designated PicU, was homologous to the Pic protein identified in Shigella flexneri and enteroaggregative E. coli. The PicU protein was expressed and investigated for functional activity.     

Analysis of genome plasticity in pathogenic and commensal Escherichia coli isolates by use of DNA arrays

Genomes of prokaryotes differ significantly in size and DNA composition. Escherichia coli is considered a model organism to analyze the processes involved in bacterial genome evolution, as the species comprises numerous pathogenic and commensal variants. Pathogenic and nonpathogenic E. coli strains differ in the presence and absence of additional DNA elements contributing to specific virulence traits and also in the presence and absence of additional genetic information. To analyze the genetic diversity of pathogenic and commensal E. coli isolates, a whole-genome approach was applied. Using DNA arrays, the presence of all translatable open reading frames (ORFs) of nonpathogenic E. coli K-12 strain MG1655 was investigated in 26 E. coli isolates, including various extraintestinal and intestinal pathogenic E. coli isolates, 3 pathogenicity island deletion mutants, and commensal and laboratory strains. Additionally, the presence of virulence-associated genes of E. coli was determined using a DNA "pathoarray" developed in our laboratory. The frequency and distributional pattern of genomic variations vary widely in different E. coli strains. Up to 10% of the E. coli K-12-specific ORFs were not detectable in the genomes of the different strains. DNA sequences described for extraintestinal or intestinal pathogenic E. coli are more frequently detectable in isolates of the same origin than in other pathotypes. Several genes coding for virulence or fitness factors are also present in commensal E. coli isolates. Based on these results, the conserved E. coli core genome is estimated to consist of at least 3,100 translatable ORFs. The absence of K-12-specific ORFs was detectable in all chromosomal regions. These data demonstrate the great genome heterogeneity and genetic diversity among E. coli strains and underline the fact that both the acquisition and deletion of DNA elements are important processes involved in the evolution of prokaryotes.     

Variation in acid resistance among shiga toxin-producing clones of pathogenic Escherichia coli

Pathogenic strains of Escherichia coli, such as E. coli O157:H7, have a low infectious dose and an ability to survive in acidic foods. These bacteria have evolved at least three distinct mechanisms of acid resistance (AR), including two amino acid decarboxylase-dependent systems (arginine and glutamate) and a glucose catabolite-repressed system. We quantified the survival rates for each AR mechanism separately in clinical isolates representing three groups of Shiga toxin-producing E. coli (STEC) clones (O157:H7, O26:H11/O111:H8, and O121:H19) and six commensal strains from ECOR group A. Members of the STEC clones were not significantly more acid resistant than the commensal strains when analyzed using any individual AR mechanism. The glutamate system provided the best protection in a highly acidic environment for all groups of isolates (<0.1 log reduction in CFU/ml per hour at pH 2.0). Under these conditions, there was notable variation in survival rates among the 30 O157:H7 strains, which depended in part on Mg(2+) concentration. The arginine system provided better protection at pH 2.5, with a range of 0.03 to 0.41 log reduction per hour, compared to the oxidative system, with a range of 0.13 to 0.64 log reduction per hour. The average survival rate for the O157:H7 clonal group was significantly less than that of the other STEC clones in the glutamate and arginine systems and significantly less than that of the O26/O111 clone in the oxidative system, indicating that this clonal group is not exceptionally acid resistant with these specific mechanisms.     

Prevalence of CS31A and F165 surface antigens in Escherichia coli isolates from animals in France, Canada and India

Bovine and porcine enterotoxigenic and non-enterotoxigenic Escherichia coli isolates from France, Canada, and India were characterized with respect to serogroup and production of fimbrial antigens CS31A and F165. Of 231 bovine isolates from the 3 countries, 20.5% produced CS31A alone, 17.7% produced F165 alone, and 17.3% produced both CS31A and F165. On the other hand, of 84 porcine isolates from Canada, 1.2% produced CS31A alone, 14.3% produced F165 alone, and no isolate produced both CS31A and F165. CS31A was found together with F5 (K99) in 7 of 16 bovine enterotoxigenic E. coli isolates of serogroups 08, 09, 020, and 023, but was not found in any of 20 F4 (K88)- or 5 F6 (987P)-positive porcine enterotoxigenic E. coli isolates. F165 was not found in enterotoxigenic E. coli. Among non-enterotoxigenic isolates, CS31A and F165 were mainly associated with serogroups 08, 09, 011, 015, 017, 023, 025, 078, 0101, 0115, 0117, 0141, and 0153.     

The use of biochemical markers, serotype and fimbriation in the detection of Escherichia coli clones

Biochemical reactions, O and K serotypes and presence of P-fimbriae were analysed in 116 Escherichia coli strains isolated in blood cultures from patients with bacteraemia and in 99 faecal strains isolated from healthy individuals. By using biochemical typing, the strains could be grouped into six main clusters with similarity index less than 0.8 (Gower, 1971) and altogether 16 subclusters with similarity index 0.82-0.89. The most discriminating tests between the clusters were fermentation of D-tagatose, saccharose, salicin and sorbose. No single biochemical property could differentiate bacteraemic isolates from faecal strains, although strains isolated from blood were significantly more often found in certain subclusters, whereas other subclusters contained mainly control strains. Bacteraemic strains possessed P-fimbriae more often, especially strains isolated from patients with E. coli in the urine concomitantly with bacteraemia. Equally, no single reaction could separate P-fimbriated from non-P-fimbriated strains. D-Tagatose was fermented more often by the P-fimbriated strains; on the other hand, melibiose and lactose fermentation tests were less often positive. Certain O serotypes (O1, O4, O6, O7, O18 and O25) were more common among bacteraemic isolates than controls. K serotypes such as K1, K5 and K52 were also more frequent among blood isolates. We conclude that a combination of biochemical tests, fimbriation and serotyping might be used to identify potentially pathogenic clusters of E. coli.     

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.     

Distribution and characterization of integrons in Escherichia coli strains of animal and human origin

One hundred and twenty clinical and commensal Escherichia coli strains isolated in Switzerland from humans and from companion and farm animals were analysed for the prevalence of integrons of classes 1, 2, and 3 and for the characterization of their gene cassettes. The relationships between integron carriage and host category, and between integron carriage and phylogenetic E. coli lineage were also analysed. Integrons were detected in 48 (40%) of the isolates and were thus widely disseminated in the human and animal E. coli strains considered. Moreover, the association between integron carriage and certain animal categories (farm animals) suggests that animals that are raised for economic purposes might be exposed to a major antibiotic pressure. Finally, our data confirm that E. coli commensal strains represent a significant source of antibiotic-resistant determinants.