Prevalence of avian-pathogenic Escherichia coli strain O1 genomic islands among extraintestinal and commensal E. coli isolates

Escherichia coli strains that cause disease outside the intestine are known as extraintestinal pathogenic E. coli (ExPEC) and include pathogens of humans and animals. Previously, the genome of avian-pathogenic E. coli (APEC) O1:K1:H7 strain O1, from ST95, was sequenced and compared to those of several other E. coli strains, identifying 43 genomic islands. Here, the genomic islands of APEC O1 were compared to those of other sequenced E. coli strains, and the distribution of 81 genes belonging to 12 APEC O1 genomic islands among 828 human and avian ExPEC and commensal E. coli isolates was determined. Multiple islands were highly prevalent among isolates belonging to the O1 and O18 serogroups within phylogenetic group B2, which are implicated in human neonatal meningitis. Because of the extensive genomic similarities between APEC O1 and other human ExPEC strains belonging to the ST95 phylogenetic lineage, its ability to cause disease in a rat model of sepsis and meningitis was assessed. Unlike other ST95 lineage strains, APEC O1 was unable to cause bacteremia or meningitis in the neonatal rat model and was significantly less virulent than uropathogenic E. coli (UPEC) CFT073 in a mouse sepsis model, despite carrying multiple neonatal meningitis E. coli (NMEC) virulence factors and belonging to the ST95 phylogenetic lineage. These results suggest that host adaptation or genome modifications have occurred either in APEC O1 or in highly virulent ExPEC isolates, resulting in differences in pathogenicity. Overall, the genomic islands examined provide targets for further discrimination of the different ExPEC subpathotypes, serogroups, phylogenetic types, and sequence types.     

Genotypic and phenotypic traits that distinguish neonatal meningitis-associated Escherichia coli from fecal E. coli isolates of healthy human hosts

Neonatal meningitis Escherichia coli (NMEC) is one of the top causes of neonatal meningitis worldwide. Here, 85 NMEC and 204 fecal E. coli isolates from healthy humans (HFEC) were compared for possession of traits related to virulence, antimicrobial resistance, and plasmid content. This comparison was done to identify traits that typify NMEC and distinguish it from commensal strains to refine the definition of the NMEC subpathotype, identify traits that might contribute to NMEC pathogenesis, and facilitate choices of NMEC strains for future study. A large number of E. coli strains from both groups were untypeable, with the most common serogroups occurring among NMEC being O18, followed by O83, O7, O12, and O1. NMEC strains were more likely than HFEC strains to be assigned to the B2 phylogenetic group. Few NMEC or HFEC strains were resistant to antimicrobials. Genes that best discriminated between NMEC and HFEC strains and that were present in more than 50% of NMEC isolates were mainly from extraintestinal pathogenic E. coli genomic and plasmid pathogenicity islands. Several of these defining traits had not previously been associated with NMEC pathogenesis, are of unknown function, and are plasmid located. Several genes that had been previously associated with NMEC virulence did not dominate among the NMEC isolates. These data suggest that there is much about NMEC virulence that is unknown and that there are pitfalls to studying single NMEC isolates to represent the entire subpathotype.     

肠外致病性大肠杆菌致病机制及公共卫生学意义

致病性大肠杆菌包括肠致病性大肠杆菌(intestinal pathogenic Escherichia coli, IPEC)和肠外致病性大肠杆菌(extraintestinalpathogenicE.coli,ExPEC),可引起人和动物多种感染性疾病。ExPEC主要在肠道外其他组织脏器定殖并导致感染,包括尿道致病性大肠杆菌(uropathogenicE.coli, UPEC)、新生儿脑膜炎大肠杆菌(newborn meningitis E. coli, NMEC)和禽致病性大肠杆菌(avian pathogenic E. coli, APEC)。人源ExPEC (UPEC和NMEC)主要引起人尿道感染、肾盂肾炎和新生儿脑膜炎,而APEC可导致禽类的大肠杆菌病,造成家禽业的巨大经济损失。另外,乳腺致病性大肠杆菌(mammary pathogenic E. coli, MPEC)和猪源ExPEC可导致奶牛乳房炎、猪的肺炎及急性败血症等病症。研究发现,ExPEC类菌株在基因组结构上很相似,与IPEC本质区别在于致病机制不同,ExPEC具有很多相同的毒力基因和耐药基因,而且动物源ExPEC...     

Zoonotic Potential of Escherichia coli Isolates from Retail Chicken Meat Products and Eggs

Chicken products are suspected as a source of extraintestinal pathogenic Escherichia coli (ExPEC), which causes diseases in humans. The zoonotic risk to humans from chicken-source E. coli is not fully elucidated. To clarify the zoonotic risk posed by ExPEC in chicken products and to fill existing knowledge gaps regarding ExPEC zoonosis, we evaluated the prevalence of ExPEC on shell eggs and compared virulence-associated phenotypes between ExPEC and non-ExPEC isolates from both chicken meat and eggs. The prevalence of ExPEC among egg-source isolates was low, i.e., 5/108 (4.7%). Based on combined genotypic and phenotypic screening results, multiple human and avian pathotypes were represented among the chicken-source ExPEC isolates, including avian-pathogenic E. coli (APEC), uropathogenic E. coli (UPEC), neonatal meningitis E. coli (NMEC), and sepsis-associated E. coli (SEPEC), as well as an undefined ExPEC group, which included isolates with fewer virulence factors than the APEC, UPEC, and NMEC isolates. These findings document a substantial prevalence of human-pathogenic ExPEC-associated genes and phenotypes among E. coli isolates from retail chicken products and identify key virulence traits that could be used for screening.     

Virulence genotypes of Escherichia coli strains from bacteremia in relation to phylogeny

Bacteremia is the principal way of dissemination of local infections to distant organs. Escherichia coli bacteremia is almost always clinically significant, suggesting an increased risk of developing sepsis syndrome. Fifty-one E. coli bloodstream human isolates were analyzed using PCR technique for several molecular markers associated with extraintestinal virulence, and their phylogenetic group assignment, taking into account the link between the phylogenetic background and the intrinsic virulence of this species. Sixteen virulence genotypes have been identified, the majority of the blood isolates carrying the association of two genes. The genes encoding type 1 fimbria and aerobactin had the highest prevalence. As a confirmation of other studies, the strains assigned to E. coli phylogenetic group B2 exhibited the highest concentration of virulence genes, and represented almost half of the clinical blood isolates. The multifactorial virulence of E. coli strains isolated from invasive infections reflects a phylogenetic inheritance, and supports the concept of ExPEC pathotype as a subset of E. coli population involved in human infectious diseases. The surveillance of geographical variation of E. coli pathogenic clones is useful for epidemiological analysis.     

A probable aculeacin A acylase from the Ralstonia solanacearum GMI1000 is N-acyl-homoserine lactone acylase with quorum-quenching activity

Background

Extraintestinal pathogenic Escherichia coli (ExPEC) strains of serotype O1:K1:H7/NM are frequently implicated in neonatal meningitis, urinary tract infections and septicemia in humans. They are also commonly isolated from colibacillosis in poultry. Studies to determine the similarities of ExPEC from different origins have indicated that avian strains potentially have zoonotic properties.

Results

A total of 59 ExPEC O1:K1:H7/NM isolates (21 from avian colibacillosis, 15 from human meningitis, and 23 from human urinary tract infection and septicemia) originated from four countries were characterized by phylogenetic PCR grouping, Multilocus Sequence Typing (MLST), Pulsed Field Gel Electrophoresis (PFGE) and genotyping based on several genes known for their association with ExPEC or avian pathogenic Escherichia coli (APEC) virulence. APEC and human ExPEC isolates differed significantly in their assignments to phylogenetic groups, being phylogroup B2 more prevalent among APEC than among human ExPEC (95% vs. 53%, P = 0.001), whereas phylogroup D was almost exclusively associated with human ExPEC (47% vs. 5%, P = 0.0000). Seven virulence genes showed significant differences, being fimAv _MT78 and sat genes linked to human isolates, while papGII, tsh, iron, cvaC and iss were significantly associated to APEC. By MLST, 39 of 40 ExPEC belonging to phylogroup B2, and 17 of 19 belonging to phylogroup D exhibited the Sequence Types (STs) ST95 and ST59, respectively. Additionally, two novel STs (ST1013 and ST1006) were established. Considering strains sharing the same ST, phylogenetic group, virulence genotype and PFGE cluster to belong to the same subclone, five subclones were detected; one of those grouped six strains of human and animal origin from two countries.

Conclusion

Present results reveal that the clonal group B2 O1:K1:H7/NM ST95, detected in strains of animal and human origin, recovered from different dates and geographic sources, provides evidence that some APEC isolates may act as potential pathogens for humans and, consequently, poultry as a foodborne source, suggesting no host specificity for this type of isolates. A novel and important finding has been the detection of the clonal group D O1:K1:H7/NM ST59 almost exclusively in humans, carrying pathogenic genes linked to the phylogenetic group D. This finding would suggest D O1:K1:H7/NM ST59 as a host specific pathotype for humans.     

Evolution of the iss gene in Escherichia coli

The increased serum survival gene iss has long been recognized for its role in extraintestinal pathogenic Escherichia coli (ExPEC) virulence. iss has been identified as a distinguishing trait of avian ExPEC but not of human ExPEC. This gene has been localized to large virulence plasmids and shares strong similarities with the bor gene from bacteriophage lambda. Here, we demonstrate that three alleles of iss occur among E. coli isolates that appear to have evolved from a common lambda bor precursor. In addition to the occurrence of iss on the ColV/BM virulence plasmids, at least two iss alleles occur within the E. coli chromosome. One of these alleles (designated type 3) was found to occur in the genomes of all currently sequenced ExPEC strains on a similar prophage element that also harbors the Sit iron and manganese transport system. When the prevalence of the three iss types was examined among 487 E. coli isolates, the iss type 3 gene was found to occur at a high frequency among ExPEC isolates, irrespective of the host source. The plasmid-borne iss allele (designated type 1) was highly prevalent among avian pathogenic E. coli and neonatal meningitis-associated E. coli isolates but not among uropathogenic E. coli isolates. This study demonstrates the evolution of iss in E. coli and provides an additional tool for discriminating among E. coli pathotypes through the differentiation of the three iss allele types and bor.     

Comparative Genomic Analysis Shows That Avian Pathogenic Escherichia coli Isolate IMT5155 (O2:K1:H5; ST Complex 95, ST140) Shares Close Relationship with ST95 APEC O1:K1 and Human ExPEC O18:K1 Strains

Avian pathogenic E. coli and human extraintestinal pathogenic E. coli serotypes O1, O2 and O18 strains isolated from different hosts are generally located in phylogroup B2 and ST complex 95, and they share similar genetic characteristics and pathogenicity, with no or minimal host specificity. They are popular objects for the study of ExPEC genetic characteristics and pathogenesis in recent years. Here, we investigated the evolution and genetic blueprint of APEC pathotype by performing phylogenetic and comparative genome analysis of avian pathogenic E. coli strain IMT5155 (O2:K1:H5; ST complex 95, ST140) with other E. coli pathotypes. Phylogeny analyses indicated that IMT5155 has closest evolutionary relationship with APEC O1, IHE3034, and UTI89. Comparative genomic analysis showed that IMT5155 and APEC O1 shared significant genetic overlap/similarities with human ExPEC dominant O18:K1 strains (IHE3034 and UTI89). Furthermore, the unique PAI I₅₁₅₅ (GI-12) was identified and found to be conserved in APEC O2 serotype isolates. GI-7 and GI-16 encoding two typical T6SSs in IMT5155 might be useful markers for the identification of ExPEC dominant serotypes (O1, O2, and O18) strains. IMT5155 contained a ColV plasmid p1ColV₅₁₅₅, which defined the APEC pathotype. The distribution analysis of 10 sequenced ExPEC pan-genome virulence factors among 47 sequenced E. coli strains provided meaningful information for B2 APEC/ExPEC-specific virulence factors, including several adhesins, invasins, toxins, iron acquisition systems, and so on. The pathogenicity tests of IMT5155 and other APEC O1:K1 and O2:K1 serotypes strains (isolated in China) through four animal models showed that they were highly virulent for avian colisepticemia and able to cause septicemia and meningitis in neonatal rats, suggesting zoonotic potential of these APEC O1:K1 and O2:K1 isolates.     

Intestine and Environment of the Chicken as Reservoirs for Extraintestinal Pathogenic Escherichia coli Strains with Zoonotic Potential

Although research has increasingly focused on the pathogenesis of avian pathogenic Escherichia coli (APEC) infections and the “APEC pathotype” itself, little is known about the reservoirs of these bacteria. We therefore compared outbreak strains isolated from diseased chickens (n = 121) with nonoutbreak strains, including fecal E. coli strains from clinically healthy chickens (n = 211) and strains from their environment (n = 35) by determining their virulence gene profiles, phylogenetic backgrounds, responses to chicken serum, and in vivo pathogenicities in a chicken infection model. In general, by examining 46 different virulence-associated genes we were able to distinguish the three groups of avian strains, but some specific fecal and environmental isolates had a virulence gene profile that was indistinguishable from that determined for outbreak strains. In addition, a substantial number of phylogenetic EcoR group B2 strains, which are known to include potent human and animal extraintestinal pathogenic E. coli (ExPEC) strains, were identified among the APEC strains (44.5%) as well as among the fecal E. coli strains from clinically healthy chickens (23.2%). Comparably high percentages (79.2 to 89.3%) of serum-resistant strains were identified for all three groups of strains tested, bringing into question the usefulness of this phenotype as a principal marker for extraintestinal virulence. Intratracheal infection of 5-week-old chickens corroborated the pathogenicity of a number of nonoutbreak strains. Multilocus sequence typing data revealed that most strains that were virulent in chicken infection experiments belonged to sequence types that are almost exclusively associated with extraintestinal diseases not only in birds but also in humans, like septicemia, urinary tract infection, and newborn meningitis, supporting the hypothesis that not the ecohabitat but the phylogeny of E. coli strains determines virulence. These data provide strong evidence for an avian intestinal reservoir hypothesis which could be used to develop intestinal intervention strategies. These strains pose a zoonotic risk because either they could be transferred directly from birds to humans or they could serve as a genetic pool for ExPEC strains.     

The genome sequence of avian pathogenic Escherichia coli strain O1:K1:H7 shares strong similarities with human extraintestinal pathogenic E. coli genomes

Escherichia coli strains that cause disease outside the intestine are known as extraintestinal pathogenic E. coli (ExPEC) and include human uropathogenic E. coli (UPEC) and avian pathogenic E. coli (APEC). Regardless of host of origin, ExPEC strains share many traits. It has been suggested that these commonalities may enable APEC to cause disease in humans. Here, we begin to test the hypothesis that certain APEC strains possess potential to cause human urinary tract infection through virulence genotyping of 1,000 APEC and UPEC strains, generation of the first complete genomic sequence of an APEC (APEC O1:K1:H7) strain, and comparison of this genome to all available human ExPEC genomic sequences. The genomes of APEC O1 and three human UPEC strains were found to be remarkably similar, with only 4.5% of APEC O1's genome not found in other sequenced ExPEC genomes. Also, use of multilocus sequence typing showed that some of the sequenced human ExPEC strains were more like APEC O1 than other human ExPEC strains. This work provides evidence that at least some human and avian ExPEC strains are highly similar to one another, and it supports the possibility that a food-borne link between some APEC and UPEC strains exists. Future studies are necessary to assess the ability of APEC to overcome the hurdles necessary for such a food-borne transmission, and epidemiological studies are required to confirm that such a phenomenon actually occurs.     

Occurrence of virulence and antimicrobial resistance genes in Escherichia coli isolates from different aquatic ecosystems within the St. Clair River and Detroit River areas

Although the number of Escherichia coli bacteria in surface waters can differ greatly between locations, relatively little is known about the distribution of E. coli pathotypes in surface waters used as sources for drinking or recreation. DNA microarray technology is a suitable tool for this type of study due to its ability to detect high numbers of virulence and antimicrobial resistance genes simultaneously. Pathotype, phylogenetic group, and antimicrobial resistance gene profiles were determined for 308 E. coli isolates from surface water samples collected from diverse aquatic ecosystems at six different sites in the St. Clair River and Detroit River areas. A higher frequency (48%) of E. coli isolates possessing virulence and antimicrobial resistance genes was observed in an urban site located downstream of wastewater effluent outfalls than in the other examined sites (average of 24%). Most E. coli pathotypes were extraintestinal pathogenic E. coli (ExPEC) pathotypes and belonged to phylogenetic groups B2 and D. The ExPEC pathotypes were found to occur across all aquatic ecosystems investigated, including riverine, estuarine, and offshore lake locations. The results of this environmental study using DNA microarrays highlight the widespread distribution of E. coli pathotypes in aquatic ecosystems and the potential public health threat of E. coli pathotypes originating from municipal wastewater sources.     

Proteome response of an extraintestinal pathogenic Escherichia coli strain with zoonotic potential to human and chicken sera

A subset of extraintestinal pathogenic Escherichia coli is zoonotic and has developed strategies to adapt to different host-specific environments. However, the underlying mechanisms of these adaptive strategies have yet to be discerned. Here, the proteomic response of an avian pathogenic E. coli strain, which appears indistinguishable from neonatal meningitis E. coli, was compared following growth in human and avian sera to determine whether it uses the same mechanisms to overcome the antibacterial effects of sera from different host species. Proteins involved in biosynthesis of iron receptors were up-regulated under both sera, suggesting that serum, regardless of the host of origin, is an iron-limited environment. However, several proteins involved in synthesis of nucleic acids, sulfur-containing amino acids and fatty acids, were differentially expressed in response to the sera from different hosts. Mutational analysis showed that this APEC strain required nucleotide biosynthesis during incubation in human, but not avian serum, and deletion of genes involved in the biosynthesis of sulfur-containing amino acids increased its resistance to human serum. Continued investigation of the proteome of 'zoonotic' ExPEC strains, grown under other 'dual' host conditions, will contribute to our understanding of ExPEC pathogenesis and host specificity and development of effective therapies and control strategies.     

Phylogenetic relationships among clonal groups of extraintestinal pathogenic Escherichia coli as assessed by multi-locus sequence analysis

The evolutionary origins of extraintestinal pathogenic Escherichia coli (ExPEC) remain uncertain despite these organisms' relevance to human disease. A valid understanding of ExPEC phylogeny is needed as a framework against which the observed distribution of virulence factors and clinical associations can be analyzed. Accordingly, phylogenetic relationships were defined by multi-locus sequence analysis among 44 representatives of selected ExPEC clonal groups and the E. coli Reference (ECOR) collection. Recombination, which significantly obscured the phylogenetic signal for several strains, was dealt with by excluding strains or specific sequences. Conflicting overall phylogenies, and internal phylogenies for virulence-associated phylogenetic group B2, were inferred depending on the specific dataset (i.e., how extensively purged of recombination), outgroup (Salmonella enterica and/or Escherichia fergusonii), and analysis method (neighbor joining, maximum parsimony, maximum likelihood, or Bayesian likelihood). Nonetheless, the major E. coli phylogenetic groups A, B1, and B2 were consistently well resolved, as was a major sub-component of group D and an ECOR 37-O157:H7 clade. Moreover, nine important ExPEC clonal groups within groups B2 and D, characterized by serotypes O6:K2:H1, O18:K1:H7, O6:H31, and O4:K+:H+ (from group B2), and O1:K1:H-, O7:K1:H-, O157:K+:H (non-7), O15:K52:H1, and O11/17/77:K52:H18 ("clonal group A") (from group D), were consistently well resolved, regardless of clinical background (cystitis, pyelonephritis, neonatal meningitis, sepsis, or fecal), host group, geographical origin, and virulence profile. Among the group B2-derived clonal groups the O6:K2:H1 clade appeared basal. Within group D, "clonal group A" and the O15:K52:H1 clonal group were consistently placed with ECOR 47 and ECOR 44, respectively, as nearest neighbors. These findings clarify phylogenetic relationships among key ExPEC clonal groups but also emphasize that recombination appears to obscure the oldest evolutionary relationships, despite extensive targeted sequencing and use of a wide range of analysis techniques.     

New Role for the ibeA Gene in H2O2 Stress Resistance of Escherichia coli

ibeA is a virulence factor found in some extraintestinal pathogenic Escherichia coli (ExPEC) strains from the B2 phylogenetic group and particularly in newborn meningitic and avian pathogenic strains. It was shown to be involved in the invasion process of the newborn meningitic strain RS218. In a previous work, we showed that in the avian pathogenic E. coli (APEC) strain BEN2908, isolated from a colibacillosis case, ibeA was rather involved in adhesion to eukaryotic cells by modulating type 1 fimbria synthesis (M. A. Cortes et al., Infect. Immun. 76:4129-4136, 2008). In this study, we demonstrate a new role for ibeA in oxidative stress resistance. We showed that an ibeA mutant of E. coli BEN2908 was more sensitive than its wild-type counterpart to H(2)O(2) killing. This phenotype was also observed in a mutant deleted for the whole GimA genomic region carrying ibeA and might be linked to alterations in the expression of a subset of genes involved in the oxidative stress response. We also showed that RpoS expression was not altered by the ibeA deletion. Moreover, the transfer of an ibeA-expressing plasmid into an E. coli K-12 strain, expressing or not expressing type 1 fimbriae, rendered it more resistant to an H(2)O(2) challenge. Altogether, these results show that ibeA by itself is able to confer increased H(2)O(2) resistance to E. coli. This feature could partly explain the role played by ibeA in the virulence of pathogenic strains.     

Adherent-invasive Escherichia coli phenotype displayed by intestinal pathogenic E. coli strains from cats, dogs, and swine

The adherent-invasive Escherichia coli (AIEC) pathotype, which has been associated with Crohn's disease, shows similar traits to human and animal extraintestinal pathogenic E. coli (ExPEC) with respect to their phylogenetic origin and virulence gene profiles. Here, we demonstrate that animal ExPEC strains generally do not share the AIEC phenotype. In contrast, this phenotype is very frequent among animal intestinal pathogenic E. coli (InPEC) strains, particularly of feline and canine origin, that genetically resemble ExPEC. These results strengthen the particular identity and disease specificity of the AIEC pathotype and the putative role animals might play in the transmission of AIEC-like strains to humans.     

Genotypic analyses of Escherichia coli isolated from chickens with colibacillosis and apparently healthy chickens in Japan

A genotypic comparison using pulsed-field gel electrophoresis (PFGE), amplified ribosomal restriction analysis (ARDRA) as well as PCRs targeting virulence associated genes reported elsewhere in avian pathogenic Escherichia coli(APEC) was made between E. coli strains isolated from chickens with colibacillosis and those from the feces of apparently healthy chickens in Japan. The majority (67%) of clinical isolates belonged to a certain phylogenetic ARDRA but not PFGE cluster, with virulence-related genes carried by ColV plasmid being markedly prevalent. The result suggests that APEC strains originated from the same "ancestor" in the course of E. coli evolution.     

ExPEC-typical virulence-associated genes correlate with successful colonization by intestinal E. coli in a small piglet group

Upon studying the transmission of Escherichia coli from a sow to five of her piglets, we observed domination of the coliform flora in piglets by a single E. coli clone, especially after weaning. This haemolytic cloneH1 did not harbour any virulence determinants typical for intestinal pathogenic E. coli isolates from swine but had a virulence gene profile very similar to extraintestinal E. coli (ExPEC), including genes coding for P fimbriae and several iron acquisition systems, besides having an affiliation to the phylogenetic B2 group. Overall, we show that the presence of higher numbers of ExPEC-typical virulence-associated genes (VAGs) in clones correlate with their successful colonization ability in piglets. We conclude that VAGs typical for ExPEC also support intestinal colonization in healthy pigs. Faeces of healthy domestic pigs can harbour high numbers of ExPEC-similar E. coli and are suggested to be a potential risk for the transmission of such bacteria to other hosts.     

Genotypes and Pathogenicity of Cellulitis Isolates Reveal Traits That Modulate APEC Virulence

We characterized 144 Escherichia coli isolates from severe cellulitis lesions in broiler chickens from South Brazil. Analysis of susceptibility to 15 antimicrobials revealed frequencies of resistance of less than 30% for most antimicrobials except tetracycline (70%) and sulphonamides (60%). The genotyping of 34 virulence-associated genes revealed that all the isolates harbored virulence factors related to adhesion, iron acquisition and serum resistance, which are characteristic of the avian pathogenic E. coli (APEC) pathotype. ColV plasmid-associated genes (cvi/cva, iroN, iss, iucD, sitD, traT, tsh) were especially frequent among the isolates (from 66.6% to 89.6%). According to the Clermont method of ECOR phylogenetic typing, isolates belonged to group D (47.2%), to group A (27.8%), to group B2 (17.4%) and to group B1 (7.6%); the group B2 isolates contained the highest number of virulence-associated genes. Clonal relationship analysis using the ARDRA method revealed a similarity level of 57% or higher among isolates, but no endemic clone. The virulence of the isolates was confirmed in vivo in one-day-old chicks. Most isolates (72.9%) killed all infected chicks within 7 days, and 65 isolates (38.1%) killed most of them within 24 hours. In order to analyze differences in virulence among the APEC isolates, we created a pathogenicity score by combining the times of death with the clinical symptoms noted. By looking for significant associations between the presence of virulence-associated genes and the pathogenicity score, we found that the presence of genes for invasins ibeA and gimB and for group II capsule KpsMTII increased virulence, while the presence of pic decreased virulence. The fact that ibeA, gimB and KpsMTII are characteristic of neonatal meningitis E. coli (NMEC) suggests that genes of NMEC in APEC increase virulence of strains.     

Colonisation dynamics and virulence of two clonal groups of multidrug-resistant Escherichia coli isolated from dogs

The study established the virulence potential of multidrug-resistant Escherichia coli (MDREC) isolates from nosocomial infections in hospitalised dogs. The isolates were resistant to fluoroquinolones, belonged to two distinct clonal groups (CG1 and CG2) and contained a plasmid-mediated AmpC (CMY-7) β-lactamase. CG1 isolates (n = 14) possessed two of 36 assayed extraintestinal virulence genes (iutA and traT) and belonged to phylogenetic group A, whereas CG2 isolates (n = 19) contained four such genes (iutA, ibeA, fimH and kpsMT K5) and belonged to group D. In a mouse gastrointestinal tract colonisation model, colonisation by index CG1 strain C1 was transient, in contrast to the index CG2 strain C2b, which persisted up to 40 days post-inoculation. In a mouse subcutaneous challenge model, both strains were less virulent than archetypal group B2 extraintestinal pathogenic E. coli (ExPEC) strain CFT073; strain C1 caused no systemic signs and strain C2b was lethal to only one of six mice. In a mouse urinary tract infection model, strain C2b colonised the mouse bladder over 2 logs higher compared to strain C1. Whilst both groups of canine MDREC appear less virulent than a reference human ExPEC strain, CG2 strains have greater capacity for colonisation and virulence.     

Detection of cytotoxic necrotizing factor types 1 and 2 among fecal Escherichia coli isolates from Brazilian children with and without diarrhea

The enteropathogenic role of cytotoxic necrotizing factor (CNF)-producing Escherichia coli was investigated by searching cnf genes among 2074 isolates from 200 children with and 200 without acute diarrhea in Brazil. Fourteen (7%) cases versus 10 (5%) control children carried at least one cnf positive isolate (P = 0.50) and most isolates expressed CNF type 1. DNA sequences of virulence factors of extraintestinal pathogenic E. coli (ExPEC) were detected in 78.6% of CNF1-producing isolates. Besides not being associated with human acute diarrhea, the CNF1-producing isolates here identified may represent potential ExPEC transitorily composing the normal intestinal flora.