Avian Pathogenic Escherichia Coli Research Articles

Recombinant Salmonella gallinarum (S. gallinarum) Vaccine Candidate Expressing Avian Pathogenic Escherichia coli Type I Fimbriae Provides Protections against APEC O78 and O161 Serogroups and S. gallinarum Infection.

Avian pathogenic Escherichia coli (APEC) is one of the leading pathogens that cause devastating economic losses to the poultry industry. Type I fimbriae are essential adhesion factors of APEC, which can be targeted and developed as a vaccine candidate against multiple APEC serogroups due to their excellent immunogenicity and high homology. In this study, the recombinant strain SG102 was developed by expressing the APEC type I fimbriae gene cluster (fim) on the cell surface of an avirulent Salmonella gallinarum (S. gallinarum) vector strain using a chromosome-plasmid-balanced lethal system. The expression of APEC type I fimbriae was verified by erythrocyte hemagglutination assays and antigen-antibody agglutination tests. In vitro, the level of the SG102 strain adhering to leghorn male hepatoma (LMH) cells was significantly higher than that of the empty plasmid control strain, SG101. At two weeks after oral immunization, the SG102 strain remained detectable in the livers, spleens, and ceca of SG102-immunized chickens, while the SG101 strain was eliminated in SG101-immunized chickens. At 14 days after the secondary immunization with 5 × 109 CFU of the SG102 strain orally, highly antigen-specific humoral and mucosal immune responses against APEC type I fimbriae protein were detected in SG102-immunized chickens, with IgG and secretory IgA (sIgA) concentrations of 221.50 μg/mL and 1.68 μg/mL, respectively. The survival rates of SG102-immunized chickens were 65% (13/20) and 60% (12/20) after challenge with 50 LD50 doses of APEC virulent strains O78 and O161 serogroups, respectively. By contrast, 95% (19/20) and 100% (20/20) of SG101-immunized chickens died in challenge studies involving APEC O78 and O161 infections, respectively. In addition, the SG102 strain effectively provided protection against lethal challenges from the virulent S. gallinarum strain. These results demonstrate that the SG102 strain, which expresses APEC type I fimbriae, is a promising vaccine candidate against APEC O78 and O161 serogroups as well as S. gallinarum infections.

ProQ binding to small RNA RyfA promotes virulence and biofilm formation in avian pathogenic Escherichia coli

Avian pathogenic Escherichia coli (APEC) is a notable subpathotype of the nonhuman extraintestinal pathogenic E. coli (ExPEC). Recognized as an extraintestinal foodborne pathogen, the zoonotic potential of APEC/ExPEC allows for cross-host transmission via APEC-contaminated poultry meat and eggs. ProQ, an RNA binding protein, is evolutionarily conserved in E. coli. However, its regulatory roles in the biofilm formation and virulence of APEC/ExPEC have not been explored. In this study, proQ deletion in the APEC strain FY26 significantly compromised its biofilm-forming ability. Furthermore, animal tests and cellular infection experiments showed that ProQ depletion significantly attenuated APEC virulence, thereby diminishing its capacity for bloodstream infection and effective adherence to and persistence within host cells. Transcriptome analysis revealed a decrease in the transcription level of the small RNA (sRNA) RyfA in the mutant FY26ΔproQ, suggesting a direct interaction between the sRNA RyfA and ProQ. This interaction might indicate that sRNA RyfA is a novel ProQ-associated sRNA. Moreover, the direct binding of ProQ to the sRNA RyfA was crucial for APEC biofilm formation, pathogenicity, adhesion, and intracellular survival. In conclusion, our findings provide detailed insight into the interaction between ProQ and sRNA RyfA and deepen our understanding of the regulatory elements that dictate APEC virulence and biofilm development. Such insights are instrumental in developing strategies to counteract APEC colonization within hosts and impede APEC biofilm establishment on food surfaces.

Effects of pomegranate (Punica granatum L.) peel on the growth performance and intestinal microbiota of broilers challenged with Escherichia coli

The effects of pomegranate peel on the growth performance, intestinal morphology, and the cecal microbial community were investigated in broilers challenged with avian pathogenic Escherichia coli (APEC) O78. A total of 240 one-day-old chicks (120 males and 120 females) were randomly and evenly allotted into 4 treatment groups (each with 6 biological replicates each of 10 chicks), i.e., negative control (NC), positive control (PC), and 2 experimental groups treated with 0.2% fermented pomegranate peel (FP) and 0.2% unfermented pomegranate peel (UFP), respectively, with PC, FP, and UFP groups challenged with APEC O78 (5 × 108 CFU) on day 14. Results showed that the challenge of APEC O78 decreased the body weight (BW) and average daily gain (ADG) of broilers from 1 to 28 d (P < 0.01). These broilers exhibited more pathological conditions in the heart and liver and higher mortality rates in 28 d compared to the NC group. Diet supplemented with pomegranate peel (either fermented or unfermented) significantly increased BW, ADG, and the villus height/crypt depth ratio (VCR) of small intestine in 28 d compared to the NC group (P < 0.05). Results of the taxonomic structure of the gut microbiota showed that compared to the NC group, the APEC challenge significantly decreased the relative abundance of Bacteroidetes and increased the relative abundance of Firmicutes (P < 0.01). Compared to the PC group, the relative abundance of Ruminococcus_torques_group in FP group was increased, while the relative abundance of Alistipes was decreased. In summary, our study showed that the dietary supplementation of pomegranate peel could maintain the intestinal microbiota at a state favorable to the host, effectively reduce the abnormal changes in the taxonomic structure of the intestinal microbiota, and improve the growth performance in broilers treated with APEC.

Role of Ibea Gene in Hbmecs Invasion and Biofilm Formation Induced By Neonatal Meningitis Escherichia coli

Background: Bacterial meningitis is a severe infectious disease that affects the central nervous system and can result in high mortality rates and neurological complications. In neonates, meningitis is mainly caused by neonatal meningitis Escherichia coli (NMEC), which invades the intestine, leading to hematogenous spreadand potential bacterial meningitis through blood-brain barrier. Previous studies indicated that the ability of NMEC to adhere and invade is associated with biofilm formation and a deficiency in ibeA may result in decreased virulence and biofilm-forming ability of avian pathogenic Escherichia coli (APEC). Furthermore, the ibeA gene is positioned downstream of ibeR. Our previous studies have shown that ibeR and ibeA genes can both regulate the virulence and relevant phenotypes in E. coli K1. However, the effects of ibeR and ibeA on the biofilm formation and invasion of K1 strain remain to be further studied. Recent studies suggest that indole may act as a signaling molecule to regulate the expression of adhesion and biofilm-promoting factors in E. coli. However, the mechanism by which indole affects biofilms is still unclear. This study aimed to investigate the mechanism of ibeA inhibition of indole-induced NMEC invasion and biofilm formation. Methods: The viability and growth of the wild strain E44 and ibeA-deletion mutant strain ZD1 were assessed by MTT cell proliferation assay kit (Coolaber). The absorbance values were measured at 490 nm and 540 nm by ELISA. The invasion activities and the biofilm formation ability of E44 and ZD1 were detected in vitro studies. We conducted a cell invasion test using the Human Brain Microvascular Endothelial Cells (HBMEC) model. The ability to form a biofilm was measured at 600 nm using a specific biofilm formation index. Biofilm phenotypic was observed by an confocal laser-scanning microscope(Olympus FV-1000, Japan). Then, we used indole as a regulatory agency to compare its biofilm inhibitory efficacy between E44 and ibeR-deletion mutant strain BR2. The experimental data were analyzed using the SPSS 19.0 statistical package. A statistically significant difference was indicated by P&amp;lt;0.05. Results: No difference was found in bacterial growth between the E. coli strains E44 and ZD1. The biofilm formation and HBMECs invasion of ZD1 was significantly lower than that of E44. After treatment with 0.5mM indole, the biofilm formation ability and the ibeA protein expressions of E44 were significantly inhibited. However, indole at different concentrations had no obvious inhibition on BR2 biofilm formation and the ibeA protein expressions. Conclusion: In summary, the study found that ibeA had a significant regulatory effect on HBMECs invasion and biofilm formation of NMEC, which may inhibit by indole via regulating ibeA through ibeR. ( Acknowledgements: Grant from School of Public Health of Southern Medical University, Guangzhou, China, No. GW202329; J.Z., Q.L. and L.W. contributed equally to this work; Corresponding author: Hong Cao, gzhcao@smu.edu.cn)

CTX-M-55-type ESBL-producing fluoroquinolone-resistant Escherichia coli sequence type 23 repeatedly caused avian colibacillosis in Kagoshima Prefecture, Japan

The production of expanded-spectrum beta-lactamases (ESBLs) and fluoroquinolone resistance in Enterobacteriaceae has become a global concern. The aim of this study was to investigate the spread of ESBL-producing and fluoroquinolone-resistant avian pathogenic Escherichia coli (APEC) in Kagoshima, a prefecture with the largest amount of poultry in Japan. The antimicrobial susceptibility and genetic characteristics of 228 APEC strains isolated from 57 farms in Kagoshima Prefecture, Japan, between 2005 and 2017 were analysed. Information about the companies with hatcheries connected to the farms was also collected, and the epidemiologic relatedness of APEC strains and the processes of adopting chicks were compared. Seven CTX-M-type ESBL genes, blaCTX-M-1, blaCTX-M-2, blaCTX-M-14, blaCTX-M-15, blaCTX-M-25, blaCTX-M-55, and blaCTX-M-65, were found in 60 (26.3%) of the 228 APEC strains. The ciprofloxacin-resistant strains belonged to 10 different sequence types (ST10, ST23, ST93, ST155, ST156, ST350, ST359, ST602, ST648, and ST9479), and the two ST602 strains showed remarkably high ciprofloxacin resistance (MIC: 128 µg/mL) and had amino acid mutations in GyrA (S83L and D87N), ParC (S80I), and ParE (E460A). A CTX-M-55-type ESBL-producing fluoroquinolone-resistant Og78-ST23 strain was isolated multiple times over two years on a farm. Furthermore, epidemiologically closely related strains were isolated from different farms that used the same common hatcheries. APEC is often transferred from hatcheries to farms via healthy chicks, and the prudent use of antimicrobials and careful monitoring of resistant strains on poultry farms and hatcheries are important in preventing the selection and spread of high-risk APEC strains such as CTX-M-55-type ESBL-producing Og78-ST23.

Avian Pathogenic Escherichia coli (APEC) in Broiler Breeders: An Overview.

Poultry meat is one of the major animal protein sources necessary to meet the global protein demand. Sustainability in broiler production is the key to achieving its continuous supply, and broiler breeders play a critical role in maintaining this sustainability by providing good quality chicks. Colibacillosis, the disease caused by avian pathogenic Escherichia coli (APEC), causes severe economic losses to the poultry industry globally. Moreover, APEC causes an additional burden among broiler breeders, such as a decrease in egg production and mortality among these birds. There is vertical transmission of APEC to the broiler chicks through eggs, resulting in increased first-week mortality and subsequent horizontal transmission at the hatchery. In this regard, the vertical transmission of antibiotic resistance genes is another concern that needs attention. Controlling several diseases in broiler breeders would possibly reduce the first-week mortality in chicks, thereby maintaining the production level. For that, constant monitoring of the bacterial populations is critical. Moreover, amidst the increased antibiotic resistance pattern, more focus on alternative treatment strategies like vaccines, probiotics, and bacteriophages is necessary. Future research focusing on strategies to mitigate APEC in broiler breeders would be one of the finest solutions for sustainable broiler production.

The Isolation and Characterization of Bacteriophages Infecting Avian Pathogenic Escherichia coli O1, O2 and O78 Strains.

Avian pathogenic Escherichia coli (APEC), such as O1, O2 and O78, are important serogroups relating to chicken health, being responsible for colibacillosis. In this study, we isolated and characterized bacteriophages (phages) from hen feces and human sewage in Alberta with the potential for controlling colibacillosis in laying hens. The lytic profile, host range, pH tolerance and morphology of seven APEC-infecting phages (ASO1A, ASO1B, ASO2A, ASO78A, ASO2B, AVIO78A and ASO78B) were assessed using a microplate phage virulence assay and transmission electron microscopy (TEM). The potential safety of phages at the genome level was predicted using AMRFinderPlus and the Virulence Factor Database. Finally, phage genera and genetic relatedness with other known phages from the NCBI GenBank database were inferred using the virus intergenomic distance calculator and single gene-based phylogenetic trees. The seven APEC-infecting phages preferentially lysed APEC strains in this study, with ECL21443 (O2) being the most susceptible to phages (n = 5). ASO78A had the broadest host range, lysing all tested strains (n = 5) except ECL20885 (O1). Phages were viable at a pH of 2.5 or 3.5-9.0 after 4 h of incubation. Based on TEM, phages were classed as myovirus, siphovirus and podovirus. No genes associated with virulence, antimicrobial resistance or lysogeny were detected in phage genomes. Comparative genomic analysis placed six of the seven phages in five genera: Felixounavirus (ASO1A and ASO1B), Phapecoctavirus (ASO2A), Tequatrovirus (ASO78A), Kayfunavirus (ASO2B) and Sashavirus (AVIO78A). Based on the nucleotide intergenomic similarity (<70%), phage ASO78B was not assigned a genus in the siphovirus and could represent a new genus in class Caudoviricetes. The tail fiber protein phylogeny revealed variations within APEC-infecting phages and closely related phages. Diverse APEC-infecting phages harbored in the environment demonstrate the potential to control colibacillosis in poultry.

The IbeA protein from adherent invasive Escherichia coli isa flavoprotein sharing structural homology with FAD-dependent oxidoreductases.

Invasion of brain endothelium protein A (IbeA) is a virulence factor specific to pathogenic Escherichia coli. Originally identified in the K1 strain causing neonatal meningitis, it was more recently found in avian pathogenic Escherichia coli (APEC) and adherent invasive Escherichia coli (AIEC). In these bacteria, IbeA facilitates host cell invasion and intracellular survival, in particular, under harsh conditions like oxidative stress. Furthermore, IbeA from AIEC contributes to intramacrophage survival and replication, thus enhancing the inflammatory response within the intestine. Therefore, this factor is a promising drug target for anti-AIEC strategies in the context of Crohn's disease. Despite such an important role, the biological function of IbeA remains largely unknown. In particular, its exact nature and cellular localization, i.e., membrane-bound invasin versus cytosolic factor, are still of debate. Here, we developed an efficient protocol for recombinant expression of IbeA under native conditions and demonstrated that IbeA from AIEC is a soluble, homodimeric flavoprotein. Using mass spectrometry and tryptophan fluorescence measurements, we further showed that IbeA preferentially binds flavin adenine dinucleotide (FAD), with an affinity in the one-hundred nanomolar range and optimal binding under reducing conditions. 3D-modeling with AlphaFold revealed that IbeA shares strong structural homology with FAD-dependent oxidoreductases. Finally, we used ligand docking, mutational analyses, and molecular dynamics simulations to identify the FAD binding pocket within IbeA and characterize possible conformational changes occurring upon ligand binding. Overall, we suggest that the role of IbeA in the survival of AIEC within host cells, notably macrophages, is linked to modulation of redox processes.

Emergence and Genomic Features of a mcr-1 Escherichia coli from Duck in Hungary.

Plasmids carrying high-risk resistance mechanisms in pathogenic E. coli have gained particular attention in veterinary medicine, especially since the discovery of the colistin resistance gene, mcr-1. Here, we provide the first evidence of its emergence and describe the complete mcr-1 plasmid sequence of a multi-resistant avian pathogenic E. coli (APEC) strain from waterfowl in Hungary. Whole-genome sequencing analysis and core-genome MLST were performed to characterize the genome structure of the mcr-1 plasmid and to reveal the phylogenetic relation between the Hungarian duck strain Ec45-2020 and the internationally circulating mcr-1-positive E. coli strains from poultry and humans. Results showed that plasmid pEc45-2020-33kb displayed a high level of genome identity with mcr-1 plasmids of IncX4 type widespread among human, animal and food reservoirs of enteric bacteria of public health. The mcr-1-positive E. coli strain Ec45-2020 belongs to the ST162 genotype, considered as one of the globally disseminated zoonotic genotypes of MDR E. coli. In accordance with international findings, our results underline the importance of continuous surveillance of enteric bacteria with high-risk antimicrobial resistance genotypes, including neglected animals, such as waterfowls, as possible reservoirs for the colistin resistance gene mcr-1.

Phenotypic and genotypic characterizations of bacteria isolated from the respiratory microbiota of healthy turkeys with potential for probiotic composition.

Desirable characteristics of Staphylococcus sp., Streptococcus sp., Bacillus sp., Klebsiella sp., Escherichia coli, and Pseudomonas pseudoalcaligenes isolated from the trachea of healthy turkeys were evaluated as probiotic candidates in the search for new alternatives to solve antimicrobial resistance issues in poultry. In current study phenotypic and genotypic capacity to produce bacteriocin-like substances, efficacy to inhibit the growth of avian pathogens, susceptibility to antimicrobials of bacteria isolated from the respiratory microbiota of healthy turkeys, and the presence of virulence-associated genes (VAGs) predictors of Avian Pathogenic Escherichia coli (APEC) were evaluated. Nine E. coli and one Klebsiella sp. strains produced bacteriocin-like substances, and all harbored the cvaA gene. Some strains also showed antagonistic activity against APEC. Multidrug-resistant profile was found in 54% of the strains. Six strains of bacteriocin-like substances producing E. coli also harbored 3-5 VAGs. The study showed that two bacterial genuses (Klebsiella sp. and E. coli) present desirable probiotic characteristics. Our results identified strains with potential for poultry's respiratory probiotic.

COLISEPTICEMIA INFECTION IN A 20 DAYS OLD BROILER CHICKEN IN TIMUHUN VILLAGE, KLUNGKUNG

Colisepticemia is one of poultry infectious disease caused by Avian pathogenic Escherichia coli. The aim of this study was identifying the causative of an infected broiler chickens aged 20 days. The research was conducted at a close house farm in Timuhun Village, Banjarangkan, Klungkung Regency, Bali with a population of 16,000 broiler chickens. The number of sick chickens was 163 and 55 died. The chicken samples were brought to the Veterinary Pathology Laboratory, Udayana Faculty of Veterinary Medicine for necropsy. Organ samples that underwent changes were taken to the Veterinary Bacteriology and Parasitology Laboratory. In bacteriological examination, organ samples were isolated using nutrient agar and eosyne methylene blue agar (EMBA) media. Furthermore, the results of bacterial isolation were identified using triple sugar iron agar (TSIA) media, Simmon Citrate Agar (SCA), Sulfid Indole Motility (SIM), Methyl Red-Voges Proskauer (MR-VP) and testing sugars (glucose and lactose). In the parasitology laboratory, fecal samples were examined using native, sedimentation and flotation methods. The results of the epidemiological examination of the disease, 1% morbidity, 0.3% mortality and 33.7% case fatality rate (CFR). Post-mortem examination found typical anatomical pathology in the form of polyserotysis and fibrin accumulation in the pericardium, perihepatic, pleural and mesentery. The results of the fecal examination did not find any agents and/or worm eggs. Bacterial isolation on EMBA media grew metallic green round colonies with black dots. Gram stain shows short rods and is colored red. Based on the results of pathology, parasitology and bacteriology examinations, the etiology of this cases caused by Avian Pathogenic Escherichia coli (APEC). Elevating sanitation was needed to reduce the cases.

Extracellular vesicles produced by avian pathogenic Escherichia coli (APEC) activate macrophage proinflammatory response and neutrophil extracellular trap (NET) formation through TLR4 signaling

BackgroundAvian pathogenic Escherichia coli (APEC) is the major pathogen causing important avian diseases in poultry. As an important subtype of extraintestinal pathogenic E. coli, APEC has zoonotic potential and is considered a foodborne pathogen. APEC extracellular vesicles (EVs) may play vital roles in the interaction of the pathogen with its host cells. However, the precise roles played by APEC EVs are still not completely clear, especially in immune cells.ResultsIn this study, we investigated the relationships between APEC EVs and immune cells. The production and characteristics of the EVs of APEC isolate CT265 were identified. Toll like receptor 4 (TLR4) triggered the cellular immune responses when it interacted with APEC EVs. APEC EVs induced a significant release of proinflammatory cytokines in THP-1 macrophages. APEC EVs induced the macrophage inflammatory response via the TLR4/MYD88/NF-κB signaling pathway, which participated in the activation of the APEC-EV-induced NLRP3 inflammasome. However, the loss of lipopolysaccharide (LPS) from APEC EVs reduced the activation of the NLRP3 inflammasome mediated by TLR4/MYD88/NF-κB signaling. Because APEC EVs activated the macrophage inflammatory response and cytokines release, we speculated that the interaction between APEC EVs and macrophages activated and promoted neutrophil migration during APEC extraintestinal infection. This study is the first to report that APEC EVs induce the formation of neutrophil extracellular traps (NETs) and chicken heterophil extracellular traps. Treatment with APEC EVs induced SAPK/JNK activation in neutrophils. The inhibition of TLR4 signaling suppressed APEC-EV-induced NET formation. However, although APEC EVs activated the immune response of macrophages and initiated NET formation, they also damaged macrophages, causing their apoptosis. The loss of LPS from APEC EVs did not prevent this process.ConclusionAPEC-derived EVs induced inflammatory responses in macrophages and NETs in neutrophils, and that TLR4 was involved in the APEC-EV-activated inflammatory response. These findings provided a basis for the further study of APEC pathogenesis.

A lytic phage to control multidrug-resistant avian pathogenic Escherichia coli (APEC) infection.

The inappropriate use of antibiotics has led to the emergence of multidrug-resistant strains. Bacteriophages (phages) have gained renewed attention as promising alternatives or supplements to antibiotics. In this study, a lytic avian pathogenic Escherichia coli (APEC) phage designated as PEC9 was isolated and purified from chicken farm feces samples. The morphology, genomic information, optimal multiplicity of infection (MOI), one-step growth curve, thermal stability, pH stability, in vitro antibacterial ability and biofilm formation inhibition ability of the phage were determined. Subsequently, the therapeutic effects of the phages were investigated in the mice model. The results showed that PEC9 was a member of the siphovirus-like by electron microscopy observation. Biological characterization revealed that it could lyse two serotypes of E. coli, including O1 (9/20) and O2 (6/20). The optimal multiplicity of infection (MOI) of phage PEC9 was 0.1. Phage PEC9 had a latent period of 20 min and a burst period of 40 min, with an average burst size of 68 plaque-forming units (PFUs)/cell. It maintained good lytic activity at pH 3-11 and 4-50°C and could efficiently inhibit the bacterial planktonic cell growth and biofilm formation, and reduce bacterial counts within the biofilm, when the MOI was 0.01, 0.1, and 1, respectively. Whole-genome sequencing showed that PEC9 was a dsDNA virus with a genome of 44379 bp and GC content of 54.39%. The genome contains 56 putative ORFs and no toxin, virulence, or resistance-related genes were detected. Phylogenetic tree analysis showed that PEC9 is closely related to E. coli phages vB_EcoS_Zar3M, vB_EcoS_PTXU06, SECphi18, ZCEC10, and ZCEC11, but most of these phages exhibit different gene arrangement. The phage PEC9 could successfully protect mice against APEC infection, including improved survival rate, reduced bacterial loads, and organ lesions. To conclude, our results suggest that phage PEC9 may be a promising candidate that can be used as an alternative to antibiotics in the control of APEC infection.

A comparative study on histopathological features of duodenum, jejunum, and ileum from broiler chicken with avian pathogenic Escherichia coli infection

Avian colibacillosis is a high-prevalence chicken farm disease caused by avian pathogen-ic Escherichia coli (APEC). It is necessary to identify and look up to the bacterial activity to stave off a decline in chicken performance. This study aimed to identify and determine the pathogenetic activity of APEC within broiler chickens on the duodenum, jejunum, and ileum histopathology comparison. The samples were collected from a total of 40 broilers, obtained from a highest-colibacillosis historical chicken house, identified with bacterial isolation swab-technique, and analyzed with nonparametric statistical lesions scoring. The identification result showed 82.5% E. coli with a 62.5% APEC con-firmed. The histopathological feature showed a significant difference (p&lt;0.05), where the jejunum ap-peared to encounter the most significant damage compared to the duodenum and ileum. Thus, it is con-cluded that APEC field infection varies in characteristics and pathogenicity, which affects intestinal organs based on the histology changes, mainly in the jejunum part of the intestine.

Therapeutics and prophylactic efficacy of novel lytic Escherichia phage vB_EcoS_PJ16 against multidrug-resistant avian pathogenic E. coli using in vivo study.

Avian pathogenic Escherichia coli (APEC) is the causative agent of avian colibacillosis, which causes significant economic losses to the poultry industry. The growing resistance of bacteria to antibiotics is a major global public health concern. However, there is limited data on the efficacy of phage therapy in effectively controlling and treating APEC infections. In this study, a novel lytic Escherichia phage, vB_EcoS_PJ16, was isolated from poultry farm wastewater and characterized in both in vitro and in vivo conditions. Transmission electron microscopy analysis revealed the presence of an icosahedral head and a long non-contractile tail, classifying the phage under the Caudoviricetes class. Host range determination showed that Escherichia phage vB_EcoS_PJ16 exhibited lytic activity against multiple strains of pathogenic E. coli, while no significant signs of lysis for Klebsiella pneumoniae, Salmonella Typhimurium, Listeria monocytogenes, and Staphylococcus aureus. Biophysical characterization revealed that the isolated phage was sturdy, as it remained viable for up to 300days at temperatures of 30°C, 37°C, and 42°C and for up to 24h at pH 5 to 11, with only minor changes in titer. Kinetic analysis at multiplicity of infection (MOI) 0.1 showed a latency period of about 20min and a burst size of 26.5 phage particles per infected cell for phage vB_EcoS_PJ16. Whole genome sequencing unveiled that the phage vB_EcoS_PJ16 genome consists of a double-stranded linear DNA molecule with 57,756bp and a GC content of 43.58%. The Escherichia phage vB_EcoS_PJ16 genome consisted of 98 predicted putative ORFs, with no transfer RNA identified in the genome. Among these 98 genes, 34 genes were predicted to have known functions. A significant reduction in APEC viability was observed at MOI 100 during in vitro bacterial challenge tests conducted at different MOIs (0.01, 1, and 100). In vivo oral evaluation of the isolated phage to limit E. coli infections in day-old chicks indicated a decrease in mortality within both the therapeutic (20%) and prophylactic (30%) groups, when compared to the control group. The findings of this study contribute to our current knowledge of Escherichia phages and suggest a potentially effective role of phages in the therapeutic and prophylactic control of antibiotic-resistant APEC strains.

Effects of the in ovo injection of an Escherichia coli vaccine on the hatchability and quality characteristics of commercial layer hatchlings

In the commercial egg industry, avian pathogenic Escherichia coli (APEC) can lead to significant economic loss. The Poulvac E. coli vaccine (PECV) is a commercially available attenuated live vaccine commonly applied via spray or drinking water to protect against losses associated with colibacillosis. The PECV has not been tested in layer hatching eggs using in ovo injection. Therefore, the purpose of this experiment was to determine the effects of injecting 50 μL of different doses of the PECV into Hy-Line W-36-layer hatching eggs on the hatchability and quality characteristics of hatchlings. At 18 d of incubation (DOI), treatments included 1 noninjected and 1 diluent-injected control. Furthermore, PECV treatments included a full dose (4.4×108E. coli CFU) or serial dilutions of the full dose to produce 4.4×106, 4.4×104, or 4.4×102 CFU doses of E. coli. In ovo injections targeted the amnion. Percent hatchability of live embryonated eggs (HI), percent residue eggs, hatchling mortality, and female chick whole and yolk-free BW, relative yolk sac weight, and body length were among the variables examined. Treatment significantly (P < 0.0001) affected HI, with HI being highest in the control groups (97.3% in the noninjected and 94.2% in the diluent-injected), and with HI values being 89.0, 88.9, 84.4, and 71.2% in the 4.4×102, 4.4×104, 4.4×106, and 4.4×108 CFU E. coli dose treatments, respectively. The percentage of live embryos that did not complete hatch but that pipped internally (P=0.024) or externally (P < 0.0001) were significantly affected by treatment, with percentages being highest in the 4.4×108 CFU treatment. Female chick body length was significantly (P < 0.0001) affected by treatment and was longer in both control groups and in the 1×102 CFU E. coli treatment in comparison to all other treatments. Yolk-free female chick BW was significantly (P=0.034) affected by treatment and was lower in the 4.4×106 CFU and 4.4×108 CFU treatments when compared to the diluent-injected control group. An increase in the E. coli concentration administered in the amnion of embryonated layer hatching eggs at 18 DOI decreased hatch success and female chick yolk-free BW and body length.

The role of AJB35136 and fdtA genes in biofilm formation by avian pathogenic Escherichia coli

BackgroundInfections caused by avian pathogenic Escherichia coli (APEC) result in significant economic losses in poultry industry. APEC strains are known to form biofilms in various conditions allowing them to thrive even under harsh and nutrient-deficient conditions on different surfaces, and this ability enables them to evade chemical and biological eradication methods. Despite knowing the whole genome sequences of various APEC isolates, little has been reported regarding their biofilm-associated genes. A random transposon mutant library of the wild-type APEC IMT 5155 comprising 1,300 mutants was analyzed for biofilm formation under nutrient deprived conditions using Videoscan technology coupled with fluorescence microscopy. Seven transposon mutants were found to have reproducibly and significantly altered biofilm formation and their mutated genes were identified by arbitrary PCR and DNA sequencing. The intact genes were acquired from the wild-type strain, cloned in pACYC177 plasmid and transformed into the respective altered biofilm forming transposon mutants, and the biofilm formation was checked in comparison to the wild type and mutant strains under the same conditions.ResultsIn this study, we report seven genes i.e., nhaA, fdeC, yjhB, lysU, ecpR, AJB35136 and fdtA of APEC with significant contribution to biofilm formation. Reintroduction of AJB35136 and fdtA, reversed the altered phenotype proving that a significant role being played by these two O-antigen related genes in APEC biofilm formation. Presence of these seven genes across nonpathogenic E. coli and APEC genomes was also analyzed showing that they are more prevalent in the latter.ConclusionsThe study has elucidated the role of these genes in APEC biofilm formation and compared them to adhesion expanding the knowledge and understanding of the economically significant pathogens.

Efficacy of zinc oxide and copper oxide nanoparticles on virulence genes of avian pathogenic E. coli (APEC) in broilers

BackgroundColibacillosis is one of the broilers’ most dominant bacterial diseases, either as a primary or a secondary infection. As E. coli antimicrobial drug resistance is rising; there is a need to develop new approaches to its control. In light of this, a comparative study of the in-vitro antibacterial activity of Arabic gum stabilized zinc and copper nanoparticles (AG-ZnNPs and AG-CuNPs) against PCR-identified field avian pathogenic E. coli (APEC) strains and virulence genes (ibeA, hlyA, iss, pap C and ompA) was applied to study the therapeutic effect of zinc and copper nanoparticles to be used as an antibiotic alternative (Nanobiotic). Furthermore, the in-vivo effects of CuNPs were evaluated. Additionally, the CuNPs liver and muscle residues with or without infection were examined. The eighty broilers were divided into four groups; G1: negative control, G2: infected control with E. coli O17, G3: non-infected treated (AG-CuNPs 50 mg/kg body weight), and G4: infected treated (AG-CuNPs 50 mg/kg body weight). AG-CuNPs treatment was given to broilers for five days in drinking water.ResultsE. coli was isolated from diseased broilers at an average incidence rate of 20% from intestinal and liver samples. All identified serotypes (O17, O78, O91, O121, and O159) were resistant to AG-ZnNPs and sensitive to AG-CuNPs. AG-CuNPs minimal inhibitory and bactericidal concentrations (MIC and MBC) for O17 were 7.5 and 60 mg/ml, respectively. Conventional uniplex PCR results showed that strain O17 contained virulence genes (ibeA, hlyA, iss, and papC), where AG-CuNPs significantly reduced the expression of all target genes when examined by Real-time quantitative PCR. Additionally, the bactericidal activity of AG-CuNPs on O17 was 100% at 20 minutes and 40 mg/ml and confirmed by transmission electron microscopy. Furthermore, no mortality was recorded in treated groups compared to G2. Subsequently, no E. coli was re-isolated from the liver in the G4 after treatment. The total protein, albumin, globulin, and lysozyme activity were significantly increased in G4 compared to G2, while the activities of liver enzymes (alanine aminotransferase (ALT), Gamma-glutamyl transferase (GGT), and alkaline phosphatase (ALP)) were markedly decreased in G4 compared to G2. Additionally, uric acid, creatinine, and C-reactive protein levels were decreased in G4 compared to G2. However, the liver enzymes, kidney functions, C-reactive protein levels, and Cu residues were non-significantly changed in G4 compared to G1.ConclusionGreen synthesized AG-CuNPs are recommended as an effective antimicrobial alternative against APEC strains.

Assessment of an Enterobactin Conjugate Vaccine in Layers to Protect Their Offspring from Colibacillosis.

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.

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.