Whole-genome analyses of extended-spectrum or AmpC ß-lactamase-producing Escherichia coli isolates from companion dogs in Japan.

The emergence and global spread of extended-spectrum or AmpC ß-lactamase (ESBL/AmpC)-producing Enterobacteriaceae in companion animals have led to the hypothesis that companion animals might be reservoirs for cross-species transmission because of their close contact with humans. However, current knowledge in this field is limited; therefore, the role of companion animals in cross-species transmission remains to be elucidated. Herein, we studied ESBL/AmpC-producing Enterobacteriaceae, Escherichia coli in particular, isolated from extraintestinal sites and feces of companion dogs. Whole-genome sequencing analysis revealed that (i) extraintestinal E. coli isolates were most closely related to those isolated from feces from the same dog, (ii) chromosomal sequences in the ST131/C1-M27 clade isolated from companion dogs were highly similar to those in the ST131/C1-M27 clade of human origin, (iii) certain plasmids, such as IncFII/pMLST F1:A2:B20/blaCTX-M-27, IncI1/pMLST16/blaCTX-M-15, or IncI1/blaCMY-2 from dog-derived E. coli isolates, shared high homology with those from several human-derived Enterobacteriaceae, (iv) chromosomal blaCTX-M-14 was identified in the ST38 isolate from a companion dog, and (v) eight out of 14 tested ESBL/AmpC-producing E. coli isolates (i.e., ST131, ST68, ST405, and ST998) belonged to the human extraintestinal pathogenic E. coli (ExPEC) group. All of the bla-coding plasmids that were sequenced genome-wide were capable of horizontal transfer. These results suggest that companion dogs can spread ESBL/AmpC-producing ExPEC via their feces. Furthermore, at least some ESBL/AmpC-producing ExPECs and bla-coding plasmids can be transmitted between humans and companion dogs. Thus, companion dogs can act as an important reservoir for ESBL/AmpC-producing E. coli in the community.

The Serine Protease Autotransporters TagB, TagC, and Sha from Extraintestinal Pathogenic Escherichia coli Are Internalized by Human Bladder Epithelial Cells and Cause Actin Cytoskeletal Disruption.

TagB, TagC (tandem autotransporter genes B and C), and Sha (Serine-protease hemagglutinin autotransporter) are recently described members of the SPATE (serine protease autotransporters of Enterobacteriaceae) family. These SPATEs can cause cytopathic effects on bladder cells and contribute to urinary tract infection in a mouse model. Bladder epithelial cells form an important barrier in the urinary tract. Some SPATEs produced by pathogenic E. coli are known to breach the bladder epithelium. The capacity of these newly described SPATEs to alter bladder epithelial cells and the role of the serine protease active site were investigated. All three SPATE proteins were internalized by bladder epithelial cells and altered the distribution of actin cytoskeleton. Sha and TagC were also shown to degrade mucin and gelatin respectively. Inactivation of the serine catalytic site in each of these SPATEs did not affect secretion of the SPATEs from bacterial cells, but abrogated entry into epithelial cells, cytotoxicity, and proteolytic activity. Thus, our results show that the serine catalytic triad of these proteins is required for internalization in host cells, actin disruption, and degradation of host substrates such as mucin and gelatin.

Serine protease autotransporters of Enterobacteriaceae (SPATEs) are largely distributed among Escherichia coli isolated from the bloodstream.

Extraintestinal pathogenic Escherichia coli (ExPEC) is the major cause of Gram-negative-related sepsis. Bacterial survival in the bloodstream is mediated by a variety of virulence traits, including those mediating immune system evasion. Serine protease autotransporters of Enterobacteriaceae (SPATE) constitute a superfamily of virulence factors that can cause tissue damage and cleavage of molecules of the complement system, which is a key feature for the establishment of infection in the bloodstream. In this study, we analyzed 278 E. coli strains isolated from human bacteremia from inpatients of both genders, different ages, and clinical conditions. These strains were screened for the presence of SPATE-encoding genes as well as for phylogenetic classification and intrinsic virulence of ExPEC. SPATE-encoding genes were detected in 61.2% of the strains and most of these strains (44.6%) presented distinct SPATE-encoding gene profiles. sat was the most frequent gene among the entire collection, found in 34.2%, followed by vat (28.4%), pic (8.3%), and tsh (4.7%). Although in low frequencies, espC (0.7%), eatA (1.1%), and espI (1.1%) were detected and are being reported for the first time in extraintestinal isolates. The presence of SPATE-encoding genes was positively associated to phylogroup B2 and intrinsic virulent strains. These findings suggest that SPATEs are highly prevalent and involved in diverse steps of the pathogenesis of bacteremia caused by E. coli.

Wastewater as a Probable Environmental Reservoir of Extended-Spectrum-ß-Lactamase Genes: Detection of Chimeric ß-Lactamases CTX-M-64 and CTX-M-123.

The presence of antimicrobial-resistant bacteria and resistance genes in aquatic environments is a serious public health concern. This study focused on Escherichia coli possessing blaCTX-M genes in wastewater inflows. Twelve crude inflow water samples from wastewater treatment plant (WWTP) A and two samples each from three other WWTPs were collected in 2017 and 2018. A total of 73 E. coli isolates with 31 different sequence types (STs) harboring distinctive blaCTX-M gene repertoires were detected. In WWTP A influents, blaCTX-M-14 (14 isolates) was dominant, followed by blaCTX-M-15 (12 isolates) and blaCTX-M-27 (10 isolates). The chimeric blaCTX-M-64 and blaCTX-M-123 genes were each identified in one of the E. coli isolates from the same WWTP A inflow port. The blaCTX-M-27 gene was associated with five of seven B2-ST131 isolates, including three isolates of the B2-O25b-ST131-H30R/non-Rx lineage. One of the remaining two isolates belonged to the B2-O25b-ST131-H30R/Rx lineage harboring the blaCTX-M-15 gene. As for the B2-O25b-ST131-H30R/non-Rx lineage, two isolates with blaCTX-M-27 were recovered from each of the WWTP B and D influents, and one isolate with blaCTX-M-174 was also recovered from WWTP B influent. Whole-genome sequencing of chimeric blaCTX-M-harboring E. coli isolates revealed that the blaCTX-M-64 gene was integrated into the chromosome of ST10 E. coli B22 via ISEcp1-mediated transposition of a 9,467-bp sequence. The blaCTX-M-123-carrying IncI1 plasmid pB64 was 109,169 bp in length with pST108. The overall findings suggest that wastewater may act as a probable reservoir of clinically significant clonal lineages mediating antimicrobial resistance genes and chimeric genes that have not yet been identified from human isolates of domestic origin in Japan.IMPORTANCE Global spread of CTX-M-type extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae is a critical concern in both clinical and community settings. This dominance of CTX-M-type ESBL producers may be largely due to the successful international spread of epidemic clones, as represented by the extraintestinal pathogenic Escherichia coli (ExPEC) ST131. Our findings highlight the worrisome presence of diverse E. coli clones associated with humans, including ExPEC lineages harboring the most common blaCTX-M variants in untreated wastewater samples. Moreover, the chimeric genes blaCTX-M-64 and blaCTX-M-123, which have not yet been identified from human isolates of domestic origin in Japan, were identified. Exposure to untreated wastewater through combined sewer overflow caused by heavy rains derived from abnormal weather change could pose a risk for human health due to ingesting those antimicrobial-resistant bacteria.

Secreted proteases: A new insight in the pathogenesis of extraintestinal pathogenic Escherichia coli.

Bacterial secreted proteases are the key factors that increase the virulence potential of different pathogens. Extraintestinal pathogenic E. coli (ExPEC) is a distinct pathotype that has unique ability to infect various body sites apart from the gastrointestinal tract causing several life-threatening diseases both in human and animals. Thus, understanding of ExPEC pathogenesis is crucial in effective management of disease caused by these pathogens. It is known that ExPEC possesses a broad spectrum of virulence factors including the secreted proteases which elude the host defence system. Recent studies have shown that high prevalence as well as the action of the secreted proteases influence the pathogenesis of ExPEC. However, literature on the secreted proteases present in ExPEC and their role in promoting virulence of ExPEC is rather limited. This review describes the distribution, characterization and the role of serine and metalloproteases secreted by diverse pathotypes of ExPEC, highlighting the significance of secreted proteases of ExPEC in pathogenesis.

Pathogenic potential and the role of clones and plasmids in beta-lactamase-producing E. coli from chicken faeces in Vietnam.

BACKGROUND: Antimicrobial resistance (AMR) in food-producing animals is a global public health issue. This study investigated AMR and virulence profiles of E. coli isolated from healthy chickens in Vietnam. E. coli were isolated from fecal samples collected in five chicken farms located in the provinces of Hoa Binh, Thai Nguyen and Bac Giang in the North of Vietnam. These isolates were examined by disk diffusion for their AMR, PCR for virulence and AMR genes, pulsed-field gel electrophoresis for relatedness between blaCMY-2/blaCTX-M-positive isolates, electroporation for transferability of blaCMY-2 or blaCTX-M genes and sequencing for mutations responsible for ciprofloxacin resistance. RESULTS: Up to 99% of indicator isolates were multidrug resistant. Resistance to third-generation cephalosporins (3GC) was encoded by both blaCTX-M and blaCMY-2 genes; blaCTX-M genes being of genotypes blaCTX-M-1, - 14, - 15, - 17, - 57 and - 87, whereas ciprofloxacin resistance was due to mutations in the gyrA and parC genes. Some isolates originating from farms located in different provinces of Vietnam were found to be closely related, suggesting they may have been disseminated from a same source of contamination. Plasmids may also have played a role in the diffusion of 3GC-resistance as the blaCMY-2 gene was located on plasmids A/C and I1, and the blaCTX-M gene variants were carried by I1, FIB, R and HI1. Plasmids carrying the blaCMY-2/blaCTX-M genes also co-transferred resistance to other antimicrobials. In addition, isolates potentially capable of infecting humans, of which some produced blaCMY-2/blaCTX-M, were identified in this study. CONCLUSIONS: Both clones and plasmids could be involved in the dissemination of 3GC-resistant E. coli within and between chicken farms in Vietnam. These results demonstrate the necessity to monitor AMR and control antimicrobial use in poultry in Vietnam.

Whole-genome sequences of multidrug-resistant Escherichia coli in South-Kivu Province, Democratic Republic of Congo: characterization of phylogenomic changes, virulence and resistance genes.

BACKGROUND: Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli are responsible for severe infections worldwide. Whereas their genotypic and pathogenic characteristics are not documented in Democratic Republic of Congo (DRC), recent studies conducted at the Bukavu General Hospital in the South Kivu province highlighted their high prevalence in extra-intestinal infections. Here we provide data on molecular characterization of ESBL producing-Escherichia coli isolates from patients with extra-intestinal infections at this provincial hospital. METHODS: Whole-genome sequencing was carried out on 21 of these ESBL-producing Extra-intestinal Pathogenic Escherichia coli (ExPEC) for analysis of phylogenomic evolution, virulence factor and antimicrobial resistance (AMR) genes. Data were compared to phylogenetically close genomes using Multi-Locus Sequence Typing and Single Nucleotide Polymorphism-based phylogenetic approaches. RESULTS: The distribution of E. coli sequence types (ST) was as follows: ST 131 (n = 7), ST405 (n = 4), ST410 (n = 2), and other STs (ST10, ST58, ST95, ST393, ST443, S617, ST648, and ST2450). All ST131 belonged to the O25b-ST131 pandemic clone. Unexpectedly, they harbored more virulence genes than their GenBank counterparts. IncF plasmid replicons included novel FIB 69, FII 105 and FII 107 alleles. ESBL-genes included the plasmid-mediated CTX-M-15 in all isolates, and the SHV-12 allele. Other AMR genes included blaOXA-1, blaTEM-1, as well as genes encoding resistance against aminoglycosides, quinolones, chloramphenicol, rifampicin, tetracyclines, sulfonamides and trimethoprim. CONCLUSION: Current data confirm the clonal spread of ESBL-producing ST131 and ST405 clones in patients from South Kivu, and the acquisition of resistance and virulence genes. A closer survey of AMR and virulence should therefore be prompted in this high-risk area.

The Periplasmic Trehalase Affects Type 1 Fimbria Production and Virulence of Extraintestinal Pathogenic Escherichia coli Strain MT78.

Extraintestinal pathogenic Escherichia coli (ExPEC) is responsible for various infections outside the gastrointestinal tract in humans and other animals. ExPEC strain MT78 is invasive to various nonphagocytic cells and highly virulent in vivo To identify genes required for invasion of nonphagocytic cells by this strain, we applied signature-tagged mutagenesis to generate a library of mutants and tested them for invasion of avian fibroblasts. Mutants showing reduced cellular invasion included those with insertions in the fim operon, encoding type 1 fimbriae. Another attenuated mutant showed a disruption in the treA gene, which encodes a periplasmic trehalase. The substrate of TreA, trehalose, can be metabolized and used as a carbon source or can serve as an osmoprotectant under conditions of osmotic stress in E. coli K-12. We generated and characterized mutant MT78ΔtreA In contrast to the wild type, MT78ΔtreA was able to grow under osmotic stress caused by 0.6 M urea but not in minimal M9 medium with trehalose as the only carbon source. It presented decreased association and invasion of avian fibroblasts, decreased yeast agglutination titer, and impaired type 1 fimbria production. In a murine model of urinary tract infection, MT78ΔtreA was less able to colonize the bladder. All phenotypes were rescued in the complemented mutant. Our results show that the treA gene is needed for optimal production of type 1 fimbriae in ExPEC strain MT78 and that loss of treA significantly reduces its cell invasion capacity and colonization of the bladder in a murine model of urinary tract infection.

In vitro efficacy of 16 antimicrobial drugs against a large collection of ß-lactamase-producing isolates of extraintestinal pathogenic Escherichia coli from dogs and cats.

PURPOSE: The aim of this study was to assess the in vitro efficacy of candidate antimicrobials against extended-spectrum ß-lactamase (ESBL)-producing isolates of extraintestinal pathogenic Escherichia coli (ExPEC) from companion animals. METHODOLOGY: A total of 90 ESBL-producing ExPEC isolates from dogs and cats were tested for susceptibility to 16 antimicrobials with the agar dilution method. We also identified the ESBLs and AmpC ß-lactamases of these isolates with PCR and DNA sequencing.Results/Key findings. All isolates were susceptible to meropenem, tebipenem and amikacin (AMK), and various proportions were susceptible to latamoxef (LMX, 97.8 %), fosfomycin (FOM, 97.8 %), faropenem (FPM, 96.7 %), nitrofurantoin (NFT, 96.7 %), flomoxef (FMX, 93.3 %), piperacillin/tazobactam (PTZ, 92.2 %), cefmetazole (CMZ, 91.1 %), chloramphenicol (80.0 %), trimethoprim/sulfamethoxazole (64.4 %), amoxicillin/clavulanic acid (63.3 %), ceftibuten (60.0 %), tetracycline (52.2 %) and enrofloxacin (10.0 %). A genetic analysis showed that 83 of the 90 (92.2 %) isolates were positive for CTX-M-type genes: CTX-M-14 (n=26), CTX-M-27 (n=20), CTX-M-55 (n=17), CTX-M-15 (n=12), CTX-M-2 (n=5), CTX-M-24 (n=2), CTX-M-104 (n=2) and CTX-M-3 (n=1). Eight isolates also expressed AmpC ß-lactamase phenotypes. CONCLUSION: This study demonstrates that the susceptibility rates to PTZ, CMZ, LMX, AMK, FOM, FPM, NFT and FMX were similar to those to carbapenems (>90 %), implying that these drugs are available alternatives to carbapenems for the treatment of companion animals infected with ExPEC-producing CTX-M-type ESBLs. Further in vivo studies of the effective use of these antimicrobials are required.

The Rhomboid Protease GlpG Promotes the Persistence of Extraintestinal Pathogenic Escherichia coli within the Gut.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains are typically benign within the mammalian gut but can disperse to extraintestinal sites to cause diseases like urinary tract infections and sepsis. As occupation of the intestinal tract is often a prerequisite for ExPEC-mediated pathogenesis, we set out to understand how ExPEC colonizes this niche. A screen using transposon sequencing (Tn-seq) was performed to search for genes within ExPEC isolate F11 that are important for growth in intestinal mucus, which is thought to be a major source of nutrients for E. coli in the gut. Multiple genes that contribute to ExPEC fitness in mucus broth were identified, with genes that are directly or indirectly associated with fatty acid beta-oxidation pathways being especially important. One of the identified mucus-specific fitness genes encodes the rhomboid protease GlpG. In vitro, we found that the disruption of glpG had polar effects on the downstream gene glpR, which encodes a transcriptional repressor of factors that catalyze glycerol degradation. Mutation of either glpG or glpR impaired ExPEC growth in mucus and on plates containing the long-chain fatty acid oleate as the sole carbon source. In contrast, in a mouse gut colonization model in which the natural microbiota is unperturbed, the disruption of glpG but not glpR significantly reduced ExPEC survival. This work reveals a novel biological role for a rhomboid protease and highlights new avenues for defining mechanisms by which ExPEC strains colonize the mammalian gastrointestinal tract.