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Colibacillosis, caused by avian pathogenic Escherichia coli (APEC), is an important infectious disease in chickens and a major cause of mortality in young chicks. Therefore, protecting young chickens from colibacillosis is important for improving welfare and productivity in the poultry industry. Recently, we developed a novel enterobactin (Ent) conjugate vaccine that could induce high titers of anti-Ent immunoglobulin Y (IgY) in chicken serum and consequently mitigate the organ lesions caused by APEC infection. Considering that maternal immunization is a practical approach to confer instant immune protection to the hatchlings, in this study, we immunized breeder hens with the Ent conjugate vaccine and evaluated the maternal immune protection on the progenies challenged with APEC. Three doses of the vaccine induced high titers of anti-Ent IgY in the hens (about 16- and 64-fold higher than the control group in the sera and egg yolks, respectively), resulting in an eight-fold of increase in anti-Ent IgY in the sera of progenies. However, the anti-Ent maternal immunity did not display significant protection against APEC challenge in the young chicks as there was no significant difference in APEC load (in liver, lung, and spleen) or organ lesions (in heart, liver, spleen, lung, and air sac) between the vaccinated and control groups. In future studies, the APEC infection model needs to be optimized to exhibit proper pathogenicity of APEC, and the maternal immunization regimen can be further improved to boost the maternally derived anti-Ent IgY in the hatchlings.

Colibacillosis is one of the most common and economically devastating infectious diseases in poultry production worldwide. Innovative universal vaccines are urgently needed to protect chickens from the infections caused by genetically diverse avian pathogenic Escherichia coli (APEC). Enterobactin (Ent) is a highly conserved siderophore required for E. coli iron acquisition and pathogenesis. The Ent-specific antibodies induced by a novel Ent conjugate vaccine significantly inhibited the in vitro growth of diverse APEC strains. In this study, White Leghorn chickens were immunized with the Ent conjugate vaccine using a crossed design with two variables, vaccination (with or without) and APEC challenge (O1, O78, or PBS control), resulting in six study groups (9 to 10 birds/group). The chickens were subcutaneously injected with the vaccine (100 μg per bird) at 7 days of age, followed by booster immunization at 21 days of age. The chickens were intratracheally challenged with an APEC strain (10 8 CFU/bird) or PBS at 28 days of age. At 5 days post infection, all chickens were euthanized to examine lesions and APEC colonization of the major organs. Immunization of chickens with the Ent vaccine elicited a strong immune response with a 64-fold increase in the level of Ent-specific IgY in serum. The hypervirulent strain O78 caused extensive lesions in lung, air sac, heart, liver, and spleen with significantly reduced lesion scores observed in the vaccinated chickens. Interestingly, the vaccination did not significantly reduce APEC levels in the examined organs. The APEC O1 with low virulence only caused sporadic lesions in the organs in both vaccination and control groups. The Ent conjugate vaccine altered the bacterial community of the ileum and cecum. Taken together, the findings from this study showed the Ent conjugate vaccine could trigger a strong specific immune response and was promising to confer protection against APEC infection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Avian pathogenic Escherichia coli (APEC) is the etiological agent of avian colibacillosis, a leading cause of economic loss to the poultry industry worldwide. APEC causes disease using a diverse repertoire of virulence factors and has the ability to form biofilms, which contributes to the survival and persistence of APEC in harsh environments. The objective of this study was to identify genes most widespread and important in APEC that contribute to APEC biofilm formation. Using the characterized APEC O18 as the template strain, a total of 15,660 mutants were randomly generated using signature tagged mutagenesis and evaluated for decreased biofilm formation ability using the crystal violet assay. Biofilm deficient mutants were sequenced, and a total of 547 putative biofilm formation genes were identified. Thirty of these genes were analyzed by PCR for prevalence among 109 APEC isolates and 104 avian fecal E. coli (AFEC) isolates, resulting in nine genes with significantly greater prevalence in APEC than AFEC. The expression of these genes was evaluated in the wild-type APEC O18 strain using quantitative real-time PCR (qPCR) in both the exponential growth phase and the mature biofilm phase. To investigate the role of these genes in biofilm formation, isogenic mutants were constructed and evaluated for their biofilm production and planktonic growth abilities. Four of the mutants (rfaY, rfaI, and two uncharacterized genes) displayed significantly decreased biofilm formation, and of those four, one (rfaI) displayed significantly decreased growth compared to the wild type. Overall, this study identified novel genes that may be important in APEC and its biofilm formation. The data generated from this study will benefit further investigation into the mechanisms of APEC biofilm formation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Young et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Avian pathogenic E. coli is the causative agent of extra-intestinal infections in birds known as colibacillosis, which can manifest as localized or systemic infections. The disease affects all stages of poultry production, resulting in economic losses that occur due to morbidity, carcass condemnation and increased mortality of the birds. APEC strains have a diverse virulence trait repertoire, which includes virulence factors involved in adherence to and invasion of the host cells, serum resistance factors, and toxins. However, the pathogenesis of APEC infections remains to be fully elucidated. The Type 6 secretion (T6SS) system has recently gained attention due to its role in the infection process and protection of bacteria from host defenses in human and animal pathogens. Previous work has shown that T6SS components are involved in the adherence to and invasion of host cells, as well as in the formation of biofilm, and intramacrophage bacterial replication. Here, we analyzed the frequency of T6SS genes hcp , impK , evpB , vasK and icmF in a collection of APEC strains and their potential role in virulence-associated phenotypes of APECO18. The T6SS genes were found to be significantly more prevalent in APEC than in fecal E. coli isolates from healthy birds. Expression of T6SS genes was analyzed in culture media and upon contact with host cells. Mutants were generated for hcp , impK , evpB , and icmF and characterized for their impact on virulence-associated phenotypes, including adherence to and invasion of host model cells, and resistance to predation by Dictyostelium discoideum. Deletion of the aforementioned genes did not significantly affect adherence and invasion capabilities of APECO18. Deletion of hcp reduced resistance of APECO18 to predation by D. discoideum , suggesting that T6SS is involved in the virulence of APECO18., Competing Interests: The authors declare there are no competing interests., (©2021 de Oliveira et al.)

Neonatal meningitis Escherichia coli (NMEC) is the second leading cause of sepsis and meningitis in neonates worldwide. Here, we report the genome sequence of NMEC15, belonging to serotype O18:K1, isolated from the cerebrospinal fluid (CSF) of an infant with neonatal bacterial meningitis (NBM) in the Netherlands.

Colisitin-associated resistance in bacteria of food producing animals has gained significant attention with the mcr gene being linked with resistance. Recently, newer variants of mcr have emerged with more than nine variants currently recognized. Reports of mcr associated resistance in Escherichia coli of poultry appear to be relatively limited, but its prevalence requires assessment since poultry is one of the most important and cheapest sources of the world's protein and the emergence of resistance could limit our ability to treat disease outbreaks. Here, 107 E. coli isolates from production poultry were screened for the presence of mcr 1-9 . The isolates were collected between April 2015 and June 2016 from broiler chickens and free-range layer hens in Rio de Janeiro, Brazil. All isolates were recovered from the trachea and cloaca of healthy birds and an additional two isolates were recovered from sick birds diagnosed with colibacillosis. All isolates were screened for the presence of mcr-1 to 9 using PCR and Sanger sequencing for confirmation of positive genes. Additionally, pulse field gel electrophoresis (PFGE) analysis, avian fecal E. coli (APEC) virulence associated gene screening, plasmid replicon typing and antimicrobial resistance phenotype and resistance gene screening, were also carried out to further characterize these isolates. The mcr-1 gene was detected in 62 (57.9%) isolates (61 healthy and 1 APEC) and the mcr-5 gene was detected in 3 (2.8%) isolates; mcr-2, mcr-3, mcr-4, mcr-6, mcr-7, mcr-8 , and mcr-9 were not detected in any isolate. In addition, mcr 1 and 5 positive isolates were phenotypically resistant to colistin using the agar dilution assay (> 8ug/ml). PFGE analysis found that most of the isolates screened had unique fingerprints suggesting that the emergence of colistin resistance was not the result of clonal dissemination. Plasmid replicon types IncI2, FIB , and B/O were found in 38, 36, and 34% of the mcr positive isolates and were the most prevalent replicon types detected; tetA and tetB (32 and 26%, respectively) were the most prevalent antimicrobial resistance genes detected and iutA , was the most prevalent APEC virulence associated gene, detected in 50% of the isolates. Approximately 32% of the isolates examined could be classified as APEC-like, based on the presence of 3 or more genes of APEC virulence associated path panel ( iroN, ompT, hlyF, iss, iutA ). This study has identified a high prevalence of mcr-1 in poultry isolates in Brazil, suggesting that animal husbandry practices could result in a potential source of resistance to the human food chain in countries where application of colistin in animal health is practiced. Emergence of the mcr gene and associated colisitin resistance in production poultry warrants continued monitoring from the animal health and human health perspective., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Barbieri, Pimenta, de Melo, Nolan, de Souza and Logue.)

Passive immunization with specific egg yolk antibodies (immunoglobulin Y, IgY) is emerging as a promising alternative to antibiotics to control bacterial infections. Recently, we developed a novel conjugate vaccine that could trigger a strong immune response in rabbits directed against enterobactin (Ent), a highly conserved siderophore molecule utilized by different Gram-negative pathogens. However, induction of Ent-specific antibodies appeared to be affected by the choice of animal host and vaccination regimen. It is still unknown if the Ent conjugate vaccine can trigger a specific immune response in layers for the purpose of production of anti-Ent egg yolk IgY. In this study, three chicken vaccination trials with different regimens were performed to determine conditions for efficient production of anti-Ent egg yolk IgY. Purified Ent was conjugated to three carrier proteins, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) and CmeC (a subunit vaccine candidate), respectively. Intramuscular immunization of Barred Rock layers with KLH-Ent conjugate four times induced strong immune response against whole conjugate vaccine but the titer of Ent-specific IgY did not change in yolk with only a 4 fold increase detected in serum. In the second trial, three different Ent conjugate vaccines were evaluated in Rhode Island Red pullets with four subcutaneous injections. The KLH-Ent or CmeC-Ent conjugate consistently induced high level of Ent-specific IgY in both serum (up to 2,048 fold) and yolk (up to 1,024 fold) in each individual chicken. However, the Ent-specific immune response was only temporarily and moderately induced using a BSA-Ent vaccination. In the third trial, ten White Leghorn layers were subcutaneously immunized three times with KLH-Ent, leading to consistent and strong immune response against both whole conjugate and the Ent molecule in each chicken; the mean titer of Ent-specific IgY increased approximately 32 and 256 fold in serum and yolk, respectively. Consistent with its potent binding to various Ent derivatives, the Ent-specific egg yolk IgY also inhibited in vitro growth of a representative Escherichia coli strain. Together, this study demonstrated that the novel Ent conjugate vaccine could induce strong, specific, and robust immune response in chickens. The Ent-specific hyperimmune egg yolk IgY has potential for passive immune intervention against Gram-negative infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zeng, Wang, Huang, Logue, Barbieri, Nolan and Lin.)

Colibacillosis caused by avian pathogenic Escherichia coli (APEC) is a devastating disease of poultry that results in multi-million-dollar losses annually to the poultry industry. Disease syndromes associated with APEC includes colisepticemia, cellulitis, air sac disease, peritonitis, salpingitis, omphalitis, and osteomyelitis among others. A total of 61 APEC isolates collected during the Fall of 2018 (Aug-Dec) from submitted diagnostic cases of poultry diagnosed with colibacillosis were assessed for the presence of 44 virulence-associated genes, 24 antimicrobial resistance genes and 17 plasmid replicon types. Each isolate was also screened for its ability to form biofilm using the crystal violet assay and antimicrobial susceptibility to 14 antimicrobials using the NARMS panel. Overall, the prevalence of virulence genes ranged from 1.6% to >90% with almost all strains harboring genes that are associated with the ColV plasmid-the defining trait of the APEC pathotype. Overall, 58 strains were able to form biofilms and only three strains formed negligible biofilms. Forty isolates displayed resistance to antimicrobials of the NARMS panel ranging from one to nine agents. This study highlights that current APEC causing disease in poultry possess virulence and resistance traits and form biofilms which could potentially lead to challenges in colibacillosis control., Competing Interests: The authors declare that they have no competing interests., (© 2021 Newman et al.)

Most Escherichia coli (E. coli) strains do not cause disease, naturally living in the lower intestine and is expelled into the environment within faecal matter. Escherichia coli can utilize citrate under anaerobic conditions but not aerobic conditions. However, the underlying regulatory mechanisms are poorly understood. In this study, we explored regulatory mechanisms of citrate fermentation genes by global regulators ArcA and Fnr under anaerobic conditions. A gel mobility shift assay showed that the regulator proteins ArcA and Fnr binded to the promoter region localized between the citAB and citCDEFXGT operons. Subsequent assays confirmed that ArcA indirectly controled the expression of citrate fermentation genes via regulating CitA-CitB system, while Fnr directly regulated but also indirectly modulated citrate fermentation genes via controling CitA-CitB system. Deletions of arcA and fnr significantly reduced the growth of Escherichia coli in M9 medium with a citrate carbon source. We conclude that both ArcA and Fnr can indirectly control the citrate utilization via CitA-CitB system, while Fnr can also directly regulate the expression of citrate fermentation genes in E. coli under anaerobic conditions., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Turkey cellulitis, also known as clostridial dermatitis is a significant cause of morbidity, mortality, and carcass condemnation at slaughter resulting in considerable losses for turkey producers. Here, we assessed the potential role of Avian Pathogenic Escherichia coli (APEC) in a cellulitis outbreak on a turkey farm in Iowa. Birds from one farm with a history of cellulitis and one farm with no history of disease (for comparison) were followed from the age of 10 weeks (before the outbreak) to 18 weeks (just prior to slaughter). E. coli recovered from the litter, from skin lesions of birds with cellulitis, and from systemic lesions of birds submitted for necropsy, were assessed. A total of 333 isolates were analyzed and screened for virulence-associated genes, antimicrobial resistance genes including heavy metal resistance, adhesins, invasins, and protectins, iron acquisition systems and their phylogenetic group through multiplex PCR. In addition, PCR was used to serogroup the isolates, and pulsed field gel electrophoresis (PFGE) was used to analyze a subset of strains from the farm environment (litter) and birds at 17 and 18 weeks of age when the cellulitis infection appeared to peak. Overall, E. coli isolates recovered from cellulitis lesions and systemic infection were identified as APEC, while a lower prevalence of E. coli recovered from the litter met the criteria of APEC-like. Direct comparison of E. coli isolates from the litter, lesions, and systemic strains using PFGE failed to find identical clones across all three sources reflecting the diversity of strains present in the poultry environment causing disease. This study highlights the role of APEC in turkey cellulitis and should not be overlooked as a significant contributor to the disease in turkeys., (Copyright © 2020 de Oliveira, Newman, Sato, Noel, Rauk, Nolan, Barbieri and Logue.)

Objective: Extraintestinal Pathogenic E. coli (ExPEC), are responsible for host diseases such as Neonatal Meningitis Escherichia coli (NMEC), the second-leading cause of neonatal bacterial meningitis, Avian Pathogenic E. coli (APEC), a cause of extraintestinal disease in poultry, and Uropathogenic E. coli (UPEC), the most common cause of urinary tract infections. Virulence factors associated with NMEC include outer membrane protein A (OmpA) and type I fimbriae (FimH), which also occur in APEC and UPEC. OmpA contributes to NMEC's ability to cross the blood-brain barrier, persist in the bloodstream and has been identified as a potential vaccine target for ExPEC, however the protein has amino acid variants, which may influence virulence of strains or alter vaccine efficacy. Although OmpA is present in virtually all E. coli, differences in its amino acid residues have yet to be surveyed in ExPEC., Results: Here the ompA gene (n = 399) from ExPEC collections were sequenced and translated in silico. Twenty-five different OmpA polymorphism patterns were identified. Seven polymorphism patterns were significantly associated with an ExPEC subpathotype, but chromosomal history most likely accounts for most differences found. The differences in OmpA protein sequences suggest that OmpA may influence variation in virulence and host specificity within ExPEC subpathotypes.

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., (Copyright © 2019 American Society for Microbiology.)

Avian-pathogenic Escherichia coli (APEC) is a subgroup of extraintestinal pathogenic E. coli (ExPEC) presumed to be zoonotic and to represent an external reservoir for extraintestinal infections in humans, including uropathogenic E. coli (UPEC) causing urinary tract infections. Comparative genomics has previously been applied to investigate whether APEC and human ExPEC are distinct entities. Even so, whole-genome-based studies are limited, and large-scale comparisons focused on single sequence types (STs) are not available yet. In this study, comparative genomic analysis was performed on 323 APEC and human ExPEC genomes belonging to sequence type 95 (ST95) to investigate whether APEC and human ExPEC are distinct entities. Our study showed that APEC of ST95 did not constitute a unique ExPEC branch and was genetically diverse. A large genetic overlap between APEC and certain human ExPEC was observed, with APEC located on multiple branches together with closely related human ExPEC, including nearly identical APEC and human ExPEC. These results illustrate that certain ExPEC clones may indeed have the potential to cause infection in both poultry and humans. Previously described ExPEC-associated genes were found to be encoded on ColV plasmids. These virulence-associated plasmids seem to be crucial for ExPEC strains to cause avian colibacillosis and are strongly associated with strains of the mixed APEC/human ExPEC clusters. The phylogenetic analysis revealed two distinct branches consisting of exclusively closely related human ExPEC which did not carry the virulence-associated plasmids, emphasizing a lower avian virulence potential of human ExPEC in relation to an avian host. IMPORTANCE APEC causes a range of infections in poultry, collectively called colibacillosis, and is the leading cause of mortality and is associated with major economic significance in the poultry industry. A growing number of studies have suggested APEC as an external reservoir of human ExPEC, including UPEC, which is a reservoir. ExPEC belonging to ST95 is considered one of the most important pathogens in both poultry and humans. This study is the first in-depth whole-genome-based comparison of ST95 E. coli which investigates both the core genomes as well as the accessory genomes of avian and human ExPEC. We demonstrated that multiple lineages of ExPEC belonging to ST95 exist, of which the majority may cause infection in humans, while only part of the ST95 cluster seem to be avian pathogenic. These findings further support the idea that urinary tract infections may be a zoonotic infection., (Copyright © 2019 Jørgensen et al.)

Extraintestinal pathogenic Escherichia coli (ExPEC) is the leading cause of bloodstream and other extraintestinal infections in human and animals. The greatest challenge encountered by ExPEC during an infection is posed by the host defense mechanisms, including lysozyme. ExPEC have developed diverse strategies to overcome this challenge. The aim of this study was to characterize the molecular mechanism of ExPEC resistance to lysozyme. For this, 15,000 transposon mutants of a lysozyme-resistant ExPEC strain NMEC38 were screened; 20 genes were identified as involved in ExPEC resistance to lysozyme-of which five were located in the gene cluster between galF and gnd, and were further confirmed to be involved in O-specific polysaccharide biosynthesis. The O-specific polysaccharide was able to inhibit the hydrolytic activity of lysozyme; it was also required by the complete lipopolysaccharide (LPS)-mediated protection of ExPEC against the bactericidal activity of lysozyme. The O-specific polysaccharide was further shown to be able to directly interact with lysozyme. Furthermore, LPS from ExPEC strains of different O serotypes was also able to inhibit the hydrolytic activity of lysozyme. Because of their cell surface localization and wide distribution in Gram-negative bacteria, O-specific polysaccharides appear to play a long-overlooked role in protecting bacteria against exogenous lysozyme.

Extraintestinal pathogenic Escherichia coli (ExPEC) is one of the leading causes of bloodstream infections. Characteristically, these organisms exhibit strong resistance to the bactericidal action of host serum. Although numerous serum resistance factors in ExPEC have been identified, their regulatory mechanisms during in vivo infection remain largely unknown. Here, RNA sequencing analyses together with quantitative reverse-transcription PCR revealed that ExPEC genes involved in the biosynthesis of extracytoplasmic polysaccharides (ECPs) including K-capsule, lipopolysaccharide (LPS), colanic acid, peptidoglycan and Yjb exopolysaccharides were significantly upregulated in response to serum under low oxygen conditions and during bloodstream infection. The oxygen sensor FNR directly activated the expression of K-capsule and colanic acid and also indirectly modulated the expression of colanic acid, Yjb exopolysaccharides and peptidoglycan via the known Rcs regulatory system. The global regulator Fur directly or indirectly repressed the expression ofECP biosynthesis genes in iron replete media, whereas the low iron conditions in the bloodstream could relieve Fur repression. Using in vitro and animal models, FNR, Fur and the Rcs system were confirmed as contributing to ExPEC ECP production, serum resistance and virulence. Altogether, these findings indicated that the global regulators FNR andFur and the signaling transduction system Rcs coordinately regulated the expression of ECP biosynthesis genes leading to increased ExPEC serum resistance in response to low oxygen and low iron levels in the bloodstream., (© 2018 John Wiley & Sons Ltd.)

Avian pathogenic Escherichia coli (APEC) is the causative agent of colibacillosis, a disease that affects poultry production worldwide and leads to multimillion-dollar losses annually. Here, we report the genome sequence of APEC O2-211, a sequence type 117 (ST117) strain isolated from a diseased chicken.

Neonatal meningitis Escherichia coli (NMEC) is the second leading cause of neonatal bacterial meningitis worldwide. We report the genome sequence of the multidrug-resistant NMEC serotype O75:H5:K1 strain mcjchv-1, which resulted in an infant's death. The O75 serogroup is rare among NMEC isolates; therefore, this strain is considered an emergent pathogen.

Extraintestinal pathogenic Escherichia coli (ExPEC) include avian pathogenic E. coli (APEC), neonatal meningitis E. coli (NMEC), and uropathogenic E. coli (UPEC) and are responsible for significant animal and human morbidity and mortality. This study sought to investigate if biofilm formation by ExPEC likely contributes to these losses since biofilms are associated with recurrent urinary tract infections, antibiotic resistance, and bacterial exchange of genetic material. Therefore, the goal of this study was to examine differences in biofilm formation among a collection of ExPEC and to ascertain if there is a relationship between their ability to produce biofilms and their assignment to phylogenetic groups in three media types - M63, diluted TSB, and BHI. Our results suggest that ExPEC produce relatively different levels of biofilm formation in the media tested as APEC (70.4%, p = 0.0064) and NMEC (84.4%, p = 0.0093) isolates were poor biofilm formers in minimal medium M63 while UPEC isolates produced significantly higher ODs under nutrient-limited conditions with 25% of strains producing strong biofilms in diluted TSB ( p = 0.0204). Additionally, E. coli phylogenetic assignment using Clermont's original and revised typing scheme demonstrated significant differences among the phylogenetic groups in the different media. When the original phylogenetic group isolates previously typed as group D were phylogenetically typed under the revised scheme and examined, they showed substantial variation in their ability to form biofilms, which may explain the significant values of revised phylogenetic groups E and F in M63 ( p = 0.0291, p = 0.0024). Our data indicates that biofilm formation is correlated with phylogenetic classification and subpathotype or commensal grouping of E. coli strains.

Avian pathogenic Escherichia coli (APEC) is the etiologic agent of colibacillosis, an important cause of morbidity and mortality in poultry. Though, many virulence factors associated with APEC pathogenicity are known, their regulation remains unclear. FNR (fumarate and nitrate reduction) is a well-known global regulator that works as an oxygen sensor and has previously been described as a virulence regulator in bacterial pathogens. The goal of this study was to examine the role of FNR in the regulation of APEC virulence factors, such as Type I fimbriae, and processes such as adherence and invasion, type VI secretion, survival during oxidative stress, and growth in iron-restricted environments. To accomplish this goal, APEC O1, a well-characterized, highly virulent, and fully sequenced strain of APEC harboring multiple virulence mechanisms, some of which are plasmid-linked, was compared to its FNR mutant for expression of various virulence traits. Deletion of FNR was found to affect APEC O1's adherence, invasion and expression of ompT , a plasmid-encoded outer membrane protein, type I fimbriae, and aatA , encoding an autotransporter. Indeed, the fnr - mutant showed an 8-fold reduction in expression of type I fimbriae and a highly significant ( P < 0.0001) reduction in expression of fimA, ompT (plasmid-borne), and aatA . FNR was also found to regulate expression of the type VI secretion system, affecting the expression of vgrG . Further, FNR was found to be important to APEC O1's growth in iron-deficient media and survival during oxidative stress with the mutant showing a 4-fold decrease in tolerance to oxidative stress, as compared to the wild type. Thus, our results suggest that FNR functions as an important regulator of APEC virulence.

Antimicrobial resistance associated with colistin has emerged as a significant concern worldwide threatening the use of one of the most important antimicrobials for treating human disease. Here, we examined a collection (n = 980) of Avian Pathogenic Escherichia coli (APEC) isolated from poultry with colibacillosis from the US and internationally for the presence of mcr-1 and mcr-2, genes known to encode colistin resistance. Included in the analysis was an additional set of avian fecal E. coli (AFEC) (n = 220) isolates from healthy birds for comparative analysis. The mcr-1 gene was detected in a total of 12 isolates recovered from diseased production birds from China and Egypt. No mcr genes were detected in the healthy fecal isolates. The full mcr-1 gene from positive isolates was sequenced using specifically designed primers and were compared with sequences currently described in NCBI. mcr-1 positive isolates were also assessed for phenotypic colistin resistance and extended spectrum beta lactam phenotypes and genotypes. This study has identified mcr-1 in APEC isolates dating back to at least 2010 and suggests that animal husbandry practices could result in a potential source of resistance to the human food chain in countries where application of colistin in animal health is practiced.

The Clermont scheme has been used for subtyping of Escherichia coli since it was initially described in early 2000. Since then, researchers have used the scheme to type and sub-type commensal E. coli and pathogenic E. coli , such as extraintestinal pathogenic E. coli (ExPEC), and compare their phylogenetic assignment by pathogenicity, serogroup, distribution among ExPEC of different host species and complement of virulence and resistance traits. Here, we compare assignments of human and avian ExPEC and commensal E. coli using the old and revised Clermont schemes to determine if the new scheme provides a refined snapshot of isolate classification. 1,996 E. coli from human hosts and poultry, including 84 human neonatal meningitis E. coli isolates, 88 human vaginal E. coli , 696 human uropathogenic E. coli , 197 healthy human fecal E. coli , 452 avian pathogenic E. coli (APEC), 200 retail poultry E. coli , 80 crop and gizzard E. coli from healthy poultry at slaughter and 199 fecal E. coli from healthy birds at slaughter. All isolates were subject to phylogenetic analysis using the Clermont et al. (2000, 2013) schemes and compared to determine the effect of the new classification on strain designation. Most of the isolates' strain designation remained where they were originally assigned. Greatest designation change occurred in APEC where 53.8% of isolates were reclassified; while classification rates among human strains ranged from 8 to 14%. However, some significant changes were observed for UPEC associated strains with significant ( P < 0.05) designation changes observed from A to C and D to E or F phylogenetic types; a similar designation change was noted among NMEC for D to F designation change. Among the APEC significant designation changes were observed from A to C and D to E and F. These studies suggest that the new scheme provides a tighter and more meaningful definition of some ExPEC; while the new typing scheme has a significant impact on APEC classification. A comparison of phylogenetic group assignment by content of virulence, resistance, replicon and pathogenicity island genes in APEC suggests that insertion of pathogenicity islands into the genome appears to correlate closely with revised phylogenetic assignment.

Avian pathogenic Escherichia coli (APEC) causes one of the most common bacterial diseases of poultry worldwide. Effective control methods are therefore desirable and will be facilitated by a better understanding of the host response to the pathogen. Currently, microRNAs (miRNAs) involved in host resistance to APEC are unknown. Here, we applied RNA sequencing to explore the changed miRNAs and deregulated genes in the spleen of three groups of broilers: nonchallenged (NC), APEC-challenged with mild pathology (CM), and APEC-challenged with severe pathology (CS). Twenty-seven differentially expressed miRNAs (fold change >1.5; P value <0.01) were identified, including 13 miRNAs between the NC and CM, 17 between the NC and CS, and 14 between the CM and CS groups. Through functional analysis of these miRNA targets, 12 immune-related biological processes were found to be significantly enriched. Based on combined analyses of differentially expressed miRNAs and mRNAs within each of the three groups, 43 miRNA-mRNA pairs displayed significantly negative correlations (r < -0.8). Notably, gga-miR-429 was greatly increased in the CS group compared to levels in both the CM and NC groups. In vitro, gga-miR-429 directly repressed luciferase reporter gene activity via binding to 3' untranslated regions of TMEFF2, NTRK2, and SHISA2. Overexpression of gga-miR-429 in the HD11 macrophage cell line significantly inhibited TMEFF2 and SHISA2 expression, which are involved in the lipopolysaccharide-induced platelet-derived growth factor (PDGF) and Wnt signaling pathways. In summary, we provide the first report characterizing the miRNA changes during APEC infection, which may help to shed light on the roles of these recently identified genetic elements in the mechanisms of host resistance and susceptibility to APEC., (Copyright © 2016 Jia et al.)

Avian pathogenic Escherichia coli (APEC) can cause significant morbidity in chickens. The thymus provides the essential environment for T cell development; however, the thymus transcriptome has not been examined for gene expression in response to APEC infection. An improved understanding of the host genomic response to APEC infection could inform future breeding programs for disease resistance and APEC control. We therefore analyzed the transcriptome of the thymus of birds challenged with APEC, contrasting susceptible and resistant phenotypes. Thousands of genes were differentially expressed in birds of the 5-day post infection (dpi) challenged-susceptible group vs. 5 dpi non-challenged, in 5 dpi challenged-susceptible vs. 5 dpi challenged-resistant birds, as well as in 5 dpi vs. one dpi challenged-susceptible birds. The Toll-like receptor signaling pathway was the major innate immune response for birds to respond to APEC infection. Moreover, lysosome and cell adhesion molecules pathways were common mechanisms for chicken response to APEC infection. The T-cell receptor signaling pathway, cell cycle, and p53 signaling pathways were significantly activated in resistant birds to resist APEC infection. These results provide a comprehensive assessment of global gene networks and biological functionalities of differentially expressed genes in the thymus under APEC infection. These findings provide novel insights into key molecular genetic mechanisms that differentiate host resistance from susceptibility in this primary lymphoid tissue, the thymus., (© The Author 2016. Published by Oxford University Press on behalf of Poultry Science Association.)

Avian-pathogenic Escherichia coli (APEC) is the causative agent of colibacillosis, a disease that affects all facets of poultry production worldwide, resulting in multimillion dollar losses annually. Here, we report the genome sequence of an APEC O18 sequence type 95 (ST95) strain associated with disease in a chicken., (Copyright © 2016 Nicholson et al.)

Neonatal meningitis Escherichia coli (NMEC) is a common agent of neonatal bacterial meningitis, causing high neonatal mortality and neurologic sequelae in its victims. Here, we present the complete genome sequence of NMEC O18 (also known as NMEC 58), a highly virulent (O18ac:K1, ST416) strain., (Copyright © 2016 Nicholson et al.)

Avian pathogenic Escherichia coli (APEC), an extraintestinal pathogenic E. coli (ExPEC), constitutes an animal health and a potential zoonotic risk. Most studies focus on the response of a single tissue to APEC infection. Understanding interactions among lymphoid tissues is of importance in controlling APEC infection. Therefore, we studied bone marrow, bursa, and thymus transcriptomes because of these tissues' crucial roles in development of pre-lymphocytes, B cells, and T cells, respectively. Using lesion scores of liver, pericardium, and air sacs, infected birds were classified as either resistant or susceptible. Little difference in gene expression was detected in resistant birds in bone marrow versus bursa or thymus, while there were large differences between tissues in susceptible birds. Phagosome, lysosome and cytokine interactions were strongly enhanced in thymus versus bone marrow in susceptible birds, and T cell receptor (TCR), cell cycle, and p53 signaling were significantly decreased. B cell receptor (BCR) was also significantly suppressed in bursa versus bone marrow in susceptible birds. This research provides novel insights into the complex developmental changes in gene expression occurring across the primary lymphoid organs and, therefore, serves as a foundation to understanding the cellular and molecular basis of host resistance to APEC infection., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Avian pathogenic Escherichia coli (APEC) is associated with colibacillosis in poultry. Here, we present the first complete sequence of an APEC strain of the O7:HNT serotype and ST73 sequence type, isolated from a broiler with cellulitis. Complete genomes of APEC with distinct genetic backgrounds may be useful for comparative analysis., (Copyright © 2016 Maluta et al.)

Neonatal Meningitis Escherichia coli (NMEC) is one of the most common causes of neonatal bacterial meningitis in the US and elsewhere resulting in mortality or neurologic deficits in survivors. Large plasmids have been shown experimentally to increase the virulence of NMEC in the rat model of neonatal meningitis. Here, 9 ExPEC-like plasmids were isolated from NMEC and sequenced to identify the core and accessory plasmid genes of ExPEC-like virulence plasmids in NMEC and create an expanded plasmid phylogeny. Results showed sequenced virulence plasmids carry a strongly conserved core of genes with predicted functions in five distinct categories including: virulence, metabolism, plasmid stability, mobile elements, and unknown genes. The major functions of virulence-associated and plasmid core genes serve to increase in vivo fitness by adding multiple iron uptake systems to the genetic repertoire to facilitate NMEC's survival in the host's low iron environment, and systems to enhance bacterial resistance to host innate immunity. Phylogenetic analysis based on these core plasmid genes showed that at least two lineages of ExPEC-like plasmids could be discerned. Further, virulence plasmids from Avian Pathogenic E. coli and NMEC plasmids could not be differentiated based solely on the genes of the core plasmid genome.

Extraintestinal pathogenic Escherichia coli (ExPEC) has major negative impacts on human and animal health. Recent research suggests food-borne links between human and animal ExPEC diseases with particular concern for poultry contaminated with avian pathogenic E. coli (APEC), the avian ExPEC. APEC is also a very important animal pathogen, causing colibacillosis, one of the world's most widespread bacterial diseases of poultry. Previous studies showed marked atrophy and lymphocytes depletion in the bursa during APEC infection. Thus, a more comprehensive understanding of the avian bursa response to APEC infection will facilitate genetic selection for disease resistance. Four-week-old commercial male broiler chickens were infected with APEC O1 or given saline as a control. Bursas were collected at 1 and 5 days post-infection (dpi). Based on lesion scores of liver, pericardium and air sacs, infected birds were classified as having mild or severe pathology, representing resistant and susceptible phenotypes, respectively. Twenty-two individual bursa RNA libraries were sequenced, each yielding an average of 27 million single-end, 100-bp reads. There were 2469 novel genes in the total of 16,603 detected. Large numbers of significantly differentially expressed (DE) genes were detected when comparing susceptible and resistant birds at 5 dpi, susceptible and non-infected birds at 5 dpi, and susceptible birds at 5 dpi and 1 dpi. The DE genes were associated with signal transduction, the immune response, cell growth and cell death pathways. These data provide considerable insight into potential mechanisms of resistance to ExPEC infection, thus paving the way to develop strategies for ExPEC prevention and treatment, as well as enhancing innate resistance by genetic selection in animals.

Antimicrobial agents of various types have important bearing on the outcomes of microbial infections. These agents may be bacteriostatic or -cidal, exert their impact via various means, originate from a living organism or a laboratory, and appropriately be used in or on living tissue or not. Though the primary focus of this chapter is on resistance to the antimicrobial agents used to treat uropathogenic Escherichia coli (UPEC)-caused urinary tract infections (UTIs), some attention will be given to UPEC's resistance to silver-containing antiseptics, which may be incorporated into catheters to prevent foreign body-associated UTIs.

Background: Avian pathogenic Escherichia coli (APEC) is a major cause of disease impacting animal health. The bone marrow is the reservoir of immature immune cells; however, it has not been examined to date for gene expression related to developmental changes (cell differentiation, maturation, programming) after APEC infection. Here, we study gene expression in the bone marrow between infected and non-infected animals, and between infected animals with mild (resistant) versus severe (susceptible) pathology, at two times post-infection., Results: We sequenced 24 bone marrow RNA libraries generated from the six different treatment groups with four replicates each, and obtained an average of 22 million single-end, 100-bp reads per library. Genes were detected as differentially expressed (DE) between APEC treatments (mild pathology, severe pathology, and mock-challenged) at a given time point, or DE between 1 and 5 days post-infection (dpi) within the same treatment group. Results demonstrate that many immune cells, genes and related pathways are key contributors to the different responses to APEC infection between susceptible and resistant birds and between susceptible and non-challenged birds, at both times post-infection. In susceptible birds, lymphocyte differentiation, proliferation, and maturation were greatly impaired, while the innate and adaptive immune responses, including dendritic cells, monocytes and killer cell activity, TLR- and NOD-like receptor signaling, as well as T helper cells and many cytokine activities, were markedly enhanced. The resistant birds' immune system, however, was similar to that of non-challenged birds., Conclusion: The DE genes in the immune cells and identified signaling models are representative of activation and resolution of infection in susceptible birds at both post-infection days. These novel results characterizing transcriptomic response to APEC infection reveal that there is combinatorial activity of multiple genes controlling myeloid cells, and B and T cell lymphopoiesis, as well as immune responses occurring in the bone marrow in these early stages of response to infection.

Avian pathogenic Escherichia coli (APEC) strains cause one of the three most significant infectious diseases in the poultry industry and are also potential food-borne pathogens threating human health. In this study, we showed that ArcA (aerobic respiratory control), a global regulator important for E. coli's adaptation from anaerobic to aerobic conditions and control of that bacterium's enzymatic defenses against reactive oxygen species (ROS), is involved in the virulence of APEC. Deletion of arcA significantly attenuates the virulence of APEC in the duck model. Transcriptome sequencing (RNA-Seq) analyses comparing the APEC wild type and the arcA mutant indicate that ArcA regulates the expression of 129 genes, including genes involved in citrate transport and metabolism, flagellum synthesis, and chemotaxis. Further investigations revealed that citCEFXG contributed to APEC's microaerobic growth at the lag and log phases when cultured in duck serum and that ArcA played a dual role in the control of citrate metabolism and transportation. In addition, deletion of flagellar genes motA and motB and chemotaxis gene cheA significantly attenuated the virulence of APEC, and ArcA was shown to directly regulate the expression of motA, motB, and cheA. The combined results indicate that ArcA controls metabolism, chemotaxis, and motility contributing to the pathogenicity of APEC., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

This study characterized 52 Escherichia coli isolates from distinct diseased organs of 29 broiler chickens with clinical symptoms of colibacillosis in the Southern Brazilian state of Rio Grande do Sul. Thirty-eight isolates were highly virulent and 14 were virtually avirulent in 1-day-old chicks, yet all isolates harbored virulence factors characteristic of avian pathogenic E. coli (APEC), including those related to adhesion, iron acquisition, and serum resistance. E. coli reference collection phylogenetic typing showed that isolates belonged mostly to group D (39%), followed by group A (29%), group B1 (17%), and group B2 (15%). Phylogenetic analyses using the Amplified Ribosomal DNA Restriction Analysis and pulse-field gel electrophoresis methods were used to discriminate among isolates displaying the same serotype, revealing that five birds were infected with two distinct APEC strains. Among the 52 avian isolates, 2 were members of the pandemic E. coli O25:H4-B2-ST131 clone.

Neonatal meningitis Escherichia coli K1 (NMEC) are thought to be transmitted from mothers to newborns during delivery or by nosocomial infections. However, the source of E. coli K1 causing these infections is not clear. Avian pathogenic E. coli (APEC) have the potential to cause infection in humans while human E. coli have potential to cause colibacillosis in poultry, suggesting that these strains may lack host specificity. APEC strains are capable of causing meningitis in newborn rats; however, it is unclear whether these bacteria use similar mechanisms to that of NMEC to establish disease. Using four representative APEC and NMEC strains that belong to serotype O18, we demonstrate that these strains survive in human serum similar to that of the prototypic NMEC strain E44, a derivative of RS218. These bacteria also bind and enter both macrophages and human cerebral microvascular endothelial cells (HCMEC/D3) with similar frequency as that of E44. The amino acid sequences of the outer membrane protein A (OmpA), an important virulence factor in the pathogenesis of meningitis, are identical within these representative APEC and NMEC strains. Further, these strains also require FcγRI-α chain (CD64) and Ecgp96 as receptors for OmpA in macrophages and HCMEC/D3, respectively, to bind and enter these cells. APEC and NMEC strains induce meningitis in newborn mice with varying degree of pathology in the brains as assessed by neutrophil recruitment and neuronal apoptosis. Together, these results suggest that serotype O18 APEC strains utilize similar pathogenic mechanisms as those of NMEC strains in causing meningitis.

Uropathogenic Escherichia coli (UPEC) is responsible for the majority of urinary tract infections (UTIs), which are some of the world's most common bacterial infections of humans. Here, we examined the role of FNR (fumarate and nitrate reduction), a well-known global regulator, in the pathogenesis of UPEC infections. We constructed an fnr deletion mutant of UPEC CFT073 and compared it to the wild type for changes in virulence, adherence, invasion, and expression of key virulence factors. Compared to the wild type, the fnr mutant was highly attenuated in the mouse model of human UTI and showed severe defects in adherence to and invasion of bladder and kidney epithelial cells. Our results showed that FNR regulates motility and multiple virulence factors, including expression of type I and P fimbriae, modulation of hemolysin expression, and expression of a novel pathogenicity island involved in α-ketoglutarate metabolism under anaerobic conditions. Our results demonstrate that FNR is a key global regulator of UPEC virulence and controls expression of important virulence factors that contribute to UPEC pathogenicity., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Avian pathogenic Escherichia coli (APEC) strains belong to a category that is associated with colibacillosis, a serious illness in the poultry industry worldwide. Additionally, some APEC groups have recently been described as potential zoonotic agents. In this work, we compared APEC strains with extraintestinal pathogenic E. coli (ExPEC) strains isolated from clinical cases of humans with extra-intestinal diseases such as urinary tract infections (UTI) and bacteremia. PCR results showed that genes usually found in the ColV plasmid (tsh, iucA, iss, and hlyF) were associated with APEC strains while fyuA, irp-2, fepC sitDchrom, fimH, crl, csgA, afa, iha, sat, hlyA, hra, cnf1, kpsMTII, clpVSakai and malX were associated with human ExPEC. Both categories shared nine serogroups (O2, O6, O7, O8, O11, O19, O25, O73 and O153) and seven sequence types (ST10, ST88, ST93, ST117, ST131, ST155, ST359, ST648 and ST1011). Interestingly, ST95, which is associated with the zoonotic potential of APEC and is spread in avian E. coli of North America and Europe, was not detected among 76 APEC strains. When the strains were clustered based on the presence of virulence genes, most ExPEC strains (71.7%) were contained in one cluster while most APEC strains (63.2%) segregated to another. In general, the strains showed distinct genetic and fingerprint patterns, but avian and human strains of ST359, or ST23 clonal complex (CC), presented more than 70% of similarity by PFGE. The results demonstrate that some "zoonotic-related" STs (ST117, ST131, ST10CC, ST23CC) are present in Brazil. Also, the presence of moderate fingerprint similarities between ST359 E. coli of avian and human origin indicates that strains of this ST are candidates for having zoonotic potential.

Avian pathogenic Escherichia coli (APEC) are responsible for heavy economic losses in poultry industry. Here we investigate DNA methylome of spleen and identify functional DNA methylation changes related to host response to APEC among groups of non-challenged chickens (NC), challenged with mild (MD) and severe pathology (SV). DNA methylation was enriched in the gene bodies and repeats. Promoter and CGIs are hypomethylated. Integration analysis revealed 22, 87, and 9 genes exhibiting inversely changed DNA methylation and gene expression in NC vs. MD, NC vs. SV, and MD vs. SV, respectively. IL8, IL2RB, and IL1RAPL1 were included. Gene network analysis suggested that besides inflammatory response, other networks and pathways such as organismal injury and abnormalities, cell signaling and molecular transport, are probably related to host response to APEC infection. Moreover, methylation changes in cell cycle processes might contribute to the lesion phenotype differences between MD and SV.

Avian pathogenic Escherichia coli (APEC) infection is responsible for great economic losses to the poultry industry worldwide and there is increasing evidence of its zoonotic importance. In this study, 219 E. coli isolates from 84 poultry flocks in Egypt, including 153 APEC, 30 avian fecal E. coli (AFEC), and 36 environmental E. coli, were subjected to phylogenetic grouping and virulence genotyping. Additionally, 50 of these isolates (30 APEC from colisepticemia and 20 AFEC) were subjected to a more-extensive characterization which included serogrouping, antimicrobial susceptibility analysis, screening for seven intestinal E. coli virulence genes (stx1, stx2, eae, espP, KatP, hlyA, and fliCh7), multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and in vivo virulence testing. More than 90% of the total APEC examined possessed iroN, ompT, hlyF, iss, and iutA, indicating that Egyptian APECs, like their counterparts from the United States, harbor plasmid pathogenicity islands (PAIs). The majority of APEC and AFEC were of phylogenetic groups A, B1, and D. For the 50-isolate subgroup, more than 70% of APEC and 80% ofAFEC were multidrug resistant. Among the subgroup of APEC, MLST analysis identified 11 sequence types (ST) while seven STs were found among AFEC. Based on PFGE, the genetic relatedness of APEC and AFEC ranged from 50%-100% and clustered into four primary groups at 50% similarity. Two of the eight APEC strains tested in chickens were able to induce 25% mortality in 1-day-old chicks. APECs were distinguished from AFECs and environmental E. coli by their content of plasmid PAI genes, whereas APEC isolated from colisepticemia and AFEC were not distinguishable based on their antimicrobial resistance patterns, as both groups were multidrug resistant. Avian E. coli strains from broiler flocks in Egypt show similar sequence types to E. coli associated with human infection.