Escherichia coli ST117: exploring the zoonotic hypothesis.

Extraintestinal pathogenic Escherichia coli (ExPEC) can lead to severe infections, with additional risks of increasing antimicrobial resistance rates. Genotypic similarities between ExPEC and avian pathogenic E. coli (APEC) support a possible role for a poultry meat reservoir in human disease. Some genomic studies have been done on the ST117 lineage which contaminates poultry meat, carries multidrug resistance, can be found in the human intestinal microbiota, and causes human extraintestinal disease. This study analyzed the genomes of 61 E. coli from Brazilian poultry outbreaks focusing on ST117, to further define its possible zoonotic characteristics by genotypic and phylogenomic analyses, along with 1,699 worldwide ST117 isolates originating from human, animal, and environment sources. A predominance of ST117 was detected in the Brazilian isolates (n = 20/61) frequently carrying resistance to critical antibiotics (>86%) linked to IncFII, IncI1, or IncX4 replicons. High similarities were found between IncX4 from Brazilian outbreaks and those from E. coli recovered from imported Brazilian poultry meat and human clinical cases. The ST117 phylogeny showed non-specificity according to host and continent and an AMR index score indicated the highest resistance in Asia and South America, with the latter statistically more resistant and overrepresented with resistance to extended-spectrum beta-lactamases (ESBL). Most ST117 human isolates were predicted to have a poultry origin (93%, 138/148). In conclusion, poultry is a likely source for zoonotic ExPEC strains, particularly the ST117 lineage which can also serve as a reservoir for resistance determinants against critical antibiotics encoded on highly transmissible plasmids. IMPORTANCE: Certain extraintestinal pathogenic Escherichia coli (ExPEC) are particularly important as they affect humans and animals. Lineages, such as ST117, are predominant in poultry and frequent carriers of antibiotic resistance, presenting a risk to humans handling or ingesting poultry products. We analyzed ExPEC isolates causing outbreaks in Brazilian poultry, focusing on the ST117 as the most detected lineage. Genomic comparisons with international isolates from humans and animals were performed describing the potential zoonotic profile. The Brazilian ST117 isolates carried resistance determinants against critical antibiotics, mainly on plasmids, in some cases identical to those carried by international isolates. South American ST117 isolates from all sources generally exhibit more resistance, including to critical antibiotics, and worldwide, the vast majority of human isolates belonging to this lineage have a predicted poultry origin. As the world's largest poultry exporter, Brazil has an important role in developing strategies to prevent the dissemination of multidrug-resistant zoonotic ExPEC strains.

Synergy between Group 2 capsules and lipopolysaccharide underpins serum resistance in extra-intestinal pathogenic Escherichia coli.

Escherichia coli (E. coli) is a major cause of urinary tract infections, bacteraemia, and sepsis. CFT073 is a prototypic, urosepsis isolate of sequence type (ST) 73. This laboratory, among others, has shown that strain CFT073 is resistant to serum, with capsule and other extracellular polysaccharides imparting resistance. The interplay of such polysaccharides remains under-explored. This study has shown that CFT073 mutants deficient in lipopolysaccharide (LPS) O-antigen and capsule display exquisite serum sensitivity. Additionally, O-antigen and LPS outer core mutants displayed significantly decreased surface K2 capsule, coupled with increased unbound K2 capsule being detected in the supernatant. The R1 core and O6 antigen are involved in the tethering of K2 capsule to the CFT073 cell surface, highlighting the importance of the R1 core in serum resistance. The dependence of capsule on LPS was shown to be post-transcriptional and related to changes in cell surface hydrophobicity. Furthermore, immunofluorescence microscopy suggested that the surface pattern of capsule is altered in such LPS core mutants, which display a punctate capsule pattern. Finally, targeting LPS biosynthesis using sub-inhibitory concentrations of a WaaG inhibitor resulted in increased serum sensitivity and decreased capsule in CFT073. Interestingly, the dependency of capsule on LPS has been observed previously in other Enterobacteria, indicating that the synergy between these polysaccharides is not just strain, serotype or species-specific but may be conserved across several pathogenic Gram-negative species. Therefore, using WaaG inhibitor derivatives to target LPS is a promising therapeutic strategy to reduce morbidity and mortality by reducing or eliminating surface capsule.

Efflux of TolC protein to different antimicrobials can be replaced by other outer membrane proteins with similar ß-barrel structures in extraintestinal pathogenic Escherichia coli.

AIM: As a major efflux pump system in Gram-negative bacteria, AcrAB-TolC plays a key role in the transport of multiple drug substrates and is considered a potential target for the development of novel antimicrobials. Our previous study found that TolC inactivation compromised the resistance to different antimicrobials in porcine extraintestinal pathogenic Escherichia coli (ExPEC) strain PPECC042 (WT). This study was designed to investigate the functional substitution of TolC by other outer membrane proteins (OMPs) with similar ß-barrel structures in pumping out different antimicrobials. METHODS AND RESULTS: In this study, we found that over-expression of several OMPs with similar ß-barrel structures, OmpX, OmpC, OmpN, OmpW, and PhoE, in the ΔtolC strain restored the resistance to macrolides, quinolones, or tetracyclines to the level of WT strain. However, the introduction of any one of the five OMPs did not affect the resistance of the strains ΔacrA, ΔacrB, and ΔacrAΔtolC. Further study revealed that the efflux activity was significantly reduced in the ΔtolC strain, but not in the WT strain and the ΔtolC strains over-expressing various OMPs. Additionally, Nile red dye test and ciprofloxacin accumulation test confirmed that the lost efflux activity and drug accumulation in bacterial periplasm by TolC inactivation was restored by the over-expression of each OMP, depending on the presence of genes acrA and acrB. CONCLUSION: All five OMPs can replace the TolC protein to play the efflux role in pumping out the drugs from the periplasm to the extracellular space with the help of proteins AcrA and AcrB.

Population structure and antibiotic resistance of swine extraintestinal pathogenic Escherichia coli from China.

Extraintestinal Pathogenic Escherichia coli (ExPEC) pose a significant threat to human and animal health. However, the diversity and antibiotic resistance of animal ExPEC, and their connection to human infections, remain largely unexplored. The study performs large-scale genome sequencing and antibiotic resistance testing of 499 swine-derived ExPEC isolates from China. Results show swine ExPEC are phylogenetically diverse, with over 80% belonging to phylogroups B1 and A. Importantly, 15 swine ExPEC isolates exhibit genetic relatedness to human-origin E. coli strains. Additionally, 49 strains harbor toxins typical of enteric E. coli pathotypes, implying hybrid pathotypes. Notably, 97% of the total strains are multidrug resistant, including resistance to critical human drugs like third- and fourth-generation cephalosporins. Correspondingly, genomic analysis unveils prevalent antibiotic resistance genes (ARGs), often associated with co-transfer mechanisms. Furthermore, analysis of 20 complete genomes illuminates the transmission pathways of ARGs within swine ExPEC and to human pathogens. For example, the transmission of plasmids co-harboring fosA3, blaCTX-M-14, and mcr-1 genes between swine ExPEC and human-origin Salmonella enterica is observed. These findings underscore the importance of monitoring and controlling ExPEC infections in animals, as they can serve as a reservoir of ARGs with the potential to affect human health or even be the origin of pathogens infecting humans.

The osmoregulated metabolism of trehalose contributes to production of type 1 fimbriae and bladder colonization by extraintestinal Escherichia coli strain BEN2908.

In Escherichia coli, the disaccharide trehalose can be metabolized as a carbon source or be accumulated as an osmoprotectant under osmotic stress. In hypertonic environments, E. coli accumulates trehalose in the cell by synthesis from glucose mediated by the cytosolic enzymes OtsA and OtsB. Trehalose in the periplasm can be hydrolyzed into glucose by the periplasmic trehalase TreA. We have previously shown that a treA mutant of extraintestinal E. coli strain BEN2908 displayed increased resistance to osmotic stress by 0.6 M urea, and reduced production of type 1 fimbriae, reduced invasion of avian fibroblasts, and decreased bladder colonization in a murine model of urinary tract infection. Since loss of TreA likely results in higher periplasmic trehalose concentrations, we wondered if deletion of otsA and otsB genes, which would lead to decreased internal trehalose concentrations, would reduce resistance to stress by 0.6 M urea and promote type 1 fimbriae production. The BEN2908ΔotsBA mutant was sensitive to osmotic stress by urea, but displayed an even more pronounced reduction in production of type 1 fimbriae, with the consequent reduction in adhesion/invasion of avian fibroblasts and reduced bladder colonization in the murine urinary tract. The BEN2908ΔtreAotsBA mutant also showed a reduction in production of type 1 fimbriae, but in contrast to the ΔotsBA mutant, resisted better than the wild type in the presence of urea. We hypothesize that, in BEN2908, resistance to stress by urea would depend on the levels of periplasmic trehalose, but type 1 fimbriae production would be influenced by the levels of cytosolic trehalose.

Distribution of papA and papG Variants among Escherichia coli Genotypes: Association with Major Extraintestinal Pathogenic Lineages.

The pyelonephritis-associated fimbria (P fimbria) is one of the most recognized adhesion determinants of extraintestinal pathogenic Escherichia coli strains (ExPECs). Twelve variants have been described for the gene encoding the P fimbria major structural subunit PapA and three variants for the gene encoding the adhesin subunit PapG. However, their distribution among the ExPEC diversity has not been comprehensively addressed. A complete landscape of that distribution might be valuable for delineating basic studies about the pathogenicity mechanisms of ExPECs and following up on the evolution of ExPEC lineages, particularly those most epidemiologically relevant. Therefore, we performed a massive descriptive study to detect the papA and papG variants along different E. coli genotypes represented by genomic sequences contained in the NCBI Assembly Refseq database. The most common papA variants were F11, F10, F48, F16, F12, and F7-2, which were found in significant association with the most relevant ExPEC genotypes, the phylogroups B2 and D, and the sequence types ST95, ST131, ST127, ST69, ST12, and ST73. On the other hand, the papGII variant was by far the most common followed by papGIII, and both were also found to have a significant association with common ExPEC genotypes. We noticed the presence of genomes, mainly belonging to the sequence type ST12, harboring two or three papA variants and two papG variants. Furthermore, the most common papA and papG variants were also detected in records representing strains isolated from humans and animals such as poultry, bovine, and dogs, supporting previous hypotheses of potential cross-transmission. Finally, we characterized a set of 17 genomes from Chilean uropathogenic E. coli strains and found that ST12 and ST73 were the predominant sequence types. Variants F7-1, F7-2, F8, F9, F11, F13, F14, F16, and F48 were detected for papA, and papGII and papGIII variants were detected for papG. Significant associations with the sequence types observed in the analysis of genomes contained in the NCBI Assembly Refseq database were also found in this collection in 16 of 19 cases for papA variants and 7 of 9 cases for the papG variants. This comprehensive characterization might support future basic studies about P fimbria-mediated ExPEC adherence and future typing or epidemiological studies to monitor the evolution of ExPECs producing P fimbria.

Vaccines against extraintestinal pathogenic Escherichia coli (ExPEC): progress and challenges.

The emergence of antimicrobial resistance (AMR) is a principal global health crisis projected to cause 10 million deaths annually worldwide by 2050. While the Gram-negative bacteria Escherichia coli is commonly found as a commensal microbe in the human gut, some strains are dangerously pathogenic, contributing to the highest AMR-associated mortality. Strains of E. coli that can translocate from the gastrointestinal tract to distal sites, called extraintestinal E. coli (ExPEC), are particularly problematic and predominantly afflict women, the elderly, and immunocompromised populations. Despite nearly 40 years of clinical trials, there is still no vaccine against ExPEC. One reason for this is the remarkable diversity in the ExPEC pangenome across pathotypes, clades, and strains, with hundreds of genes associated with pathogenesis including toxins, adhesins, and nutrient acquisition systems. Further, ExPEC is intimately associated with human mucosal surfaces and has evolved creative strategies to avoid the immune system. This review summarizes previous and ongoing preclinical and clinical ExPEC vaccine research efforts to help identify key gaps in knowledge and remaining challenges.

First Isolation of the Heteropathotype Shiga Toxin-Producing and Extra-Intestinal Pathogenic (STEC-ExPEC) E. coli O80:H2 in French Healthy Cattle: Genomic Characterization and Phylogenetic Position.

The emerging heteropathotype shigatoxigenic (STEC) and extra-intestinal pathogenic Escherichia coli (ExPEC) O80:H2 has been the second leading cause of pediatric HUS in France since the mid-2010s. In contrast with other highly pathogenic STEC serotypes, for which ruminants have clearly been identified as the main human infection source, this heteropathotype's reservoir remains unknown. In this context, we describe for the first time the isolation of seven STEC O80:H2 strains from healthy cattle on a single cattle farm in France. This study aimed at (i) characterizing the genome and (ii) investigating the phylogenetic positions of these O80:H2 STEC strains. The virulomes, resistomes, and phylogenetic positions of the seven bovine isolates were investigated using in silico typing tools, antimicrobial susceptibility testing and cgMLST analysis after short-read whole genome sequencing (WGS). One representative isolate (A13P112V1) was also subjected to long-read sequencing. The seven isolates possessed ExPEC-related virulence genes on a pR444_A-like mosaic plasmid, previously described in strain RDEx444 and known to confer multi-drug resistance. All isolates were clonally related and clustered with human clinical strains from France and Switzerland with a range of locus differences of only one to five. In conclusion, our findings suggest that healthy cattle in France could potentially act as a reservoir of the STEC-ExPEC O80:H2 pathotype.

Baicalin Weakens the Virulence of Porcine Extraintestinal Pathogenic Escherichia coli by Inhibiting the LuxS/AI-2 Quorum-Sensing System.

Porcine extraintestinal pathogenic Escherichia coli (ExPEC) is a pathogenic bacterium that causes huge economic losses to the pig farming industry and considerably threatens human health. The quorum sensing (QS) system plays a crucial role in the survival and pathogenesis of pathogenic bacteria. Hence, it is a viable approach to prevent ExPEC infection by compromising the QS system, particularly the LuxS/AI-2 system. In this study, we investigated the effects of baicalin on the LuxS/AI-2 system of ExPEC. Baicalin at concentrations of 25, 50, and 100 µg/mL significantly diminished the survival ability of ExPEC in hostile environments and could inhibit the biofilm formation and autoagglutination ability in ExPEC. Moreover, baicalin dose-dependently decreased the production of AI-2 and down-regulated the expression level of luxS in PCN033. These results suggest that baicalin can weaken the virulence of PCN033 by inhibiting the LuxS/AI-2 system. After the gene luxS was deleted, AI-2 production in PCN033 was almost completely eliminated, similar to the effect of baicalin on the production of AI-2 in PCN033. This indicates that baicalin reduced the production of AI-2 by inhibiting the expression level of luxS in ExPEC. In addition, the animal experiment further showed the potential of baicalin as a LuxS/AI-2 system inhibitor to prevent ExPEC infection. This study highlights the potential of baicalin as a natural quorum-sensing inhibitor for therapeutic applications in preventing ExPEC infection by targeting the LuxS/AI-2 system.

Progress toward a vaccine for extraintestinal pathogenic E. coli (ExPEC) II: efficacy of a toxin-autotransporter dual antigen approach.

Extraintestinal pathogenic Escherichia coli (ExPEC) is a leading cause of worldwide morbidity and mortality, the top cause of antimicrobial-resistant (AMR) infections, and the most frequent cause of life-threatening sepsis and urinary tract infections (UTI) in adults. The development of an effective and universal vaccine is complicated by this pathogen's pan-genome, its ability to mix and match virulence factors and AMR genes via horizontal gene transfer, an inability to decipher commensal from pathogens, and its intimate association and co-evolution with mammals. Using a pan virulome analysis of >20,000 sequenced E. coli strains, we identified the secreted cytolysin α-hemolysin (HlyA) as a high priority target for vaccine exploration studies. We demonstrate that a catalytically inactive pure form of HlyA, expressed in an autologous host using its own secretion system, is highly immunogenic in a murine host, protects against several forms of ExPEC infection (including lethal bacteremia), and significantly lowers bacterial burdens in multiple organ systems. Interestingly, the combination of a previously reported autotransporter (SinH) with HlyA was notably effective, inducing near complete protection against lethal challenge, including commonly used infection strains ST73 (CFT073) and ST95 (UTI89), as well as a mixture of 10 of the most highly virulent sequence types and strains from our clinical collection. Both HlyA and HlyA-SinH combinations also afforded some protection against UTI89 colonization in a murine UTI model. These findings suggest recombinant, inactive hemolysin and/or its combination with SinH warrant investigation in the development of an E. coli vaccine against invasive disease.

Antibacterial activity of the antimicrobial peptide PMAP-36 in combination with tetracycline against porcine extraintestinal pathogenic Escherichia coli in vitro and in vivo.

The increase in the emergence of antimicrobial resistance has led to great challenges in controlling porcine extraintestinal pathogenic Escherichia coli (ExPEC) infections. Combinations of antimicrobial peptides (AMPs) and antibiotics can synergistically improve antimicrobial efficacy and reduce bacterial resistance. In this study, we investigated the antibacterial activity of porcine myeloid antimicrobial peptide 36 (PMAP-36) in combination with tetracycline against porcine ExPEC PCN033 both in vitro and in vivo. The minimum bactericidal concentrations (MBCs) of AMPs (PMAP-36 and PR-39) against the ExPEC strains PCN033 and RS218 were 10 µM and 5 µM, respectively. Results of the checkerboard assay and the time-kill assay showed that PMAP-36 and antibiotics (tetracycline and gentamicin) had synergistic bactericidal effects against PCN033. PMAP-36 and tetracycline in combination led to PCN033 cell wall shrinkage, as was shown by scanning electron microscopy. Furthermore, PMAP-36 delayed the emergence of PCN033 resistance to tetracycline by inhibiting the expression of the tetracycline resistance gene tetB. In a mouse model of systemic infection of PCN033, treatment with PMAP-36 combined with tetracycline significantly increased the survival rate, reduced the bacterial load and dampened the inflammatory response in mice. In addition, detection of immune cells in the peritoneal lavage fluid using flow cytometry revealed that the combination of PMAP-36 and tetracycline promoted the migration of monocytes/macrophages to the infection site. Our results suggest that AMPs in combination with antibiotics may provide more therapeutic options against multidrug-resistant porcine ExPEC.

The faecal microbiome of the Australian silver gull contains phylogenetically diverse ExPEC, aEPEC and Escherichia coli carrying the transmissible locus of stress tolerance.

Wildlife are implicated in the dissemination of antimicrobial resistance, but their roles as hosts for Escherichia coli that pose a threat to human and animal health is limited. Gulls (family Laridae) in particular, are known to carry diverse lineages of multiple-antibiotic resistant E. coli, including extra-intestinal pathogenic E. coli (ExPEC). Whole genome sequencing of 431 E. coli isolates from 69 healthy Australian silver gulls (Chroicocephalus novaehollandiae) sampled during the 2019 breeding season, and without antibiotic selection, was undertaken to assess carriage in an urban wildlife population. Phylogenetic analysis and genotyping resolved 123 sequence types (STs) representing most phylogroups, and identified diverse ExPEC, including an expansive phylogroup B2 cluster comprising 103 isolates (24 %; 31 STs). Analysis of the mobilome identified: i) widespread carriage of the Yersinia High Pathogenicity Island (HPI), a key ExPEC virulence determinant; ii) broad distribution of two novel phage elements, each carrying sitABCD and iii) carriage of the transmissible locus of stress tolerance (tLST), an element linked to sanitation resistance. Of the 169 HPI carrying isolates, 49 (48 %) represented diverse B2 isolates hosting FII-64 ColV-like plasmids that lacked iutABC and sitABC operons typical of ColV plasmids, but carried the serine protease autotransporter gene, sha. Diverse E. coli also carried archetypal ColV plasmids (52 isolates; 12 %). Clusters of closely related E. coli (<50 SNVs) from ST58, ST457 and ST746, sourced from healthy gulls, humans, and companion animals, were frequently identified. In summary, anthropogenically impacted gulls host an expansive E. coli population, including: i) putative ExPEC that carry ColV virulence gene cargo (101 isolates; 23.4 %) and HPI (169 isolates; 39 %); ii) atypical enteropathogenic E. coli (EPEC) (17 isolates; 3.9 %), and iii) E. coli that carry the tLST (20 isolates; 4.6 %). Gulls play an important role in the evolution and transmission of E. coli that impact human health.

Detection and characterization of potentially hybrid enteroaggregative Escherichia coli (EAEC) strains isolated from urinary tract infection.

Uropathogenic Escherichia coli (UPEC) have the potential to receive the virulence markers of intestinal pathotypes and transform into various important hybrid pathotypes. This study aimed to investigate the frequency and characteristics of hybrid enteroaggregative E. coli (EAEC)/UPEC strains. Out of 202 UPEC strains, nine (4.5%) were detected as hybrid EAEC/UPEC. These strains carried one to four iron uptake systems. Among nine investigated pathogenicity islands (PAIs), PAI IV536, PAI II536, and PAI ICFT073 were found in 9 (100%), 3 (33.3%), and 1 (11.1%) strains, respectively. The chuA and sitA genes were detected in 5 (55.5%) and 3 (33.3%) hybrid strains, respectively. Six hybrid strains were found to be typical extraintestinal pathogenic E. coli (ExPEC) according to their virulence traits. Most of the hybrid strains belonged to the phylogenetic group E (6/9). Among the hybrid strains, seven (7/9) were able to form biofilm and adhere to cells; however, only two strains penetrated into the HeLa cells. Our findings reveal some of the virulence characteristics of hybrid strains that lead to fitness and infection in the urinary tract. These strains, with virulence factors of intestinal and non-intestinal pathotypes, may become emerging pathogens in clinical settings; therefore, further studies are needed to reveal their pathogenicity mechanisms and so that preventive measures can be taken.

Genetic features of antimicrobial drug-susceptible extraintestinal pathogenic Escherichia coli pandemic sequence type 95.

IMPORTANCE: Despite the increasing prevalence of antibiotic-resistant Escherichia coli strains that cause urinary tract and bloodstream infections, a major pandemic lineage of extraintestinal pathogenic E. coli (ExPEC) ST95 has a comparatively low frequency of drug resistance. We compared the genomes of 1,749 ST95 isolates to identify genetic features that may explain why most strains of ST95 resist becoming drug-resistant. Identification of such genomic features could contribute to the development of novel strategies to prevent the spread of antibiotic-resistant genes and devise new measures to control antibiotic-resistant infections.

Can Extraintestinal Pathogenic Escherichia coli with Heat Resistance Profile Overcome Nonthermal Technologies?

Ultraviolet-C light-emitting diode (UVC-LED) and ultrasound (US) are two nonthermal technologies with the potential to destroy pathogens. However, little is known about their effectiveness in strains with a history of heat resistance. Thus, this study aimed to evaluate the phenotype and genotype of heat-resistant extraintestinal pathogenic Escherichia coli (ExPEC) with heat resistance genes after the application of US, UVC-LED, and UVC-LED+US. For this, two central composite rotatable designs were used to optimize the UVC-LED and US conditions in four ExPEC isolated from beef. From the genome of these isolates obtained in a previous study, possible genes for UVC resistance were analyzed. Results showed that US was ineffective in reducing >0.30 log colony-forming unit/mL, and that when used after UVC-LED, it showed a nonsynergic or antagonistic effect. Also, UVC-LED had the greatest effect at the maximum dose (4950 mJ/cm2 from 1.65 mW/cm2 for 50 min). However, the strains showed some recovery after that, which could be implicated in the expression of genes included in SOS system genes, some others present in the transmissible Locus of Stress Tolerance (trxBC and degP), and others (terC). Thus, ExPEC can overcome the conditions used in this study for US, UVC-LED, and UVC-LED+US, probably due to the history of resistance to other cellular damage. The result of this study will contribute to future studies that aim to find better treatment conditions for each food product.

Strain belonging to an emerging, virulent sublineage of ST131 Escherichia coli isolated in fresh spinach, suggesting that ST131 may be transmissible through agricultural products.

Food contamination with pathogenic Escherichia coli can cause severe disease. Here, we report the isolation of a multidrug resistant strain (A23EC) from fresh spinach. A23EC belongs to subclade C2 of ST131, a virulent clone of Extraintestinal Pathogenic E. coli (ExPEC). Most A23EC virulence factors are concentrated in three pathogenicity islands. These include PapGII, a fimbrial tip adhesin linked to increased virulence, and CsgA and CsgB, two adhesins known to facilitate spinach leaf colonization. A23EC also bears TnMB1860, a chromosomally-integrated transposon with the demonstrated potential to facilitate the evolution of carbapenem resistance among non-carbapenemase-producing enterobacterales. This transposon consists of two IS26-bound modular translocatable units (TUs). The first TU carries aac(6')-lb-cr, bla OXA-1, ΔcatB3, aac(3)-lle, and tmrB, and the second one harbors bla CXT-M-15. A23EC also bears a self-transmissible plasmid that can mediate conjugation at 20°C and that has a mosaic IncF [F(31,36):A(4,20):B1] and Col156 origin of replication. Comparing A23EC to 86 additional complete ST131 sequences, A23EC forms a monophyletic cluster with 17 other strains that share the following four genomic traits: (1) virotype E (papGII+); (2) presence of a PAI II536-like pathogenicity island with an additional cnf1 gene; (3) presence of chromosomal TnMB1860; and (4) frequent presence of an F(31,36):A(4,20):B1 plasmid. Sequences belonging to this cluster (which we named "C2b sublineage") are highly enriched in septicemia samples and their associated genetic markers align with recent reports of an emerging, virulent sublineage of the C2 subclade, suggesting significant pathogenic potential. This is the first report of a ST131 strain belonging to subclade C2 contaminating green leafy vegetables. The detection of this uropathogenic clone in fresh food is alarming. This work suggests that ST131 continues to evolve, gaining selective advantages and new routes of transmission. This highlights the pressing need for rigorous epidemiological surveillance of ExPEC in vegetables with One Health perspective.

Occurrence of Major Human Extraintestinal Pathogenic Escherichia coli Sequence Types Among Diarrheic Pet Animals: A Potential Public Health Threat.

Background: Extraintestinal pathogenic Escherichia coli (ExPEC) has become a mounting public health concern. The present study was conducted to address the role of diarrheic pet animals as potential reservoirs for major human ExPEC sequence types (STs). Materials and Methods: Rectal swabs were collected from 145 diarrheic pet animals (75 dogs and 70 cats). Samples were processed for isolation and identification of E. coli by culture methods. Afterward, ExPEC isolates were identified on a molecular basis through detection of ExPEC phylogroups (B2 and D) coupled with carriage of two or more of the virulence genes associated with ExPEC (papAH, papC, sfa/focDE, afa/draBC, iutA, and kpsMT II). ExPEC STs 131, 73, 69, and 95 were identified among ExPEC isolates by quadruplex PCR and tested for their antimicrobial susceptibility. Eventually, two isolates underwent gene sequencing for the phylogenetic analysis. Results: Of 145 pet animals, 16 (11%) E. coli strains were identified as ExPEC, in which 15 (10.3%) isolates belonged to phylogroup B2 and 1 (0.69%) strain belonged to phylogroup D. The major human ExPEC STs were detected in 13 (9%) isolates, whereas the prevalence rates were 5.3% and 12.9% for dogs and cats, respectively. The isolation rates of ExPEC STs were 4.8%, 2.8%, 0.69%, and 0.69% for ST73, ST131, ST95, and ST69, respectively. Regarding the prevalence of virulence genes among ExPEC STs, the most prevalent ones were papC and sfa/focDE (92.3%), followed by papAH (76.9%), iutA (53.8%), afa/draBC (30.8%), and kpsMT II (30.8%). Moreover, 38.5% of the obtained human ExPEC STs were multidrug resistant. The phylogenetic analysis of two ExPEC ST73 gene sequences showed high genetic relatedness to those isolated from humans in different countries. Conclusions: The fecal carriage of major human ExPEC STs among diarrheic dogs and cats poses a potential zoonotic hazard with serious public health implications.

Detection of Extended-spectrum ß-lactamase-producing Escherichia coli isolates by isothermal amplification and association of their virulence genes and phylogroups with extraintestinal infection.

Extraintestinal pathogenic Escherichia coli (ExPEC) producing extended-spectrum ß-lactamases (ESBL) cause serious human infections due to their virulence and multidrug resistance (MDR) profiles. We characterized 144 ExPEC strains (collected from a tertiary cancer institute) in terms of antimicrobial susceptibility spectrum, ESBL variants, virulence factors (VF) patterns, and Clermont's phylogroup classification. The developed multiplex recombinase polymerase amplification and thermophilic helicase-dependent amplification (tHDA) assays for blaCTX-M, blaOXA, blaSHV, and blaTEM detection, respectively, were validated using PCR-sequencing results. All ESBL-ExPEC isolates carried blaCTX-M genes with following prevalence frequency of variants: blaCTX-M-15 (50.5%) > blaCTX-M-55 (17.9%) > blaCTX-M-27 (16.8%) > blaCTX-M-14 (14.7%). The multiplex recombinase polymerase amplification assay had 100% sensitivity, and specificity for blaCTX-M, blaOXA, blaSHV, while tHDA had 86.89% sensitivity, and 100% specificity for blaTEM. The VF genes showed the following prevalence frequency: traT (67.4%) > ompT (52.6%) > iutA (50.5%) > fimH (47.4%) > iha (33.7%) > hlyA (26.3%) > papC (12.6%) > cvaC (3.2%), in ESBL-ExPEC isolates which belonged to phylogroups A (28.4%), B2 (28.4%), and F (22.1%). The distribution of traT, ompT, and hlyA and phylogroup B2 were significantly different (P < 0.05) between ESBL-ExPEC and non-ESBL-ExPEC isolates. Thus, these equipment-free isothermal resistance gene amplification assays contribute to effective treatment and control of virulent ExPEC, especially antimicrobial resistance strains.

New Genetic Markers Differentiating IPEC and ExPEC Pathotypes-A New Approach to Genome-Wide Analysis Using a New Bioinformatics Tool.

The increasingly expanding genomic databases generate the need for new tools for their processing and further use. In the paper, a bioinformatics tool, which is a search engine of microsatellite elements-trinucleotide repeat sequences (TRS) in files of FASTA type-is presented. An innovative approach was applied in the tool, which consists of connecting-within one search engine-both mapping of TRS motifs and extracting sequences that are found between the mapped TRS motifs. Accordingly, we present hereby the tool called TRS-omix, which comprises a new engine for searching information on genomes and enables generation of sets of sequences and their number, providing the basis for making comparisons between genomes. In our paper, we showed one of the possibilities of using the software. Using TRS-omix and other IT tools, we showed that we were able to extract sets of DNA sequences that can be assigned only to the genomes of the extraintestinal pathogenic Escherichia coli strains or to the genomes of the intestinal pathogenic Escherichia coli strains, as well as providing the basis for differentiation of the genomes/strains belonging to each of these clinically essential pathotypes.

E. coli Common pili promote the fitness and virulence of a hybrid aEPEC/ExPEC strain within diverse host environments.

Pathogenic subsets of Escherichia coli include diarrheagenic (DEC) strains that cause disease within the gut and extraintestinal pathogenic E. coli (ExPEC) strains that are linked with urinary tract infections, bacteremia, and other infections outside of intestinal tract. Among DEC strains is an emergent pathotype known as atypical enteropathogenic E. coli (aEPEC), which can cause severe diarrhea. Recent sequencing efforts revealed that some E. coli strains possess genetic features that are characteristic of both DEC and ExPEC isolates. BA1250 is a newly reclassified hybrid strain with characteristics of aEPEC and ExPEC. This strain was isolated from a child with diarrhea, but its genetic features indicate that it might have the capacity to cause disease at extraintestinal sites. The spectrum of adhesins encoded by hybrid strains like BA1250 are expected to be especially important in facilitating colonization of diverse niches. E. coli common pilus (ECP) is an adhesin expressed by many E. coli pathogens, but how it impacts hybrid strains has not been ascertained. Here, using zebrafish larvae as surrogate hosts to model both gut colonization and extraintestinal infections, we found that ECP can act as a multi-niche colonization and virulence factor for BA1250. Furthermore, our results indicate that ECP-related changes in activation of envelope stress response pathways may alter the fitness of BA1250. Using an in silico approach, we also delineated the broader repertoire of adhesins that are encoded by BA1250, and provide evidence that the expression of at least a few of these varies in the absence of functional ECP.