Efficacy of passive immunization in broiler chicks via an inactivated Escherichia coli autogenous vaccine administered to broiler breeder hens.

Avian pathogenic Escherichia coli (APEC) cause extra-intestinal infections called colibacillosis, which is the dominant bacterial disease in broilers. To date, given the diversity of APEC strains and the need for an acceptable level of protection in day-old chicks, no satisfactory commercial vaccine is available. As part of a French nationwide project, we selected three representative strains among several hundred APEC that cause colibacillosis disease. We first performed experiments to develop colibacillosis in vivo models, using an inoculum of 3 × 107 CFU of each E. coli strain per chick. Two APEC strains (19-381 and 19-383-M1) were found to be highly virulent for day-old chicks, whereas the third strain (19-385-M1) induced no mortality nor morbidity.We then produced an autogenous vaccine using the (Llyod, 1982; MaCQueen, 1967) 19-381 and 19-383-M1 APEC strains and a passive immunization trial was undertaken. Specific-pathogen-free Leghorn hens were vaccinated twice 2 weeks apart, the control group receiving a saline solution. The vaccinated and control hens exhibited no clinical signs, and egg production and fertility of both groups were similar. Fertile eggs were collected for 2 weeks after the second vaccination and chicks were obtained. After challenge with each APEC (19-381 and 19-383-M1), chicks appeared to be partially protected from infection with the 19-383-M1 strain, with 40% mortality compared with 80% for the non-vaccinated chicks. No protection was found when the chicks were challenged with the 19-381 strain. Now, further work is needed to consider some aspects: severity of the pathogen challenge model, persistence of the protection, number of APEC strains in the autogenous vaccine, choice of adjuvants, and heterologous protection by the vaccine made from strain 19-383-M1.RESEARCH HIGHLIGHTS Three APEC strains were characterized and selected to develop in vivo models of colibacillosis.A bivalent autogenous vaccine was produced and a passive immunization trial was carried out.Protection of chicks was demonstrated when challenged with the 19-383-M1 APEC strain (homologous challenge).Further work is needed in particular to evaluate the protection against heterologous challenge.

Escherichia coli O80 hybrid pathotype strains producing Shiga toxin and ESBL: molecular characterization and potential therapeutic options.

OBJECTIVES: Enterohaemorrhagic Escherichia coli (EHEC) infections may be complicated by haemolytic uraemic syndrome (HUS). The emerging worldwide EHEC serogroup O80 has acquired a mosaic plasmid combining extraintestinal virulence and antibiotic resistance. This hybrid pathotype is associated with invasive infections that require antibiotic therapy, classically not recommended in EHEC infections, increasing the risk of HUS. We characterized two ESBL-producing O80 EHEC strains, which is an unusual resistance mechanism among EHECs, and determined the safest therapy to be used for invasive infections. METHODS: WGS of two strains isolated from the stools of an asymptomatic carrier and a patient with HUS was performed using Illumina and Nanopore technologies. Generated reads were combined to assemble genomes. We determined the safest therapy by comparing Shiga toxin (Stx) production by the two strains in the presence of several antibiotics. RESULTS: The strains were genetically close to the O80 EHEC clone, belonging to ST301 and harbouring stx2d, eae-ξ, ehxA and genes characteristic of the extraintestinal virulence plasmid pS88. Long-read sequencing identified the acquisition of an additional plasmid harbouring CTX-M-type genes (blaCTX-M-14 and blaCTX-M-1). Azithromycin decreased Stx production at subinhibitory concentrations, ciprofloxacin increased it and imipenem had no major effect. The combination of azithromycin and imipenem overall reduced Stx production. CONCLUSIONS: Acquisition of an additional plasmid harbouring ESBL genes is a step towards increasing the risk of O80 EHEC dissemination and represents a serious public health concern. The combination of azithromycin and imipenem reduced Stx production and suggests that this combination could be tested in clinical trials.

Variations of the Escherichia coli population in the digestive tract of broilers.

We explored the between-group and temporal variations in the intestinal Escherichia coli populations of broilers under experimental conditions, taking both antimicrobial resistance and virulence into consideration. Four replicates of 45 commercial chicks were reared in four animal facilities. On their first day of life (Day 0), they were orally inoculated with two extended-spectrum-cephalosporin-resistant (ESCR) E. coli (2.72 log10 CFU of a bla CMY-2- and 2.55 log10 CFU of a bla CTX-M-carrying E. coli). Faecal samples were then collected weekly and caecal samples were obtained from birds sacrificed on Days 21 or 42. The total, ESC-, ciprofloxacin- and gentamicin-resistant E. coli populations were enumerated on MacConkey (MC) and MC-supplemented media, and eight virulence-associated genes (VAGs) (iroN, iutA, iss, ompT, hlyF, vat, frzorf4 , and fyuA) were sought by PCR on isolates obtained on MC agar. The results showed significant between-group differences in the size of the resistant sub-populations and the presence of VAGs. Contrary to bla CTX-M-positive strains, bla CMY-positive strains persisted up to Day 42, but represented only a minor fraction of the total E. coli population. The ESC-, gentamicin- and ciprofloxacin-resistant populations decreased over time. Isolates obtained during the first week contained a mean of 5.1 VAGs. The percentages of some VAG profiles differed between faecal isolates on Day 41 and caecal isolates on Day 42. The fluctuations or differences between E. coli isolates according to group, age, and faecal or caecal origin need to be considered when designing experimental protocols and seeking to improve colibacillosis control. RESEARCH HIGHLIGHTS Temporal variations in the intestinal E. coli populations of broilers was studied. The antibiotic-resistant populations decreased over time. Virulence profiles differed between faecal isolates on Day 41 and caecal isolates on Day 42. Strains with the highest numbers of virulence genes were present during the first days.

Emerging Multidrug-Resistant Hybrid Pathotype Shiga Toxin-Producing Escherichia coli O80 and Related Strains of Clonal Complex 165, Europe.

Enterohemorrhagic Escherichia coli serogroup O80, involved in hemolytic uremic syndrome associated with extraintestinal infections, has emerged in France. We obtained circularized sequences of the O80 strain RDEx444, responsible for hemolytic uremic syndrome with bacteremia, and noncircularized sequences of 35 O80 E. coli isolated from humans and animals in Europe with or without Shiga toxin genes. RDEx444 harbored a mosaic plasmid, pR444_A, combining extraintestinal virulence determinants and a multidrug resistance-encoding island. All strains belonged to clonal complex 165, which is distantly related to other major enterohemorrhagic E. coli lineages. All stx-positive strains contained eae-ξ, ehxA, and genes characteristic of pR444_A. Among stx-negative strains, 1 produced extended-spectrum ß-lactamase, 1 harbored the colistin-resistance gene mcr1, and 2 possessed genes characteristic of enteropathogenic and pyelonephritis E. coli. Because O80-clonal complex 165 strains can integrate intestinal and extraintestinal virulence factors in combination with diverse drug-resistance genes, they constitute dangerous and versatile multidrug-resistant pathogens.

Shiga Toxin-Producing Serogroup O91 Escherichia coli Strains Isolated from Food and Environmental Samples.

Shiga toxin-producing Escherichia coli (STEC) strains of the O91:H21 serotype have caused severe infections, including hemolytic-uremic syndrome. Strains of the O91 serogroup have been isolated from food, animals, and the environment worldwide but are not well characterized. We used a microarray and other molecular assays to examine 49 serogroup O91 strains (environmental, food, and clinical strains) for their virulence potential and phylogenetic relationships. Most of the isolates were identified to be strains of the O91:H21 and O91:H14 serotypes, with a few O91:H10 strains and one O91:H9 strain being identified. None of the strains had the eae gene, which codes for the intimin adherence protein, and many did not have some of the genetic markers that are common in other STEC strains. The genetic profiles of the strains within each serotype were similar but differed greatly between strains of different serotypes. The genetic profiles of the O91:H21 strains that we tested were identical or nearly identical to those of the clinical O91:H21 strains that have caused severe diseases. Multilocus sequence typing and clustered regularly interspaced short palindromic repeat analyses showed that the O91:H21 strains clustered within the STEC 1 clonal group but the other O91 serotype strains were phylogenetically diverse.IMPORTANCE This study showed that food and environmental O91:H21 strains have similar genotypic profiles and Shiga toxin subtypes and are phylogenetically related to the O91:H21 strains that have caused hemolytic-uremic syndrome, suggesting that these strains may also have the potential to cause severe illness.

Targeted Amplicon Sequencing for Single-Nucleotide-Polymorphism Genotyping of Attaching and Effacing Escherichia coli O26:H11 Cattle Strains via a High-Throughput Library Preparation Technique.

Enterohemorrhagic Escherichia coli (EHEC) O26:H11, a serotype within Shiga toxin-producing E. coli (STEC) that causes severe human disease, has been considered to have evolved from attaching and effacing E. coli (AEEC) O26:H11 through the acquisition of a Shiga toxin-encoding gene. Targeted amplicon sequencing using next-generation sequencing technology of 48 phylogenetically informative single-nucleotide polymorphisms (SNPs) and three SNPs differentiating Shiga toxin-positive (stx-positive) strains from Shiga toxin-negative (stx-negative) strains were used to infer the phylogenetic relationships of 178 E. coli O26:H11 strains (6 stx-positive strains and 172 stx-negative AEEC strains) from cattle feces to 7 publically available genomes of human clinical strains. The AEEC cattle strains displayed synonymous SNP genotypes with stx2-positive sequence type 29 (ST29) human O26:H11 strains, while stx1 ST21 human and cattle strains clustered separately, demonstrating the close phylogenetic relatedness of these Shiga toxin-negative AEEC cattle strains and human clinical strains. With the exception of seven stx-negative strains, five of which contained espK, three stx-related SNPs differentiated the STEC strains from non-STEC strains, supporting the hypothesis that these AEEC cattle strains could serve as a potential reservoir for new or existing pathogenic human strains. Our results support the idea that targeted amplicon sequencing for SNP genotyping expedites strain identification and genetic characterization of E. coli O26:H11, which is important for food safety and public health.

Variable tellurite resistance profiles of clinically-relevant Shiga toxin-producing Escherichia coli (STEC) influence their recovery from foodstuffs.

Tellurite (Tel)-amended selective media and resistance (Tel-R) are widely used for detecting Shiga toxin-producing Escherichia coli (STEC) from foodstuffs. Tel-R of 81 O157 and non-O157 STEC strains isolated from animal, food and human was thus investigated. Variations of STEC tellurite minimal inhibitory concentration (MIC) values have been observed and suggest a multifactorial and variable tellurite resistome between strains. Some clinically-relevant STEC were found highly susceptible and could not be recovered using a tellurite-based detection scheme. The ter operon was highly prevalent among highly Tel-R STEC but was not always detected among intermediately-resistant strains. Many STEC serogroup strains were found to harbor sublines showing a gradient of MIC values. These Tel-R sublines showed statistically significant log negative correlations with increasing tellurite concentration. Whatever the tellurite concentration, the highest number of resistant sublines was observed for STEC belonging to the O26 serogroup. Variations in the number of these Tel-R sublines could explain the poor recovery of some STEC serogroups on tellurite-amended media especially from food products with low levels of contamination. Comparison of tellurite MIC values and distribution of virulence-related genes showed Tel-R and virulence to be related.

Characteristics of emerging human-pathogenic Escherichia coli O26:H11 strains isolated in France between 2010 and 2013 and carrying the stx2d gene only.

Strains of Escherichia coli O26:H11 that were positive for stx2 alone (n = 23), which were not epidemiologically related or part of an outbreak, were isolated from pediatric patients in France between 2010 and 2013. We were interested in comparing these strains with the new highly virulent stx2a-positive E. coli O26 clone sequence type 29 (ST29) that has emerged recently in Europe, and we tested them by multilocus sequence typing (MLST), stx2 subtyping, clustered regularly interspaced short palindromic repeat (CRISPR) sequencing, and plasmid (ehxA, katP, espP, and etpD) and chromosomal (Z2098, espK, and espV) virulence gene profiling. We showed that 16 of the 23 strains appeared to correspond to this new clone, but the characteristics of 12 strains differed significantly from the previously described characteristics, with negative results for both plasmid and chromosomal genetic markers. These 12 strains exhibited a ST29 genotype and related CRISPR arrays (CRISPR2a alleles 67 or 71), suggesting that they evolved in a common environment. This finding was corroborated by the presence of stx2d in 7 of the 12 ST29 strains. This is the first time that E. coli O26:H11 carrying stx2d has been isolated from humans. This is additional evidence of the continuing evolution of virulent Shiga toxin-producing E. coli (STEC) O26 strains. A new O26:H11 CRISPR PCR assay, SP_O26_E, has been developed for detection of these 12 particular ST29 strains of E. coli O26:H11. This test is useful to better characterize the stx2-positive O26:H11 clinical isolates, which are associated with severe clinical outcomes such as bloody diarrhea and hemolytic uremic syndrome.

Molecular profiling of Escherichia coli O157:H7 and non-O157 strains isolated from humans and cattle in Alberta, Canada.

Virulence markers in Shiga toxin-producing Escherichia coli (STEC) and their association with diseases remain largely unknown. This study determines the importance of 44 genetic markers for STEC (O157 and non-O157) from human clinical cases and their correlation to disease outcome. STEC isolated from a cattle surveillance program were also included. The virulence genes tested were present in almost all O157:H7 isolates but highly variable in non-O157 STEC isolates. Patient age was a significant determinant of clinical outcome.

Genetic Diversity of the fliC Genes Encoding the Flagellar Antigen H19 of Escherichia coli and Application to the Specific Identification of Enterohemorrhagic E. coli O121:H19.

Enterohemorrhagic Escherichia coli (EHEC) O121:H19 belong to a specific clonal type distinct from other classical EHEC and major enteropathogenic E. coli groups and is regarded as one of the major EHEC serogroups involved in severe infections in humans. Sequencing of the fliC genes associated with the flagellar antigen H19 (fliCH19) revealed the genetic diversity of the fliCH19 gene sequences in E. coli. A cluster analysis of 12 fliCH19 sequences, 4 from O121 and 8 from non-O121 E. coli strains, revealed five different genotypes. All O121:H19 strains fell into one cluster, whereas a second cluster was formed by five non-O121:H19 strains. Cluster 1 and cluster 2 strains differ by 27 single nucleotide exchanges in their fliCH19 genes (98.5% homology). Based on allele discrimination of the fliCH19 genes, a real-time PCR test was designed for specific identification of EHEC O121:H19. The O121 fliCH19 PCR tested negative in 73 E. coli H19 strains that belonged to serogroups other than O121, including 28 different O groups, O-nontypeable H19, and O-rough:H19 strains. The O121 fliCH19 PCR reacted with all 16 tested O121:H19 strains and 1 O-rough:H19 strain which was positive for the O121 wzx gene. A cross-reaction was observed only with E. coli H32 strains which share sequence similarities in the target region of the O121 fliCH19 PCR. The combined use of O-antigen genotyping (O121 wzx) and the detection of O121 fliCH19 allele type contributes to improving the identification and molecular serotyping of EHEC O121:H19 motile and nonmotile strains and variants of these strains lacking stx genes.

Genetic Diversity and Pathogenic Potential of Attaching and Effacing Escherichia coli O26:H11 Strains Recovered from Bovine Feces in the United States.

Escherichia coli O26 has been identified as the most common non-O157 Shiga toxin-producing E. coli (STEC) serogroup to cause human illnesses in the United States and has been implicated in outbreaks around the world. E. coli has high genomic plasticity, which facilitates the loss or acquisition of virulence genes. Attaching and effacing E. coli (AEEC) O26 strains have frequently been isolated from bovine feces, and there is a need to better characterize the relatedness of these strains to defined molecular pathotypes and to describe the extent of their genetic diversity. High-throughput real-time PCR was used to screen 178 E. coli O26 isolates from a single U.S. cattle feedlot, collected from May to July 2011, for the presence or absence of 25 O26 serogroup-specific and virulence-associated markers. The selected markers were capable of distinguishing these strains into molecularly defined groups (yielding 18 unique marker combinations). Analysis of the clustered regularly interspaced short palindromic repeat 1 (CRISPR1) and CRISPR2a loci further discriminated isolates into 24 CRISPR types. The combination of molecular markers and CRISPR typing provided 20.8% diversity. The recent CRISPR PCR target SP_O26-E, which was previously identified only in stx2-positive O26:H11 human clinical strains, was identified in 96.4% (161/167 [95% confidence interval, 99.2 to 93.6%]) of the stx-negative AEEC O26:H11 bovine fecal strains. This supports that these stx-negative strains may have previously contained a prophage carrying stx or could acquire this prophage, thus possibly giving them the potential to become pathogenic to humans. These results show that investigation of specific genetic markers may further elucidate our understanding of the genetic diversity of AEEC O26 strains in bovine feces.

The utility of multiple molecular methods including whole genome sequencing as tools to differentiate Escherichia coli O157:H7 outbreaks.

A standardised method for determining Escherichia coli O157:H7 strain relatedness using whole genome sequencing or virulence gene profiling is not yet established. We sought to assess the capacity of either high-throughput polymerase chain reaction (PCR) of 49 virulence genes, core-genome single nt variants (SNVs) or k-mer clustering to discriminate between outbreak-associated and sporadic E. coli O157:H7 isolates. Three outbreaks and multiple sporadic isolates from the province of Alberta, Canada were included in the study. Two of the outbreaks occurred concurrently in 2014 and one occurred in 2012. Pulsed-field gel electrophoresis (PFGE) and multilocus variable-number tandem repeat analysis (MLVA) were employed as comparator typing methods. The virulence gene profiles of isolates from the 2012 and 2014 Alberta outbreak events and contemporary sporadic isolates were mostly identical; therefore the set of virulence genes chosen in this study were not discriminatory enough to distinguish between outbreak clusters. Concordant with PFGE and MLVA results, core genome SNV and k-mer phylogenies clustered isolates from the 2012 and 2014 outbreaks as distinct events. k-mer phylogenies demonstrated increased discriminatory power compared with core SNV phylogenies. Prior to the widespread implementation of whole genome sequencing for routine public health use, issues surrounding cost, technical expertise, software standardisation, and data sharing/comparisons must be addressed.

Diverse virulence gene content of Shiga toxin-producing Escherichia coli from finishing swine.

Shiga toxin-producing Escherichia coli (STEC) infections are a critical public health concern because they can cause severe clinical outcomes, such as hemolytic uremic syndrome, in humans. Determining the presence or absence of virulence genes is essential in assessing the potential pathogenicity of STEC strains. Currently, there is limited information about the virulence genes carried by swine STEC strains; therefore, this study was conducted to examine the presence and absence of 69 virulence genes in STEC strains recovered previously from finishing swine in a longitudinal study. A subset of STEC strains was analyzed by pulsed-field gel electrophoresis (PFGE) to examine their genetic relatedness. Swine STEC strains (n = 150) were analyzed by the use of a high-throughput real-time PCR array system, which included 69 virulence gene targets. Three major pathotypes consisted of 16 different combinations of virulence gene profiles, and serotypes were determined in the swine STEC strains. The majority of the swine STEC strains (n = 120) belonged to serotype O59:H21 and carried the same virulence gene profile, which consisted of 9 virulence genes: stx2e, iha, ecs1763, lpfAO113, estIa (STa), ehaA, paa, terE, and ureD. The eae, nleF, and nleH1-2 genes were detected in one swine STEC strain (O49:H21). Other genes encoding adhesins, including iha, were identified (n = 149). The PFGE results demonstrated that swine STEC strains from pigs raised in the same finishing barn were closely related. Our results revealed diverse virulence gene contents among the members of the swine STEC population and enhance understanding of the dynamics of transmission of STEC strains among pigs housed in the same barn.

Genetic diversity and virulence potential of shiga toxin-producing Escherichia coli O113:H21 strains isolated from clinical, environmental, and food sources.

Shiga toxin-producing Escherichia coli strains of serotype O113:H21 have caused severe human diseases, but they are unusual in that they do not produce adherence factors coded by the locus of enterocyte effacement. Here, a PCR microarray was used to characterize 65 O113:H21 strains isolated from the environment, food, and clinical infections from various countries. In comparison to the pathogenic strains that were implicated in hemolytic-uremic syndrome in Australia, there were no clear differences between the pathogens and the environmental strains with respect to the 41 genetic markers tested. Furthermore, all of the strains carried only Shiga toxin subtypes associated with human infections, suggesting that the environmental strains have the potential to cause disease. Most of the O113:H21 strains were closely related and belonged in the same clonal group (ST-223), but CRISPR analysis showed a great degree of genetic diversity among the O113:H21 strains.

Distribution of infectious and parasitic agents among three sentinel bee species across European agricultural landscapes.

Infectious and parasitic agents (IPAs) and their associated diseases are major environmental stressors that jeopardize bee health, both alone and in interaction with other stressors. Their impact on pollinator communities can be assessed by studying multiple sentinel bee species. Here, we analysed the field exposure of three sentinel managed bee species (Apis mellifera, Bombus terrestris and Osmia bicornis) to 11 IPAs (six RNA viruses, two bacteria, three microsporidia). The sentinel bees were deployed at 128 sites in eight European countries adjacent to either oilseed rape fields or apple orchards during crop bloom. Adult bees of each species were sampled before their placement and after crop bloom. The IPAs were detected and quantified using a harmonised, high-throughput and semi-automatized qPCR workflow. We describe differences among bee species in IPA profiles (richness, diversity, detection frequencies, loads and their change upon field exposure, and exposure risk), with no clear patterns related to the country or focal crop. Our results suggest that the most frequent IPAs in adult bees are more appropriate for assessing the bees' IPA exposure risk. We also report positive correlations of IPA loads supporting the potential IPA transmission among sentinels, suggesting careful consideration should be taken when introducing managed pollinators in ecologically sensitive environments.

Towards a molecular definition of enterohemorrhagic Escherichia coli (EHEC): detection of genes located on O island 57 as markers to distinguish EHEC from closely related enteropathogenic E. coli strains.

Among strains of Shiga-toxin (Stx) producing Escherichia coli (STEC), seven serogroups (O26, O45, O103, O111, O121, O145, and O157) are associated with severe clinical illness in humans. These strains are also called enterohemorrhagic E. coli (EHEC), and the development of methods for their reliable detection from food has been challenging thus far. PCR detection of major EHEC virulence genes stx1, stx2, eae, and O-serogroup-specific genes is useful but does not identify EHEC strains specifically. Searching for the presence of additional genes issued from E. coli O157:H7 genomic islands OI-122 and OI-71 increases the specificity but does not clearly discriminate EHEC from enteropathogenic E. coli (EPEC) strains. Here, we identified two putative genes, called Z2098 and Z2099, from the genomic island OI-57 that were closely associated with EHEC and their stx-negative derivative strains (87% for Z2098 and 91% for Z2099). Z2098 and Z2099 were rarely found in EPEC (10% for Z2098 and 12% for Z2099), STEC (2 and 15%), and apathogenic E. coli (1% each) strains. Our findings indicate that Z2098 and Z2099 are useful genetic markers for a more targeted diagnosis of typical EHEC and new emerging EHEC strains.

Discrimination of enterohemorrhagic Escherichia coli (EHEC) from non-EHEC strains based on detection of various combinations of type III effector genes.

Enterohemorrhagic Escherichia coli (EHEC) strains comprise a subgroup of Shiga-toxin (Stx)-producing E. coli (STEC) and are characterized by a few serotypes. Among these, seven priority STEC serotypes (O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7) are most frequently implicated in severe clinical illness worldwide. Currently, standard methods using stx, eae, and O-serogroup-specific gene sequences for detecting the top 7 EHEC serotypes bear the disadvantage that these genes can be found in non-EHEC strains as well. Here, we explored the suitability of ureD, espV, espK, espN, Z2098, and espM1 genes and combinations thereof as candidates for a more targeted EHEC screening assay. For a very large panel of E. coli strains (n = 1,100), which comprised EHEC (n = 340), enteropathogenic E. coli (EPEC) (n = 392), STEC (n = 193), and apathogenic strains (n = 175), we showed that these genetic markers were more prevalent in EHEC (67.1% to 92.4%) than in EPEC (13.3% to 45.2%), STEC (0.5% to 3.6%), and apathogenic E. coli strains (0 to 2.9%). It is noteworthy that 38.5% of the EPEC strains that tested positive for at least one of these genetic markers belonged to the top 7 EHEC serotypes, suggesting that such isolates might be Stx-negative derivatives of EHEC. The associations of espK with either espV, ureD, or Z2098 were the best combinations for more specific and sensitive detection of the top 7 EHEC strains, allowing detection of 99.3% to 100% of these strains. In addition, detection of 93.7% of the EHEC strains belonging to other serotypes than the top 7 offers a possibility for identifying new emerging EHEC strains.

Genotypes and virulence characteristics of Shiga toxin-producing Escherichia coli O104 strains from different origins and sources.

Sixty-two Escherichia coli strains carrying the wzxO104-gene from different sources, origins and time periods were analyzed for their serotypes, virulence genes and compared for genomic similarity by pulsed-field gel-electrophoresis (PFGE). The O104 antigen was present in 55 strains and the structurally and genetically related capsular antigen K9 in five strains. The presence of 49 genes associated with enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC) was investigated. Fifty-four strains of serotypes O104:H2 (n=1), O104:H4 (n=37), O104:H7 (n=5) and O104:H21 (n=11) produced Shiga-toxins (Stx). Among STEC O104, a close association between serotype, virulence gene profile and genomic similarity was found. EAEC virulence genes were only present in STEC O104:H4 strains. EHEC-O157 plasmid-encoded genes were only found in STEC O104:H2, O104:H7 and O104:H21 strains. None of the 62 O104 or K9 strains carried an eae-gene involved in the attaching and effacing phenotype. The 38 O104:H4 strains formed a single PFGE-cluster (>83.7% similarity). Thirty-one of these strains were from the European O104:H4 outbreak in 2011. The outbreak strains and older O104:H4 strains from Germany (2001), Georgia and France (2009) clustered together at>86.2% similarity. O104:H4 strains isolated between 2001 and 2009 differed for some plasmid-encoded virulence genes compared to the outbreak strains from 2011. STEC O104:H21 and STEC O104:H7 strains isolated in the U.S. and in Europe showed characteristic differences in their Stx-types, virulence gene and PFGE profiles indicating that these have evolved separately. E. coli K9 strains were not associated with virulence and were heterogeneous for their serotypes and PFGE profiles.

Molecular and phenotypic characterization of Escherichia coli O26:H8 among diarrheagenic E. coli O26 strains isolated in Brazil.

Escherichia coli strains of serogroup O26 comprise two distinct groups of pathogens, characterized as enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC). Among the several genes related to type III secretion system-secreted effector proteins, espK was found to be highly specific for EHEC O26:H11 and its stx-negative derivative strains isolated in European countries. E. coli O26 strains isolated in Brazil from infant diarrhea, foods, and the environment have consistently been shown to lack stx genes and are thus considered atypical EPEC. However, no further information related to their genetic background is known. Therefore, in this study, we aimed to discriminate and characterize these Brazilian O26 stx-negative strains by phenotypic, genetic, and biochemical approaches. Among 44 isolates confirmed to be O26 isolates, most displayed flagellar antigen H11 or H32. Out of the 13 nonmotile isolates, 2 tested positive for fliCH11, and 11 were fliCH8 positive. The identification of genetic markers showed that several O26:H11 and all O26:H8 strains tested positive for espK and could therefore be discriminated as EHEC derivatives. The presence of H8 among EHEC O26 and its stx-negative derivative isolates is described for the first time. The interaction of three isolates with polarized Caco-2 cells and with intestinal biopsy specimen fragments ex vivo confirmed the ability of the O26 strains analyzed to cause attaching-and-effacing (A/E) lesions. The O26:H32 strains, isolated mostly from meat, were considered nonvirulent. Knowledge of the virulence content of stx-negative O26 isolates within the same serotype helped to avoid misclassification of isolates, which certainly has important implications for public health surveillance.