Genome Sequence Analysis and Characterization of Shiga Toxin 2 Production by Escherichia coli O157:H7 Strains Associated With a Laboratory Infection.

A laboratory-acquired E. coli O157:H7 infection with associated severe sequelae including hemolytic uremic syndrome occurred in an individual working in the laboratory with a mixture of nalidixic acid-resistant (NalR) O157:H7 mutant strains in a soil-biochar blend. The patient was hospitalized and treated with an intravenous combination of metronidazole and levofloxacin. The present study investigated the source of this severe laboratory acquired infection and further examined the influence of the antibiotics used during treatment on the expression and production of Shiga toxin. Genomes of two Stx2a-and eae-positive O157:H7 strains isolated from the patient's stool were sequenced along with two pairs of the wt strains and their derived NalR mutants used in the laboratory experiments. High-resolution SNP typing determined the strains' individual genetic relatedness and unambiguously identified the two laboratory-derived NalR mutant strains as the source of the researcher's life-threatening disease, rather than a conceivable ingestion of unrelated O157:H7 isolates circulating at the same time. It was further confirmed that in sublethal doses, the antibiotics increased toxin expression and production. Our results support a simultaneous co-infection with clinical strains in the laboratory, which were the causative agents of previous O157:H7 outbreaks, and further that the administration of antibiotics may have impacted the outcome of the infection.

Involvement of a putative ATP-Binding Cassette (ABC) Involved in manganese transport in virulence of Listeria monocytogenes.

Listeria monocytogenes is a foodborne pathogen and the causative agent of listeriosis, a disease associated with high fatality (20-30%) and hospitalization rates (>95%). ATP-Binding Cassette (ABC) transporters have been demonstrated to be involved in the general stress response. In previous studies, in-frame deletion mutants of the ABC transporter genes, LMOf2365_1875 and LMOf2365_1877, were constructed and analyzed; however, additional work is needed to investigate the virulence potential of these deletion mutants. In this study, two in vitro methods and one in vivo model were used to investigate the virulence potential of in-frame deletion mutants of ABC transporter genes. First, the invasion efficiency in host cells was measured using the HT-29 human cell line. Second, cell-to-cell spread activity was measured using a plaque forming assay. Lastly, virulence potential of the mutants was tested in the Galleria mellonella wax moth model. Our results demonstrated that the deletion mutant, ⊿LMOf2365_1875, displayed decreased invasion and cell-to-cell spread efficiency in comparison to the wild-type, LMOf2365, indicating that LMOf2365_1875 may be required for virulence. Furthermore, the reduced virulence of these mutants was confirmed using the Galleria mellonella wax moth model. In addition, the expression levels of 15 virulence and stress-related genes were analyzed by RT-PCR assays using stationary phase cells. Our results showed that virulence-related gene expression levels from the deletion mutants were elevated (15/15 genes from ⊿LMOf2365_1877 and 7/15 genes from ⊿LMOf2365_1875) compared to the wild type LMOf2365, suggesting that ABC transporters may negatively regulate virulence gene expression under specific conditions. The expression level of the stress-related gene, clpE, also was increased in both deletion mutants, indicating the involvement of ABC transporters in the stress response. Taken together, our findings suggest that ABC transporters may be used as potential targets to develop new therapeutic strategies to control L. monocytogenes.

Two homologous Salmonella serogroup C1-specific genes are required for flagellar motility and cell invasion.

BACKGROUND: Salmonella is a major bacterial pathogen associated with a large number of outbreaks of foodborne diseases. Many highly virulent serovars that cause human illness belong to Salmonella serogroup C1, and Salmonella ser. Choleraesuis is a prominent cause of invasive infections in Asia. Comparative genomic analysis in our previous study showed that two homologous genes, SC0368 and SC0595 in Salmonella ser. Choleraesuis were unique to serogroup C1. In this study, two single-deletion mutants (Δ0368 and Δ0595) and one double-deletion mutant (Δ0368Δ0595) were constructed based on the genome. All these mutants and the wild-type strain were subjected to RNA-Seq analysis to reveal functional relationships of the two serogroup C1-specific genes. RESULTS: Data from RNA-Seq indicated that deletion of SC0368 resulted in defects in motility through repression of σ28 in flagellar regulation Class 3. Consistent with RNA-Seq data, results from transmission electron microcopy (TEM) showed that flagella were not present in △0368 and △0368△0595 mutants resulting in both swimming and swarming defects. Interestingly, the growth rates of two non-motile mutants △0368 and △0368△0595 were significantly greater than the wild-type, which may be associated with up-regulation of genes encoding cytochromes, enhancing bacterial proliferation. Moreover, the △0595 mutant was significantly more invasive in Caco-2 cells as shown by bacterial enumeration assays, and the expression of lipopolysaccharide (LPS) core synthesis-related genes (rfaB, rfaI, rfaQ, rfaY, rfaK, rfaZ) was down-regulated only in the △0368△0595 mutant. In addition, this study also speculated that these two genes might be contributing to serotype conversion for Salmonella C1 serogroup based on their apparent roles in biosynthesis of LPS and the flagella. CONCLUSION: A combination of biological and transcriptomic (RNA-Seq) analyses has shown that the SC0368 and SC0595 genes are involved in biosynthesis of flagella and complete LPS, as well as in bacterial growth and virulence. Such information will aid to revealing the role of these specific genes in bacterial physiology and evolution within the serogroup C1.

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.

Detection of Hepatitis A Virus and Other Enteric Viruses in Shellfish Collected in the Gulf of Naples, Italy.

To assess the quality of shellfish harvest areas, bivalve mollusk samples from three coastal areas of the Campania region in Southwest Italy were evaluated for viruses over a three-year period (2015-2017). Screening of 289 samples from shellfish farms and other locations by qPCR and RT-qPCR identified hepatitis A virus (HAV; 8.9%), norovirus GI (NoVGI; 10.8%) and GII (NoVGII; 39.7%), rotavirus (RV; 9.0%), astrovirus (AsV; 20.8%), sapovirus (SaV; 18.8%), aichivirus-1 (AiV-1; 5.6%), and adenovirus (AdV, 5.6%). Hepatitis E virus (HEV) was never detected. Sequence analysis identified HAV as genotype IA and AdV as type 41. This study demonstrates the presence of different enteric viruses within bivalve mollusks, highlighting the limitations of the current EU classification system for shellfish growing waters.

Draft Genome Sequences of Seven Strains of Shiga Toxin-Producing Escherichia coli O111 with Variation in Their Sensitivity to Novobiocin.

Inclusion of novobiocin as a selective agent for enrichment media and selective agars inhibits the growth of some Shiga toxin-producing Escherichia coli (STEC) strains, particularly non-O157 STEC, which can yield false-negative detection results. Here, we report the draft genomic sequences of seven STEC O111 isolates with different sensitivities to novobiocin.

Draft Whole-Genome Sequences of Seven Listeria monocytogenes Strains with Variations in Virulence and Stress Responses.

Listeria monocytogenes is an important foodborne pathogen that causes listeriosis. Here, we report the draft genome sequences of seven L. monocytogenes strains isolated from food, environmental, and clinical sources. Sequence differences at the genome level may help in understanding why these strains displayed different virulence and stress response characteristics.

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.

Prevalence and characteristics of Shiga toxin-producing Escherichia coli in finishing pigs: Implications on public health.

Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens, which can cause serious illnesses, including hemorrhagic colitis and hemolytic uremic syndrome. To study the epidemiology of STEC in finishing pigs and examine the potential risks they pose for human STEC infections, we conducted a longitudinal cohort study in three finishing sites. Six cohorts of pigs (2 cohorts/site, 20 pigs/cohort) were randomly selected, and fecal samples (n=898) were collected every two weeks through their finishing period. Eighty-two pigs (68.3%) shed STEC at least once, and the proportion of STEC-positive pigs varied across sites (50-97.5%) and cohorts (15-100%). Clinically important serotypes, O157:H7 (stx2c, eae) and O26:H11 (stx1a, eae), were recovered from two pigs at sites C and A, respectively. The most common serotype isolated was O59:H21 (stx2e), which was particularly prevalent in site B as it was recovered from all STEC positive pigs (n=39). Each cohort showed different patterns of STEC shedding, which were associated with the prevalent serotype. The median shedding duration of STEC in pigs was 28days, consistent with our prior study. However, among pigs shedding O59:H21 at least once, pigs in cohort B2 had a significantly longer shedding duration of 42days (P<0.05) compared to other cohorts. Stx2e was the most commonly observed stx variant in finishing pigs (93.9%), in accordance with the previous studies. Stx2e has been reported to be significantly associated with edema disease in pigs, however, the pathogenicity in humans warrants further investigations. Nonetheless, our findings affirm that pigs are an important reservoir for human STEC infections, and that the circulating serotypes in a cohort and site management factors may significantly affect the prevalence of STEC. Molecular characterization of STEC isolates and epidemiological studies to identify risk factors for shedding in pigs are strongly warranted to further address the significance to public health and to develop mitigation strategies.

Genome Sequences of 34 Shiga Toxin-Producing Escherichia coli Isolates from Swine and Other Sources.

Shiga toxin-producing Escherichia coli (STEC) bacteria are foodborne pathogens that can be carried by various animals. The swine STEC population is partially composed of host-specific strains that are often not well characterized. In this work, the genome sequences of a number of swine STEC strains are presented.

Escherichia coli O157:H7 Acid Sensitivity Correlates with Flocculation Phenotype during Nutrient Limitation.

Shiga toxin producing Escherichia coli (STEC) strains vary in acid resistance; however, little is known about the underlying mechanisms that result in strain specific differences. Among 25 STEC O157:H7 strains tested, 7 strains flocculated when grown statically for 18 h in minimal salts medium at 37°C, while 18 strains did not. Interestingly, the flocculation phenotype (cells came out of suspension) was found to correlate with degree of acid sensitivity in an assay with 400 mM acetic acid solution at pH 3.3 targeting acidified foods. Strains exhibiting flocculation were more acid sensitive and were designated FAS, for flocculation acid sensitive, while the acid resistant strain designated PAR for planktonic acid resistant. Flocculation was not observed for any strains during growth in complex medium (Luria Bertani broth). STEC strains B201 and B241 were chosen as representative FAS (2.4 log reduction) and PAR (0.15 log reduction) strains, respectively, due to differences in acid resistance and flocculation phenotype. Results from electron microscopy showed evidence of fimbriae production in B201, whereas fimbriae were not observed in B241.Curli fimbriae production was identified through plating on Congo red differential medium, and all FAS strains showed curli fimbriae production. Surprisingly, 5 PAR strains also had evidence of curli production. Transcriptomic and targeted gene expression data for B201 and B241indicated that csg and hde (curli and acid induced chaperone genes, respectively) expression positively correlated with the phenotypic differences observed for these strains. These data suggest that FAS strains grown in minimal medium express curli, resulting in a flocculation phenotype. This may be regulated by GcvB, which positively regulates curli fimbriae production and represses acid chaperone proteins. RpoS and other regulatory mechanisms may impact curli fimbriae production, as well. These findings may help elucidate mechanisms underlying differences among STEC strains in relating acid resistance and biofilm formation.

Detection and Isolation of the "Top Seven" Shiga Toxin-Producing Escherichia coli in Ground Beef: Comparison of RapidFinder Kits to the U.S. Department of Agriculture Microbiology Laboratory Guidebook Method.

Shiga toxin-producing Escherichia coli (STEC) O157:H7 and serogroups O26, O45, O103, O111, O121, and O145 are often referred to as the "top seven" STEC, and these have been declared to be adulterants in beef by the U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS). The aim of this work was to compare the methods described in the USDA FSIS Microbiology Laboratory Guidebook (MLG) to a two-stage Applied Biosystems RapidFinder STEC real-time PCR method to test for the top seven STEC in raw ground beef. The specificity of the RapidFinder workflow that targets non-O157 STEC O-antigen genes, stx1, stx2, and eae, and E. coli O157-specific targets was determined with 132 top seven STEC strains and 283 exclusion strains. All inclusion strains were positive, and all exclusion strains gave negative results with the RapidFinder assay. Strains carrying all of the known variants of stx1 and stx2, including stx2f and stx2g, were also detected. For RapidFinder analysis, 375-g ground beef samples spiked with ≥4 CFU of representative STEC strains were enriched in 1 L of tryptic soy broth (TSB) for 10 h at 42 ± 1°C, and for the MLG method, 325-g samples were similarly spiked and enriched in 975 mL of modified TSB for 15 h at 42 ± 1°C. Following DNA extraction, real-time PCR was performed using RapidFinder Express software, and for the MLG method, the BAX Real-Time PCR STEC Suite and the BAX Real-Time E. coli O157:H7 assay were used with the BAX System Q7 software. Following immunomagnetic separation, presumptive colonies from modified Rainbow agar O157 plates were confirmed by the real-time PCR assays. Results of the RapidFinder and BAX assays were similar; all samples were positive after 10 and 15 h of enrichment, respectively. Isolation and confirmation of isolates was possible on all samples, except that two O111:NM strains could not be isolated from a portion of the inoculated samples. Thus, the RapidFinder system can be used for routine and rapid detection of the top seven STEC in beef.

Disinfectant and Antimicrobial Susceptibility Profiles of the Big Six Non-O157 Shiga Toxin-Producing Escherichia coli Strains from Food Animals and Humans.

The disinfectant and antimicrobial susceptibility profiles of 138 non-O157 Shiga toxin-producing Escherichia coli strains (STECs) from food animals and humans were determined. Antimicrobial resistance (AMR) was moderate (39.1% of strains) in response to 15 antimicrobial agents. Animal strains had a lower AMR prevalence (35.6%) than did human strains (43.9%) but a higher prevalence of the resistance profile GEN-KAN-TET. A decreasing prevalence of AMR was found among animal strains from serogroups O45 > O145 > O121 > O111 > O26 > O103 and among human strains from serogroups O145 > O103 > O26 > O111 > O121 > O45. One animal strain from serogroups O121 and O145 and one human strain from serogroup O26 had extensive drug resistance. A high prevalence of AMR in animal O45 and O121 strains and no resistance or a low prevalence of resistance in human strains from these serogroups suggests a source other than food animals for human exposure to these strains. Among the 24 disinfectants evaluated, all strains were susceptible to triclosan. Animal strains had a higher prevalence of resistance to chlorhexidine than did human strains. Both animal and human strains had a similar low prevalence of low-level benzalkonium chloride resistance, and animal and human strains had similar susceptibility profiles for most other disinfectants. Benzyldimethylammonium chlorides and C10AC were the primary active components in disinfectants DC&R and P-128, respectively, against non-O157 STECs. A disinfectant FS512 MIC ≥ 8 µg/ml was more prevalent among animal O121 strains (61.5%) than among human O121 strains (25%), which may also suggest a source of human exposure to STEC O121 other than food animals. Bacterial inhibition was not dependent solely on pH but was correlated with the presence of dissociated organic acid species and some undissociated acids.

Complete Genome Sequences of Escherichia coli O157:H7 Strains SRCC 1675 and 28RC, Which Vary in Acid Resistance.

The level of acid resistance among Escherichia coli O157:H7 strains varies, and strains with higher resistance to acid may have a lower infectious dose. The complete genome sequences belonging to two strains of Escherichia coli O157:H7 with different levels of acid resistance are presented here.

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.

Characterization of Shiga Toxin Subtypes and Virulence Genes in Porcine Shiga Toxin-Producing Escherichia coli.

Similar to ruminants, swine have been shown to be a reservoir for Shiga toxin-producing Escherichia coli (STEC), and pork products have been linked with outbreaks associated with STEC O157 and O111:H-. STEC strains, isolated in a previous study from fecal samples of late-finisher pigs, belonged to a total of 56 serotypes, including O15:H27, O91:H14, and other serogroups previously associated with human illness. The isolates were tested by polymerase chain reaction (PCR) and a high-throughput real-time PCR system to determine the Shiga toxin (Stx) subtype and virulence-associated and putative virulence-associated genes they carried. Select STEC strains were further analyzed using a Minimal Signature E. coli Array Strip. As expected, stx 2e (81%) was the most common Stx variant, followed by stx 1a (14%), stx 2d (3%), and stx 1c (1%). The STEC serogroups that carried stx 2d were O15:H27, O159:H16 and O159:H-. Similar to stx 2a and stx 2c, the stx 2d variant is associated with development of hemorrhagic colitis and hemolytic uremic syndrome, and reports on the presence of this variant in STEC strains isolated from swine are lacking. Moreover, the genes encoding heat stable toxin (estIa) and enteroaggregative E. coli heat stable enterotoxin-1 (astA) were commonly found in 50 and 44% of isolates, respectively. The hemolysin genes, hlyA and ehxA, were both detected in 7% of the swine STEC strains. Although the eae gene was not found, other genes involved in host cell adhesion, including lpfAO113 and paa were detected in more than 50% of swine STEC strains, and a number of strains also carried iha, lpfAO26, lpfAO157, fedA, orfA, and orfB. The present work provides new insights on the distribution of virulence factors among swine STEC strains and shows that swine may carry Stx1a-, Stx2e-, or Stx2d-producing E. coli with virulence gene profiles associated with human infections.

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.].

Genome-Scale Screening and Validation of Targets for Identification of Salmonella enterica and Serovar Prediction.

Salmonella enterica is the most common foodborne pathogen worldwide, with 2,500 recognized serovars. Detection of S. enterica and its classification into serovars are essential for food safety surveillance and clinical diagnosis. The PCR method is useful for these applications because of its rapidity and high accuracy. We obtained 412 candidate detection targets for S. enterica using a comparative genomics mining approach. Gene ontology (GO) functional enrichment analysis of these candidate targets revealed that the GO term with the largest number of unigenes with known function (38 of 177, 21.5%) was significantly involved in pathogenesis (P < 10(-24)). All the candidate targets were then evaluated by PCR assays. Fifteen targets showed high specificity for the detection of S. enterica by verification with 151 S. enterica strains and 34 non-Salmonella strains. The phylogenetic trees of verified targets were highly comparable with those of housekeeping genes, especially for differentiating S. enterica strains into serovars. The serovar prediction ability was validated by sequencing one target (S9) for 39 S. enterica strains belonging to six serovars. Identical mutation sites existed in the same serovar, and different mutation sites were found in diverse serovars. Our findings revealed that 15 verified targets can be potentially used for molecular detection, and some of them can be used for serotyping of S. enterica strains.

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.