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.     

Changes in Pulsed-Field Gel Electrophoresis Patterns in Clinical Isolates of Enterohemorrhagic Escherichia coli O157:H7 Associated with Loss of Shiga Toxin Genes

Pulsed-field gel electrophoresis (PFGE) of XbaI-digested DNA fragments of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains showed disappearance of a 70- or 80-kb fragment in their patterns associated with loss of Shiga toxin genes during maintenance or subcultivation. Hybridization experiments with a DNA probe complementary to Shiga toxin sequences revealed that the Shiga toxin genes in the parental strain were located on fragments the same size as the lost fragments from the toxin-negative derivatives. The evidence indicates that PFGE pattern of EHEC O157:H7 may change due to loss of Shiga toxin genes, which is likely to be associated with curing of Shiga toxin gene carrying phages in vitro. Received: 4 May 1998 / Accepted: 19 August 1998     

Presence of enterohemorrhagic Escherichia coli ST678/O104:H4 in France prior to 2011

Two isolates of enterohemorrhagic Escherichia coli (EHEC) O104:H4 were isolated in France in 2004 and 2009. Both were characterized and compared to the strain which caused the German outbreak in 2011 and to other O104:H4 strains. This suggests that different O104:H4 EHEC strains were present several years prior to the 2011 outbreak.     

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.     

The Gut Bacterium Bacteroides thetaiotaomicron Influences the Virulence Potential of the Enterohemorrhagic Escherichia coli O103:H25

Enterohemorrhagic E. coli (EHEC) is associated with severe gastrointestinal disease. Upon entering the gastrointestinal tract, EHEC is exposed to a fluctuating environment and a myriad of other bacterial species. To establish an infection, EHEC strains have to modulate their gene expression according to the GI tract environment. In order to explore the interspecies interactions between EHEC and an human intestinal commensal, the global gene expression profile was determined of EHEC O103:H25 (EHEC NIPH-11060424) co-cultured with B. thetaiotaomicron (CCUG 10774) or grown in the presence of spent medium from B. thetaiotaomicron. Microarray analysis revealed that approximately 1% of the EHEC NIPH-11060424 genes were significantly up-regulated both in co-culture (30 genes) and in the presence of spent medium (44 genes), and that the affected genes differed between the two conditions. In co-culture, genes encoding structural components of the type three secretion system were among the most affected genes with an almost 4-fold up-regulation, while the most affected genes in spent medium were involved in chemotaxis and were more than 3-fold up-regulated. The operons for type three secretion system (TTSS) are located on the Locus of enterocyte effacement (LEE) pathogenicity island, and qPCR showed that genes of all five operons (LEE1-LEE5) were up-regulated. Moreover, an increased adherence to HeLa cells was observed in EHEC NIPH-11060424 exposed to B. thetaiotaomicron. Expression of stx2 genes, encoding the main virulence factor of EHEC, was down-regulated in both conditions (co-culture/spent medium). These results show that expression of EHEC genes involved in colonization and virulence is modulated in response to direct interspecies contact between cells, or to diffusible factors released from B. thetaiotaomicron. Such interspecies interactions could allow the pathogen to recognize its predilection site and modulate its behaviour accordingly, thus increasing the efficiency of colonization of the colon mucosa, facilitating its persistence and increasing its virulence potential.     

The Complete Genome Sequence of Escherichia coli EC958: A High Quality Reference Sequence for the Globally Disseminated Multidrug Resistant E. coli O25b:H4-ST131 Clone

Escherichia coli ST131 is now recognised as a leading contributor to urinary tract and bloodstream infections in both community and clinical settings. Here we present the complete, annotated genome of E. coli EC958, which was isolated from the urine of a patient presenting with a urinary tract infection in the Northwest region of England and represents the most well characterised ST131 strain. Sequencing was carried out using the Pacific Biosciences platform, which provided sufficient depth and read-length to produce a complete genome without the need for other technologies. The discovery of spurious contigs within the assembly that correspond to site-specific inversions in the tail fibre regions of prophages demonstrates the potential for this technology to reveal dynamic evolutionary mechanisms. E. coli EC958 belongs to the major subgroup of ST131 strains that produce the CTX-M-15 extended spectrum β-lactamase, are fluoroquinolone resistant and encode the fimH30 type 1 fimbrial adhesin. This subgroup includes the Indian strain NA114 and the North American strain JJ1886. A comparison of the genomes of EC958, JJ1886 and NA114 revealed that differences in the arrangement of genomic islands, prophages and other repetitive elements in the NA114 genome are not biologically relevant and are due to misassembly. The availability of a high quality uropathogenic E. coli ST131 genome provides a reference for understanding this multidrug resistant pathogen and will facilitate novel functional, comparative and clinical studies of the E. coli ST131 clonal lineage.     

Correlation between In Vivo Biofilm Formation and Virulence Gene Expression in Escherichia coli O104:H4

The emergence of novel pathogens poses a major public health threat causing widespread epidemics in susceptible populations. The Escherichia coli O104:H4 strain implicated in a 2011 outbreak in northern Germany caused the highest frequency of hemolytic uremic syndrome (HUS) and death ever recorded in a single E. coli outbreak. Therefore, it has been suggested that this strain is more virulent than other pathogenic E. coli (e.g., E. coli O157:H7). The E. coli O104:H4 outbreak strain possesses multiple virulence factors from both Shiga toxin (Stx)-producing E. coli (STEC) and enteroaggregative E. coli (EAEC), though the mechanism of pathogenesis is not known. Here, we demonstrate that E. coli O104:H4 produces a stable biofilm in vitro and that in vivo virulence gene expression is highest when E. coli O104:H4 overexpresses genes required for aggregation and exopolysaccharide production, a characteristic of bacterial cells residing within an established biofilm. Interrupting exopolysaccharide production and biofilm formation may therefore represent effective strategies for combating future E. coli O104:H4 infections.     

Vesicle-Mediated Transfer of Virulence Genes from Escherichia coli O157:H7 to Other Enteric Bacteria

Membrane vesicles are released from the surfaces of many gram-negative bacteria during growth. Vesicles consist of proteins, lipopolysaccharide, phospholipids, RNA, and DNA. Results of the present study demonstrate that membrane vesicles isolated from the food-borne pathogen Escherichia coli O157:H7 facilitate the transfer of genes, which are then expressed by recipient Salmonella enterica serovar Enteritidis or E. coli JM109. Electron micrographs of purified DNA from E. coli O157:H7 vesicles showed large rosette-like structures, linear DNA fragments, and small open-circle plasmids. PCR analysis of vesicle DNA demonstrated the presence of specific genes from host and recombinant plasmids (hly, L7095, mobA, and gfp), chromosomal DNA (uidA and eaeA), and phage DNA (stx1 and stx2). The results of PCR and the Vero cell assay demonstrate that genetic material, including virulence genes, is transferred to recipient bacteria and subsequently expressed. The cytotoxicity of the transformed enteric bacteria was sixfold higher than that of the parent isolate (E. coli JM109). Utilization of the nonhost plasmid (pGFP) permitted the evaluation of transformation efficiency (ca. 10³ transformants μg of DNA⁻¹) and demonstrated that vesicles can deliver antibiotic resistance. Transformed E. coli JM109 cells were resistant to ampicillin and fluoresced a brilliant green. The role vesicles play in genetic exchange between different species in the environment or host has yet to be defined.     

Assessment of Virulence Factors Characteristic of Human Escherichia coli Pathotypes and Antimicrobial Resistance in O157:H7 and Non-O157:H7 Isolates from Livestock in Spain

The distribution of virulence factors (VFs) typical of diarrheagenic Escherichia coli and the antimicrobial resistance (AMR) profiles were assessed in 780 isolates from healthy pigs, broilers, and cattle from Spain. VF distribution was broader than expected, although at low prevalence for most genes, with AMR being linked mainly to host species.     

Intimin Gene (eae) Subtype-Based Real-Time PCR Strategy for Specific Detection of Shiga Toxin-Producing Escherichia coli Serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 in Cattle Feces

Shiga toxin-producing Escherichia coli (STEC) strains belonging to serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 are known to be associated with particular subtypes of the intimin gene (eae), namely, γ1, β1, ε, θ, and γ1, respectively. This study aimed at evaluating the usefulness of their detection for the specific detection of these five main pathogenic STEC serotypes in cattle feces. Using real-time PCR assays, 58.7% of 150 fecal samples were found positive for at least one of the four targeted eae subtypes. The simultaneous presence of stx, eae, and one of the five O group markers was found in 58.0% of the samples, and the five targeted stx plus eae plus O genetic combinations were detected 143 times. However, taking into consideration the association between eae subtypes and O group markers, the resulting stx plus eae subtype plus O combinations were detected only 46 times. The 46 isolation assays performed allowed recovery of 22 E. coli strains belonging to one of the five targeted STEC serogroups. In contrast, only 2 of 39 isolation assays performed on samples that were positive for stx, eae and an O group marker, but that were negative for the corresponding eae subtype, were successful. Characterization of the 24 E. coli isolates showed that 6 were STEC, including 1 O157:H7, 3 O26:H11, and 2 O145:H28. The remaining 18 strains corresponded to atypical enteropathogenic E. coli (aEPEC). Finally, the more discriminating eae subtype-based PCR strategy described here may be helpful for the specific screening of the five major STEC in cattle feces.     

Detection and Characterization of the Fimbrial sfp Cluster in Enterohemorrhagic Escherichia coli O165:H25/NM Isolates from Humans and Cattle

The sfp cluster, encoding Sfp fimbriae and located in the large plasmid of sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157 (pSFO157), has been considered a unique characteristic of this organism. We discovered and then characterized the sfp cluster in EHEC O165:H25/NM (nonmotile) isolates of human and bovine origin. All seven strains investigated harbored a complete sfp cluster (carrying sfpA, sfpH, sfpC, sfpD, sfpJ, sfpF, and sfpG) of 6,838 bp with >99% nucleotide sequence homology to the sfp cluster of SF EHEC O157:NM. The sfp cluster in EHEC O165:H25/NM strains was located in an ~80-kb (six strains) or ~120-kb (one strain) plasmid which differed in structure, virulence genes, and sfp flanks from pSFO157. All O165:H25/NM strains belonged to the same multilocus sequence type (ST119) and were only distantly phylogenetically related to SF EHEC O157:NM (ST11). The highly conserved sfp cluster in different clonal backgrounds suggests that this segment was acquired independently by EHEC O165:H25 and SF EHEC O157:NM. Its presence in an additional EHEC serotype extends the diagnostic utility of PCR targeting sfpA as an easy and efficient approach to seek EHEC in patients' stools. The reasons for the convergence of pathogenic EHEC strains on a suite of virulence loci remain unknown.     

Variation in Resistance to High Hydrostatic Pressure and rpoS Heterogeneity in Natural Isolates of Escherichia coli O157:H7

Several natural isolates of Escherichia coli O157:H7 have previously been shown to exhibit stationary-phase-dependent variation in their resistance to inactivation by high hydrostatic pressure. In this report we demonstrate that loss of the stationary-phase-inducible sigma factor RpoS resulted in decreased resistance to pressure in E. coli O157:H7 and in a commensal strain. Furthermore, variation in the RpoS activity of the natural isolates of O157:H7 correlated with the pressure resistance of those strains. Heterogeneity was noted in the rpoS alleles of the natural isolates that may explain the differences in RpoS activity. These results are consistent with a role for rpoS in mediating resistance to high hydrostatic pressure in E. coli O157:H7.     

Genetic Diversity and Virulence Potential of Shiga Toxin-Producing Escherichia coli O113:H21 Strains Isolated from Clinical,Environmental, and Food Sources

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

Comparative Genomic Analysis Shows That Avian Pathogenic Escherichia coli Isolate IMT5155 (O2:K1:H5; ST Complex 95, ST140) Shares Close Relationship with ST95 APEC O1:K1 and Human ExPEC O18:K1 Strains

Avian pathogenic E. coli and human extraintestinal pathogenic E. coli serotypes O1, O2 and O18 strains isolated from different hosts are generally located in phylogroup B2 and ST complex 95, and they share similar genetic characteristics and pathogenicity, with no or minimal host specificity. They are popular objects for the study of ExPEC genetic characteristics and pathogenesis in recent years. Here, we investigated the evolution and genetic blueprint of APEC pathotype by performing phylogenetic and comparative genome analysis of avian pathogenic E. coli strain IMT5155 (O2:K1:H5; ST complex 95, ST140) with other E. coli pathotypes. Phylogeny analyses indicated that IMT5155 has closest evolutionary relationship with APEC O1, IHE3034, and UTI89. Comparative genomic analysis showed that IMT5155 and APEC O1 shared significant genetic overlap/similarities with human ExPEC dominant O18:K1 strains (IHE3034 and UTI89). Furthermore, the unique PAI I₅₁₅₅ (GI-12) was identified and found to be conserved in APEC O2 serotype isolates. GI-7 and GI-16 encoding two typical T6SSs in IMT5155 might be useful markers for the identification of ExPEC dominant serotypes (O1, O2, and O18) strains. IMT5155 contained a ColV plasmid p1ColV₅₁₅₅, which defined the APEC pathotype. The distribution analysis of 10 sequenced ExPEC pan-genome virulence factors among 47 sequenced E. coli strains provided meaningful information for B2 APEC/ExPEC-specific virulence factors, including several adhesins, invasins, toxins, iron acquisition systems, and so on. The pathogenicity tests of IMT5155 and other APEC O1:K1 and O2:K1 serotypes strains (isolated in China) through four animal models showed that they were highly virulent for avian colisepticemia and able to cause septicemia and meningitis in neonatal rats, suggesting zoonotic potential of these APEC O1:K1 and O2:K1 isolates.     

Survival of Escherichia coli O157:H7, Listeria monocytogenes 4b and Yersinia enterocolitica O3 in different yogurt and kefir combinations as prefermentation contaminant

AIMS: To compare microbiological safety of yogurt, kefir and different combinations of yogurt and kefir samples by using three foodborne pathogenic strains (Escherichia coli O157:H7, Listeria monocytogenes 4b and Yersinia enterocolitica O3) as indicators. METHODS AND RESULTS: Fresh yogurt and kefir drinks were added to pasteurized milk at a 5% rate either separately or together, and then incubated at different temperatures (43 degrees C for yogurt and 30 degrees C for kefir), depending on appropriate growth temperature of their starter microflora. While traditional yogurt was found to be the least suppressive on the three pathogenic micro-organisms, samples obtained from two subsequent fermentation process (samples fermented at 43 degrees C for 3 h and at 30 degrees C for 21 h) were more suppressive than that of traditional kefir. There was no significant survival difference between E. coli O157:H7 and L. monocytogenes 4b in samples tested (P > 0.05), but Y. enterocolitica O3 was more susceptible than other two test strains (P < 0.05). CONCLUSIONS: The microbiological safety of the dairy product fermented at two consecutive periods was superior than that of traditional yogurt or kefir alone. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments may mimic what happens when yogurt and kefir starter micro-organisms are combined in a milk fermentation process with different time and temperature periods.