Avian Pathogenic Escherichia Coli Pathogenicity Research Articles

Isolation and Characterization of Escherichia coli from Brazilian Broilers.

Avian pathogenic Escherichia coli (APEC) causes colibacillosis, one of the main diseases leading to economic losses in industrial poultry farming due to high morbidity and mortality and its role in the condemnation of chicken carcasses. This study aimed to isolate and characterize APEC obtained from necropsied chickens on Brazilian poultry farms. Samples from birds already necropsied by routine inspection were collected from 100 batches of broiler chickens from six Brazilian states between August and November 2021. Three femurs were collected per batch, and characteristic E. coli colonies were isolated on MacConkey agar and characterized by qualitative PCR for minimal predictive APEC genes, antimicrobial susceptibility testing, and whole genome sequencing to identify species, serogroups, virulence genes, and resistance genes. Phenotypic resistance indices revealed significant resistance to several antibiotics from different antimicrobial classes. The isolates harbored virulence genes linked to APEC pathogenicity, including adhesion, iron acquisition, serum resistance, and toxins. Aminoglycoside resistance genes were detected in 79.36% of isolates, 74.6% had sulfonamide resistance genes, 63.49% showed β-lactam resistance genes, and 49.2% possessed at least one tetracycline resistance gene. This study found a 58% prevalence of avian pathogenic E. coli in Brazilian poultry, with strains showing notable antimicrobial resistance to commonly used antibiotics.

Galleria mellonella larvae as an alternative model to determine the pathogenicity of avian pathogenic Escherichia coli

ABSTRACT The pathogenicity of avian pathogenic Escherichia coli (APEC) can be reliably determined using a biological model with 1-day-old chicks. However, public concerns regarding the use of vertebrates in research have led to the exploration of other biological models. Alternative infection models, including Galleria mellonella larvae, have also been studied. This study aimed to evaluate whether G. mellonella larvae are a viable in vivo model for evaluating the pathogenicity of APEC isolates and to determine the best protocol for this model. First, the pathogenicity index (PI) in vivo was determined for 40 APEC isolates from 1-day-old chicks. The same isolates were then used to establish the best protocol for the determination of PI in larvae of G. mellonella by determining the LD50, melanization, cytotoxicity test, total haemocyte count, mortality rate, construction of survival curves, and larval disease score. Cytotoxicity tests and total haemocyte counts were not suitable for distinguishing the pathogenicity groups. LD50 was a useful tool for differentiating between high and low pathogenicity but not for intermediate pathogenicity. Similarly, melanization could be used as a variable to characterize the pathogenicity of APEC isolates but should be associated with other variables. Mortality rates and survival curves were useful tools for characterizing nonpathogenic and highly pathogenic isolates. In this study, G. mellonella larvae were a viable model, and the larval disease score was the main variable that determined the pathogenicity of APEC isolates in the G. mellonella model. RESEARCH HIGHLIGHTS Galleria mellonella larvae are a viable model for determining APEC pathogenicity. Larval disease score is the main variable for determining APEC pathogenicity. Response variables should be evaluated up to 24 h post-inoculation.

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.

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 transcriptional regulator EtrA mediates ompW contributing to the pathogenicity of avian pathogenic Escherichia coli

Avian pathogenic Escherichia coli (APEC) causes avian colibacillosis and leads to high mortality in poultry and huge economic losses. Therefore, it is important to investigate the pathogenic mechanisms of APEC. Outer membrane protein OmpW is involved in the environmental adaptation and pathogenesis of Gram-negative bacteria. OmpW is regulated by many proteins, including FNR, ArcA, and NarL. In previous studies, regulator EtrA is involved in the pathogenicity of APEC and affects the transcript levels of ompW. However, the function of OmpW in APEC and its regulation remain unclear. In this study, we constructed mutant strains with altered etrA and/or ompW genes to evaluate the roles of EtrA and OmpW in the biological characteristics and pathogenicity of APEC. Compared with wild-type strain AE40, mutant strains ∆etrA, ∆ompW, and ∆etrA∆ompW showed significantly lower motility, lower survival under external environmental stress, and lower resistance to serum. Biofilm formation by ∆etrA and ∆etrA∆ompW was significantly enhanced relative to that of AE40. The transcript levels of TNF-α, IL1β, and IL6 were also significantly enhanced in DF-1 cells infected with these mutant strains. Animal infection assays showed that deletion of etrA and ompW genes attenuated the virulence of APEC in chick models, and damage to the trachea, heart, and liver caused by these mutant strains was attenuated relative to that caused by the wild-type strain. RT-qPCR and β-galactosidase assay showed that EtrA positively regulates the expression of the ompW gene. These findings demonstrate that regulator EtrA positively regulates the expression of OmpW, and that they both contribute to APEC motility, biofilm formation, serum resistance, and pathogenicity.

Biodistribution of 89Zr-DFO-labeled avian pathogenic Escherichia coli outer membrane vesicles by PET imaging in chickens

Avian pathogenic Escherichia coli (APEC) is a serious systemic infectious disease in poultry infections, causing severe economic losses to the poultry industry. Previous studies have shown that secretion of virulence proteins was required for the pathogenicity of APEC through the secretion system. Outer membrane vesicles (OMVs) are a generalized secretion system of Gram-negative bacteria that play a key role in the long-distance delivery of virulence factors, but whether they are associated with the pathogenic mechanism of APEC has not been determined. In this study, OMVs were purified and characterized from AE17 (O2 serotype) by ultracentrifugation and density gradient centrifugation and their protein cargo was identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, 89Zr was labeled after chelating AE17 OMVs by DFO and positron emission tomography PET imaging was used to track 89Zr-DFO-OMVs in chickens and to pathologically analyze the distribution sites. This study showed that AE17 OMVs were membrane vesicles ranging in size from 20 to 200 nm and proteomic analysis revealed the presence of virulence proteins, including adhesion proteins OmpA, OmpC, OmpF, OmpX, FimH, FimC and FigE, and serum resistance proteins OmpT and MliC and immune response regulator proteins (FliC). In addition, in vivo PET imaging to track the biodistribution of AE17 OMVs showed that AE17 OMVs were taken up by the lung region and the gastrointestinal and renal regions but were not detected in other areas. Pathological analysis of the tissue sites where AE17 OMVs were ingested showed inflammatory responses and damage. These findings suggested that AE17 OMVs not only contained a group of virulence proteins associated with AE17 infection but can also deliver these virulence proteins over long distances and caused tissue inflammatory damage. Our study revealed a previously unidentified causative microbial signal in the pathogenesis of APEC that could aid in the development of vaccines and antibiotics effective against APEC.

Effects of duck circovirus on immune function and secondary infection of Avian Pathogenic Escherichia coli

Duck circovirus (DuCV) infection occurs frequently in ducks in China and is generally believed to lead to immunosuppression and secondary infection, though there has been a lack of detailed research and direct evidence. In this study, one-day-old Cherry Valley ducklings were artificially infected with DuCV alone and co-infected with DuCV and Avian Pathogenic Escherichia coli (APEC). The immune indexes at 32 d old were systematically monitored, including immune organ weight, lymphocyte transformation rate, IL-10, IL-12, soluble CD4 (sCD4), soluble CD8 (sCD8), IFN-γ, viral loads in each organ, APEC colonization, and so on. The results showed the development of immune organs in ducklings was affected, resulting in a decrease in the lymphocyte transformation rate (LTR), IL-12, sCD4, sCD8, IFN-γ and an increase in IL-10 content at 8 to 32 d postinfection (dpi). In the detection of virus loads in some organs, it was found that 8 dpi, DuCV existed stably in various organs, suggesting the importance of preventing and controlling the virus in the early stage of culture. The results of exploring the DuCV infection that shows some influence on secondary infection by APEC. The results showed that DuCV infection could significantly enhance the pathogenicity of APEC and the colonization ability of APEC in vivo. DuCV can induce more serious APEC infection in 24 dpi than in 14 dpi. Based on the above results, it can be concluded that DuCV infection will affect the immune system, cause immunosuppression, and lead to more serious secondary infection.

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

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

The response regulator OmpR contributes to the pathogenicity of avian pathogenic Escherichia coli

Avian colibacillosis is a serious systemic infectious disease in poultry and caused by avian pathogenic Escherichia coli (APEC). Previous studies have shown that 2-component systems (TCSs) are involved in the pathogenicity of APEC. OmpR, a response regulator of OmpR/EnvZ TCS, plays an important role in E. coli K-12. However, whether OmpR correlates with APEC pathogenesis has not been established. In this study, we constructed an ompR gene mutant and complement strains by using the CRISPR-Cas9 system and found that the inactivation of the ompR gene attenuated bacterial motility, biofilm formation, and the production of curli. The resistance to environmental stress, serum sensitivity, adhesion, and invasion of DF-1 cells, and pathogenicity in chicks were all significantly reduced in the mutant strain AE17ΔompR. These phenotypes were restored in the complement strain AE17C-ompR. The qRT-PCR results showed that OmpR influences the expression of genes associated with the flagellum, biofilm formation, and virulence. These findings indicate that the regulator OmpR contributes to APEC pathogenicity by affecting the expression and function of virulence factors.

The Escherichia coli type III secretion system 2 Is involved in the biofilm formation and virulence of avian Pathogenic Escherichia coli

The Escherichia coli type III secretion system 2 (ETT2) is found in most pathogenic E. coli strains. Although many ETT2 gene clusters carry multiple genetic mutations or deletions, ETT2 is known to be involved in bacterial virulence. To date, no studies have been conducted on the role of ETT2 in the virulence of avian pathogenic Escherichia coli (APEC), which harbours ETT2. Thus, we deleted the ETT2 of APEC strain and evaluated the phenotypes and pathogenicities of the mutant. The results showed that deletion of ETT2 had no effect on APEC growth, but significantly promoted biofilm formation. In addition, as compared to the wild-type （WT） strain, the ETT2 deletion significantly promoted adherence to and invasion of DF-1 chicken fibroblasts and facilitated survival in the sera of specific-pathogen-free chickens. Analysis of the role of ETT2 in animal infection models demonstrated that the distribution of viable bacteria in the blood and organs of chicks infected with the ΔETT2 was significantly higher than those infected with WT. The results of RNA sequencing indicated that multiple genes involved in biofilm formation, lipopolysaccharide components, fimbrial genes and virulence effector proteins are regulated by ETT2. Collectively, these results implicated ETT2 is involved in the biofilm formation and pathogenicity of APEC.

ClpV1 in avian pathogenic Escherichia coli is a crucial virulence factor contributing to meningitis in a mouse model in vivo

Meningitis associated with avian pathogenic Escherichia coli (APEC) is an infectious disease of poultry that has gained significant attention because of its potential to infect humans. APEC can utilize two type Ⅵ secretion systems (T6SSs) to efficiently transport toxin effectors into hosts. ClpV1 is one of the core components of the T6SS1. To our knowledge, it has not been clarified how the clpV1 gene contributes to the pathogenicity of meningitis-associated APEC. To investigate the function of the clpV1 gene in the process of Escherichia coli meningitis, a mutant TW-XMΔclpV1 strain was constructed and characterized. In this study, the clpV1 deleted strain displayed a significant decrease in both motility and biofilm formation as well as a reduction in the expression of virulence genes fliC, luxS and ibeA. In vivo studies using mouse and duck models found that the clpV1 deleted groups showed decreased proliferation, fewer lesions and lower expression of inflammatory cytokines in the brain suggesting that clpV1 is involved in the pathogenicity of TWXM. Besides, the decreased quantity of Evans Blue (EB) and the down-regulation of tight junctions (TJs) proteins in the mouse clpV1 deleted group demonstrating a more intact blood-brain barrier (BBB). In conclusion, these results suggest that the clpV1 gene is associated with motility and biofilm formation of TWXM strain and contributes to meningitis by damaging the BBB and brain tissues.

The ETT2 transcriptional regulator EivF affects the serum resistance and pathogenicity of avian pathogenic Escherichia coli

Avian pathogenic Escherichia coli (APEC), a pathotype of extraintestinal pathogenic Escherichia coli (ExPEC), can cause serious systemic infectious diseases in poultry. Escherichia coli type III secretion system 2 (ETT2) is widely distributed in E. coli strains, including ExPEC and Enterohemorrhagic Escherichia coli (EHEC). The transcriptional regulator EivF, which is located at the ETT2 cluster, affects the secretion of LEE-encoded proteins and increases bacterial adhesion to human intestinal epithelial cells in EHEC O157:H7. In a previous study, we demonstrated the transcriptional regulator can affect APEC's motility and biofilm formation. Here, we evaluated whether EivF is involved in the pathogenicity of APEC, and we found that inactivation of eivF significantly enhanced resistance to the serum, adherence to chicken embryo fibroblast (DF-1) cells, and the colonization ability of APEC in chicks. To further clarify the regulation mechanism of transcriptional regulator EivF, we performed transcriptome sequencing to analyze the differentially expressed genes and pathways, showing that EivF regulates membrane, adhesion, environmental stress, and secretion protein genes, and EivF is involved in the localization, biological adhesion, biological regulation, membrane, and toxin activity. These findings indicated that the ETT2 transcriptional regulator EivF plays a crucial role in the pathogenicity of APEC as a negative repressor.

Flagellar rotor protein FliG is involved in the virulence of avian pathogenic Escherichia coli

Avian pathogenic Escherichia coli (APEC), a type of extraintestinal pathogenic E. coli, causes avian colibacillosis, a disease of significant economic importance to poultry producers worldwide, which is characterized by systemic infection. However, the pathogenesis of avian pathogenic E. coli strains is not well defined. Here, the role of a flagellar rotor protein encoded by the fliG gene of avian pathogenic E. coli strain AE17 was investigated. To study the role of FliG in the pathogenicity of APEC, fliG mutant and complemented strains were constructed and characterized. The inactivation of fliG had no effect on APEC growth, but significantly reduced bacterial motility. Compared with the wild type, the fliG mutant was highly attenuated in a chick infection model and showed severe defects in its adherence to and invasion of chicken embryo fibroblast DF-1 cells. The fliG mutant also showed reduced resistance to serum in chicks. The expression of the inflammatory cytokines interleukin 1β (IL1β), IL6, and IL8 was reduced in HD-11 macrophages infected with the fliG mutant strain compared with their expression in the wild-type strain. These results demonstrate that the FliG contributes to the virulence of APEC.

The role of Escherichia coli type III secretion system 2 chaperone protein ygeG in pathogenesis of avian pathogenic Escherichia coli

The Escherichia coli type III secretion system 2 (ETT2) is present in most E. coli strains, carries a 29.9-kb ETT2 pathogenicity island (PAI) and is involved in the virulence of avian pathogenic Escherichia coli (APEC). A chaperone protein is essential for the bacterial secretion system, but the function of the ETT2 chaperone protein has not been determined. This study showed that ygeG had sequence homology with the identified bacterial chaperone protein and it possessed tetratri-copeptide repeats (TPR) containing protein. To investigate the role of ygeG in the ETT2 of APEC, ygeG mutant and complemented strains were constructed and characterized. Inactivation of ygeG had no effect on APEC growth, but significantly promoted biofilm formation, and the adherence to and invasion of DF-1 cells, especially the survival abilities in specific-pathogen-free (SPF) chicken sera serum. Analysis of the role of ygeG in chicken infection models revealed that the deletion of ygeG increased bacterial virulence. RNA Sequencing (RNA-Seq) analyses comparing the APEC wild type and the ygeG mutant indicated that multiple genes encoding biofilm formation, outer membrane proteins, fimbrial genes and virulence effector protein genes were regulated by ygeG. These results revealed the role of ygeG as a chaperone protein that affected the virulence and pathogenicity of APEC.

The two-component system, BasSR, is involved in the regulation of biofilm and virulence in avian pathogenic Escherichia coli

ABSTRACT Avian pathogenic Escherichia coli (APEC) is a subgroup of extra-intestinal pathogenic E. coli (ExPEC) strains that cause avian colibacillosis, resulting in significant economic losses to the poultry industry worldwide. It has been reported that a few two-component signal transduction systems (TCS) participate in the regulation of the virulence factors of APEC infection. In this study, a basSR-deficient mutant strain was constructed from its parent strain APECX40 (WT), and high-throughput sequencing (RNA-seq) was performed to analyse the transcriptional profile of WT and its mutant strain XY1. Results showed that the deletion of basSR down-regulated the transcript levels of a series of biofilm- and virulence-related genes. Results of biofilm formation assays and bird model experiments indicated that the deletion of basSR inhibited biofilm formation in vitro and decreased bacterial virulence and colonization in vivo. In addition, electrophoretic mobility shift assays confirmed that the BasR protein could bind to the promoter regions of several biofilm- and virulence-related genes, including ais, opgC and fepA. This study suggests that the BasSR TCS might be a global regulator in the pathogenesis of APEC infection. RESEARCH HIGHLIGHTS Transcriptional profiling showed that BasSR might be a global regulator in APEC. BasSR increases APEC pathogenicity in vivo. BasSR positively regulates biofilm- and the virulence-associated genes. BasSR can bind to the promoter regions of virulence-associated genes ais, opgC and fepA.

Hcp2a of type VI secretion system contributes to IL8 and IL1β expression of chicken tracheal epithelium by affecting APEC colonization

Avian pathogenic Escherichia coli (APEC) is an important pathogen that causes avian colibacillosis in poultry. APEC infection can lead to pathological changes in chicken trachea. The type VI secretion system (T6SS) of APEC contribute to the pathogenicity of APEC. However, whether T6SS plays a role in infection of the trachea remains unclear. We constructed mutant strain Δhcp2a by the Red recombination method system. The role of hcp2a (the structural secretion components and secretory protein of the T6SS) in the infection of trachea was investigated. The mutation strain displayed a significant increase in biofilm formation and a decrease in resistance to chicken serum. Moreover, RNA sequencing analyses showed that infection of chicken tracheal epithelium by the mutant strain Δhcp2a induced differential expression of genes. The result also showed that 14 genes (13 genes were downregulated) were enriched in cytokine-cytokine receptor interaction signalling pathway at 12 and 24 h post infection. The mutation Δhcp2a resulted in significant decreases in the bacterial loads in trachea at 6 and 12 h post infection. Real-time PCR analyses showed that the hcp2a mutation downregulated the expression of IL8 and IL1β at mRNA level in chicken tracheal epithelium. Our results indicate that mutation of hcp2a influenced genes expression of the cytokine-cytokine receptor interaction pathway by decreasing APEC colonization in the trachea.

The transcriptional regulator PhoP mediates the tolC molecular mechanism on APEC biofilm formation and pathogenicity

ABSTRACTAs a transcriptional regulator of the classical binary regulatory system, PhoP plays an important role in the life activities of avian pathogenic Escherichia coli (APEC). In previous experiments, we found that the absence of phoP affects APEC biofilm formation and pathogenicity. To further explore the molecular mechanism of phoP regulation of these phenomena, the differentially expressed gene tolC was screened based on phoP-derived transcriptional data, and the specific sequence identity of the PhoP binding sequence was predicted by bioinformatics and verified by electrophoretic mobility shift assay (EMSA). The results showed that PhoP can directly bind to the tolC promoter. On this basis, tolC deletion and complementary strains were constructed. Biofilm formation was quantified by crystal violet staining and rdar morphology change was observed in these strains. Loss of tolC reduced biofilm formation. We also examined pathological changes in organ paraffin sections by challenging chicks with the strains. After loss of tolC, the clinical signs of pericarditis and liver and spleen enlargement in chicks were alleviated, and pathogenicity to the host cells was decreased. Additionally, quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that tolC deletion downregulated secA/secB/secE/secY transcript levels, which are part of the type II secretion system secreting virulence effector element. These results indicate that tolC contributes to the phoP-mediated effect on APEC biofilm formation and pathogenicity.

Anti-bacterial activity of baicalin against APEC through inhibition of quorum sensing and inflammatory responses

Avian pathogenic Escherichia coli (APEC), collectively known as causative agent of extraintestinal infections, is an important cause of morbidity and mortality in poultry. Currently, quorum sensing (QS), biofilm formation and virulence factors are considered as novel prospective targets for antimicrobial therapy to control APEC invasion. In addition, inflammatory responses are also served as the major pathological features of APEC invasion. This study was aimed to explore the effect of baicalin on APEC and APEC-induced inflammatory responses. After treatment with baicalin, we mainly examined the AI-2 secretion, biofilm formation, expression of virulence genes of APEC, and the levels of inflammatory cytokines, as well as the expression of NF-κB pathway. Our results showed that baicalin significantly inhibited the QS via decreasing the AI-2 secretion, biofilm formation, and the expression of virulence genes of APEC such as LsrB, LsrK, LuxS, pfs, H-NS, fimA, fimB, fyuA, csgA, csgB, and rpoS. Moreover, baicalin significantly attenuated the release of lactate dehydrogenase (LDH), and the adhesion of APEC to chicken type II pneumocytes to reduce cell damage. Furthermore, baicalin also inhibited the expression of pro-inflammatory cytokines and NF-κB activation. Thus, our data revealed that baicalin could interfere with the quorum sensing, biofilm formation and virulence genes expression to relieve the APEC pathogenicity. Additionally, baicalin decreased the inflammatory responses of chicken type II pneumocytes induced by APEC. Taken together, these data provide a novel potential pharmaco-therapeutic approach to chicken colibacillosis.

Role of hypothetical protein YicS in the pathogenicity of Avian Pathogenic Escherichia coli in vivo and in vitro

Avian Pathogenic Escherichia coli (APEC) strains belong to the extra-intestinal pathogenic group of E. coli (ExPEC) that causes colibacillosis in poultry. A variety of putative virulence factors of APEC are recognized as potent causes of pathogenicity, the mechanisms underlying their pathogenicity are still not fully understood. The role of yicS in the virulence of pathogenic E. coli is still unclear. Thus, yicS may be related to biofilm formation, which in some bacteria plays a role in pathogenicity. Therefore, the fact that this gene appears to be under positive selection pressure suggests that yicS may be associated with the pathogenicity of APEC. To better understand the role of yicS protein in APEC biological characteristics and pathogenicity, we deleted yicS in an APEC Swollen Head Syndrome strain (APEC strain SCI-07) and studied its effects by comparing wild type and isogenic mutants through comprehensive in vitro and in vivo assays. We demonstrated that yicS plays a role in pathogenicity of APEC. We suggest that the yicS gene, which encodes an exporter protein, has a significant role in biofilm formation, motility, invasion of CEC-32 and Hep-2 cells and APEC pathogenicity in a day-old chick model.

FNR Regulates the Expression of Important Virulence Factors Contributing to the Pathogenicity of Avian Pathogenic Escherichia coli.

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.