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.

Genetic diversity and multidrug resistance of phylogenic groups B2 and D in InPEC and ExPEC isolated from chickens in Central China.

BACKGROUND: Avian colibacillosis is an infectious bacterial disease caused by avian pathogenic Escherichia coli (APEC). APEC causes a wide variety of intestinal and extraintestinal infections, including InPEC and ExPEC, which result in enormous losses in the poultry industry. In this study, we investigated the prevalence of InPEC and ExPEC in Central China, and the isolates were characterized using molecular approaches and tested for virulence factors and antibiotic resistance. RESULTS: A total of 200 chicken-derived E. coli isolates were collected for study from 2019 and 2020. The prevalence of B2 and D phylogenic groups in the 200 chicken-derived E. coli was verified by triplex PCR, which accounted for 50.53% (48/95) and 9.52% (10/105) in ExPEC and InPEC, respectively. Additionally, multilocus sequence typing method was used to examine the genetic diversity of these E. coli isolates, which showed that the dominant STs of ExPEC included ST117 (n = 10, 20.83%), ST297 (n = 5, 10.42%), ST93 (n = 4, 8.33%), ST1426 (n = 4, 8.33%) and ST10 (n = 3, 6.25%), while the dominant ST of InPEC was ST117 (n = 2, 20%). Furthermore, antimicrobial susceptibility tests of 16 antibiotics for those strains were conducted. The result showed that more than 60% of the ExPEC and InPEC were resistant to streptomycin and nalidixic acid. Among these streptomycin resistant isolates (n = 49), 99.76% harbored aminoglycoside resistance gene strA, and 63.27% harbored strB. Among these nalidixic acid resistant isolates (n = 38), 94.74% harbored a S83L mutation in gyrA, and 44.74% harbored a D87N mutation in gyrA. Moreover, the prevalence of multidrug-resistant (MDR) in the isolates of ExPEC and InPEC was 31.25% (15/48) and 20% (2/10), respectively. Alarmingly, 8.33% (4/48) of the ExPEC and 20% (2/10) of the InPEC were extensively drug-resistant (XDR). Finally, the presence of 13 virulence-associated genes was checked in these isolates, which over 95% of the ExPEC and InPEC strains harbored irp2, feoB, fimH, ompT, ompA. 10.42% of the ExPEC and 10% of the InPEC were positive for kpsM. Only ExPEC isolates carried ibeA gene, and the rate was 4.17%. All tested strains were negative to LT and cnf genes. The carrying rate of iss and iutA were significantly different between the InPEC and ExPEC isolates (P < 0.01). CONCLUSIONS: To the best of our knowledge, this is the first report on the highly pathogenic groups of InPEC and ExPEC in Central China. We find that 50.53% (48/95) of the ExPEC belong to the D/B2 phylogenic group. The emergence of XDR and MDR strains and potential virulence genes may indicate the complicated treatment of the infections caused by APEC. This study will improve our understanding of the prevalence and pathogenicity of APEC.

Development of an O-polysaccharide based recombinant glycoconjugate vaccine in engineered E. coli against ExPEC O1.

Extraintestinal pathogenic Escherichia coli O1 is a frequently identified serotype that causes serious infections and is often refractory to antimicrobial therapy. Glycoconjugate vaccine represents a promising measure to reduce ExPEC infections. Herein, we designed an O1-specific glyco-optimized chassis strain for manufacture of O-polysaccharide (OPS) antigen and OPS-based bioconjugate. Specifically, OPS and OPS-based glycoprotein were synthesized in glyco-optimized chassis strain, when compared to the unmeasurable level of the parent strain. The optimal expression of oligosaccharyltransferase and carrier protein further improved the titer. MS analysis elucidated the correct structure of resulting bioconjugate at routine and unreported glycosylation sequons of carrier protein, with a higher glycosylation efficiency. Finally, purified bioconjugate stimulated mouse to generate specific IgG antibodies and protected them against virulent ExPEC O1 challenge. The plug-and-play glyco-optimized platform is suitable for bioconjugate synthesis, thus providing a potential platform for future medical applications.

Novel Small Molecule Growth Inhibitor Affecting Bacterial Outer Membrane Reduces Extraintestinal Pathogenic Escherichia coli (ExPEC) Infection in Avian Model.

Avian pathogenic Escherichia coli (APEC), a subgroup of extraintestinal pathogenic E. coli (ExPEC), causes colibacillosis in chickens and is reportedly implicated in urinary tract infections and meningitis in humans. A major limitation for the current ExPEC antibiotic therapy is the development of resistance, and antibacterial drugs that can circumvent this problem are critically needed. Here, we evaluated eight novel membrane-affecting anti-APEC small molecule growth inhibitors (GIs), identified in our previous study, against APEC infection in chickens. Among the GIs tested, GI-7 (the most effective), when administered orally (1 mg/kg of body weight), reduced the mortality (41.7%), severity of lesions (62.9%), and APEC load (2.6 log) in chickens. Furthermore, GI-7 administration at an optimized dose (60 mg/liter) in drinking water also reduced the mortality (14.7%), severity of lesions (29.5%), and APEC load (2.2 log) in chickens. The abundances of Lactobacillus and oleate were increased in the cecum and serum, respectively, of GI-7-treated chickens. Pharmacokinetic analysis revealed that GI-7 was readily absorbed with minimal accumulation in the tissues. Earlier, we showed that GI-7 induced membrane blebbing and increased membrane permeability in APEC, suggesting an effect on the APEC membrane. Consistent with this finding, the expression of genes essential for maintaining outer membrane (OM) integrity was downregulated in GI-7-treated APEC. Furthermore, decreased levels of lipopolysaccharide (LPS) transport (Lpt) proteins and LPS were observed in GI-7-treated APEC. However, the mechanism of action of GI-7 currently remains unknown and needs further investigation. Our studies suggest that GI-7 represents a promising novel lead compound that can be developed to treat APEC infection in chickens and related human ExPEC infections. IMPORTANCE APEC is a subgroup of ExPEC, and genetic similarities of APEC with human ExPECs, including uropathogenic E. coli (UPEC) and neonatal meningitis E. coli (NMEC), have been reported. Our study identified a novel small molecule growth inhibitor, GI-7, effective in reducing APEC infection in chickens with an efficacy similar to that of the currently used antibiotic sulfadimethoxine, notably with an 8-times-lower dose. GI-7 affects the OM integrity and decreases the Lpt protein and LPS levels in APEC, an antibacterial mechanism that can overcome the antibiotic resistance problem. Overall, GI-7 represents a promising lead molecule/scaffold for the development of novel antibacterial therapies that could have profound implications for treating APEC infections in chickens, as well as human infections caused by ExPECs and other related Gram-negative bacteria. Further elucidation of the mechanism of action of GI-7 and identification of its target(s) in APEC will benefit future novel antibacterial development efforts.

Peptides Affecting the Outer Membrane Lipid Asymmetry System (MlaA-OmpC/F) Reduce Avian Pathogenic Escherichia coli (APEC) Colonization in Chickens.

Avian pathogenic Escherichia coli (APEC), an extraintestinal pathogenic E. coli (ExPEC), causes colibacillosis in chickens and is reportedly associated with urinary tract infections and meningitis in humans. Development of resistance is a major limitation of current ExPEC antibiotic therapy. New antibacterials that can circumvent resistance problem such as antimicrobial peptides (AMPs) are critically needed. Here, we evaluated the efficacy of Lactobacillus rhamnosus GG (LGG)-derived peptides against APEC and uncovered their potential antibacterial targets. Three peptides (NPSRQERR [P1], PDENK [P2], and VHTAPK [P3]) displayed inhibitory activity against APEC. These peptides were effective against APEC in biofilm and chicken macrophage HD11 cells. Treatment with these peptides reduced the cecum colonization (0.5 to 1.3 log) of APEC in chickens. Microbiota analysis revealed two peptides (P1 and P2) decreased Enterobacteriaceae abundance with minimal impact on overall cecal microbiota of chickens. Bacterial cytological profiling showed peptides disrupt APEC membranes either by causing membrane shedding, rupturing, or flaccidity. Furthermore, gene expression analysis revealed that peptides downregulated the expression of ompC (>13.0-fold), ompF (>11.3-fold), and mlaA (>4.9-fold), genes responsible for the maintenance of outer membrane (OM) lipid asymmetry. Consistently, immunoblot analysis also showed decreased levels of OmpC and MlaA proteins in APEC treated with peptides. Alanine scanning studies revealed residues crucial (P1, N, E, R and P; P2, D and E; P3, T, P, and K) for their activity. Overall, our study identified peptides with a new antibacterial target that can be developed to control APEC infections in chickens, thereby curtailing poultry-originated human ExPEC infections. IMPORTANCE Avian pathogenic Escherichia coli (APEC) is a subgroup of extraintestinal pathogenic E. coli (ExPEC) and considered a foodborne zoonotic pathogen transmitted through consumption of contaminated poultry products. APEC shares genetic similarities with human ExPECs, including uropathogenic E. coli (UPEC) and neonatal meningitis E. coli (NMEC). Our study identified Lactobacillus rhamnosus GG (LGG)-derived peptides (P1 [NPSRQERR], P2 [PDENK], and P3 [VHTAPK]) effective in reducing APEC infection in chickens. Antimicrobial peptides (AMPs) are regarded as ideal candidates for antibacterial development because of their low propensity for resistance development and ability to kill resistant bacteria. Mechanistic studies showed peptides disrupt the APEC membrane by affecting the MlaA-OmpC/F system responsible for the maintenance of outer membrane (OM) lipid asymmetry, a promising new druggable target to overcome resistance problems in Gram-negative bacteria. Altogether, these peptides can provide a valuable approach for development of novel anti-ExPEC therapies, including APEC, human ExPECs, and other related Gram-negative pathogens. Furthermore, effective control of APEC infections in chickens can curb poultry-originated ExPEC infections in humans.

Surviving Serum: the Escherichia coli iss Gene of Extraintestinal Pathogenic E. coli Is Required for the Synthesis of Group 4 Capsule.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains constitute a serious and emerging clinical problem, as they cause a variety of infections and are usually highly antibiotic resistant. Many ExPEC strains are capable of evading the bactericidal effects of serum and causing sepsis. One critical factor for the development of septicemia is the increased serum survival (iss) gene, which is highly correlated with complement resistance and lethality. Although it is very important, the function of the iss gene has not been elucidated so far. We have been studying the serum survival of a septicemic strain of E. coli serotype O78, which has a group 4 capsule. Here, we show that the iss gene is required for the synthesis of capsules, which protect the bacteria from the bactericidal effect of complement. Moreover, we show that the deletion of the iss gene results in significantly increased binding of the complement proteins that constitute the membrane attack complex to the bacterial surface.

Extraintestinal Pathogenic Escherichia coli ST405 Isolate Coharboring blaNDM-5 and blaCTXM-15: A New Threat in Mozambique.

The development of carbapenem resistance in extraintestinal pathogenic Escherichia coli (ExPEC) has significant clinical implications, particularly in countries where second-line antimicrobials are not readily available, rendering treatments ineffective, and ExPEC infections untreatable. Thus, early detection of high-risk ExPEC lineages and raising awareness of the specific mechanisms underlying carbapenem resistance are mandatory for the selection of appropriate treatment options and the prevention of E. coli spread. This study aims to investigate the phenotypic and genotypic features of the first NDM-5 carbapenemase-producing ExPEC strain isolated from the blood of a patient admitted to the Maputo Central Hospital (MCH), in Mozambique. E. coli SSM100 isolate was identified by MALDI-TOF, it displayed high-level resistance to third generation cephalosporins, carbapenems, fluoroquinolones, and aminoglycosides, performing antimicrobial susceptibilities testing by VITEK 2 system. E. coli SSM100 isolate was classified through whole-genome sequencing as ST405-D-O102: H6, a globally distributed lineage associated with antimicrobial resistance, carrying the blaNDM-5 gene located on an F1:A1:B49 plasmid, coharboring blaCTX-M-15, blaTEM-1, aadA2, sul1, and dfrA12 genes. In addition, mutations in gyrA (S83L and D87N), parC (S80I and E84V), and parE (I529L) conferring fluoroquinolone resistance were also found. Moreover, SSM100 isolate carried 88 virulence genes, of which 28 are reported to be associated with UPEC. The emergence of NDM-5 carbapenemase in a pandemic ST405-D-O102:H6 clone in Mozambique is of great concern. Locations of extended-spectrum ß-lactamase determinants and NDM-5 carbapenemase gene on IncF-plasmid can increase their spread reinforcing the need for antimicrobial surveillance and the urgent introduction of carbapenemase detection tests in diagnostic laboratories of the country.

Antiviral Resistance and Phage Counter Adaptation to Antibiotic-Resistant Extraintestinal Pathogenic Escherichia coli.

Extraintestinal pathogenic Escherichia coli (ExPEC), often multidrug resistant (MDR), is a leading cause of urinary tract and systemic infections. The crisis of emergent MDR pathogens has led some to propose bacteriophages as a therapeutic. However, bacterial resistance to phage is a concerning issue that threatens to undermine phage therapy. Here, we demonstrate that E. coli sequence type 131, a circulating pandemic strain of ExPEC, rapidly develops resistance to a well-studied and therapeutically active phage (ϕHP3). Whole-genome sequencing of the resisters revealed truncations in genes involved in lipopolysaccharide (LPS) biosynthesis, the outer membrane transporter ompA, or both, implicating them as phage receptors. We found ExPEC resistance to phage is associated with a loss of fitness in host microenvironments and attenuation in a murine model of systemic infection. Furthermore, we constructed a novel phage-bacterium bioreactor to generate an evolved phage isolate with restored infectivity to all LPS-truncated ExPEC resisters. This study suggests that although the resistance of pandemic E. coli to phage is frequent, it is associated with attenuation of virulence and susceptibility to new phage variants that arise by directed evolution.IMPORTANCE In response to the rising crisis of antimicrobial resistance, bacteriophage (phage) therapy has gained traction. In the United States, there have been over 10 cases of largely successful compassionate-use phage therapy to date. The resilience of pathogens allowing their broad antibiotic resistance means we must also consider resistance to therapeutic phages. This work fills gaps in knowledge regarding development of phage resisters in a model of infection and finds critical fitness losses in those resisters. We also found that the phage was able to rapidly readapt to these resisters.

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.

Genomic analysis of trimethoprim-resistant extraintestinal pathogenic Escherichia coli and recurrent urinary tract infections.

Urinary tract infections (UTIs) are the most common bacterial infections requiring medical attention and a leading justification for antibiotic prescription. Trimethoprim is prescribed empirically for uncomplicated cases. UTIs are primarily caused by extraintestinal pathogenic Escherichia coli (ExPEC) and ExPEC strains play a central role in disseminating antimicrobial-resistance genes worldwide. Here, we describe the whole-genome sequences of trimethoprim-resistant ExPEC and/or ExPEC from recurrent UTIs (67 in total) from patients attending a regional Australian hospital from 2006 to 2008. Twenty-three sequence types (STs) were observed, with ST131 predominating (28 %), then ST69 and ST73 (both 7 %). Co-occurrence of trimethoprim-resistance genes with genes conferring resistance to extended-spectrum ß-lactams, heavy metals and quaternary ammonium ions was a feature of the ExPEC described here. Seven trimethoprim-resistance genes were identified, most commonly dfrA17 (38 %) and dfrA12 (18 %). An uncommon dfrB4 variant was also observed. Two blaCTX-M variants were identified - blaCTX-M-15 (16 %) and blaCTX-M-14 (10 %). The former was always associated with dfrA12, the latter with dfrA17, and all blaCTX-M genes co-occurred with chromate-resistance gene chrA. Eighteen class 1 integron structures were characterized, and chrA featured in eight structures; dfrA genes featured in seventeen. ST131 H30Rx isolates possessed distinct antimicrobial gene profiles comprising aac(3)-IIa, aac(6)-Ib-cr, aph(3')-Ia, aadA2, blaCTX-M-15, blaOXA-1 and dfrA12. The most common virulence-associated genes (VAGs) were fimH, fyuA, irp2 and sitA (all 91 %). Virulence profile clustering showed ST131 H30 isolates carried similar VAGs to ST73, ST405, ST550 and ST1193 isolates. The sole ST131 H27 isolate carried molecular predictors of enteroaggregative E. coli/ExPEC hybrid strains (aatA, aggR, fyuA). Seven isolates (10 %) carried VAGs suggesting ColV plasmid carriage. Finally, SNP analysis of serial UTI patients experiencing worsening sequelae demonstrated a high proportion of point mutations in virulence factors.

Virulence and resistance properties of E. coli isolated from urine samples of hospitalized patients in Rio de Janeiro, Brazil - The role of mobile genetic elements.

Extraintestinal pathogenic E. coli (ExPEC) is the most frequent etiological agent of urinary tract infections (UTIs). Particular evolutionary successful lineages are associated with severe UTIs and higher incidences of multidrug resistance. Most of the resistance genes are acquired by horizontal transfer of plasmids and other mobile genetic elements (MGEs), and this process has been associated with the successful dissemination of particular lineages. Here, we identified the presence of MGEs and their role in virulence and resistance profiles of isolates obtained from the urine of hospitalized patients in Brazil. Isolates belonging to the successful evolutionary lineages of sequence type (ST) 131, ST405, and ST648 were found to be multidrug-resistant, while those belonging to ST69 and ST73 were often not. Among the ST131, ST405, and ST648 isolates with a resistant phenotype, a high number of mainly IncFII plasmids was identified. The plasmids contained resistance cassettes, and these were also found within phage-related sequences and the chromosome of the isolates. The resistance cassettes were found to harbor several resistance genes, including blaCTX-M-15. In addition, in ST131 isolates, diverse pathogenicity islands similar to those found in highly virulent ST73 isolates were detected. Also, a new genomic island associated with several virulence genes was identified in ST69 and ST131 isolates. In addition, several other MGEs present in the ST131 reference strain EC958 were identified in our isolates, most of them exclusively in ST131 isolates. In contrast, genomic islands present in this reference strain were only partially present or completely absent in our ST131 isolates. Of all isolates studied, ST73 and ST131 isolates had the most similar virulence profile. Overall, no clear association was found between the presence of specific MGEs and virulence profiles. Furthermore, the interplay between virulence and resistance by acquiring MGEs seemed to be lineage dependent. Although the acquisition of IncF plasmids, specific PAIs, GIs, and other MGEs seemed to be involved in the success of some lineages, it cannot explain the success of different lineages, also indicating other (host) factors are involved in this process. Nevertheless, the detection, identification, and surveillance of lineage-specific MGEs may be useful to monitor (new) emerging clones.

Success of Escherichia coli O25b:H4 Sequence Type 131 Clade C Associated with a Decrease in Virulence.

Escherichia coli O25b:H4 sequence type 131 (ST131), which is resistant to fluoroquinolones and which is a producer of CTX-M-15, is globally one of the major extraintestinal pathogenic E. coli (ExPEC) lineages. Phylogenetic analyses showed that multidrug-resistant ST131 strains belong to clade C, which recently emerged from clade B by stepwise evolution. It has been hypothesized that features other than multidrug resistance could contribute to this dissemination since other major global ExPEC lineages (ST73 and ST95) are mostly antibiotic susceptible. To test this hypothesis, we compared early biofilm production, presence of ExPEC virulence factors (VFs), and in vivo virulence in a mouse sepsis model in 19 and 20 epidemiologically relevant strains of clades B and C, respectively. Clade B strains were significantly earlier biofilm producers (P < 0.001), carriers of more VFs (P = 4e-07), and faster killers of mice (P = 2e-10) than clade C strains. Gene inactivation experiments showed that the H30-fimB and ibeART genes were associated with in vivo virulence. Competition assays in sepsis, gut colonization, and urinary tract infection models between the most anciently diverged strain (B1 subclade), one C1 subclade strain, and a B4 subclade recombining strain harboring some clade C-specific genetic events showed that the B1 strain always outcompeted the C1 strain, whereas the B4 strain outcompeted the C1 strain, depending on the mouse niches. All these findings strongly suggest that clade C evolution includes a progressive loss of virulence involving multiple genes, possibly enhancing overall strain fitness by avoiding severe infections, even if it comes at the cost of a lower colonization ability.

Genomic analysis of fluoroquinolone-susceptible phylogenetic group B2 extraintestinal pathogenic Escherichia coli causing infections in cats.

Extraintestinal pathogenic Escherichia coli (ExPEC) can cause urinary tract and other types of infection in cats, but the relationship of cat ExPEC to human ExPEC remains equivocal. This study investigated the prevalence of ExPEC-associated sequence types (STs) from phylogenetic group B2 among fluoroquinolone-susceptible cat clinical isolates. For this, 323 fluoroquinolone-susceptible cat clinical E. coli isolates from Australia underwent PCR-based phylotyping and random amplified polymorphic DNA analysis to determine clonal relatedness. Of the 274 group B2 isolates, 53 underwent whole genome sequencing (WGS), whereas 221 underwent PCR-based screening for (group B2) sequence type complexes (STc) STc12, STc73, ST131, and STc372. Group B2 was the dominant phylogenetic group (274/323, 85 %), whereas within group B2 ST73 dominated, according to both WGS (43 % of 53; followed by ST127, ST12, and ST372 [4/53, 8 % each]) and ST-specific PCR (20 % of 221). In WGS-based comparisons of cat and reference human ST73 isolates, cat isolates had a relatively conserved virulence gene profile but were phylogenetically diverse. Although in the phylogram most cat and human ST73 isolates occupied host species-specific clusters within serotype-specific clades (O2:H1, O6:H1, O25:H1, O50/O2:H1), cat and human isolates were intermingled within two serotype-specific clades: O120:H31 (3 cat and 2 human isolates) and O22:H1 (3 cat and 5 human isolates). These findings confirm the importance of human-associated group B2 lineages as a cause of urinary tract infections in cats. The close genetic relationship of some cat and human ST73 strains suggests bi-directional transmission may be possible.

Diversity and trends in population structure of ESBL-producing Enterobacteriaceae in febrile urinary tract infections in children in France from 2014 to 2017.

BACKGROUND: The population structure of extraintestinal pathogenic Escherichia coli evolves over time, notably due to the emergence of antibiotic-resistant clones such as ESBL-producing Enterobacteriaceae (ESBL-E). OBJECTIVES: To analyse by WGS the genetic diversity of a large number of ESBL-E isolated from urinary tract infections in children from paediatric centres across France between 2014 and 2017 and collected by the National Observatory of febrile urinary tract infection (FUTI) caused by ESBL-E. METHODS: A total of 40 905 Enterobacteriaceae-positive urine cultures were identified. ESBL-E were found in 1983 samples (4.85%). WGS was performed on 251 ESBL-E causing FUTI. STs, core genome MLST (cgMLST), serotype, fimH allele, ESBL genes and presence of papGII key virulence factor were determined. RESULTS: E. coli and Klebsiella pneumoniae were found in 86.9% (218/251) and 11.2% (28/251) of cases, respectively. Several STs predominate among E. coli such as ST131, ST38, ST69, ST73, ST95, ST405, ST12 and ST1193, while no ST emerged in K. pneumoniae. E. coli ST131, ST38 and ST1193 increased during the study period, with a heterogeneity in papGII prevalence (64.5%, 35% and 20% respectively). Most isolates harboured the CTX-M type (97%) with a predominance of blaCTX-M-15. blaCTX-M-27, an emerging variant in E. coli, is found in various STs. cgMLST enabled discrimination of clusters within the main STs. CONCLUSIONS: The predominance of ST131, and the emergence of other STs such as ST38 and ST1193 combined with ESBL genes deserves close epidemiological surveillance considering their high threat in infectious disease. cgMLST could be a discriminant complementary tool for the analyses.

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.

Escherichia coli from Commercial Broiler and Backyard Chickens Share Sequence Types, Antimicrobial Resistance Profiles, and Resistance Genes with Human Extraintestinal Pathogenic Escherichia coli.

Escherichia coli recovered from poultry, and extraintestinal pathogenic E. coli (ExPEC), responsible for most cases of urinary tract infection (UTI) and bloodstream infection (BSI) in humans, may share genetic characteristics, suggesting that poultry are a potential source of ExPEC. Here, we compared E. coli isolated from commercial broiler and backyard chickens (n = 111) with ExPEC isolated from patients with community- or hospital-acquired UTI or BSI (n = 149) from Southeast Brazil. Isolates were genotyped by multilocus sequence typing, tested for susceptibility to antimicrobial agents, and screened for ß-lactamase genes. We found that 10 genotypes were shared among poultry and human isolates: sequence type (ST) 10, ST48, ST58, ST88, ST90, ST93, ST131, ST602, ST617, and ST1018. Thirty-five (23%) ExPEC and 35 (31%) poultry E. coli isolates belonged to the shared STs. ST58 and ST88 isolates from human and poultry sources shared identical antimicrobial resistance profiles. blaTEM-1 was the most prevalent ß-lactamase gene, identified in 65 (92%) of 71 ExPEC and 29 (67%) of 43 poultry E. coli that tested positive for ß-lactamase genes. Commercial broiler chicken isolates shared the extended-spectrum ß-lactamase (ESBL) genes blaCTX-M-2,blaCTX-M-8, and blaSHV-2 with human isolates; backyard chicken isolates lacked ESBL genes. In conclusion, several genotypic and phenotypic characteristics were shared between human and poultry E. coli; this suggests that there is potential for transmission of E. coli and antimicrobial resistance genes from poultry to humans, perhaps through environmental contamination, direct contact, or consumption. Additional research is needed to understand the potential direction and pathways of transmission.

Effects of two-dose ceftiofur treatment for metritis on the temporal dynamics of antimicrobial resistance among fecal Escherichia coli in Holstein-Friesian dairy cows.

A pair-matched longitudinal study conducted on three dairy farms in the U.S. High-Plains explored the temporal effects of two-dose ceftiofur crystalline-free acid (CCFA) treatment for metritis on third-generation cephalosporin (3GC) resistance among enteric E. coli in Holstein-Friesian cows. The current 13-day slaughter withholding period does not account for rising populations of third-generation cephalosporin (3GC) resistant bacteria in feces of animals following CCFA treatment. A total of 124 matched-pairs of cows were enrolled in the study. Cows diagnosed with postpartum metritis received the product twice at the labeled dose of 6.6 mg/kg subcutaneously at the base of alternating ears. Untreated cows-absent clinical metritis-were matched on lactation number and calving date. Feces were collected per rectum on days 0 (baseline), 6, 16, 28, and 56. Environmental samples, from watering troughs as well as surface manure from fresh-cow, hospital, maternity, and milking pens, and from the compost pile were collected prior to the animal sample collection period. Historical data on metritis rates and CCFA use were compiled from herd records. On day 0, cows exhibited an overall mean difference of over 4 log10 colony forming units (CFU) comparing 3GC resistant E. coli to the general E. coli population. At the first eligible slaughter date, the difference declined to 3.31 log10 CFU among cows in the CCFA group (P<0.01 compared to control cows). Such differences were no longer observed between the treated and control groups by day 28. Results suggest a 13-day withholding period following the final treatment is insufficient to allow levels of 3GC resistant E. coli to return to baseline. This effect varied by farm and was dependent upon the starting level of resistance. A farm-specific extended slaughter-withholding period could reduce the microbial risk to food products at slaughter.

Resistance to critically important antimicrobials in Australian silver gulls (Chroicocephalus novaehollandiae) and evidence of anthropogenic origins.

OBJECTIVES: Antimicrobial resistance (AMR) to critically important antimicrobials (CIAs) amongst Gram-negative bacteria can feasibly be transferred amongst wildlife, humans and domestic animals. This study investigated the ecology, epidemiology and origins of CIA-resistant Escherichia coli carried by Australian silver gulls (Chroicocephalus novaehollandiae), a gregarious avian wildlife species that is a common inhabitant of coastal areas with high levels of human contact. METHODS: Sampling locations were widely dispersed around the perimeter of the Australian continent, with sites separated by up to 3500 km. WGS was used to study the diversity and molecular characteristics of resistant isolates to ascertain their epidemiological origin. RESULTS: Investigation of 562 faecal samples revealed widespread occurrence of extended-spectrum cephalosporin-resistant (21.7%) and fluoroquinolone-resistant (23.8%) E. coli. Genome sequencing revealed that CIA-resistant E. coli isolates (n = 284) from gulls predominantly belonged to human-associated extra-intestinal pathogenic E. coli (ExPEC) clones, including ST131 (17%), ST10 (8%), ST1193 (6%), ST69 (5%) and ST38 (4%). Genomic analysis revealed that gulls carry pandemic ExPEC-ST131 clades (O25:H4 H30-R and H30-Rx) and globally emerging fluoroquinolone-resistant ST1193 identified among humans worldwide. Comparative analysis revealed that ST131 and ST1193 isolates from gulls overlapped extensively with human clinical isolates from Australia and overseas. The present study also detected single isolates of carbapenem-resistant E. coli (ST410-blaOXA-48) and colistin-resistant E. coli (ST345-mcr-1). CONCLUSIONS: The carriage of diverse CIA-resistant E. coli clones that strongly resemble pathogenic clones from humans suggests that gulls can act as ecological sponges indiscriminately accumulating and disseminating CIA-resistant bacteria over vast distances.

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.

Diversification of Colonization Factors in a Multidrug-Resistant Escherichia coli Lineage Evolving under Negative Frequency-Dependent Selection.

Escherichia coli is a major cause of bloodstream and urinary tract infections globally. The wide dissemination of multidrug-resistant (MDR) strains of extraintestinal pathogenic E. coli (ExPEC) poses a rapidly increasing public health burden due to narrowed treatment options and increased risk of failure to clear an infection. Here, we present a detailed population genomic analysis of the ExPEC ST131 clone, in which we seek explanations for its success as an emerging pathogenic strain beyond the acquisition of antimicrobial resistance (AMR) genes. We show evidence for evolution toward separate ecological niches for the main clades of ST131 and differential evolution of anaerobic metabolism, key colonization, and virulence factors. We further demonstrate that negative frequency-dependent selection acting across accessory loci is a major mechanism that has shaped the population evolution of this pathogen.IMPORTANCE Infections with multidrug-resistant (MDR) strains of Escherichia coli are a significant global public health concern. To combat these pathogens, we need a deeper understanding of how they evolved from their background populations. By understanding the processes that underpin their emergence, we can design new strategies to limit evolution of new clones and combat existing clones. By combining population genomics with modelling approaches, we show that dominant MDR clones of E. coli are under the influence of negative frequency-dependent selection, preventing them from rising to fixation in a population. Furthermore, we show that this selection acts on genes involved in anaerobic metabolism, suggesting that this key trait, and the ability to colonize human intestinal tracts, is a key step in the evolution of MDR clones of E. coli.