Clonal relationships among Escherichia coli serogroup O78 isolates from human and animal infections.

We investigated the clonal relationships among 63 Escherichia coli strains of antigen serogroup O78 isolated from infections in humans, cattle, sheep, pigs, and chickens. Both septicemic and enterotoxigenic isolates were included in the study. A main group of 55 E. coli strains consisting of 52 septicemic isolates and 3 human enterotoxigenic E. coli isolates were clustered in related clones. The remaining eight strains, four human and four animal isolates, were clonally heterogeneous. The main group of 55 clonally related strains included isolates from human and animal infections. This result indicates that animals are a possible source of serogroup O78 septicemic E. coli infections in humans.     

Replicon typing of virulence plasmids of enterotoxigenic Escherichia coli isolates from cattle.

Plasmid DNA hybridization with probes for virulence factors used for basic replicons of plasmids was used to identify the virulence plasmids of a collection of enterotoxigenic Escherichia coli isolates from cattle. The virulence probes were derived from the genes coding for the heat-stable enterotoxin STaP and for the F5 (K99) and F41 fimbrial adhesins. The replicon probes were derived from 16 different basic replicons of plasmids (probes repFIA, repFIB, repFIC, repFIIA, repI1, repHI1, repHI2, repL/M, repN, repP, repQ, repT, repU, repW, repX, and repY). The virulence genes coding for the STaP enterotoxin and for the F5 adhesin were located on a single plasmid band in each isolate. The sizes of most of these virulence plasmids were from 65 to 95 MDa. The F41 probe failed to hybridize with any plasmid band. The virulence plasmids had multireplicon types typical of plasmids of the IncF groups. The most common basic replicon association was the triple RepFIA-RepFIB-RepFIC family association.     

Colony incompatibility studies of enterotoxigenic Escherichia coli O126 isolated during one outbreak.

Strains of heat-stable enterotoxin-producing Escherichia coli belonging to O group O126 isolated during an outbreak of gastroenteritis were studied by the colony incompatibility phenomenon. They were compared with other isolates of O126. All of the outbreak strains except one appeared related in these studies and were different from all other strains studied. The one different outbreak strain was isolated 6 weeks after all of the others and may represent some new factor. The study demonstrated the potential usefulness of monitoring strains during an outbreak for relatedness.     

Clonal relationships and variation in virulence among Escherichia coli strains of avian origin

Average genetic relatedness among 44 Escherichia coli strains of serotypes O1, O2, and O78 isolated mainly from birds with colibacillosis or swollen-head syndrome from France or Saudi Arabia was estimated based on allelic variation detected by multilocus enzyme electrophoresis. For 20 enzyme-encoding loci, we resolved 2.8 alleles per locus and distinguished 17 electrophoretic types (ETs) that were used to mark naturally occurring cell lineages or clones. On average, ETs differed at 37% of their loci. Forty-eight percent of the isolates represent three ETs, two of which belong to previously defined complexes of clones identified in avian disease in North America and Europe. Virulence of strains, assessed in experimental infections of day-old chicks, showed little variation among isolates of a clone, but was significantly variable among isolates of different clone complexes. These findings add support to the evidence that a majority of avian isolates that cause colibacillosis belong to a few cosmopolitan pathogenic clones and indicate a substantial between-clone component of pathogenicity.     

Identification of a cluster of strains bearing a new adhesin among genetically diverse enterotoxigenic Escherichia coli isolates of serogroup O20

About one-third of the enterotoxigenic Escherichia coli isolates lack any of the known colonization factors. Among this group, those of serogroup O20 are the most frequently isolated in Argentina. By combining analysis of adhesion to Caco-2 cells, random amplified polymorphic DNA, and pulsed-field gel electrophoresis techniques, we were able to identify three sets of closely related strains with different binding properties. Further analysis of the most prevalent group revealed that all these isolates expressed the recently described adhesin CS22.     

Clonal relationships among bloodstream isolates of Escherichia coli.

The clonal relationships among 187 bloodstream isolates of Escherichia coli from 179 patients at Boston, Mass., Long Beach, Calif., and Nairobi, Kenya, were determined by multilocus enzyme electrophoresis (MLEE), analysis of polymorphisms associated with the ribosomal operon (ribotyping), and serotyping. MLEE based on 20 enzymes resolved 101 electrophoretic types (ETs), forming five clusters; ribotyping resolved 56 distinct patterns concordant with the analysis by MLEE. The isolates at each study site formed a genetically diverse group and demonstrated similar clonal structures, with the same small subset of lineages accounting for the majority of isolates at each site. Moreover, two ribotypes accounted for approximately 30% of the isolates at each study site. One cluster contained the majority (65%) of isolates and, by direct comparison of the ETs and ribotypes of individual isolates, was genetically indistinguishable from the largest cluster for each of two other collections of E. coli causing pyelonephritis and neonatal meningitis (R. K. Selander, T. K. Korhonen, V. Väisänen-Rhen, P. H. Williams, P. E. Pattison, and D. A. Caugent, Infect. Immun. 52:213-222, 1986; M. Arthur, C. E. Johnson, R. H. Rubin, R. D. Arbeit, C. Campanelli, C. Kim, S. Steinbach, M. Agarwal, R. Wilkinson, and R. Goldstein, Infect. Immun. 57:303-313, 1989), thus defining a virulent set of lineages. The isolates within these virulent lineages typically carried DNA homologous to the adhesin operon pap or sfa and the hemolysin operon hly and expressed O1, O2, O4, O6, O18, O25, or O75 antigens. DNA homologous to pap was distributed among isolates of each major cluster, whereas hly was restricted to isolates of two clusters, typically detected in pap-positive strains, and sfa was restricted to isolates of one cluster, typically detected in pap- and hly-positive strains. The occurrence of pap-positive isolates in the same geographically and genetically divergent lineages suggests that this operon was acquired early in the radiation of E. coli, while hly and sfa were acquired subsequently, most likely by pap-positive and pap- and hly-positive precursors, respectively.     

Evaluation of ribotyping as epidemiologic tool for typing Escherichia coli serogroup O157 isolates.

A total of 121 representative Escherichia coli O157:H7 and O157:NM (nonmotile) isolates were characterized by ribotype, phage type, verotoxin genotype, and genomic fingerprints generated by pulsed-field gel electrophoresis. Ribotyping was not able to discriminate between O157:H7 isolates, and phage typing and pulsed-field gel electrophoresis were the most valuable and discriminatory techniques.     

Virulence factors and biochemical characteristics of serotypes of Escherichia coli serogroup O29.

Escherichia coli strains belonging to serogroup O29 were studied. Invasiveness was the most common virulence factor described in this serogroup, but a few papers also reported production of heat-stable (ST) enterotoxin. In the present study invasive ability was found in O29:H- strains, whereas production of ST-I enterotoxin was observed only in serotype O29:H21 strains, showing that virulence was a characteristic of specific serotypes or bioserotypes within the O29 serogroup. Different serotypes were found among strains that were neither invasive nor toxigenic. Invasive strains were biochemically less active than the toxigenic ones and presented the invasiveness plasmid (pINV) of about 120 to 140 megadaltons, whereas hybridization tests showed that ST-I production was related to a plasmid of about 90 megadaltons. A diffuse adherence pattern to HeLa cells was observed in all ST-I isolates, but the role of this adherence in the pathogenicity of these strains was not determined. Thus, a unique biochemical pattern and plasmid profile may be useful characteristics to distinguish between pathogenic (toxigenic or invasive) and nonpathogenic O29 strains.     

Enterotoxigenic Escherichia coli strains belonging to a new serogroup, Escherichia coli O166.

Thirty-two strains of Escherichia coli belonging to a new O group, O166, were examined. Twenty-one strains had the flagella antigen H27, five had the H15 antigen, five had the H7 antigen, and one was nonmotile. All the H27 strains and the nonmotile strain produced heat-stable enterotoxin but not heat-labile enterotoxin. All the H7 strains produced heat-labile enterotoxin but not heat-stable enterotoxin. The remaining strains were nonenterotoxigenic. None of the strains possessed colonization factor antigens CFA/I, CFA/II, or PCF8775.     

Common virulence factors and genetic relationships between O18:K1:H7 Escherichia coli isolates of human and avian origin

Extraintestinal pathogenic (ExPEC) Escherichia coli strains of serotype O18:K1:H7 are mainly responsible for neonatal meningitis and sepsis in humans and belong to a limited number of closely related clones. The same serotype is also frequently isolated from the extraintestinal lesions of colibacillosis in poultry, but it is not well known to what extent human and avian strains of this particular serotype are related. Twenty-two ExPEC isolates of human origin and 33 isolates of avian origin were compared on the basis of their virulence determinants, lethality for chicks, pulsed-field gel electrophoresis (PFGE) patterns, and classification in the main phylogenetic groups. Both avian and human isolates were lethal for chicks and harbored similar virulence genotypes. A major virulence pattern, identified in 75% of the isolates, was characterized by the presence of F1 variant fimbriae; S fimbriae; IbeA; the aerobactin system; and genomic fragments A9, A12, D1, D7, D10, and D11 and by the absence of P fimbriae, F1C fimbriae, Afa adhesin, and CNF1. All but one of the avian and human isolates also belonged to major phylogenetic group B2. However, various subclonal populations could be distinguished by PFGE in relation to animal species and geographical origin. These results demonstrate that very closely related clones can be recovered from extraintestinal infections in humans and chickens and suggest that avian pathogenic E. coli isolates of serotype O18:K1:H7 are potential human pathogens.     

Mutual enhancement of virulence by enterotoxigenic and enteropathogenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) and enteropathogenic E. coli (EPEC) are common causes of diarrhea in children in developing countries. Dual infections with both pathogens have been noted fairly frequently in studies of diarrhea around the world. In previous laboratory work, we noted that cholera toxin and forskolin markedly potentiated EPEC-induced ATP release from the host cell, and this potentiated release was found to be mediated by the cystic fibrosis transmembrane conductance regulator. In this study, we examined whether the ETEC heat-labile toxin (LT) or the heat-stable toxin (STa, also known as ST) potentiated EPEC-induced ATP release. We found that crude ETEC culture filtrates, as well as purified ETEC toxins, did potentiate EPEC-induced ATP release in cultured T84 cells. Coinfection of T84 cells with live ETEC plus EPEC bacteria also resulted in enhanced ATP release compared to EPEC alone. In Ussing chamber studies of chloride secretion, adenine nucleotides released from the host by EPEC also significantly enhanced the chloride secretory responses that were triggered by crude ETEC filtrates, purified STa, and the peptide hormone guanylin. In addition, adenosine and LT had additive or synergistic effects in inducing vacuole formation in T84 cells. Therefore, ETEC toxins and EPEC-induced damage to the host cell both enhance the virulence of the other type of E. coli. Our in vitro data demonstrate a molecular basis for a microbial interaction, which could result in increased severity of disease in vivo in individuals who are coinfected with ETEC and EPEC.     

Decreased prevalence of virulence factors among ciprofloxacin-resistant uropathogenic Escherichia coli isolates

Ciprofloxacin resistance was identified in 18% and 6% of consecutively collected, clinically significant urinary tract isolates of Escherichia coli from inpatients and outpatients, respectively. In comparison to ciprofloxacin-susceptible isolates, there were fewer resistant isolates that expressed beta-hemolysis (outpatient, 9% versus 87%, P < 0.0001; inpatient, 4% versus 76%, P < 0.0001) and that had a papEF genotype, genes encoding P fimbriae (outpatient, 30% versus 70%, P = 0.0004; inpatient, 26% versus 70%, P < 0.0001).     

Distribution of virulence factors,antimicrobial resistance genes and phylogenetic relatedness among Shiga toxin-producing Escherichia coli serogroup O91 from human infections

Shiga toxin-producing Escherichia coli (STEC) belonging to the serogroup O91 are among the most common non-O157 STEC serogroups associated with human illness in Europe. This study aimed to analyse the virulence factors, antimicrobial resistance genes and phylogenetic relatedness among 48 clinical STEC O91 isolates collected during 2003–2019 in Switzerland. The isolates were subjected to whole genome sequencing using short-read sequencing technologies and a subset of isolates additionally to long-read sequencing. They belonged to O91:H10 (n = 6), O91:H14 (n = 40), and O91:H21 (n = 2). Multilocus sequence typing showed that the O91:H10 isolates all belonged to sequence type (ST)641, while the O91:H14 isolates were assigned to ST33, ST9700, or were non-typeable. Both O91:H21 isolates belonged to ST442. Shiga toxin gene stx1a was the most common Shiga toxin gene subtype among the isolates, followed by stx2b, stx2d and stx2a. All isolates were LEE-negative and carried one or two copies of the IrgA adhesin gene iha. In a subset of long-read sequenced isolates, modules of the Locus of Adhesion and Autoaggregation pathogenicity island (LAA-PAI) carrying iha and other genes such as hes, lesP or agn43 were identified. A large proportion of STEC O91:H14 carried the subtilase cytotoxin gene subA, colicin genes (cba, cea, cib and cma) or microcin genes (mcmA, mchB, mchC and mchF). STEC O91:H14 were further distinguished from STEC O91:H10/H21 by one or more virulence factors found in extraintestinal pathogenic E. coli (ExPEC), including hlyF, iucC/iutA, kpsE and traT. The hlyF gene was identified on a novel mosaic plasmid that was unrelated to hlyF+ plasmids described previously in STEC. Core genome phylogenetic analysis revealed that STEC O91:H10 and STEC O91:H21 were clonally conserved, whereas STEC O91:H14 were clonally diverse. Among three STEC O91:H14 isolates, a number of resistance genes were identified, including genes that mediate resistance to aminoglycosides (aadA, aadA2, aadA9, aadA23, aph(3'')-Ib and aph(6)-Id), chloramphenicol (cmlA), sulphonamides (sul2 and sul3), and trimethoprim (drfA12). Our data contribute to understanding the genetic diversity and differing levels of virulence potential within the STEC O91 serogroup.     

Genetic characterization and virulence of enterotoxigenic Escherichia coli mutants which have lost virulence genes in vivo.

Loss of K99 and STaP genes from enterotoxigenic Escherichia coli 431 during infection occurred by either plasmid curing or plasmid deletion. These mutants expressed the F41 adhesion and colonized neonatal pigs, but only those mutants that retained STaP caused diarrhea with significant weight loss.     

Virulence genotypes and phylogenetic background of Escherichia coli serogroup O6 isolates from humans, dogs, and cats

Molecular evidence is limited for the hypothesis that humans, dogs, and cats can become colonized and infected with similar virulent Escherichia coli strains. To further assess this possibility, archived E. coli O6 isolates (n = 130) from humans (n = 55), dogs (n = 59), and cats (n = 16), representing the three main H (flagellar) types within serogroup O6 (H1, H7, and H31), were analyzed, along with selected reference strains. Isolates underwent PCR-based phylotyping, multilocus sequence typing, PCR-based detection of 55 virulence-associated genes, and XbaI pulsed-field gel electrophoresis (PFGE) profiling. Three major sequence types (STs), which corresponded closely with H types, accounted for 99% of the 130 O6 isolates. Each ST included human, dog, and cat isolates; two included reference pyelonephritis isolates CFT073 (O6:K2:H1) and 536 (O6:K15:H31). Virulence genotypes overlapped considerably among host species, despite statistically significant differences between human and pet isolates. Several human and dog isolates from ST127 (O6:H31) exhibited identical virulence genotypes and highly similar PFGE profiles, consistent with cross-species exchange of specific E. coli clones. In conclusion, the close similarity in the genomic backbone and virulence genotype between certain human- and animal-source E. coli isolates within serogroup O6 supports the hypothesis of zoonotic potential.     

Identification of enterotoxigenic Escherichia coli isolates with enzyme-labeled synthetic oligonucleotide probes.

Commercially available kits containing alkaline phosphatase-labeled oligonucleotide probes for Escherichia coli heat-stable enterotoxins (STI-H, STI-P, and STII) and the heat-labile enterotoxin were compared with bioassays and radiolabeled recombinant DNA probes to identify enterotoxigenic E. coli from 100 clinical isolates. There was very good agreement between the three methods.     

Colonization factors associated with enterotoxigenic Escherichia coli isolated in Thailand.

Eighty-six percent (72 of 84) of heat-labile and heat-stable, none of 141 heat-labile, and 24% (27 of 111) heat-stable enterotoxigenic Escherichia coli isolates from Thailand aggregated in less than 1 M (NH4)2SO4, hemagglutinated human group A and bovine erythrocytes in 1% D-mannose, and possessed either colonization factor I or colonization factor II. No other colonization factors were identified by these two methods.