Extraintestinal Pathogenic Escherichia Coli Strains Research Articles

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.

Detection of Extended-spectrum β-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 β-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.

Broad protective vaccination against systemic Escherichia coli with autotransporter antigens.

Extraintestinal pathogenic Escherichia coli (ExPEC) is the leading cause of adult life-threatening sepsis and urinary tract infections (UTI). The emergence and spread of multidrug-resistant (MDR) ExPEC strains result in a considerable amount of treatment failure and hospitalization costs, and contribute to the spread of drug resistance amongst the human microbiome. Thus, an effective vaccine against ExPEC would reduce morbidity and mortality and possibly decrease carriage in healthy or diseased populations. A comparative genomic analysis demonstrated a gene encoding an invasin-like protein, termed sinH, annotated as an autotransporter protein, shows high prevalence in various invasive ExPEC phylogroups, especially those associated with systemic bacteremia and UTI. Here, we evaluated the protective efficacy and immunogenicity of a recombinant SinH-based vaccine consisting of either domain-3 or domains-1,2, and 3 of the putative extracellular region of surface-localized SinH. Immunization of a murine host with SinH-based antigens elicited significant protection against various strains of the pandemic ExPEC sequence type 131 (ST131) as well as multiple sequence types in two distinct models of infection (colonization and bacteremia). SinH immunization also provided significant protection against ExPEC colonization in the bladder in an acute UTI model. Immunized cohorts produced significantly higher levels of vaccine-specific serum IgG and urinary IgG and IgA, findings consistent with mucosal protection. Collectively, these results demonstrate that autotransporter antigens such as SinH may constitute promising ExPEC phylogroup-specific and sequence-type effective vaccine targets that reduce E. coli colonization and virulence.

Virulence potential of antimicrobial-resistant extraintestinal pathogenic Escherichia coli from retail poultry meat in a Caenorhabditis elegans model

Healthy poultry can be a reservoir for extraintestinal pathogenic Escherichia coli (ExPEC), some of which could be multidrug resistant to antimicrobials. These ExPEC strains could contaminate the environment and/or food chain representing thus, food safety and human health risk. However, few studies have shown the virulence of poultry-source antimicrobial-resistant (AMR) ExPEC in humans. This study characterized AMR ExPEC and investigated the virulence potential of some of their isolates in a Caenorhabditis elegans infection model. A total of 46 E. coli isolates from poultry (chicken, n = 29; turkey, n = 12) retail meats and chicken feces (n = 4), or humans (n = 1) were sequenced and identified as ExPEC. Except eight, all remaining 38 ExPEC isolates were resistant to at least one antibiotic and carried corresponding antimicrobial resistance genes (ARGs). About 27 of the 46 ExPEC isolates were multidrug-resistant (≥3 antibiotic classes). Seven ExPEC isolates from chicken or turkey meats were of serotype O25:H4 and sequence type (ST) 131 which clustered with an isolate from a human urinary tract infection (UTI) case having the same serotype and ST. The C. elegans challenge model using eight of studied ExPEC isolates harboring various ARGs and virulence genes (VGs) showed that regardless of their ARG or VG numbers in tested poultry meat and feces, ExPEC significantly reduced the life span of the nematode (P < 0.05) similarly to a human UTI isolate. This study indicated the pathogenic potential of AMR ExPEC from retail poultry meat or feces, but more studies are warranted to establish their virulence in poultry and human. Furthermore, relationships between specific resistance profiles and/or VGs in these E. coli isolates for their pathogenicity deserve investigations.

Extraintestinal pathogenic Escherichia coli (ExPEC) reservoirs, and antibiotics resistance trends: a one-health surveillance for risk analysis from "farm-to-fork".

Extraintestinal pathogenic Escherichia coli (ExPEC) associated infections are significant health concerns for both animals and humans. ExPEC strains are associated with various infections in humans, i.e. urinary tract infections, meningitis, septicemia, and other infections. Over the few years, several studies revealed, food animals act as a reservoir for ExPEC pathovars, but there is no information about the agricultural sector. In particular, the extensive use of antibiotics in food animals and agricultural settings could be significantly contributed to the emergence of antibiotic-resistant pathogens. However, global outbreaks of food-borne illnesses from contaminated food have made a significant concern for both public health and food safety. This review focuses on the reservoirs for ExPEC and their potential circulation between animals, humans, and environment. In this, we first report that the agricultural setting could be the reservoir of ExPEC and can play a role in disseminating antimicrobial-resistant ExPEC. A thorough understanding of ExPEC ecology, reservoirs, and transmission dynamics can significantly contribute to reducing the burden of ExPEC-associated infections. Overall, the study provides the important data on the current state of knowledge for different reservoirs with dynamic, dissemination, and transmission of antimicrobial-resistance ExPEC in animals, humans, and environment in the "One-Health" context.

Virulence Profile, Antibiotic Resistance, and Phylogenetic Relationships among Escherichia coli Strains Isolated from the Feces and Urine of Hospitalized Patients.

Extra-intestinal pathogenic Escherichia coli (ExPEC) may inhabit the human gut microbiota without causing disease. However, if they reach extra-intestinal sites, common cystitis to bloodstream infections may occur, putting patients at risk. To examine the human gut as a source of endogenous infections, we evaluated the E. coli clonal diversity of 18 inpatients' guts and their relationship with strains isolated from urinary tract infection (UTI) in the same hospital. Random amplified polymorphic DNA evaluated the clonal diversity, and the antimicrobial susceptibility was determined by disk diffusion. One isolate of each clone detected was sequenced, and their virulome and resistome were determined. Overall, 177 isolates were screened, among which 32 clones were identified (mean of two clones per patient), with ExPEC strains found in over 75% of the inpatients' guts. Endogenous infection was confirmed in 75% of the cases. ST10, ST59, ST69, ST131, and ST1193 clones and critical mobile drug-resistance encoding genes (blaCTX-M-15, blaOXA-1, blaDHA-1, aac(6')-lb-cr, mcr-1.26, qnrB4, and qnrB19) were identified in the gut of inpatients. The genomic analysis highlighted the diversity of the fecal strains, colonization by lactose-negative E. coli, the high frequency of ExPEC in the gut of inpatients without infections, and the presence of β-lactamase producing E. coli in the gut of inpatients regardless of the previous antibiotics' usage. Considering that we found more than one ExPEC clone in the gut of several inpatients, surveillance of inpatients' fecal pathogens may prevent UTI caused by E. coli in the hospital and dissemination of risk clones.

Reductions in bacterial viability stimulate the production of Extra-intestinal Pathogenic Escherichia coli (ExPEC) cytoplasm-carrying Extracellular Vesicles (EVs).

Extra-intestinal Pathogenic Escherichia coli (ExPEC) is defined as an extra-intestinal foodborne pathogen, and several dominant sequence types (STs) ExPEC isolates are highly virulent, with zoonotic potential. Bacteria extracellular vesicles (EVs) carry specific subsets of molecular cargo, which affect various biological processes in bacteria and host. The mechanisms of EVs formation in ExPEC remains to be elucidated. Here, the purified EVs of ExPEC strains of different STs were isolated with ultracentrifugation processes. A comparative analysis of the strain proteomes showed that cytoplasmic proteins accounted for a relatively high proportion of the proteins among ExPEC EVs. The proportion of cytoplasm-carrying vesicles in ExPEC EVs was calculated with a simple green fluorescent protein (GFP) expression method. The RecA/LexA-dependent SOS response is a critical mediator of generation of cytoplasm-carrying EVs. The SOS response activates the expression of prophage-associated endolysins, Epel1, Epel2.1, and Epel2.2, which triggered cell lysis, increasing the production of ExPEC cytoplasm-carrying EVs. The repressor LexA controlled directly the expression of these endolysins by binding to the SOS boxes in the endolysin promoter regions. Reducing bacterial viability stimulated the production of ExPEC EVs, especially cytoplasm-carrying EVs. The imbalance in cell division caused by exposure to H2O2, the deletion of ftsK genes, or t6A synthesis defects activated the RecA/LexA-dependent SOS response, inducing the expression of endolysins, and thus increasing the proportion of cytoplasm-carrying EVs in the total ExPEC EVs. Antibiotics, which decreased bacterial viability, also increase the production of ExPEC cytoplasm-carrying EVs through the SOS response. Changes in the proportion of cytoplasm-carrying EVs affected the total DNA content of ExPEC EVs. When macrophages are exposed to a higher proportion of cytoplasm-carrying vesicles, ExPEC EVs were more cytotoxic to macrophages, accompanied with more-severe mitochondrial disruption and a higher level of induced intrinsic apoptosis. In summary, we offered comprehensive insight into the proteome analysis of ExPEC EVs. This study demonstrated the novel formation mechanisms of E. coli cytoplasm-carrying EVs.

Molecular Characterization of Extended-Spectrum Beta-Lactamase-Producing Extra-Intestinal Pathogenic Escherichia coli Isolated in a University Teaching Hospital Dakar-Senegal

Extra-Intestinal Pathogenic Escherichia coli (ExPEC) is a predominant Gram-negative bacterial pathogen and is responsible of several diseases including Urinary Tract Infections (UTI), nosocomial pneumonia, and neonatal meningitis. ExPEC isolates are often multidrug resistant and clones producing Extended-Spectrum Beta-Lactamases (ESBL) are increasingly reported all over the world. Seventy-eight clinical ExPEC strains (49 Community-Acquired (CA) and 29 Hospital-Acquired (HA)) were selected for this study. The majority was from UTIs (n=51), while the rest (n=27) was from pus, sputum, bronchial fluid and vaginal samples (non-uropathogenic ExPEC). Antibiotic susceptibility testing was performed using the Kirby-Bauer disc diffusion method. Standard polymerase chain reaction was used to screen major ESBL genes (blaCTX-M, blaOXA-1, blaTEM, blaSHV) and blaCTX-M variants (blaCTX-M-1, blaCTX-M-9, blaCTX-M-15, blaCTX-M-25). All the tested isolates were resistant to ampicillin, ticarcillin, amoxicillin/ clavulanic acid combination, cefalotin, cefotaxime, ceftazidime, cefepime and aztreonam, but showed a high susceptibityto fosfomycin (98.7%, n = 77), ertapenem (96.2%, n = 75), and imipenem (100%). Moreover, isolates harbored at least one ESBL gene, including blaCTX-M (98.7%), blaOXA-1 (78.2%), blaTEM (44.9%) and blaSHV (3.8%). The CTX-M variants were also found with the predominance of blaCTX-M-1 (89.7%) and blaCTX-M-15 (89.7%) followed by blaCTX-M-9 (11.5%). Despite the resistance to most of the tested antibiotics, ExPEC isolates showed fortunately good susceptibility to fosfomycin and carbapenems. blaCTX-M1, blaCTX-M15 and blaOXA-1 seem to be E.coli major ESBL genes circulating in Senegal. The high level of resistance to antimicrobials observed stresses the need of establishing an epidemiological surveillance of antimicrobial resistance in both community and hospital settings.

Use of Large-Scale Genomics to Identify the Role of Animals and Foods as Potential Sources of Extraintestinal Pathogenic Escherichia coli That Cause Human Illness

Extraintestinal pathogenic Escherichia coli (ExPEC) cause urinary tract and potentially life-threatening invasive infections. Unfortunately, the origins of ExPEC are not always clear. We used genomic data of E. coli isolates from five U.S. government organizations to evaluate potential sources of ExPEC infections. Virulence gene analysis of 38,032 isolates from human, food animal, retail meat, and companion animals classified the subset of 8142 non-diarrheagenic isolates into 40 virulence groups. Groups were identified as low, medium, and high relative risk of containing ExPEC strains, based on the proportion of isolates recovered from humans. Medium and high relative risk groups showed a greater representation of sequence types associated with human disease, including ST-131. Over 90% of food source isolates belonged to low relative risk groups, while >60% of companion animal isolates belonged to medium or high relative risk groups. Additionally, 18 of the 26 most prevalent antimicrobial resistance determinants were more common in high relative risk groups. The associations between antimicrobial resistance and virulence potentially limit treatment options for human ExPEC infections. This study demonstrates the power of large-scale genomics to assess potential sources of ExPEC strains and highlights the importance of a One Health approach to identify and manage these human pathogens.

Extraintestinal Pathogenic Escherichia coli: Beta-Lactam Antibiotic and Heavy Metal Resistance.

Multiple-antibiotic-resistant (MAR) extra-intestinal pathogenic Escherichia coli (ExPEC) represents one of the most frequent causes of human nosocomial and community-acquired infections, whose eradication is of major concern for clinicians. ExPECs may inhabit indefinitely as commensal the gut of humans and other animals; from the intestine, they may move to colonize other tissues, where they are responsible for a number of diseases, including recurrent and uncomplicated UTIs, sepsis and neonatal meningitis. In the pre-antibiotic era, heavy metals were largely used as chemotherapeutics and/or as antimicrobials in human and animal healthcare. As with antibiotics, the global incidence of heavy metal tolerance in commensal, as well as in ExPEC, has increased following the ban in several countries of antibiotics as promoters of animal growth. Furthermore, it is believed that extensive bacterial exposure to heavy metals present in soil and water might have favored the increase in heavy-metal-tolerant microorganisms. The isolation of ExPEC strains with combined resistance to both antibiotics and heavy metals has become quite common and, remarkably, it has been recently shown that heavy metal resistance genes may co-select antibiotic-resistance genes. Despite their clinical relevance, the mechanisms underlining the development and spread of heavy metal tolerance have not been fully elucidated. The aim of this review is to present data regarding the development and spread of resistance to first-line antibiotics, such as beta-lactams, as well as tolerance to heavy metals in ExPEC strains.

Third-generation cephalosporin (3GC) resistance and its association with Extra-intestinal pathogenic Escherichia coli (ExPEC). Focus on broiler carcasses

The worldwide spread of Extra-intestinal Pathogenic Escherichia coli (ExPEC), together with the antimicrobial resistance linked with extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC β-lactamases (pAmpCs) are pressing threats for public health. This study aimed to investigate the presence of ExPEC genes in third-generation cephalosporin (3 GC)-resistant E. coli and to study their distribution in broiler carcasses at the slaughterhouse after the chilling process. To this purpose, isolates from a collection of 3 GC-resistant E. coli from carcasses of broilers originating from twelve broiler farms and three production chains were investigated. Several multivariate statistical approaches were adopted to elucidate the relationships among features. Phylogroup F was predominant in all broiler batches and was mainly associated with blaTEM and ESBL genes but less correlated to ExPEC genes. Another remarkable finding was the predominance of ExPEC strains assigned to uncommon phylogroups, such as B2, D, E and Clade I, commonly found into the environment. This study represents a first step for a comprehensive characterization of ExPEC genes harboured by 3 GC-resistant E. coli. These findings may be valuable for the identification of potential risks associated to broiler carcasses as source of uncommon E. coli phylogroups.

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.

The causal relationship between O2:K7:H6 extra-intestinal pathogenic Escherichia coli (ExPEC) and native valve endocarditis: a case report

BackgroundNative valves infective endocarditis due to Escherichia coli is still a rare disease and a particular virulence of some E.coli isolate may be suspected.Case presentationA 79-year-old woman presented during the post-operative period of an orthopedic surgery a urinary tract infection following obstructive ureteral lithiasis. E. coli was isolated from a pure culture of urine and blood sampled simultaneously. After evidence of sustained E.coli septicemia, further investigations revealed acute cholecystitis with the same micro-organism in biliary drainage and a native valve mitral endocarditis. E.coli was identified as O2:K7:H6, phylogenetic group B2, ST141, and presented several putative and proven virulence genes. The present isolate can be classified as both extra-intestinal pathogenic E.coli (ExPECJJ) and uropathogenic E. coli (UPECHM).ConclusionsThe relationship between the virulent factors present in ExPEC strains and some serotypes of E. coli that could facilitate the adherence to cardiac valves warrants further investigation.

The development and characterization of an E. coli O25B bioconjugate vaccine.

Extraintestinal pathogenic Escherichia coli (ExPEC) cause a wide range of clinical diseases such as bacteremia and urinary tract infections. The increase of multidrug resistant ExPEC strains is becoming a major concern for the treatment of these infections and E. coli has been identified as a critical priority pathogen by the WHO. Therefore, the development of vaccines has become increasingly important, with the surface lipopolysaccharide constituting a promising vaccine target. This study presents genetic and structural analysis of clinical urine isolates from Switzerland belonging to the serotype O25. Approximately 75% of these isolates were shown to correspond to the substructure O25B only recently described in an emerging clone of E. coli sequence type 131. To address the high occurrence of O25B in clinical isolates, an O25B glycoconjugate vaccine was prepared using an E. coli glycosylation system. The O antigen cluster was integrated into the genome of E. coli W3110, thereby generating an E. coli strain able to synthesize the O25B polysaccharide on a carrier lipid. The polysaccharide was enzymatically conjugated to specific asparagine side chains of the carrier protein exotoxin A (EPA) of Pseudomonas aeruginosa by the PglB oligosaccharyltransferase from Campylobacter jejuni. Detailed characterization of the O25B-EPA conjugate by use of physicochemical methods including NMR and GC-MS confirmed the O25B polysaccharide structure in the conjugate, opening up the possibility to develop a multivalent E. coli conjugate vaccine containing O25B-EPA.

Antibiotic Resistance and Virulence of Extraintestinal Pathogenic Escherichia coli (ExPEC) Vary According to Molecular Types

Extraintestinal pathogenic Escherichia coli (ExPEC) can cause many human extraintestinal infections. Resistance and virulence of ExPEC are inextricably linked to its phylogenetic background. However, studies on type-specific distribution of resistance and virulence and the connection between resistance/virulence and molecular typing are lacking. Here, 411 ExPEC strains were collected and characterized using antimicrobial susceptibility testing and molecular typing. Among these, 74 representative strains were selected for whole genome sequencing and the Galleria mellonella killing assay. CH40-30-ST131, CH37-27-ST405, CH40-41-ST131, and CH13-5-ST12 isolates had high resistance rates to all antimicrobials tested. BlaCTX–M played a significant role in the β-lactam resistance of ExPEC isolates. CH14-64-ST1193, CH40-30-ST131, and CH35-27-ST69 isolates were highly virulent in the G. mellonella model. Virulence factors (VFs) involved in adherence (papB, papI, papX, and fimA), autotransporter (sat), invasion (aslA, kpsD), iron uptake (except for entD), or toxin (senB) might be responsible for pathogenicity in vivo. Specific antibiotic resistance genes (ARGs) or VFs were prevalent in specific types of strains, including papB, papI, fimA, sat, kpsD, senB, and aerobactin genes in CH14-64-ST1193 isolates; blaCTX–M–15, aac(6′)-Ib-cr, papB, papI, sat, iucA, iucB, iucC, chuT, chuX, and shuU in CH40-30-ST131 isolates; tetB in CH35-27-ST69 and CH13-5-ST12 isolates. Type distribution also differed by VF score. CH37-27-ST405 and CH26-5-ST38 isolates carried more ARGs and VFs indicating that they had a high resistance and virulence potential. This study demonstrates the type-specific distribution of resistance and virulence thus providing a basis for further research, prevention and treatment of ExPEC infections.

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 β-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.

Poultry-origin extraintestinal Escherichia coli strains carrying the traits associated with urinary tract infection, sepsis, meningitis and avian colibacillosis in India.

In-depth 'One Health' risk assessment of extraintestinal pathogenic Escherichia coli (ExPEC) strains carrying the traits of urinary tract infection, sepsis, meningitis and avian colibacillosis in poultry of India. A total of 230 E. coli isolates were recovered from chicken samples representing the different sources (faeces vs caeca), stages (poultry farms vs retails butcher shop) or environments (rural vs urban) of poultry in India. Among all poultry-origin E. coli isolates, 49 (21·1%) strains were identified as ExPEC possessing multiple virulence determinants regardless of their association with any specific phylogenetic lineages. Of particular, potentially virulent ExPEC pathotypes, that is, uropathogenic E.coli (UPEC, 20·4%), avian pathogenic E. coli (APEC, 34·6%), septicaemia-associated E. coli (SEPEC, 47·0%) and neonatal meningitis-causing E.39 coli (NMEC, 2·0%) were also detected among all ExPEC strains. Our study is the first to assess ExPEC strains circulating in the different settings of poultry in India and significantly demonstrates their potential ability to cause multiple extraintestinal infections both in humans and animals. The data of our study are in favour of the possibility that poultry-origin putative virulent ExPEC pathotypes consequently constitute a threat risk to 'One Health' or for food safety and a great concern for poultry production of India.

Escherichia coli strains of chicken and human origin: Characterization of antibiotic and heavy-metal resistance profiles, phylogenetic grouping, and presence of virulence genetic markers

Multiple antibiotic-resistant extra-intestinal pathogenic Escherichia coli (ExPEC) strains represent a serious health care problem both for poultry and humans. Recently isolates with combined resistance to both antibiotics and heavy metals have been increased worldwide, with growing concern for possible co-selection of antimicrobial resistant genes. In the present study we characterized, at a phenotypic and genetic level, 80 E. coli isolates: forty independent isolates were collected from manure samples of healthy chickens and 40 from independent human extra-intestinal infections (ExPEC strains). The results obtained indicated that i) compared to chicken, human isolates presented a broader spectrum of antibiotic resistance and virulence potentials; ii) although at a lower extent, ExPEC-associated virulence genes were also present in chicken isolates, suggesting they may be potentially pathogens; iii) that arsenic (As) and zinc (Zn) tolerance genetic determinants were significantly more prevalent among chicken and human isolates respectively, while those responsible for tolerance to cadmium (Cd), silver (Ag) and copper (Cu) were equally distributed among the two groups of strains; iv) a very strong correlation was found between chicken gentamicin (GM) resistance and cadmium (Cd) tolerance. Elucidating the role of heavy metals in the selection and spread of highly pathogenic E. coli strains (co-selection) is of primary importance to lower the potential risk of infections in poultry and humans. The control of bacterial zoonotic agents, that commonly occur in livestock and that may be transmitted, directly or via the food chain, to human populations, could be of relevant interest.

EntE, EntS and TolC synergistically contributed to the pathogenesis of APEC strain E058

Extraintestinal pathogenic Escherichia coli (ExPEC) shows an enhanced ability to cause infection outside the intestinal tract. Avian pathogenic E. coli (APEC), one type of ExPEC, causes avian colibacillosis, a disease of significant economic importance to poultry producers worldwide that is characterized by systemic infection. Some ExPEC strains as well as other pathogenic enterobacteria produce enterobactin, a catecholate siderophore used to sequester iron during infection. Here, we showed that disruption of enterobactin efflux via outer membrane protein TolC significantly decreased the pathogenicity of APEC strain E058. Furthermore, colonization and persistence assays performed using a chicken infection model showed that the ΔtolC mutant was obviously attenuated (p˂0.001). In contrast, disruption of enterobactin synthesis gene entE and/or the inner membrane transporter gene entS had little effect on pathogenicity. Analysis of growth kinetics revealed a significant reduction in the growth of triple mutant strain E058ΔentEΔentSΔtolC in iron-deficient medium compared with the wild-type strain (p˂0.001), while no growth impairment was noted for the E058ΔtolC mutant in either Luria-Bertani broth or iron-deficient medium. The E058ΔentEΔentSΔtolC mutant also showed significantly decreased virulence compared with single mutant strain E058ΔtolC. Low-copy complementation of strains E058ΔtolC and E058ΔentEΔentSΔtolC with plasmid-borne tolC restored virulence to wild-type levels in the chicken infection model. Macrophage infection assays showed that ingestion of E058ΔtolC by macrophage cell line HD11 cells was reduced compared with ingestion of the E058ΔentEΔentSΔtolC mutant. However, no significant differences were observed between the mutants and the wild-type in a chicken serum resistance assay. Together, these results suggest that EntE, EntS and TolC synergistically contributed to the pathogenesis of APEC strain E058 in an iron-deficient environment.

Identification of Host Adaptation Genes in Extraintestinal Pathogenic Escherichia coli during Infection in Different Hosts

Extraintestinal pathogenic Escherichia coli (ExPEC) is an important human and animal pathogen. Despite the apparent similarities in their known virulence attributes, some ExPEC strains can cross the host species barrier and present a zoonotic potential, whereas other strains exhibit host specificity, suggesting the existence of unknown mechanisms that remain to be identified. We applied a transposon-directed insertion site sequencing (TraDIS) strategy to investigate the ExPEC XM strain, which is capable of crossing the host species barrier, and to screen for virulence-essential genes in both mammalian (mouse) and avian (duck) models of E. coli-related septicemia. We identified 151 genes essential for systemic infection in both mammalian and avian models, 97 required only in the mammalian model, and 280 required only in the avian model. Ten genes/gene clusters were selected for further validation, and their contributions to ExPEC virulence in both mammalian and avian models or mammalian- or avian-only models were confirmed by animal tests. This represents the first comprehensive genome-wide analysis of virulence-essential genes required for systemic infections in two different host species and provides a further comprehensive understanding of ExPEC-related virulence, host specificity, and adaptation.