Global Distribution of O Serotypes and Antibiotic Resistance in Extraintestinal Pathogenic Escherichia coli Collected From the Blood of Patients With Bacteremia Across Multiple Surveillance Studies.

BACKGROUND: Extraintestinal pathogenic Escherichia coli (ExPEC) is the leading cause of bacteremia worldwide, with older populations having increased risk of invasive bacterial disease. Increasing resistance to first-line antibiotics and emergence of multidrug-resistant (MDR) strains represent major treatment challenges. ExPEC O serotypes are key targets for potential multivalent conjugate vaccine development. Therefore, we evaluated the O serotype distribution and antibiotic resistance profiles of ExPEC strains causing bloodstream infections across 4 regions. METHODS: Blood culture isolates from patients aged ≥60 years collected during 5 retrospective E. coli surveillance studies in Europe, North America, Asia-Pacific, and South America (2011-2017) were analyzed. Isolates were O serotyped by agglutination; O genotyping was performed for nontypeable isolates. Antimicrobial susceptibility testing was also conducted. RESULTS: Among 3217 ExPEC blood culture isolates, the most ubiquitous O serotype was O25 (n = 737 [22.9%]), followed by O2, O6, O1, O75, O15, O8, O16, O4, O18, O77 group, O153, O9, O101/O162, O86, and O13 (prevalence of ≥1%). The prevalence of these O serotypes was generally consistent across regions, apart from South America; together, these 16 O serotypes represented 77.6% of all ExPEC bacteremia isolates analyzed. The overall MDR frequency was 10.7%, with limited variation between regions. Within the MDR subset (n = 345), O25 showed a dominant prevalence of 63.2% (n = 218). CONCLUSIONS: Predominant O serotypes among ExPEC bacteremia isolates are widespread across different regions. O25 was the most prevalent O serotype overall and particularly dominant among MDR isolates. These findings may inform the design of multivalent conjugate vaccines that can target the predominant O serotypes associated with invasive ExPEC disease in older adults.

Molecular Characteristics, Ecology, and Zoonotic Potential of Escherichia coli Strains That Cause Hemorrhagic Pneumonia in Animals.

Hemorrhagic pneumonia (HP) is a rare but highly lethal disease, mainly of dogs and cats, caused by hemolytic Escherichia coli strains that contain cnf1 (encoding cytotoxic necrotizing factor 1). After encountering fatal HP in two dogs, we used contemporary molecular methods, including multilocus sequence typing and whole-genome sequencing, to compare the corresponding case isolates with published HP clinical isolates and newly obtained fecal E. coli isolates from 20 humans and animals in the index HP case household. We also compared the aggregated HP clinical isolates, which represented 13 discrete strains, by pulsotype with a large, private pulsotype library of diverse-source E. coli. The HP clinical isolates represented a narrow range of phylogenetic group B2 lineages (mainly sequence types 12 and 127), O types (mainly O4 and O6), and H types (mainly H5 and H31), but diverse fimH alleles (type-1 fimbriae adhesin). Their extensive, highly conserved virulence genotypes, which qualified as extraintestinal pathogenic E. coli (ExPEC), encoded diverse adhesins, toxins, iron uptake systems, and protectins. Household surveillance identified multiple HP-like fecal strains, plus abundant between-host strain sharing, including of the household's index HP strain. The pulsotype library search identified, for five HP clinical strains, same-pulsotype human and animal fecal and clinical (predominantly urine) isolates, from diverse locales and time periods. Thus, E. coli strains that cause HP derive from a narrow range of ExPEC lineages within phylogroup B2, contain multiple virulence genes other than cnf1, are shared extensively between hosts, and likely function in nature mainly as intestinal colonizers and uropathogens. IMPORTANCE This study clarifies the clonal background and extensive virulence genotypes of the E. coli strains that cause hemorrhagic pneumonia in domestic animals (mainly dogs and cats), shows that such strains circulate among animals and humans, identifies a substantial intestinal colonization component to their lifestyle, and extends their known clinical manifestations to include bacteremia and urinary tract infection. The findings place these strains better into context vis-à-vis current understandings of E. coli phylogeny, ecology, and pathogenesis; identify questions for future research; and may prove relevant for surveillance and prevention efforts.

Strain and host-cell dependent role of type-1 fimbriae in the adherence phenotype of super-shed Escherichia coli O157:H7.

Super-shed (SS) Escherichia coli O157 (E. coli O157) demonstrate a strong, aggregative, locus of enterocyte effacement (LEE)-independent adherence phenotype on bovine recto-anal junction squamous epithelial (RSE) cells, and harbor polymorphisms in non-LEE-adherence-related loci, including in the type 1 fimbriae operon. To elucidate the role of type 1 fimbriae in strain- and host-specific adherence, we evaluated the entire Fim operon (FimB-H) and its adhesion (FimH) deletion mutants in four E. coli O157 strains, SS17, SS52, SS77 and EDL933, and evaluated the adherence phenotype in bovine RSE and human HEp-2 adherence assays. Consistent with the prevailing dogma that fimH expression is genetically switched off in E. coli O157, the ΔfimHSS52, ΔfimB-HSS52, ΔfimB-HSS17, and ΔfimHSS77 mutants remained unchanged in adherence phenotype to RSE cells. In contrast, the ΔfimHSS17 and ΔfimB-HSS77 mutants changed from a wild-type strong and aggregative, to a moderate and diffuse adherence phenotype, while both ΔfimHEDL933 and ΔfimB-HEDL933 mutants demonstrated enhanced binding to RSE cells (p < 0.05). Additionally, both ΔfimHSS17 and ΔfimHEDL933 were non-adherent to HEp-2 cells (p < 0.05). Complementation of the mutant strains with their respective wild-type genes restored parental phenotypes. Microscopy revealed that the SS17 and EDL933 strains indeed carry type 1 fimbriae-like structures shorter than those seen in uropathogenic E. coli. Taken together, these results provide compelling evidence for a strain and host cell type-dependent role of fimH and the fim operon in E. coli O157 adherence that needs to be further evaluated.

Identification, Shiga toxin subtypes and prevalence of minor serogroups of Shiga toxin-producing Escherichia coli in feedlot cattle feces.

Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens that cause illnesses in humans ranging from mild to hemorrhagic enteritis with complications of hemolytic uremic syndrome and even death. Cattle are a major reservoir of STEC, which reside in the hindgut and are shed in the feces, a major source of food and water contaminations. Seven serogroups, O26, O45, O103, O111, O121, O145 and O157, called 'top-7', are responsible for the majority of human STEC infections in North America. Additionally, 151 serogroups of E. coli are known to carry Shiga toxin genes (stx). Not much is known about fecal shedding and prevalence and virulence potential of STEC other than the top-7. Our primary objectives were to identify serogroups of STEC strains, other than the top-7, isolated from cattle feces and subtype stx genes to assess their virulence potential. Additional objective was to develop and validate a novel multiplex PCR assay to detect and determine prevalence of six serogroups, O2, O74, O109, O131, O168, and O171, in cattle feces. A total of 351 strains, positive for stx gene and negative for the top-7 serogroups, isolated from feedlot cattle feces were used in the study. Of the 351 strains, 291 belonged to 16 serogroups and 60 could not be serogrouped. Among the 351 strains, 63 (17.9%) carried stx1 gene and 300 (82.1%) carried stx2, including 12 strains positive for both. The majority of the stx1 and stx2 were of stx1a (47/63; 74.6%) and stx2a subtypes (234/300; 78%), respectively, which are often associated with human infections. A novel multiplex PCR assay developed and validated to detect six serogroups, O2, O74, O109, O131, O168, and O171, which accounted for 86.9% of the STEC strains identified, was utilized to determine their prevalence in fecal samples (n = 576) collected from a commercial feedlot. Four serogroups, O2, O109, O168, and O171 were identified as the dominant serogroups prevalent in cattle feces. In conclusion, cattle shed in the feces a number of STEC serogroups, other than the top-7, and the majority of the strains isolated possessed stx2, particularly of the subtype 2a, suggesting their potential risk to cause human infections.

Multiplex PCR Assays for the Detection of One Hundred and Thirty Seven Serogroups of Shiga Toxin-Producing Escherichia coli Associated With Cattle.

Escherichia coli carrying prophage with genes that encode for Shiga toxins are categorized as Shiga toxin-producing E. coli (STEC) pathotype. Illnesses caused by STEC in humans, which are often foodborne, range from mild to bloody diarrhea with life-threatening complications of renal failure and hemolytic uremic syndrome and even death, particularly in children. As many as 158 of the total 187 serogroups of E. coli are known to carry Shiga toxin genes, which makes STEC a major pathotype of E. coli. Seven STEC serogroups, called top-7, which include O26, O45, O103, O111, O121, O145, and O157, are responsible for the majority of the STEC-associated human illnesses. The STEC serogroups, other than the top-7, called "non-top-7" have also been associated with human illnesses, more often as sporadic infections. Ruminants, particularly cattle, are principal reservoirs of STEC and harbor the organisms in the hindgut and shed in the feces, which serves as a major source of food and water contaminations. A number of studies have reported on the fecal prevalence of top-7 STEC in cattle feces. However, there is paucity of data on the prevalence of non-top-7 STEC serogroups in cattle feces, generally because of lack of validated detection methods. The objective of our study was to develop and validate 14 sets of multiplex PCR (mPCR) assays targeting serogroup-specific genes to detect 137 non-top-7 STEC serogroups previously reported to be present in cattle feces. Each assay included 7-12 serogroups and primers were designed to amplify the target genes with distinct amplicon sizes for each serogroup that can be readily identified within each assay. The assays were validated with 460 strains of known serogroups. The multiplex PCR assays designed in our study can be readily adapted by most laboratories for rapid identification of strains belonging to the non-top-7 STEC serogroups associated with cattle.

A Targeted Sequencing Assay for Serotyping Escherichia coli Using AgriSeq Technology.

The gold standard method for serotyping Escherichia coli has relied on antisera-based typing of the O- and H-antigens, which is labor intensive and often unreliable. In the post-genomic era, sequence-based assays are potentially faster to provide results, could combine O-serogrouping and H-typing in a single test, and could simultaneously screen for the presence of other genetic markers of interest such as virulence factors. Whole genome sequencing is one approach; however, this method has limited multiplexing capabilities, and only a small fraction of the sequence is informative for subtyping or identifying virulence potential. A targeted, sequence-based assay and accompanying software for data analysis would be a great improvement over the currently available methods for serotyping. The purpose of this study was to develop a high-throughput, molecular method for serotyping E. coli by sequencing the genes that are required for production of O- and H-antigens, as well as to develop software for data analysis and serotype identification. To expand the utility of the assay, targets for the virulence factors, Shiga toxins (stx 1, and stx 2) and intimin (eae) were included. To validate the assay, genomic DNA was extracted from O-serogroup and H-type standard strains and from Shiga toxin-producing E. coli, the targeted regions were amplified, and then sequencing libraries were prepared from the amplified products followed by sequencing of the libraries on the Ion S5™ sequencer. The resulting sequence files were analyzed via the SeroType Caller™ software for identification of O-serogroup, H-type, and presence of stx 1 , stx 2, and eae. We successfully identified 169 O-serogroups and 41 H-types. The assay also routinely detected the presence of stx 1a,c,d (3 of 3 strains), stx 2c-e,g (8 of 8 strains), stx 2f (1 strain), and eae (6 of 6 strains). Taken together, the high-throughput, sequence-based method presented here is a reliable alternative to antisera-based serotyping methods for E. coli.

Interlaboratory Evaluation of the U.S. Food and Drug Administration Escherichia coli Identification Microarray for Profiling Shiga Toxin-Producing Escherichia coli.

The U.S. Food and Drug Administration Escherichia coli Identification (FDA-ECID) microarray provides rapid molecular characterization of E. coli. The effectiveness of the FDA-ECID for characterizing Shiga toxin-producing E. coli (STEC) was evaluated by three federal laboratories and one reference laboratory with a panel of 54 reference E. coli strains from the External Quality Assurance program. Strains were tested by FDA-ECID for molecular serotyping (O and H antigens), Shiga toxin subtyping, and the presence of the ehxA and eae genes for enterohemolysin and intimin, respectively. The FDA-ECID O typing was 96% reproducible among the four laboratories and 94% accurate compared with the reference External Quality Assurance data. Discrepancies were due to the absence of O41 target loci on the array and to two pairs of O types with identical target sequences. H typing was 96% reproducible and 100% accurate, with discrepancies due to two strains from one laboratory that were identified as mixed by FDA-ECID. Shiga toxin (Stx) type 1 subtyping was 100% reproducible and accurate, and Stx2 subtyping was 100% reproducible but only 64% accurate. FDA-ECID identified most Stx2 subtypes but had difficulty distinguishing among stx2a, stx2c, and stx2d genes because of close similarities of these sequences. FDA-ECID was 100% effective for detecting ehxA and eae and accurately subtyped the eae alleles. This interlaboratory study revealed that FDA-ECID for STEC characterization was highly reproducible for molecular serotyping, stx and eae subtyping, and ehxA detection. However, the array was less useful for distinguishing among the highly homologous O antigen genes and the stx2a, stx2c, and stx2d subtypes.

Molecular serogrouping of Escherichia coli.

O-antigens present on the surface of Escherichia coli provide antigenic specificity for the strain and are the main components for O-serogroup designation. Serotyping using O-group-specific antisera for the identification of E. coli O-serogroups has been traditionally the gold-standard for distinguishing E. coli strains. Knowledge of the O-group is important for determining pathogenic lineage, classifying E. coli for epidemiological studies, for determining virulence, and for tracing outbreaks of diseases and sources of infection. However, serotyping has limitations, as the antisera generated against each specific O-group may cross-react, many strains are non-typeable, and others can autoagglutinate or be rough (lacking an O-antigen). Currently, the nucleotide sequences are available for most of the 187 designated E. coli O-groups. Public health and other laboratories are considering whole genome sequencing to develop genotypic methods to determine O-groups. These procedures require instrumentation and analysis that may not be accessible and may be cost-prohibitive at this time. In this review, we have identified unique gene sequences within the O-antigen gene clusters and have targeted these genes for identification of O-groups using the polymerase chain reaction. This information can be used to distinguish O-groups by developing other platforms for E. coli diagnostics in the future.

Comparative genomics of two super-shedder isolates of Escherichia coli O157:H7.

Shiga toxin-producing Escherichia coli O157:H7 (O157) are zoonotic foodborne pathogens and of major public health concern that cause considerable intestinal and extra-intestinal illnesses in humans. O157 colonize the recto-anal junction (RAJ) of asymptomatic cattle who shed the bacterium into the environment through fecal matter. A small subset of cattle, termed super-shedders (SS), excrete O157 at a rate (≥ 104 CFU/g of feces) that is several orders of magnitude greater than other colonized cattle and play a major role in the prevalence and transmission of O157. To better understand microbial factors contributing to super-shedding we have recently sequenced two SS isolates, SS17 (GenBank accession no. CP008805) and SS52 (GenBank accession no. CP010304) and shown that SS isolates display a distinctive strongly adherent phenotype on bovine rectal squamous epithelial cells. Here we present a detailed comparative genomics analysis of SS17 and SS52 with other previously characterized O157 strains (EC4115, EDL933, Sakai, TW14359). The results highlight specific polymorphisms and genomic features shared amongst SS strains, and reveal several SNPs that are shared amongst SS isolates, including in genes involved in motility, adherence, and metabolism. Finally, our analyses reveal distinctive patterns of distribution of phage-associated genes amongst the two SS and other isolates. Together, the results of our comparative genomics studies suggest that while SS17 and SS52 share genomic features with other lineage I/II isolates, they likely have distinct recent evolutionary histories. Future comparative and functional genomic studies are needed to decipher the precise molecular basis for super shedding in O157.

Association of Atypical Enteropathogenic Escherichia coli with Diarrhea and Related Mortality in Kittens.

Diarrhea is responsible for the death of approximately 900,000 children per year worldwide. In children, typical enteropathogenic Escherichia coli (EPEC) is a common cause of diarrhea and is associated with a higher hazard of death. Typical EPEC infection is rare in animals and poorly reproduced in experimental animal models. In contrast, atypical EPEC (aEPEC) infection is common in both children and animals, but its role in diarrhea is uncertain. Mortality in kittens is often attributed to diarrhea, and we previously identified enteroadherent EPEC in the intestines of deceased kittens. The purpose of this study was to determine the prevalence and type of EPEC in kittens and whether infection was associated with diarrhea, diarrhea-related mortality, gastrointestinal pathology, or other risk factors. Kittens with and without diarrhea were obtained from two shelter facilities and determined to shed atypical EPEC at a culture-based prevalence of 18%. In contrast, quantitative PCR detected the presence of the gene for intimin (eae) in feces from 42% of kittens. aEPEC was isolated from kittens with and without diarrhea. However, kittens with diarrhea harbored significantly larger quantities of aEPEC than kittens without diarrhea. Kittens with aEPEC had a significantly greater severity of small intestinal and colonic lesions and were significantly more likely to have required subcutaneous fluid administration. These findings identify aEPEC to be prevalent in kittens and a significant primary or contributing cause of intestinal inflammation, diarrhea, dehydration, and associated mortality in kittens.

Prevalence and Phylogenetic Characterization of Escherichia coli and Hygiene Indicator Bacteria Isolated from Leafy Green Produce, Beef, and Pork Obtained from Farmers' Markets in Pennsylvania.

The popularity of farmers' markets in the United States has led to over 8,400 farmers' markets being in operation in 2015. As farmers' markets have increased in size and complexity in the kinds of foods sold at these venues, so have the potential food safety risks. Since 2008, seven major foodborne illness outbreaks and two recalls associated with food products from farmers' markets have occurred, causing 80 known reported illnesses and one death. Various researchers also have observed vendors performing high-risk food safety retail behaviors, and others have identified microbiological hazards in foods sold at farmers' markets. In this study, the presence of hygiene indicators (coliforms, fecal coliforms, Listeria spp., and Escherichia coli ) was assessed in select samples of leafy green produce and meat obtained from farmers' markets in Pennsylvania. E. coli isolates were further characterized by phylogenetic profile and virulence potential. E. coli was present in 40% (20 of 50) and 18% (9 of 50) of beef and pork samples, respectively, and in 28% (15 of 54), 29% (15 of 52), and 17% (8 of 46) of kale, lettuce, and spinach samples, respectively. Listeria spp. was found in 8% (4 of 50) of beef samples, 2% (1 of 54) of kale samples, 4% (2 of 52) of lettuce samples, and 7% (3 of 46) of spinach samples. Among the 10 Listeria spp. isolates, 3 were identified as L. monocytogenes . E. coli isolated from meat samples mainly clustered into phylogroup B1 (66%; 19 of 29), whereas produce isolates clustered into phylogroups B2 (36%; 14 of 39) and B1 (33%; 13 of 39). These E. coli isolates possessed the fimH, iroN, hlyD, and eae genes associated with extraintestinal pathogenic E. coli and Shiga toxin-producing E. coli . The high prevalence but low levels of E. coli and Listeria spp. found on both produce and meat products obtained from farmers' markets in this study strongly indicate that farmers' market vendors would benefit greatly from food safety training and increased public health oversight.

Captive and free-living urban pigeons (Columba livia) from Brazil as carriers of multidrug-resistant pathogenic Escherichia coli.

Thirty Escherichia coli isolates from captive and free-living pigeons in Brazil were characterised. Virulence-associated genes identified in pigeons included those which occur relatively frequently in avian pathogenic E. coli (APEC) from commercial poultry worldwide. Eleven of 30 E. coli isolates from pigeons, belonging mainly to B1 and B2 phylogenetic groups, had high or intermediate pathogenicity for 1-day-old chicks. The frequency of multi-drug resistant (MDR) E. coli in captive pigeons was relatively high and included one isolate positive for the extended-spectrum ß-lactamase (ESBL) gene blaCTX-M-8. Pulsed field gel electrophoresis (PFGE) showed high heterogeneity among isolates. There is potential for pigeons to transmit antibiotic resistant pathogenic E. coli to other species through environmental contamination or direct contact.

Household Clustering of Escherichia coli Sequence Type 131 Clinical and Fecal Isolates According to Whole Genome Sequence Analysis.

Background. Within-household sharing of strains from the resistance-associated H30R1 and H30Rx subclones of Escherichia coli sequence type 131 (ST131) has been inferred based on conventional typing data, but it has been assessed minimally using whole genome sequence (WGS) analysis. Methods. Thirty-three clinical and fecal isolates of ST131-H30R1 and ST131-H30Rx, from 20 humans and pets in 6 households, underwent WGS analysis for comparison with 52 published ST131 genomes. Phylogenetic relationships were inferred using a bootstrapped maximum likelihood tree based on core genome sequence polymorphisms. Accessory traits were compared between phylogenetically similar isolates. Results. In the WGS-based phylogeny, isolates clustered strictly by household, in clades that were distributed widely across the phylogeny, interspersed between H30R1 and H30Rx comparison genomes. For only 1 household did the core genome phylogeny place epidemiologically unlinked isolates together with household isolates, but even there multiple differences in accessory genome content clearly differentiated these 2 groups. The core genome phylogeny supported within-household strain sharing, fecal-urethral urinary tract infection pathogenesis (with the entire household potentially providing the fecal reservoir), and instances of host-specific microevolution. In 1 instance, the household's index strain persisted for 6 years before causing a new infection in a different household member. Conclusions. Within-household sharing of E coli ST131 strains was confirmed extensively at the genome level, as was long-term colonization and repeated infections due to an ST131-H30Rx strain. Future efforts toward surveillance and decolonization may need to address not just the affected patient but also other human and animal household members.

Advances in Molecular Serotyping and Subtyping of Escherichia coli.

Escherichia coli plays an important role as a member of the gut microbiota; however, pathogenic strains also exist, including various diarrheagenic E. coli pathotypes and extraintestinal pathogenic E. coli that cause illness outside of the GI-tract. E. coli have traditionally been serotyped using antisera against the ca. 186 O-antigens and 53 H-flagellar antigens. Phenotypic methods, including bacteriophage typing and O- and H- serotyping for differentiating and characterizing E. coli have been used for many years; however, these methods are generally time consuming and not always accurate. Advances in next generation sequencing technologies have made it possible to develop genetic-based subtyping and molecular serotyping methods for E. coli, which are more discriminatory compared to phenotypic typing methods. Furthermore, whole genome sequencing (WGS) of E. coli is replacing established subtyping methods such as pulsed-field gel electrophoresis, providing a major advancement in the ability to investigate food-borne disease outbreaks and for trace-back to sources. A variety of sequence analysis tools and bioinformatic pipelines are being developed to analyze the vast amount of data generated by WGS and to obtain specific information such as O- and H-group determination and the presence of virulence genes and other genetic markers.

Correction: Comparison of O-Antigen Gene Clusters of All O-Serogroups of Escherichia coli and Proposal for Adopting a New Nomenclature for O-Typing.

[This corrects the article DOI: 10.1371/journal.pone.0147434.].

Rapid Detection of Escherichia coli O157 and Shiga Toxins by Lateral Flow Immunoassays.

Shiga toxin-producing Escherichia coli O157:H7 (STEC) cause food-borne illness that may be fatal. STEC strains enumerate two types of potent Shiga toxins (Stx1 and Stx2) that are responsible for causing diseases. It is important to detect the E. coli O157 and Shiga toxins in food to prevent outbreak of diseases. We describe the development of two multi-analyte antibody-based lateral flow immunoassays (LFIA); one for the detection of Stx1 and Stx2 and one for the detection of E. coli O157 that may be used simultaneously to detect pathogenic E. coli O157:H7. The LFIA strips were developed by conjugating nano colloidal gold particles with monoclonal antibodies against Stx1 and Stx2 and anti-lipid A antibodies to capture Shiga toxins and O157 antigen, respectively. Our results indicate that the LFIA for Stx is highly specific and detected Stx1 and Stx2 within three hours of induction of STEC with ciprofloxacin at 37 °C. The limit of detection for E. coli O157 LFIA was found to be 105 CFU/mL in ground beef spiked with the pathogen. The LFIAs are rapid, accurate and easy to use and do not require sophisticated equipment or trained personnel. Following the assay, colored bands on the membrane develop for end-point detection. The LFIAs may be used for screening STEC in food and the environment.

Potential pathogens, antimicrobial patterns and genotypic diversity of Escherichia coli isolates in constructed wetlands treating swine wastewater.

Escherichia coli populations originating from swine houses through constructed wetlands were analyzed for potential pathogens, antimicrobial susceptibility patterns, and genotypic diversity. Escherichia coli isolates (n = 493) were screened for the presence of the following virulence genes: stx1, stx2 and eae (Shiga toxin-producing E. coli [STEC]), heat-labile enterotoxin (LT) genes and heat stable toxin STa and STb (enterotoxigenic E. coli (ETEC), cytotoxin necrotizing factors 1 and 2 (cnf1 and cnf2 [necrotoxigenic E. coli- NTEC]), as well as O and H antigens, and the presence of the antibiotic resistance genes blaTEM, blaSHV, blaCMY-2, tet A, tet B, tet C, mph(A), aadA, StrA/B, sul1, sul2 and sul3. The commensal strains were further screened for 16 antimicrobials and characterized by BOX AIR-1 PCR for unique genotypes. The highest antibiotic resistance prevalence was for tetracycline, followed by erythromycin, ampicillin, streptomycin, sulfisoxazole and kanamycin. Our data showed that most of the isolates had high distribution of single or multidrug-resistant (MDR) genotypes. Therefore, the occurrence of MDR E. coli in the wetland is a matter of great concern due to possible transfer of resistance genes from nonpathogenic to pathogenic strains or vice versa in the environment.

Comparison of O-Antigen Gene Clusters of All O-Serogroups of Escherichia coli and Proposal for Adopting a New Nomenclature for O-Typing.

Escherichia coli strains are classified based on O-antigens that are components of the lipopolysaccharide (LPS) in the cell envelope. O-antigens are important virulence factors, targets of both the innate and adaptive immune system, and play a role in host-pathogen interactions. Because they are highly immunogenic and display antigenic specificity unique for each strain, O-antigens are the biomarkers for designating O-types. Immunologically, 185 O-serogroups and 11 OX-groups exist for classification. Conventional serotyping for O-typing entails agglutination reactions between the O-antigen and antisera generated against each O-group. The procedure is labor intensive, not always accurate, and exhibits equivocal results. In this report, we present the sequences of 71 O-antigen gene clusters (O-AGC) and a comparison of all 196 O- and OX-groups. Many of the designated O-types, applied for classification over several decades, exhibited similar nucleotide sequences of the O-AGCs and cross-reacted serologically. Some O-AGCs carried insertion sequences and others had only a few nucleotide differences between them. Thus, based on these findings, it is proposed that several of the E. coli O-groups may be merged. Knowledge of the O-AGC sequences facilitates the development of molecular diagnostic platforms that are rapid, accurate, and reliable that can replace conventional serotyping. Additionally, with the scientific knowledge presented, new frontiers in the discovery of biomarkers, understanding the roles of O-antigens in the innate and adaptive immune system and pathogenesis, the development of glycoconjugate vaccines, and other investigations, can be explored.

Whole genome sequencing of diverse Shiga toxin-producing and non-producing Escherichia coli strains reveals a variety of virulence and novel antibiotic resistance plasmids.

The genomes of a diverse set of Escherichia coli, including many Shiga toxin-producing strains of various serotypes were determined. A total of 39 plasmids were identified among these strains, and many carried virulence or putative virulence genes of Shiga toxin-producing E. coli strains, virulence genes for other pathogenic E. coli groups, and some had combinations of these genes. Among the novel plasmids identified were eight that carried resistance genes to aminoglycosides, carbapenems, penicillins, cephalosporins, chloramphenicol, dihydrofolate reductase inhibitors, sulfonamides, tetracyclines and resistance to heavy metals. Two of the plasmids carried six of these resistance genes and two novel IncHI2 plasmids were also identified. The results of this study showed that plasmids carrying diverse resistance and virulence genes of various pathogenic E. coli groups can be found in E. coli strains and serotypes regardless of the isolate's source and therefore, is consistent with the premise that these mobile elements carrying these traits may be broadly disseminated among E. coli.