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.

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.

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.

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.

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.

Complete nucleotide sequence of pRS218, a large virulence plasmid, that augments pathogenic potential of meningitis-associated Escherichia coli strain RS218.

BACKGROUND: Escherichia coli is the most predominant Gram-negative bacterial pathogen associated with neonatal meningitis. Previous studies indicated that the prototypic neonatal meningitis E. coli (NMEC) strain RS218 (O18:K1:H7) harbors one large plasmid. Objectives of the present study were to analyze the complete nucleotide sequence of this large plasmid (pRS218) and its contribution to NMEC pathogenesis using in vitro and in vivo models of neonatal meningitis. RESULTS: The plasmid is 114,231 bp in size, belongs to the incompatibility group FIB/IIA (IncFIB/IIA), and contains a genetic load region that encodes several virulence and fitness traits such as enterotoxicity, iron acquisition and copper tolerance. The nucleotide sequence of pRS218 showed a 41- 46% similarity to other neonatal meningitis-causing E. coli (NMEC) plasmids and remarkable nucleotide sequence similarity (up to 100%) to large virulence plasmids of E. coli associated with acute cystitis. Some genes located on pRS218 were overly represented by NMEC strains compared to fecal E. coli isolated from healthy individuals. The plasmid-cured strain was significantly attenuated relative to the RS218 wild-type strain as determined in vitro by invasion potential to human cerebral microvascular endothelial cells and in vivo by mortalities, histopathological lesions in the brain tissue, and bacterial recovery from the cerebrospinal fluid of infected rat pups. CONCLUSIONS: The pRS218 is an IncFIB/IIA plasmid which shares a remarkable nucleotide sequence similarity to large plasmids of E. coli associated with cystitis. Both in vitro and in vivo experiments indicated that pRS218 plays an important role in NMEC pathogenesis.

Investigation and control of an attaching and effacing Escherichia coli outbreak in a colony of captive budgerigars (Melopsittacus undulatus).

An increase in mortality in a captive flock of budgerigars (Melopsittacus undulatus) coincided with the isolation of attaching and effacing Escherichia coli from postmortem samples. Common histologic lesions included hepatitis, enteritis, and in one case attaching and effacing lesions along the intestinal tract. Retrospective review of necropsy records and increased sampling led to the identification of several cases of E. coli with the attaching and effacing (eae) virulence gene. Factors such as environment, nutrition, and concomitant pathogens were thought to contribute to mortality in the flock. Although it is not clear whether E. coli was a primary pathogen during the period of increased mortality, the presence of the eae gene combined with associated histologic lesions supports the conclusion that this organism was a significant contributor to mortality. Manipulation of diet, environment, and the addition of probiotic supplementation resulted in a decline in mortality rate and decreased shedding of E. coli based on negative follow-up cultures of intestines, liver, and feces.

Abrupt emergence of a single dominant multidrug-resistant strain of Escherichia coli.

BACKGROUND: Fluoroquinolone-resistant Escherichia coli are increasingly prevalent. Their clonal origins--potentially critical for control efforts--remain undefined. METHODS: Antimicrobial resistance profiles and fine clonal structure were determined for 236 diverse-source historical (1967-2009) E. coli isolates representing sequence type ST131 and 853 recent (2010-2011) consecutive E. coli isolates from 5 clinical laboratories in Seattle, Washington, and Minneapolis, Minnesota. Clonal structure was resolved based on fimH sequence (fimbrial adhesin gene: H subclone assignments), multilocus sequence typing, gyrA and parC sequence (fluoroquinolone resistance-determining loci), and pulsed-field gel electrophoresis. RESULTS: Of the recent fluoroquinolone-resistant clinical isolates, 52% represented a single ST131 subclonal lineage, H30, which expanded abruptly after 2000. This subclone had a unique and conserved gyrA/parC allele combination, supporting its tight clonality. Unlike other ST131 subclones, H30 was significantly associated with fluoroquinolone resistance and was the most prevalent subclone among current E. coli clinical isolates, overall (10.4%) and within every resistance category (11%-52%). CONCLUSIONS: Most current fluoroquinolone-resistant E. coli clinical isolates, and the largest share of multidrug-resistant isolates, represent a highly clonal subgroup that likely originated from a single rapidly expanded and disseminated ST131 strain. Focused attention to this strain will be required to control the fluoroquinolone and multidrug-resistant E. coli epidemic.

Identification and characterization of spontaneous deletions within the Sp11-Sp12 prophage region of Escherichia coli O157:H7 Sakai.

Prophages make up 12% of the enterohemorrhagic Escherichia coli genome and play prominent roles in the evolution and virulence of this food-borne pathogen. Acquisition and loss of and rearrangements within prophage regions are the primary causes of differences in pulsed-field gel electrophoresis (PFGE) patterns among strains of E. coli O157:H7. Sp11 and Sp12 are two tandemly integrated and putatively defective prophages carried by E. coli O157:H7 strain Sakai. In this study, we identified 3 classes of deletions that occur within the Sp11-Sp12 region, at a frequency of ca. 7.74 × 10(-4). One deletion resulted in a precise excision of Sp11, and the other two spanned the junction of Sp11 and Sp12. All deletions resulted in shifts in the XbaI fragment pattern observed by PFGE. We sequenced the inducible prophage pool of Sakai but did not identify any mature phage particles corresponding to either Sp11 or Sp12. Deletions containing pchB and psrC, which are Sp11-carried genes encoding proteins known or suspected to regulate type III secretion, did not affect the secretion levels of the EspA or EspB effector. Alignment of the Sp11-Sp12 DNA sequence with its corresponding regions in other E. coli O157:H7 and O55:H7 strains suggested that homologous recombination rather than integrase-mediated excision is the mechanism behind these deletions. Therefore, this study provides a mechanism behind the previously observed genetic instability of this genomic region of E. coli O157:H7.

The evolutionary divergence of Shiga toxin-producing Escherichia coli is reflected in clustered regularly interspaced short palindromic repeat (CRISPR) spacer composition.

The Shiga toxin-producing Escherichia coli (STEC) strains, including those of O157:H7 and the "big six" serogroups (i.e., serogroups O26, O45, O103, O111, O121, and O145), are a group of pathogens designated food adulterants in the United States. The relatively conserved nature of clustered regularly interspaced short palindromic repeats (CRISPRs) in phylogenetically related E. coli strains makes them potential subtyping markers for STEC detection, and a quantitative PCR (qPCR)-based assay was previously developed for O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7 isolates. To better evaluate the sensitivity and specificity of this qPCR method, the CRISPR loci of 252 O157 and big-six STEC isolates were sequenced and analyzed along with 563 CRISPR1 and 624 CRISPR2 sequences available in GenBank. General conservation of spacer content and order was observed within each O157 and big-six serogroup, validating the qPCR method. Meanwhile, it was found that spacer deletion, the presence of an insertion sequence, and distinct alleles within a serogroup are sources of false-negative reactions. Conservation of CRISPR arrays among isolates expressing the same flagellar antigen, specifically, H7, H2, and H11, suggested that these isolates share an ancestor and provided an explanation for the false positives previously observed in the qPCR results. An analysis of spacer distribution across E. coli strains provided limited evidence for temporal spacer acquisition. Conversely, comparison of CRISPR sequences between strains along the stepwise evolution of O157:H7 from its O55:H7 ancestor revealed that, over this ∼7,000-year span, spacer deletion was the primary force generating CRISPR diversity.

Presence of Shiga toxin-producing Escherichia coli O-groups in small and very-small beef-processing plants and resulting ground beef detected by a multiplex polymerase chain reaction assay.

Shiga toxin-producing Escherichia coli (STEC) are associated with foodborne illnesses, including hemolytic uremic syndrome in humans. Cattle and consequently, beef products are considered a major source of STEC. E. coli O157:H7 has been regulated as an adulterant in ground beef since 1996. The United States Department of Agriculture Food Safety and Inspection Service began regulating six additional STEC (O145, O121, O111, O103, O45, and O26) as adulterants in beef trim and raw ground beef in June 2012. Little is known about the presence of STEC in small and very-small beef-processing plants. Therefore, we propose to determine whether small and very-small beef-processing plants are a potential source of non-O157:H7 STEC. Environmental swabs, carcass swabs, hide swabs, and ground beef from eight small and very-small beef-processing plants were obtained from October 2010 to December 2011. A multiplex polymerase chain reaction assay was used to determine the presence of STEC O-groups: O157, O145, O121, O113, O111, O103, O45, and O26 in the samples. Results demonstrated that 56.6% (154/272) of the environmental samples, 35.0% (71/203) of the carcass samples, 85.2% (23/27) of the hide samples, and 17.0% (20/118) of the ground beef samples tested positive for one or more of the serogroups. However, only 7.4% (20/272) of the environmental samples, 4.4% (9/203) of the carcass samples, and 0% (0/118) ground beef samples tested positive for both the serogroup and Shiga toxin genes. Based on this survey, small and very-small beef processors may be a source of non-O157:H7 STEC. The information from this study may be of interest to regulatory officials, researchers, public health personnel, and the beef industry that are interested in the presence of these pathogens in the beef supply.

A high-throughput antibody-based microarray typing platform.

Many rapid methods have been developed for screening foods for the presence of pathogenic microorganisms. Rapid methods that have the additional ability to identify microorganisms via multiplexed immunological recognition have the potential for classification or typing of microbial contaminants thus facilitating epidemiological investigations that aim to identify outbreaks and trace back the contamination to its source. This manuscript introduces a novel, high throughput typing platform that employs microarrayed multiwell plate substrates and laser-induced fluorescence of the nucleic acid intercalating dye/stain SYBR Gold for detection of antibody-captured bacteria. The aim of this study was to use this platform for comparison of different sets of antibodies raised against the same pathogens as well as demonstrate its potential effectiveness for serotyping. To that end, two sets of antibodies raised against each of the "Big Six" non-O157 Shiga toxin-producing E. coli (STEC) as well as E. coli O157:H7 were array-printed into microtiter plates, and serial dilutions of the bacteria were added and subsequently detected. Though antibody specificity was not sufficient for the development of an STEC serotyping method, the STEC antibody sets performed reasonably well exhibiting that specificity increased at lower capture antibody concentrations or, conversely, at lower bacterial target concentrations. The favorable results indicated that with sufficiently selective and ideally concentrated sets of biorecognition elements (e.g., antibodies or aptamers), this high-throughput platform can be used to rapidly type microbial isolates derived from food samples within ca. 80 min of total assay time. It can also potentially be used to detect the pathogens from food enrichments and at least serve as a platform for testing antibodies.

Comparison of Escherichia coli ST131 pulsotypes, by epidemiologic traits, 1967-2009.

Escherichia coli sequence type 131 (ST131), an emerging disseminated public health threat, causes multidrug-resistant extraintestinal infections. Among 579 diverse E. coli ST131 isolates from 1967-2009, we compared pulsotypes (>94% similar XbaI pulsed-field gel electrophoresis profiles) by collection year, geographic origin, source, and antimicrobial drug-resistance traits. Of 170 pulsotypes, 65 had >2 isolates and accounted for 85% of isolates. Although extensively dispersed geographically, pulsotypes were significantly source specific (e.g., had little commonality between humans vs. foods and food animals). The most prevalent pulsotypes were associated with recent isolation, humans, and antimicrobial drug resistance. Predominant pulsotype 968 was associated specifically with fluoroquinolone resistance but not with extended-spectrum ß-lactamase production or bla(CTX-M-15). Thus, several highly successful antimicrobial drug-resistant lineages within E. coli ST131 have recently emerged and diffused extensively among locales while maintaining a comparatively restricted host/source range. Identification of factors contributing to this behavior of ST131 could help protect public health.

Rapid detection of the top six non-O157 Shiga toxin-producing Escherichia coli O groups in ground beef by flow cytometry.

Rapid, sensitive, and highly specific flow-cytometric assays were developed for the detection of the top six non-O157 Shiga toxin-producing Escherichia coli (STEC) O groups in ground beef. The analytical sensitivity of the assays was 2 × 10(3) target cells in a bacterial mixture of 10(5) CFU/ml, and the limit of detection in ground beef was 1 to 10 CFU following 8 h of enrichment. The assays may be utilized for rapid detection of STEC O groups in meat.

Expansion of the IncX plasmid family for improved identification and typing of novel plasmids in drug-resistant Enterobacteriaceae.

IncX plasmids are narrow host range plasmids of Enterobactericeae that have been isolated for over 50years. They are known to encode type IV fimbriae enabling their own conjugative transfer, and to provide accessory functions to their host bacteria such as resistance towards antimicrobial agents and biofilm formation. Previous plasmid-based replicon typing procedures have indicated that the prevalence of IncX plasmids is low among members of the Enterobacteriaceae. However, examination of a number of IncX-like plasmid sequences and their occurrence in various organisms suggests that IncX plasmid diversity and prevalence is underappreciated. To address these possible shortcomings, we generated additional plasmid sequences of IncX plasmids of interest and compared them to the genomes of all sequenced IncX-like plasmids. These comparisons revealed that IncX plasmids possess a highly syntenic plasmid backbone, but that they are quite divergent with respect to nucleotide and amino acid similarity. Based on phylogenetic comparisons of the sequenced IncX plasmids, the IncX plasmid group has been expanded to include at least four subtypes, IncX1-IncX4. A revised IncX plasmid replicon typing procedure, based upon these sequences and subtypes, was then developed. Use of this revised typing procedure revealed that IncX plasmid occurrence among bacterial populations is much more common than had previously been acknowledged. Thus, this revised procedure can be used to better discern the occurrence of IncX type plasmids among enterobacterial populations.

Detection of the top six non-O157 Shiga toxin-producing Escherichia coli O groups by ELISA.

There is a growing concern of a public health risk associated with non-O157 Shiga toxin-producing Escherichia coli (STEC) since E. coli serogroups O26, O45, O103, O111, O121, and O145 are frequently implicated in outbreaks of human illness worldwide. Recently, the Food Safety and Inspection Service of the U.S. Department of Agriculture declared these six STEC O groups to be adulterants in beef. We describe here a rapid, sensitive, and highly specific enzyme-linked immunosorbent assay (ELISA) for the detection of these top six non-O157 STEC O groups. The assays were tested against 174 reference E. coli O groups, with 60 clinical isolates belonging to the target O groups and 10 non-E coli strains belonging to the family Enterobacteriaceae. Assays for serogroups O103, O111, and O121 exhibited 100% specificity, while assays for serogroups O26 and O45 had 98.2% specificity, and O145 had 99.1% specificity. ELISA conducted using artificially inoculated ground beef samples displayed 100% accuracy. The sensitivity of the assay was 5×10(5) colony-forming unit (CFU)/mL, with limits of detection in the range of 1-10 CFU/25 g of ground beef sample following enrichment. The findings of the study suggest that the assay described is simple and rapid, and can be employed to detect target STEC O groups in beef and other food samples. In addition, the assay provides a conceptual framework that can be adapted for the development of similar tests for the rapid detection of other serogroups of E. coli.

Associations between multidrug resistance, plasmid content, and virulence potential among extraintestinal pathogenic and commensal Escherichia coli from humans and poultry.

The emergence of plasmid-mediated multidrug resistance (MDR) among enteric bacteria presents a serious challenge to the treatment of bacterial infections in humans and animals. Recent studies suggest that avian Escherichia coli commonly possess the ability to resist multiple antimicrobial agents, and might serve as reservoirs of MDR for human extraintestinal pathogenic Escherichia coli (ExPEC) and commensal E. coli populations. We determined antimicrobial susceptibility profiles for 2202 human and avian E. coli isolates, then sought for associations among resistance profile, plasmid content, virulence factor profile, and phylogenetic group. Avian-source isolates harbored greater proportions of MDR than their human counterparts, and avian ExPEC had higher proportions of MDR than did avian commensal E. coli. MDR was significantly associated with possession of the IncA/C, IncP1-α, IncF, and IncI1 plasmid types. Overall, inferred virulence potential did not correlate with drug susceptibility phenotype. However, certain virulence genes were positively associated with MDR, including ireA, ibeA, fyuA, cvaC, iss, iutA, iha, and afa. According to the total dataset, isolates segregated significantly according to host species and clinical status, thus suggesting that avian and human ExPEC and commensal E. coli represent four distinct populations with limited overlap. These findings suggest that in extraintestinal E. coli, MDR is most commonly associated with plasmids, and that these plasmids are frequently found among avian-source E. coli from poultry production systems.

Detection of Shiga toxin-producing Escherichia coli O26, O45, O103, O111, O113, O121, O145, and O157 serogroups by multiplex polymerase chain reaction of the wzx gene of the O-antigen gene cluster.

O-antigens on the surface of Escherichia coli are important virulence factors that are targets of both the innate and adaptive immune system and play a major role in pathogenicity. O-antigens that are responsible for antigenic specificity of the strain determine the O-serogroup. E. coli O26, O45, O103, O111, O113, O121, O145, and O157 have been the most commonly identified O-serogroups associated with Shiga toxin-producing E. coli (STEC) implicated in outbreaks of human illness all over the world. A multiplex polymerase chain reaction assay was developed to simultaneously detect the eight STEC O-serogroups targeting the wzx (O-antigen-flippase) genes of all O-antigen gene clusters. The sensitivity of the multiplex polymerase chain reaction was found to be 10 colony forming units for each O-group when enriched in broth and 100 colony forming units when enriched in artificially inoculated apple juice diluted with tryptic soy broth for 16 h at 37°C. The method can be used for detecting STEC O-groups simultaneously and may be exploited for improving the safety of food products.

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.

Escherichia coli sequence type ST131 as an emerging fluoroquinolone-resistant uropathogen among renal transplant recipients.

Among 40 Escherichia coli urine isolates from renal transplant recipients (Galveston, TX, 2003 to 2005), sequence type ST131 (O25:H4) was highly prevalent (representing 35% of isolates overall and 60% of fluoroquinolone-resistant isolates), virulent appearing, antimicrobial resistant (but extended-spectrum-cephalosporin susceptible), and associated with black race. Pulsotypes were diverse; some were linked to other locales. ST131 emerged significantly during the study period. These findings suggest that E. coli ST131 may constitute an important new multidrug-resistant threat to renal transplant recipients.