The Serum Resistome of a Globally Disseminated Multidrug Resistant Uropathogenic Escherichia coli Clone

Escherichia coli ST131 is a globally disseminated, multidrug resistant clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with antibiotic resistance; however, this phenotype alone is unlikely to explain its dominance amongst multidrug resistant uropathogens circulating worldwide in hospitals and the community. Thus, a greater understanding of the molecular mechanisms that underpin the fitness of E. coli ST131 is required. In this study, we employed hyper-saturated transposon mutagenesis in combination with multiplexed transposon directed insertion-site sequencing to define the essential genes required for in vitro growth and the serum resistome (i.e. genes required for resistance to human serum) of E. coli EC958, a representative of the predominant E. coli ST131 clonal lineage. We identified 315 essential genes in E. coli EC958, 231 (73%) of which were also essential in E. coli K-12. The serum resistome comprised 56 genes, the majority of which encode membrane proteins or factors involved in lipopolysaccharide (LPS) biosynthesis. Targeted mutagenesis confirmed a role in serum resistance for 46 (82%) of these genes. The murein lipoprotein Lpp, along with two lipid A-core biosynthesis enzymes WaaP and WaaG, were most strongly associated with serum resistance. While LPS was the main resistance mechanism defined for E. coli EC958 in serum, the enterobacterial common antigen and colanic acid also impacted on this phenotype. Our analysis also identified a novel function for two genes, hyxA and hyxR, as minor regulators of O-antigen chain length. This study offers novel insight into the genetic make-up of E. coli ST131, and provides a framework for future research on E. coli and other Gram-negative pathogens to define their essential gene repertoire and to dissect the molecular mechanisms that enable them to survive in the bloodstream and cause disease.     

The CTX-M-15-Producing Escherichia coli Clone O25b: H4-ST131 Has High Intestine Colonization and Urinary Tract Infection Abilities

Increasing numbers of pyelonephritis-associated uropathogenic Escherichia coli (UPEC) are exhibiting high resistance to antibiotic therapy. They include a particular clonal group, the CTX-M-15-producing O25b:H4-ST131 clone, which has been shown to have a high dissemination potential. Here we show that a representative isolate of this E. coli clone, referred to as TN03, has enhanced metabolic capacities, acts as a potent intestine- colonizing strain, and displays the typical features of UPEC strains. In a modified streptomycin-treated mouse model of intestinal colonization where streptomycin was stopped 5 days before inoculation, we show that TN03 outcompetes the commensal E. coli strains K-12 MG1655, IAI1, and ED1a at days 1 and 7. Using an experimental model of ascending UTI in C3H/HeN mice, we then show that TN03 colonized the urinary tract. One week after the transurethral inoculation of the TN03 isolates, the bacterial loads in the bladder and kidneys were significantly greater than those of two other UPEC strains (CFT073 and HT7) belonging to the same B2 phylogenetic group. The differences in bacterial loads did not seem to be directly linked to differences in the inflammatory response, since the intrarenal expression of chemokines and cytokines and the number of polymorphonuclear neutrophils attracted to the site of inflammation was the same in kidneys colonized by TN03, CFT073, or HT7. Lastly, we show that in vitro TN03 has a high maximum growth rate in both complex (Luria-Bertani and human urine) and minimum media. In conclusion, our findings indicate that TN03 is a potent UPEC strain that colonizes the intestinal tract and may persist in the kidneys of infected hosts.     

Molecular Characterization of a Multidrug Resistance IncF Plasmid from the Globally Disseminated Escherichia coli ST131 Clone

Escherichia coli sequence type 131 (E. coli ST131) is a recently emerged and globally disseminated multidrug resistant clone associated with urinary tract and bloodstream infections. Plasmids represent a major vehicle for the carriage of antibiotic resistance genes in E. coli ST131. In this study, we determined the complete sequence and performed a comprehensive annotation of pEC958, an IncF plasmid from the E. coli ST131 reference strain EC958. Plasmid pEC958 is 135.6 kb in size, harbours two replicons (RepFIA and RepFII) and contains 12 antibiotic resistance genes (including the bla _CTX-M-15 gene). We also carried out hyper-saturated transposon mutagenesis and multiplexed transposon directed insertion-site sequencing (TraDIS) to investigate the biology of pEC958. TraDIS data showed that while only the RepFII replicon was required for pEC958 replication, the RepFIA replicon contains genes essential for its partitioning. Thus, our data provides direct evidence that the RepFIA and RepFII replicons in pEC958 cooperate to ensure their stable inheritance. The gene encoding the antitoxin component (ccdA) of the post-segregational killing system CcdAB was also protected from mutagenesis, demonstrating this system is active. Sequence comparison with a global collection of ST131 strains suggest that IncF represents the most common type of plasmid in this clone, and underscores the need to understand its evolution and contribution to the spread of antibiotic resistance genes in E. coli ST131.     

A Commensal Gone Bad: Complete Genome Sequence of the Prototypical Enterotoxigenic Escherichia coli Strain H10407

In most cases, Escherichia coli exists as a harmless commensal organism, but it may on occasion cause intestinal and/or extraintestinal disease. Enterotoxigenic E. coli (ETEC) is the predominant cause of E. coli-mediated diarrhea in the developing world and is responsible for a significant portion of pediatric deaths. In this study, we determined the complete genomic sequence of E. coli {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407, a prototypical strain of enterotoxigenic E. coli, which reproducibly elicits diarrhea in human volunteer studies. We performed genomic and phylogenetic comparisons with other E. coli strains, revealing that the chromosome is closely related to that of the nonpathogenic commensal strain E. coli HS and to those of the laboratory strains E. coli K-12 and C. Furthermore, these analyses demonstrated that there were no chromosomally encoded factors unique to any sequenced ETEC strains. Comparison of the E. coli {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 plasmids with those from several ETEC strains revealed that the plasmids had a mosaic structure but that several loci were conserved among ETEC strains. This study provides a genetic context for the vast amount of experimental and epidemiological data that have been published.Current dogma suggests the Gram-negative motile bacterium Escherichia coli colonizes the infant gut within hours of birth and establishes itself as the predominant facultative anaerobe of the colon for the remainder of life (3, 59). While the majority of E. coli strains maintain this harmless existence, some strains have adopted a pathogenic lifestyle. Contemporary tenets suggest that pathogenic strains of E. coli have acquired genetic elements that encode virulence factors and enable the organism to cause disease (12). The large repertoire of virulence factors enables E. coli to cause a variety of clinical manifestations, including intestinal infections mediating diarrhea and extraintestinal infections, such as urinary tract infections, septicemia, and meningitis. Based on clinical manifestation of disease, the repertoire of virulence factors, epidemiology, and phylogenetic profiles, the strains causing intestinal infections can be divided into six separate pathotypes, viz., enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), diffuse adhering E. coli (DAEC), and enterotoxigenic E. coli (ETEC) (33, 35, 39).ETEC is responsible for the majority of E. coli-mediated cases of human diarrhea worldwide. It is particularly prevalent among children in developing countries, where sanitation and clean supplies of drinking water are inadequate, and in travelers to such regions. It is estimated that there are 200 million incidences of ETEC infection annually, resulting in hundreds of thousands of deaths in children under the age of 5 (55, 64). The essential determinants of ETEC virulence are traditionally considered to be colonization of the host small-intestinal epithelium via plasmid-encoded colonization factors (CFs) and subsequent release of plasmid-encoded heat-stable (ST) and/or heat-labile (LT) enterotoxins that induce a net secretory state leading to profuse watery diarrhea (20, 62). More recently, additional plasmid-encoded factors have been implicated in the pathogenesis of ETEC, namely, the EatA serine protease autotransporter (SPATE) and the EtpA protein, which acts as an intermediate in the adhesion between bacterial flagella and host cells (23, 32, 42, 46). Furthermore, a number of chromosomal factors are thought to be involved in virulence, e.g., the invasin Tia; the TibA adhesin/invasin; and LeoA, a GTPase with unknown function (14, 21, 22). E. coli {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 is considered a prototypical ETEC strain; it expresses colonization factor antigen 1 (CFA/I) and the heat-stable and heat labile toxins. Loss of a 94.8-kb plasmid encoding CFA/I and a gene for ST enterotoxin from E. coli strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 leads to reduced ability to cause diarrhea (17).Here, we report the complete genome sequence and virulence factor repertoire of the prototypical ETEC strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 and the nucleotide sequence and gene repertoire of the plasmids from ETEC strain E1392/75, and we describe a novel conserved secretion system associated with the sequenced ETEC strains.     

Genome analysis of bovine-mastitis-associated Escherichia coli O32:H37 strain P4

Escherichia coli is a major pathogen of bovine intramammary infections. Here we report the first draft of the genome sequence of the E. coli O32:H37 P4 strain, which is widely used in experimental bovine mastitis studies.     

Recent Emergence of Clonal Group O25b:K1:H4-B2-ST131 ibeA Strains among Escherichia coli Poultry Isolates,Including CTX-M-9-Producing Strains,and Comparison with Clinical Human Isolates

To discern the possible spread of the Escherichia coli O25b:H4-ST131 clonal group in poultry and the zoonotic potential of avian strains, we made a retrospective search of our strain collection and compared the findings for those strains with the findings for current strains. Thus, we have characterized a collection of 19 avian O25b:H4-ST131 E. coli strains isolated from 1995 to 2010 which, interestingly, harbored the ibeA gene. Using this virulence gene as a criterion for selection, we compared those 19 avian strains with 33 human O25b:H4-ST131 ibeA-positive E. coli strains obtained from patients with extraintestinal infections (1993 to 2009). All 52 O25b:H4-ST131 ibeA-positive E. coli strains shared the fimH, kpsMII, malX, and usp genes but showed statistically significant differences in nine virulence factors, namely, papGIII, cdtB, sat, and kpsMII K5, which were associated with human strains, and iroN, kpsMII K1, cvaC, iss, and tsh, which were associated with strains of avian origin. The XbaI macrorestriction profiles of the 52 E. coli O25b:H4-ST131 ibeA-positive strains revealed 11 clusters (clusters I to XI) of >85% similarity, with four clusters including strains of human and avian origin. Cluster VII (90.9% similarity) grouped 10 strains (7 avian and 3 human strains) that mostly produced CTX-M-9 and that also shared the same virulence profile. Finally, we compared the macrorestriction profiles of the 12 CTX-M-9-producing O25b:H4-ST131 ibeA strains (7 avian and 5 human strains) identified among the 52 strains with those of 15 human O25b:H4-ST131 CTX-M-14-, CTX-M-15-, and CTX-M-32-producing strains that proved to be negative for ibeA and showed that they clearly differed in the level of similarity from the CTX-M-9-producing strains. In conclusion, E. coli clonal group O25b:H4-ST131 ibeA has recently emerged among avian isolates with the new acquisition of the K1 capsule antigen and includes CTX-M-9-producing strains. This clonal group represents a real zoonotic risk that has crossed the barrier between human and avian hosts.Strains of the extensively antimicrobial-resistant Escherichia coli clonal group of sequence type (ST) 131 (ST131) belonging to serotype O25b:H4 have recently been recognized to be important human pathogens worldwide (9, 33). Although it is commonly associated with the dissemination of CTX-M-15 extended-spectrum cephalosporin resistance, E. coli O25b:H4-ST131 also occurs as a fluoroquinolone (FQ)-resistant but cephalosporin-susceptible pathogen (5, 22, 26, 27). Currently, it is assumed that O25b:H4-ST131 strains circulate not only among humans but also among animal hosts (13, 21, 37), which would contribute to the ongoing global emergence of O25b:H4-ST131, in the case of regular transmission between animals and humans. Even though CTX-M-15 is the most widely distributed extended-spectrum beta-lactamase (ESBL) linked to this clonal group, other, different variants of CTX-M have recently been reported, such as CTX-M-9, CTX-M-14, and CTX-M-32 (4, 34, 36, 39). Noteworthy was the detection, for the first time on poultry farms, of this clonal group producing CTX-M-9 that had macrorestriction profiles and virulence genes very similar to those observed in clinical human isolates (10).Extraintestinal pathogenic E. coli (ExPEC) strains, which include avian pathogenic E. coli (APEC) and human uropathogenic E. coli (UPEC), septicemic E. coli, and newborn meningitis-causing E. coli (NMEC) strains, exhibit considerable genome diversity and have a wide range of virulence-associated factors (12, 18). While infections caused by APEC strains initially start as a respiratory tract disease which evolves to a systemic infection of the internal organs and, finally, to sepsis, the most frequent origin of human sepsis is urinary tract infection (UTI), especially pyelonephritis (2, 3, 11). However, APEC strains have been recognized to share common traits with human isolates (29, 30, 31), including the K1 capsule antigen (23, 24, 29) and the ibeA gene (14). In addition, retail chicken products have been found to carry nalidixic-resistant ExPEC strains (17, 19), and although it is drug susceptible, an E. coli strain belonging to the O25b:H4-ST131 clonal group has even recently been detected in retail chicken (41), supporting the urgent necessity for the implementation of food control measures.The aim of the present study was to discern the possible spread of the O25b:H4-ST131 clonal group, especially CTX-M-9-producing strains, in poultry and the zoonotic potential of avian isolates. For this purpose, we made a retrospective search of our human and avian strain collections and compared the findings for those strains with the findings for current strains. Identification of this emerging clone among avian sources and comparison of the clone with clinical human isolates will shed new light on the epidemiology of the O25b:H4-ST131 clonal group.     

Virulence Patterns in a Murine Sepsis Model of ST131 Escherichia coli Clinical Isolates Belonging to Serotypes O25b:H4 and O16:H5 Are Associated to Specific Virotypes

Escherichia coli sequence type (ST)131 is an emerging disseminated public health threat implicated in multidrug-resistant extraintestinal infections worldwide. Although the majority of ST131 isolates belong to O25b:H4 serotype, new variants with different serotypes, STs using the discriminative multilocus sequence typing scheme of Pasteur Institute, and virulence-gene profiles (virotypes) have been reported with unknown implications on the pattern of spread, persistence and virulence. The aim of the present study was to compare virulence in a mouse subcutaneous sepsis model of representative ST131 clinical isolates belonging to 2 serotypes (O25b:H4, O16:H5) and nine virotypes and subtypes (A, B, C, D1, D2, D3, D4, D5 and E). Fourteen out of the 23 ST131 isolates tested (61%) killed 90 to 100% of mice challenged, and 18 of 23 (78%) at least 50%. Interestingly, different virulence patterns in association with virotypes were observed, from highly rapid lethality (death in less than 24 h) to low final lethality (death at 7 days) but with presence of an acute inflammation. This is the first study to assess virulence of ST131 isolates belonging to serotype O16:H5, which exhibited virotype C. In spite of their low virulence-gene score, O16:H5 isolates did not show significant differences in final lethality compared with highly virulent O25b:H4 isolates of virotypes A, B and C, but killed mice less rapidly. Significant differences were found, however, between virotypes A, B, C (final lethality ≥80% of mice challenged) and virotypes D, E. Particularly unexpected was the low lethality of the newly assigned virotype E taking into account that it exhibited high virulence-gene score, and the same clonotype H30 as highly virulent O25b:H4 isolates of virotypes A, B and C. In vivo virulence diversity reported in this study would reflect the genetic variability within ST131 clonal group evidenced by molecular typing.     

Characterization of uropathogenic E. coli O25b-B2-ST131, O15:K52:H1, and CGA: Neutrophils apoptosis,serum bactericidal assay,biofilm formation,and virulence typing

Pathogenic and drug-resistant strains of Escherichia coli (E. coli) O25b-B2-ST131, O15:H1-D-ST393, and CGA (clonal group A) clonal groups have spread worldwide. This study aimed at determining E. coli epidemic clonal groups, their virulence factors, biofilm formation, neutrophils apoptosis, and antimicrobial resistance pattern of uropathogenic E. coli. A total of 95 CTX-M-1-producing E. coli clinical isolates were enrolled. E. coli O25b-B2-ST131, CGA, and O15:K52:H1 were identified by serotyping and phylogrouping and allele-specific polymerase chain reaction-based assay. Antibiotic susceptibility, biofilm formation, hemolysis, and human serum bactericidal assay were performed. Neutrophil apoptosis was assayed by flow cytometry. Nine E. coli clonal groups including six O25b-B2-ST131 strains, two CGA, and one O15:K52:H1-D-ST393 strains were detected. One O25b-B2-ST131 isolate was a strong biofilm-producer. Three ST131 isolates had type I fimbriae. Furthermore, all the CGA and O15:K52:H1 and three of ST131 isolates harbored the P fimbriae. The virulence genes ompT, fimH, and traT were detected among all the clonal groups. The apoptosis was induced by O25b-B2-ST131, CGA, and O15:K52:H1 E. coli. There was no significant difference regarding apoptosis induction among clonal groups. Furthermore, the presence of the cdt, usp, and vat genes was significantly associated with the apoptosis of neutrophils by O25b-B2-ST131, CGA, and O15:K52:H1-D-ST393 clonal groups.     

Sequence Variations in the Flagellar Antigen Genes fliCH25 and fliCH28 of Escherichia coli and Their Use in Identification and Characterization of Enterohemorrhagic E. coli (EHEC) O145:H25 and O145:H28

Enterohemorrhagic E. coli (EHEC) serogroup O145 is regarded as one of the major EHEC serogroups involved in severe infections in humans. EHEC O145 encompasses motile and non-motile strains of serotypes O145:H25 and O145:H28. Sequencing the fliC-genes associated with the flagellar antigens H25 and H28 revealed the genetic diversity of the fliC_H25 and fliC_H28 gene sequences in E. coli. Based on allele discrimination of these fliC-genes real-time PCR tests were designed for identification of EHEC O145:H25 and O145:H28. The fliC_H25 genes present in O145:H25 were found to be very similar to those present in E. coli serogroups O2, O100, O165, O172 and O177 pointing to their common evolution but were different from fliC_H25 genes of a multiple number of other E. coli serotypes. In a similar way, EHEC O145:H28 harbor a characteristic fliC_H28 allele which, apart from EHEC O145:H28, was only found in enteropathogenic (EPEC) O28:H28 strains that shared some common traits with EHEC O145:H28. The real time PCR-assays targeting these fliC_H25[O145] and fliC_H28[O145] alleles allow better characterization of EHEC O145:H25 and EHEC O145:H28. Evaluation of these PCR assays in spiked ready-to eat salad samples resulted in specific detection of both types of EHEC O145 strains even when low spiking levels of 1–10 cfu/g were used. Furthermore these PCR assays allowed identification of non-motile E. coli strains which are serologically not typable for their H-antigens. The combined use of O-antigen genotyping (O145wzy) and detection of the respective fliC_H25[O145] and fliC_H28[O145] allele types contributes to improve identification and molecular serotyping of E. coli O145 isolates.     

Assessment of resistance to colicinogenic Escherichia coli by E. coli O157:H7 strains

AIMS: To assess a collection of 96 Escherichia coli O157:H7 strains for their resistance potential against a set of colicinogenic E. coli developed as a probiotic for use in cattle. METHODS AND RESULTS: Escherichia coli O157:H7 strains were screened for colicin production, types of colicins produced, presence of colicin resistance and potential for resistance development. Thirteen of 14 previously characterized colicinogenic E. coli strains were able to inhibit 74 serotype O157:H7 strains. Thirteen E. coli O157:H7 strains were found to be colicinogenic and 11 had colicin D genes. PCR products for colicins B, E-type, Ia/Ib and M were also detected. During in vitro experiments, the ability to develop colicin resistance against single-colicin producing E. coli strains was observed, but rarely against multiple-colicinogenic strains. The ability of serotype O157:H7 strains to acquire colicin plasmids or resistance was not observed during a cattle experiment. CONCLUSIONS: Escherichia coli O157:H7 has the potential to develop single-colicin resistance, but simultaneous resistance against multiple colicins appears to be unlikely. Colicin D is the predominant colicin produced by colicinogenic E. coli O157:H7 strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential for resistance development against colicin-based strategies for E. coli O157:H7 control may be very limited if more than one colicin type is used.     

Persistent Colonization of Sheep by Escherichia coli O157:H7 and Other E. coli Pathotypes

Shiga toxin-producing Escherichia coli (STEC) is an important cause of food-borne illness in humans. Ruminants appear to be more frequently colonized by STEC than are other animals, but the reason(s) for this is unknown. We compared the frequency, magnitude, duration, and transmissibility of colonization of sheep by E. coli O157:H7 to that by other pathotypes of E. coli. Young adult sheep were simultaneously inoculated with a cocktail consisting of two strains of E. coli O157:H7, two strains of enterotoxigenic E. coli (ETEC), and one strain of enteropathogenic E. coli. Both STEC strains and ETEC 2041 were given at either 10⁷ or 10¹⁰ CFU/strain/animal. The other strains were given only at 10¹⁰ CFU/strain. We found no consistent differences among pathotypes in the frequency, magnitude, and transmissibility of colonization. However, the STEC strains tended to persist to 2 weeks and 2 months postinoculation more frequently than did the other pathotypes. The tendency for persistence of the STEC strains was apparent following an inoculation dose of either 10⁷ or 10¹⁰ CFU. One of the ETEC strains also persisted when inoculated at 10¹⁰ CFU. However, in contrast to the STEC strains, it did not persist when inoculated at 10⁷ CFU. These results support the hypothesis that STEC is better adapted to persist in the alimentary tracts of sheep than are other pathotypes of E. coli.     

Persistent colonization of sheep by Escherichia coli O157:H7 and other E. coli pathotypes

Shiga toxin-producing Escherichia coli (STEC) is an important cause of food-borne illness in humans. Ruminants appear to be more frequently colonized by STEC than are other animals, but the reason(s) for this is unknown. We compared the frequency, magnitude, duration, and transmissibility of colonization of sheep by E. coli O157:H7 to that by other pathotypes of E. coli. Young adult sheep were simultaneously inoculated with a cocktail consisting of two strains of E. coli O157:H7, two strains of enterotoxigenic E. coli (ETEC), and one strain of enteropathogenic E. coli. Both STEC strains and ETEC 2041 were given at either 10(7) or 10(10) CFU/strain/animal. The other strains were given only at 10(10) CFU/strain. We found no consistent differences among pathotypes in the frequency, magnitude, and transmissibility of colonization. However, the STEC strains tended to persist to 2 weeks and 2 months postinoculation more frequently than did the other pathotypes. The tendency for persistence of the STEC strains was apparent following an inoculation dose of either 10(7) or 10(10) CFU. One of the ETEC strains also persisted when inoculated at 10(10) CFU. However, in contrast to the STEC strains, it did not persist when inoculated at 10(7) CFU. These results support the hypothesis that STEC is better adapted to persist in the alimentary tracts of sheep than are other pathotypes of E. coli.     

Complete genome sequence and comparative genome analysis of enteropathogenic Escherichia coli O127:H6 strain E2348/69

Enteropathogenic Escherichia coli (EPEC) was the first pathovar of E. coli to be implicated in human disease; however, no EPEC strain has been fully sequenced until now. Strain E2348/69 (serotype O127:H6 belonging to E. coli phylogroup B2) has been used worldwide as a prototype strain to study EPEC biology, genetics, and virulence. Studies of E2348/69 led to the discovery of the locus of enterocyte effacement-encoded type III secretion system (T3SS) and its cognate effectors, which play a vital role in attaching and effacing lesion formation on gut epithelial cells. In this study, we determined the complete genomic sequence of E2348/69 and performed genomic comparisons with other important E. coli strains. We identified 424 E2348/69-specific genes, most of which are carried on mobile genetic elements, and a number of genetic traits specifically conserved in phylogroup B2 strains irrespective of their pathotypes, including the absence of the ETT2-related T3SS, which is present in E. coli strains belonging to all other phylogroups. The genome analysis revealed the entire gene repertoire related to E2348/69 virulence. Interestingly, E2348/69 contains only 21 intact T3SS effector genes, all of which are carried on prophages and integrative elements, compared to over 50 effector genes in enterohemorrhagic E. coli O157. As E2348/69 is the most-studied pathogenic E. coli strain, this study provides a genomic context for the vast amount of existing experimental data. The unexpected simplicity of the E2348/69 T3SS provides the first opportunity to fully dissect the entire virulence strategy of attaching and effacing pathogens in the genomic context.     

Characterization of rpoS alleles in Escherichia coli O157:H7 and in other E. coli serotypes

The rpoS nucleotide and predicted amino acid sequences from three Escherichia coli O157:H7 isolates were compared with those from three other E. coli isolates, including the likely O157:H7 progenitor, E. coli O55:H7. These clinical and environmental isolates all had identical sigma S amino acid sequences, while laboratory strains K12 and DH1 had three and one amino acid alterations, respectively, in comparison with the majority sequence. To extend the analysis of sigma S sequence conservation to include other Gram-negative bacteria, the E. coli sigma S sequences were compared with those from diverse Gram-negative organisms; sigma S sequence identities ranged from 50.2 to 99.7% among the available sequences. The results further confirm the existence of rpoS alleles among different E. coli strains, although all strains were classified as acid-resistant with survival rates > 10% after 2 h exposure to pH 2.5. It was also found that all E. coli O157:H7 isolates tested had a unique nucleotide at position 543, thus differentiating these strains from other E. coli serotypes.     

Measurements of fitness and competition in commensal Escherichia coli and E. coli O157:H7 strains

Although the main reservoirs for pathogenic Escherichia coli O157:H7 are cattle and the cattle environment, factors that affect its tenure in the bovine host and its survival outside humans and cattle have not been well studied. It is also not understood what physiological properties, if any, distinguish these pathogens from commensal counterparts that live as normal members of the human and bovine gastrointestinal tracts. To address these questions, individual and competitive fitness experiments, indirect antagonism assays, and antibiotic resistance and carbon utilization analyses were conducted using a strain set consisting of 122 commensal and pathogenic strains. The individual fitness experiments, under four different environments (rich medium, aerobic and anaerobic; rumen medium, anaerobic; and a minimal medium, aerobic) revealed no differences in growth rates between commensal E. coli and E. coli O157:H7 strains. Indirect antagonism assays revealed that E. coli O157:H7 strains more frequently produced inhibitory substances than commensal strains did, under the conditions tested, although both groups displayed moderate sensitivity. Only minor differences were noted in the antibiotic resistance patterns of the two groups. In contrast, several differences between commensal and O157:H7 groups were observed based on their carbon utilization profiles. Of 95 carbon sources tested, 27 were oxidized by commensal E. coli strains but not by the E. coli O157:H7 strains. Despite the observed physiological and biochemical differences between these two groups of E. coli strains, however, the O157:H7 strains did not appear to possess traits that would confer advantages in the bovine or extraintestinal environment.     

Measurements of Fitness and Competition in Commensal Escherichia coli and E. coli O157:H7 Strains

Although the main reservoirs for pathogenic Escherichia coli O157:H7 are cattle and the cattle environment, factors that affect its tenure in the bovine host and its survival outside humans and cattle have not been well studied. It is also not understood what physiological properties, if any, distinguish these pathogens from commensal counterparts that live as normal members of the human and bovine gastrointestinal tracts. To address these questions, individual and competitive fitness experiments, indirect antagonism assays, and antibiotic resistance and carbon utilization analyses were conducted using a strain set consisting of 122 commensal and pathogenic strains. The individual fitness experiments, under four different environments (rich medium, aerobic and anaerobic; rumen medium, anaerobic; and a minimal medium, aerobic) revealed no differences in growth rates between commensal E. coli and E. coli O157:H7 strains. Indirect antagonism assays revealed that E. coli O157:H7 strains more frequently produced inhibitory substances than commensal strains did, under the conditions tested, although both groups displayed moderate sensitivity. Only minor differences were noted in the antibiotic resistance patterns of the two groups. In contrast, several differences between commensal and O157:H7 groups were observed based on their carbon utilization profiles. Of 95 carbon sources tested, 27 were oxidized by commensal E. coli strains but not by the E. coli O157:H7 strains. Despite the observed physiological and biochemical differences between these two groups of E. coli strains, however, the O157:H7 strains did not appear to possess traits that would confer advantages in the bovine or extraintestinal environment.     

The lipopolysaccharide core type of Escherichia coli O157:H7 and other non-O157 verotoxin-producing E. coli

A mouse monoclonal antibody specific for the R3 lipopolysaccharide core type of Escherichia coli was used to determine the core type of E. coli O157:H7 and other non-O157 verotoxin-producing E. coli strains. Lipopolysaccharide extracts from 28 clinical isolates were examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting and all were found to have the R3 core. None of the core lipopolysaccharide from the strains tested reacted with the control R1 and R2 specific monoclonal antibodies. A common core type between all the verotoxin-producing E. coli strains tested may be significant when considering the immune response to these bacteria, and to the receptor for the VT bacteriophage.