Human Extraintestinal Pathogenic Escherichia Coli Research Articles

NhaA facilitates the maintenance of bacterial envelope integrity and the evasion of complement attack contributing to extraintestinal pathogenic Escherichia coli virulence.

Extraintestinal pathogenic Escherichia coli (ExPEC) is responsible for severe bloodstream infections in humans and animals. However, the mechanisms underlying ExPEC's serum resistance remain incompletely understood. Through the transposon-directed insertion-site sequencing approach, our previous study identified nhaA, the gene encoding a Na+/H+ antiporter, as a crucial factor for infection in vivo. In this study, we investigated the role of NhaA in ExPEC virulence utilizing both in vitro models and systemic infection models involving avian and mammalian animals. Genetic mutagenesis analysis revealed that nhaA deletion resulted in filamentous bacterial morphology and rendered the bacteria more susceptible to sodium dodecyl sulfate, suggesting the role of nhaA in maintaining cell envelope integrity. The nhaA mutant also displayed heightened sensitivity to complement-mediated killing compared to the wild-type strain, attributed to augmented deposition of complement components (C3b and C9). Remarkably, NhaA played a more crucial role in virulence compared to several well-known factors, including Iss, Prc, NlpI, and OmpA. Our findings revealed that NhaA significantly enhanced virulence across diverse human ExPEC prototype strains within B2 phylogroups, suggesting widespread involvement in virulence. Given its pivotal role, NhaA could serve as a potential drug target for tackling ExPEC infections.

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

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

The virulence regulator AbsR in avian pathogenic Escherichia coli has pleiotropic effects on bacterial physiology

Avian pathogenic Escherichia coli (APEC) belonging to extraintestinal pathogenic E. coli (ExPEC) can cause severe infections in extraintestinal tissues in birds and humans, such as the lungs and blood. MprA (microcin production regulation, locus A, herein renamed AbsR, a blood survival regulator), a member of the MarR (multiple antibiotic resistance regulator) transcriptional regulator family, governs the expression of capsule biosynthetic genes in human ExPEC and represents a promising druggable target for antimicrobials. However, a deep understanding of the AbsR regulatory mechanism as well as its regulon is lacking. In this study, we present a systems-level analysis of the APEC AbsR regulon using ChIP-Seq (chromatin immunoprecipitation sequencing) and RNA-Seq (RNA sequencing) methods. We found that AbsR directly regulates 99 genes and indirectly regulates 667 genes. Furthermore, we showed that: 1) AbsR contributes to antiphagocytotic effects by macrophages and virulence in a mouse model for systemic infection by directly activating the capsular gene cluster; 2) AbsR positively impacts biofilm formation via direct regulation of the T2SS (type II secretion system) but plays a marginal role in virulence; and 3) AbsR directly upregulates the acid tolerance signaling system EvgAS to withstand acid stress but is dispensable in ExPEC virulence. Finally, our data indicate that the role of AbsR in virulence gene regulation is relatively conserved in ExPEC strains. Altogether, this study provides a comprehensive analysis of the AbsR regulon and regulatory mechanism, and our data suggest that AbsR likely influences virulence primarily through the control of capsule production. Interestingly, we found that AbsR severely represses the expression of the type I-F CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated) systems, which could have implications in CRISPR biology and application.

Characteristics, pathogenic mechanism, zoonotic potential, drug resistance, and prevention of avian pathogenic Escherichia coli (APEC).

Although most Escherichia coli (E. coli) strains are commensal and abundant, certain pathogenic strains cause severe diseases from gastroenteritis to extraintestinal infections. Extraintestinal pathogenic E. coli (ExPEC) contains newborn meningitis E. coli (NMEC), uropathogenic E. coli (UPEC), avian pathogenic E. coli (APEC), and septicemic E. coli (SEPEC) based on their original host and clinical symptom. APEC is a heterogeneous group derived from human ExPEC. APEC causes severe respiratory and systemic diseases in a variety of avians, threatening the poultry industries, food security, and avian welfare worldwide. APEC has many serotypes, and it is a widespread pathogenic bacterium in poultry. In addition, ExPEC strains share significant genetic similarities and similar pathogenic mechanisms, indicating that APEC potentially serves as a reservoir of virulence and resistance genes for human ExPEC, and the virulence and resistance genes can be transferred to humans through food animals. Due to economic losses, drug resistance, and zoonotic potential, APEC has attracted heightened awareness. Various virulence factors and resistance genes involved in APEC pathogenesis and drug resistance have been identified. Here, we review the characteristics, epidemiology, pathogenic mechanism zoonotic potential, and drug resistance of APEC, and summarize the current status of diagnosis, alternative control measures, and vaccine development, which may help to have a better understanding of the pathogenesis and resistance of APEC, thereby reducing economic losses and preventing the spread of multidrug-resistant APEC to humans.

Hcp Proteins of the Type VI Secretion System Promote Avian Pathogenic E. coli DE205B (O2:K1) to Induce Meningitis in Rats.

Avian pathogenic Escherichia coli (APEC) is an important extra-intestinal pathogenic E. coli (ExPEC), which often causes systemic infection in poultry and causes great economic loss to the breeding industry. In addition, as a major source of human ExPEC infection, the potential zoonotic risk of APEC has been an ongoing concern. Previous studies have pointed out that APEC is a potential zoonotic pathogen, which has high homology with human pathogenic E. coli such as uro-pathogenic E. coli (UPEC) and neonatal meningitis E. coli (NMEC), shares multiple virulence factors and can cause mammalian diseases. Previous studies have reported that O18 and O78 could cause different degrees of meningitis in neonatal rats, and different serotypes had different degrees of zoonotic risk. Here, we compared APEC DE205B (O2:K1) with NMEC RS218 (O18:K1:H7) by phylogenetic analysis and virulence gene identification to analyze the potential risk of DE205B in zoonotic diseases. We found that DE205B possessed a variety of virulence factors associated with meningitis and, through phylogenetic analysis, had high homology with RS218. DE205B could colonize the cerebrospinal fluid (CSF) of rats, and cause meningitis and nerve damage. Symptoms and pathological changes in the brain were similar to RS218. In addition, we found that DE205B had a complete T6SS, of which Hcp protein was its important structural protein. Hcp1 induced cytoskeleton rearrangement in human brain microvascular endothelial cells (HBMECs), and Hcp2 was mainly involved in the invasion of DE205B in vitro. In the meningitis model of rats, deletion of hcp2 gene reduced survival in the blood and the brain invasiveness of DE205B. Compared with WT group, Δhcp2 group induced lower inflammation and neutrophils infiltration in brain tissue, alleviating the process of meningitis. Together, these results suggested that APEC DE205B had close genetic similarities to NMEC RS218, and a similar mechanism in causing meningitis and being a risk for zoonosis. This APEC serotype provided a basis for zoonotic research.

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.

Genomic and Temporal Trends in Canine ExPEC Reflect Those of Human ExPEC.

ABSTRACTCompanion animals and humans are known to share extraintestinal pathogenic Escherichia coli (ExPEC), but the extent of E. coli sequence types (STs) that cause extraintestinal diseases in dogs is not well understood. Here, we generated whole-genome sequences of 377 ExPEC collected by the University of Melbourne Veterinary Hospital from dogs over an 11-year period from 2007 to 2017. Isolates were predominantly from urogenital tract infections (219, 58.1%), but isolates from gastrointestinal specimens (51, 13.5%), general infections (72, 19.1%), and soft tissue infections (34, 9%) were also represented. A diverse collection of 53 STs were identified, with 18 of these including at least five sequences. The five most prevalent STs were ST372 (69, 18.3%), ST73 (31, 8.2%), ST127 (22, 5.8%), ST80 (19, 5.0%), and ST58 (14, 3.7%). Apart from ST372, all of these are prominent human ExPEC STs. Other common ExPEC STs identified included ST12, ST131, ST95, ST141, ST963, ST1193, ST88, and ST38. Virulence gene profiles, antimicrobial resistance carriage, and trends in plasmid carriage for specific STs were generally reflective of those seen in humans. Many of the prominent STs were observed repetitively over an 11-year time span, indicating their persistence in the dogs in the community, which is most likely driven by household sharing of E. coli between humans and their pets. The case of ST372 as a dominant canine lineage observed sporadically in humans is flagged for further investigation.IMPORTANCE Pathogenic E. coli that causes extraintestinal infections (ExPEC) in humans and canines represents a significant burden in hospital and veterinary settings. Despite the obvious interrelationship between dogs and humans favoring both zoonotic and anthropozoonotic infections, whole-genome sequencing projects examining large numbers of canine-origin ExPEC are lacking. In support of anthropozoonosis, we found that most STs from canine infections are dominant human ExPEC STs (e.g., ST73, ST127, ST131) with similar genomic traits, such as plasmid carriage and virulence gene burden. In contrast, we identified ST372 as the dominant canine ST and a sporadic cause of infection in humans, supporting zoonotic transfer. Furthermore, we highlight that, as is the case in humans, STs in canine disease are consistent over time, implicating the gastrointestinal tract as the major community reservoir, which is likely augmented by exposure to human E. coli via shared diet and proximity.

Genomic analysis of the zoonotic ST73 lineage containing avian and human extraintestinal pathogenic Escherichia coli (ExPEC)

Extraintestinal pathogenic Escherichia coli (ExPEC) is a globally distributed pathogen, with uropathogenic E. coli (UPEC) and sepsis-associated E. coli (SEPEC) pathotypes particularly involved in human and companion animal disease, while avian pathogenic pathotype (APEC) severely impact poultry health and production. Similarities between APEC from poultry/meat and human ExPEC suggest that some APEC lineages may have zoonotic potential. ExPEC sequence type 73 (ST73) and its clonal complex (CC) are increasing causes of urinary tract infections and sepsis, but its role in zoonotic disease is less well understood. Here, we analyzed the genome sequences of 25 E. coli isolates from Brazil (11 APEC and 14 UPEC) from two time periods, from poultry producing areas and hospitals in the same geographical regions. Isolates were compared to 558 publicly available ST73/CC73 global sequences. Brazilian APEC harbored virulence factors associated with UPEC/SEPEC such as sfa, cnf1, vat, usp, hlyA, iron acquisition and protectins/serum resistance systems, while lacking some common APEC markers and widespread multidrug resistance. Analysis of core genome MLST and SNP phylogenetic trees indicated evolutionary relationships between subgroups of the Brazilian APEC to two contemporary Brazilian UPEC isolates from the same region, and one Brazilian UPEC available from another study. Phylogenies showed a non-host, geographical, or pathotype specificity, with APEC isolates clustering closely with international human UPEC, SEPEC. The remaining Brazilian UPEC grouped within human clusters. Collectively, this suggests a zoonotic potential for subgroups of Brazilian APEC from the ST73 lineage that could contaminate poultry products and subsequently cause human infection.

F Plasmid Lineages in Escherichia coli ST95: Implications for Host Range, Antibiotic Resistance, and Zoonoses.

ABSTRACTEscherichia coli sequence type 95 (ST95) is an extraintestinal pathogenic E. coli (ExPEC) renowned for its ability to cause significant morbidity and mortality in humans and poultry. A core genome analysis of 668 ST95 isolates generated 10 clades (A to J), 5 of which are reported here for the first time. F plasmid replicon sequence typing showed that almost a third (178/668 [27%]) of the collection carry pUTI89 (F29:B10) and were restricted to clade A and a sublineage of clade B. In contrast, almost half (328/668 [49%]) of the collection across multiple clades harbor ColV plasmids (multiple F types). Strikingly, ST95 lineages with pUTI89 were almost exclusively from humans, while ColV+ ST95 lineages were sourced from poultry and humans. Clade I was notable because it comprises temporally and geographically matched ColV+ isolates sourced from human and retail poultry meat, suggesting interspecies transmission via food. Clade F contained ST95 isolates of bovine origin, none of which carried ColV or pUTI89 plasmids. Remarkably, an analysis of a cohort of 34,176 E. coli isolates comprising 2,570 sequence types mirrored what was observed in ST95: (i) pUTI89 was overwhelmingly linked to E. coli sourced from humans but almost entirely absent from 13,027 E. coli isolates recovered from poultry, pigs, and cattle, and (ii) E. coli isolates harboring ColV plasmids were from multiple sources, including humans, poultry, and swine. Overall, our data suggest that F plasmids influence E. coli host range, clade structure, and zoonotic potential in ST95 and ExPEC more broadly.IMPORTANCE E. coli ST95 is one of five dominant ExPEC lineages globally and noted for causing urinary tract and bloodstream infections and neonatal meningitis in humans and colibacillosis in poultry. Using high-resolution phylogenomics, we show that F replicon sequence type is linked to ST95 clade structure and zoonotic potential. Specifically, human centric ST95 clades overwhelmingly harbor F29:B10 (pUTI89) plasmids, while clades carrying both human- and poultry-sourced isolates are typically ColV+ with multiple replicon types. Importantly, several clades identified clonal ColV+ ST95 isolates from human and poultry sources, but clade I, which housed temporally and spatially matched isolates, provided the most robust evidence. Notably, patterns of association of F replicon types with E. coli host were mirrored within a diverse collection of 34,176 E. coli genomes. Our studies indicate that the role of food animals as a source of human ExPEC disease is complex and warrants further investigation.

154 Virulence Potential of Antimicrobial Resistant Extraintestinal Pathogenic Escherichia coli from Poultry in a Caenorhabditis Elegans Model

Abstract This study investigated virulence potential of poultry antimicrobial resistant extraintestinal pathogenic E. coli (ExPEC). A total of 46 E. coli isolates from poultry meat, feces, or humans were sequenced and identified as ExPEC. Based on their characteristics, eight of these ExPEC isolates were evaluated for their potentials using a Caenorhabditis elegans infection model. The life-span of C. elegans in response to these eight isolates was examined in three life-span experiments: 1) E. coli OP 50 (negative control), K88+ enterotoxigenic E. coli strain JG280 (positive control), and an ExPEC isolate from human urinal tract infection; 2) three ExPEC isolates from chicken and turkey retail meats; 3) four ExPEC isolates from chicken feces with different antimicrobial resistance (AMR) profiles or a various number of virulence genes (VGs). All 46 isolates belonged to 24 serotypes among which 6 were of serotype O25:H4 Sequence Type 131 (ST131). Interestingly, all ST 131 isolates from chicken or turkey retail meats clustered with a human UTI isolate belonging to the similar serotype and ST type. The types and numbers of AGRs and VGs varied among the eight selected isolates for C. elegans model. The human ExPEC induced a similar effect as the JG280 on reducing (P < 0.05) survival of C. elegans. Interestingly, chicken and turkey meat ExPEC isolates, caused similar negative impacts on the survival of worms as the human ExPEC. Additionally, fecal ExPEC isolates reduced (P < 0.05) the survival of C. elegans compared to OP50. However, the survival of C. elegans was not reduced with an increasing number of VGs and did not seem to be affected by AMR profiles. This study indicated the virulence potential of ExPEC isolates from retail poultry meat or feces. The relationship between specific AMR profiles and/or numbers of VGs with pathogenicity in these E. coli isolates deserves further investigations.

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

ABSTRACTAvian 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.

Bile Salts Regulate Zinc Uptake and Capsule Synthesis in a Mastitis-Associated Extraintestinal Pathogenic Escherichia coli Strain.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains are major causes of urinary and bloodstream infections. ExPEC reservoirs are not completely understood. Some mastitis-associated E. coli (MAEC) strains carry genes associated with ExPEC virulence, including metal scavenging, immune avoidance, and host attachment functions. In this study, we investigated the role of the high-affinity zinc uptake (znuABC) system in the MAEC strain M12. Elimination of znuABC moderately decreased fitness during mouse mammary gland infections. The ΔznuABC mutant strain exhibited an unexpected growth delay in the presence of bile salts, which was alleviated by the addition of excess zinc. We isolated suppressor mutants with improved growth in bile salts, several of which no longer produced the K96 capsule made by strain M12. The addition of bile salts also reduced capsule production by strain M12 and ExPEC strain CP9, suggesting that capsule synthesis may be detrimental when bile salts are present. To better understand the role of the capsule, we compared the virulence of mastitis strain M12 with that of its unencapsulated ΔkpsCS mutant in two models of ExPEC disease. The wild-type strain successfully colonized mouse bladders and kidneys and was highly virulent in intraperitoneal infections. Conversely, the ΔkpsCS mutant was unable to colonize kidneys and was unable to cause sepsis. These results demonstrate that some MAEC strains may be capable of causing human ExPEC illness. The virulence of strain M12 in these infections is dependent on its capsule. However, capsule may interfere with zinc homeostasis in the presence of bile salts while in the digestive tract.

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.

Prevalence and Antibiotic Resistance Characteristics of Extraintestinal Pathogenic Escherichia coli among Healthy Chickens from Farms and Live Poultry Markets in China.

Simple SummaryChicken meat has been proved to be a suspected source of extraintestinal pathogenic Escherichia coli (ExPEC), causing several diseases in humans, and bacteria in healthy chickens can contaminate chicken carcasses at the slaughter; however, reports about the prevalence and molecular characteristics of ExPEC in healthy chickens are still rare. In this study, among 926 E. coli isolates from healthy chickens in China, 22 (2.4%) were qualified as ExPEC and these ExPEC isolates were clonally unrelated. A total of six serogroups were identified in this study, with O78 being the most predominant type, and all the six serogroups had been frequently reported in human ExPEC isolates in many countries. All the 22 ExPEC isolates were multidrug-resistant and most isolates carried both blaCTX-M and fosA3 resistance genes. Notably, plasmid-borne colistin resistance gene mcr-1 was identified in six ExPEC isolates, among which two carried additional carbapenemase gene blaNDM, compromising both the efficacies of the two critically important drugs for humans, carbapenems and colistin. These results highlight that healthy chickens can serve as a potential reservoir for multidrug resistant ExPEC isolates, including mcr-1-containing ExPEC.Chicken products and chickens with colibacillosis are often reported to be a suspected source of extraintestinal pathogenic Escherichia coli (ExPEC) causing several diseases in humans. Such pathogens in healthy chickens can also contaminate chicken carcasses at the slaughter and then are transmitted to humans via food supply; however, reports about the ExPEC in healthy chickens are still rare. In this study, we determined the prevalence and characteristics of ExPEC isolates in healthy chickens in China. A total of 926 E. coli isolates from seven layer farms (371 isolates), one white-feather broiler farm (78 isolates) and 17 live poultry markets (477 isolates from yellow-feather broilers) in 10 cities in China, were isolated and analyzed for antibiotic resistance phenotypes and genotypes. The molecular detection of ExPEC among these healthy chicken E. coli isolates was performed by PCRs, and the serogroups and antibiotic resistance characteristics of ExPEC were also analyzed. Pulsed-field gel electrophoresis (PFGE) and Multilocus sequence typing (MLST) were used to analyze the genetic relatedness of these ExPEC isolates. We found that the resistance rate for each of the 15 antimicrobials tested among E. coli from white-feather broilers was significantly higher than that from brown-egg layers and that from yellow-feather broilers in live poultry markets (p < 0.05). A total of 22 of the 926 E. coli isolates (2.4%) from healthy chickens were qualified as ExPEC, and the detection rate (7.7%, 6/78) of ExPEC among white-feather broilers was significantly higher than that (1.6%, 6/371) from brown-egg layers and that (2.1%, 10/477) from yellow-feather broilers (p < 0.05). PFGE and MLST analysis indicated that clonal dissemination of these ExPEC isolates was unlikely. Serogroup O78 was the most predominant type among the six serogroups identified in this study, and all the six serogroups had been frequently reported in human ExPEC isolates in many countries. All the 22 ExPEC isolates were multidrug-resistant (MDR) and the resistance rates to ampicillin (100%) and sulfamethoxazole-trimethoprim (100%) were the highest, followed by tetracycline (95.5%) and doxycycline (90.9%). blaCTX-M was found in 15 of the 22 ExPEC isolates including 10 harboring additional fosfomycin resistance gene fosA3. Notably, plasmid-borne colistin resistance gene mcr-1 was identified in six ExPEC isolates in this study. Worryingly, two ExPEC isolates were found to carry both mcr-1 and blaNDM, compromising both the efficacies of carbapenems and colistin. The presence of ExPEC isolates in healthy chickens, especially those carrying mcr-1 and/or blaNDM, is alarming and will pose a threat to the health of consumers. To our knowledge, this is the first report of mcr-1-positive ExPEC isolates harboring blaNDM from healthy chickens.

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.

Phylogenetic group determination and plasmid virulence gene profiles of colistin-resistant Escherichia coli originated from the broiler meat supply chain in Bogor, Indonesia.

Background and Aim:Pathogenic Escherichia coli contamination along the broiler meat supply chain is a serious public health concern. This bacterial infection with multidrug-resistant can lead to treatment failure. Several studies have revealed that avian pathogenic E. coli (APEC) and human extraintestinal pathogenic E. coli (ExPEC) showed a close genetic relationship and may share virulence genes. This study aimed to determine the phylogenetic group and virulence gene profiles in colistin-resistant E. coli obtained from the broiler meat supply chain in Bogor, West Java, Indonesia.Materials and Methods:Fifty-eight archive isolates originated from the cloacal swab, litter, drinking water, inside plucker swab, fresh meat at small scale poultry slaughterhouses, and traditional markets were used in this study. All the isolates were characterized by a polymerase chain reaction to determine the phylogenetic group (A, B1, B2, or D) and virulence gene profiles with APEC marker genes (iutA, hlyF, iss, iroN, and ompT).Results:Phylogenetic grouping revealed that the isolates belong to A group (34.48%), D group (34.48%), B1 group (17.24%), and B2 group (13.79%). The virulence gene prevalence was as follows: iutA (36%), hlyF (21%), ompT (21%), iroN (10%), and iss (9%). The B2 group presented with more virulence genes combinations. iroN, hlyF, and ompT genes were positively associated with the B2 group (p≤0.05).Conclusion:Our results highlight the role of colistin-resistant E. coli originated from the broiler meat supply chain as a potential reservoir for human ExPEC virulence genes.

Prevalence of antimicrobial resistance and potential pathogenicity, and possible spread of third generation cephalosporin resistance, in Escherichia coli isolated from healthy chicken farms in the region of Dakar, Senegal.

Escherichia coli is a normal inhabitant of the intestinal microbiota of chickens, a small proportion of which may be avian pathogenic E. coli (APEC) or potential extraintestinal pathogenic E. coli (ExPEC), capable of causing disease in humans. These E. coli may also be resistant to antimicrobials of critical importance in human or veterinary health. This study aims to 1) determine the prevalence of antimicrobial resistance (AMR) and resistance genes, multidrug resistance (MDR), chromosomal mechanisms of quinolone-resistance and virulence profiles of E. coli isolated from healthy chicken farms in the region of Dakar, Senegal, 2) investigate the spread of third-generation cephalosporins (3GC) resistance in E. coli isolated from healthy chicken farms with respect to virulence and resistance genes, serogroups, Pulsed-Field Gel Electrophoresis (PFGE), phylogenetic groups, plasmid types and transferability and 3) determine whether nonsusceptibility against 3GC on farms could be linked to risk factors. More than 68% of isolates from environmental faecal and drinking water samples, carcasses and carcass washes collected on 32 healthy chicken farms were multidrug resistant (MDR), resistance to antimicrobials critical in human health (3GC or ciprofloxacin) being found in all types of samples. Ciprofloxacin resistance was due to mutations in the gyrA and parC genes, 95% of tested farms harboring isolates carrying three mutations, in gyrA (Ser83Ile and Asp87Asn) and parC (Ser80Ile). Nine of the 32 farms (28.1%) demonstrated the presence of one or more 3GC-nonsusceptible indicator isolates but none of the potential risk factors were significantly associated with this presence on farms. Following ceftriaxone enrichment, presumptive extended-spectrum beta-lactamase/AmpC-beta-lactamase (ESBL/AmpC)-producer isolates were found in 17 of the 32 farms. 3GC resistance was mediated by blaCMY-2 or blaCTX-M genes, blaCTX-M being of genotypes blaCTX-M-1, blaCTX-M-8 and for the first time in chickens in Senegal, the genotype blaCTX-M-15. Clonally related ESBL/AmpC-producer isolates were found on different farms. In addition, blaCTX-M genes were identified on replicon plasmids I1 and K/B and blaCMY-2 on K/B, I1 and B/O. These plasmids were found in isolates of different clusters. In addition, 18 isolates, some of which were ESBL/AmpC-producers, were defined as potential human ExPEC. In conclusion, E. coli isolates potentially pathogenic for humans and demonstrating MDR, with resistance expressed against antimicrobials of critical importance in human health were found in healthy chickens in Senegal. Our results suggest that both clonal spreading and horizontal gene transfer play a role in the spread of 3GC-resistance and that chickens in Senegal could be a reservoir for AMR and ExPEC for humans. These results highlight the importance of raising awareness about compliance with biosecurity measures and prudent use of antimicrobials.

Multi-drug resistant Escherichia coli in diarrhoeagenic foals: Pulsotyping, phylotyping, serotyping, antibiotic resistance and virulence profiling

Extraintestinal pathogenic E. coli (ExPEC) possess the ability to cause extraintestinal infections such as urinary tract infections, neonatal meningitis and sepsis. While information is readily available describing pathogenic E. coli populations in food-producing animals, studies in companion/sports animals such as horses are limited. In addition, many antimicrobial agents used in the treatment of equine infections are also utilised in human medicine, potentially contributing to the spread of antibiotic resistance determinants among pathogenic strains. The aim of this study was to phenotypically and genotypically characterise the multidrug resistance and virulence associated with 83 equine E. coli isolates recovered from foals with diarrhoeal disease. Serotyping was performed by both PCR and sequencing. Antibiotic resistance was assessed by disc diffusion. Phylogenetic groups, virulence genes, antibiotic resistance genes and integrons were determined by PCR. Thirty-nine (46%) of the isolates were classified as ExPEC and hence considered to be potentially pathogenic to humans and animals. Identified serogroups O1, O19a, O40, O101 and O153 are among previously reported human clinical ExPEC isolates. Over a quarter of the E. coli were assigned to pathogenic phylogroups B2 (6%) and D (23%). Class 1 and class 2 integrons were detected in 85% of E. coli, revealing their potential to transfer MDR to other pathogenic and non-pathogenic bacteria. With 65% of potentially pathogenic isolates harbouring one or more TEM, SHV and CTX-M-2 group β-lactamases, in addition to the high levels of resistance to fluoroquinolones observed, our findings signal the need for increased attention to companion/sport animal reservoirs as public health threats.

Evaluation of Escherichia coli isolates from healthy chickens to determine their potential risk to poultry and human health.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains are important pathogens that cause diverse diseases in humans and poultry. Some E. coli isolates from chicken feces contain ExPEC-associated virulence genes, so appear potentially pathogenic; they conceivably could be transmitted to humans through handling and/or consumption of contaminated meat. However, the actual extraintestinal virulence potential of chicken-source fecal E. coli is poorly understood. Here, we assessed whether fecal E. coli isolates from healthy production chickens could cause diseases in a chicken model of avian colibacillosis and three rodent models of ExPEC-associated human infections. From 304 E. coli isolates from chicken fecal samples, 175 E. coli isolates were screened by PCR for virulence genes associated with human-source ExPEC or avian pathogenic E. coli (APEC), an ExPEC subset that causes extraintestinal infections in poultry. Selected isolates genetically identified as ExPEC and non-ExPEC isolates were assessed in vitro for virulence-associated phenotypes, and in vivo for disease-causing ability in animal models of colibacillosis, sepsis, meningitis, and urinary tract infection. Among the study isolates, 13% (40/304) were identified as ExPEC; the majority of these were classified as APEC and uropathogenic E. coli, but none as neonatal meningitis E. coli. Multiple chicken-source fecal ExPEC isolates resembled avian and human clinical ExPEC isolates in causing one or more ExPEC-associated illnesses in experimental animal infection models. Additionally, some isolates that were classified as non-ExPEC were able to cause ExPEC-associated illnesses in animal models, and thus future studies are needed to elucidate their mechanisms of virulence. These findings show that E. coli isolates from chicken feces contain ExPEC-associated genes, exhibit ExPEC-associated in vitro phenotypes, and can cause ExPEC-associated infections in animal models, and thus may pose a health threat to poultry and consumers.

Pandemic extra-intestinal pathogenic Escherichia coli (ExPEC) clonal group O6-B2-ST73 as a cause of avian colibacillosis in Brazil.

Extra-intestinal pathogenic Escherichia coli (ExPEC) represent an emerging pathogen, with pandemic strains increasingly involved in cases of urinary tract infections (UTIs), bacteremia, and meningitis. In addition to affecting humans, the avian pathotype of ExPEC, avian pathogenic E. coli (APEC), causes severe economic losses to the poultry industry. Several studies have revealed overlapping characteristics between APEC and human ExPEC, leading to the hypothesis of a zoonotic potential of poultry strains. However, the description of certain important pandemic clones, such as Sequence Type 73 (ST73), has not been reported in food sources. We characterized 27 temporally matched APEC strains from diverse poultry farms in Brazil belonging to the O6 serogroup because this serogroup is frequently described as a causal factor in UTI and septicemia in humans in Brazil and worldwide. The isolates were genotypically characterized by identifying ExPEC virulence factors, phylogenetically tested by phylogrouping and multilocus sequence type (MLST) analysis, and compared to determine their similarity employing the pulsed field gel electrophoresis (PFGE) technique. The strains harbored a large number of virulence determinants that are commonly described in uropathogenic E. coli (UPEC) and sepsis associated E. coli (SEPEC) strains and, to a lesser extent in neonatal meningitis associated E. coli (NMEC), such as pap (85%), sfa (100%), usp (100%), cnf1 (22%), kpsMTII (66%), hlyA (52%), and ibeA (4%). These isolates also yielded a low prevalence of some genes that are frequently described in APEC, such as iss (37%), tsh, ompT, and hlyF (8% each), and cvi/cva (0%). All strains were classified as part of the B2 phylogroup and sequence type 73 (ST73), with a cluster of 25 strains showing a clonal profile by PFGE. These results further suggest the zoonotic potential of some APEC clonal lineages and their possible role in the epidemiology of human ExPEC, in addition to providing the first description of the O6-B2-ST73 clonal group in poultry.