Avian Pathogenic Escherichia Coli Strains Research Articles

First report of mobile colistin resistance gene mcr-1 in avian pathogenic Escherichia coli isolated from turkeys in the Gaza Strip, Palestine.

Colistin is used to treat avian pathogenic Escherichia coli (APEC), a microorganism that affects turkey meat production in the Gaza Strip and worldwide. However, the recent emergence of plasmid-borne mobile colistin resistance (mcr) genes in pathogenic E. coli strains is a serious antimicrobial resistance (AMR) challenge for both human and animal health. In December 2018, colistin was banned as a veterinary antimicrobial in the Gaza Strip. This study aimed to detect and track the prevalence of colistin-resistant APEC isolated from turkey flocks in the Gaza Strip. This study investigated 239 APEC isolates from turkey flocks in the Gaza Strip between October 2018 and December 2021 (at 6-month intervals). The colistin-resistant APEC strains were detected using the broth microdilution method. The mcr-1 gene was identified using a polymerase chain reaction. The overall colistin resistance among the isolated APECs was 32.2% during the study period. The average resistance in the first interval was 37.5%, which significantly decreased to 9.3% in the last interval. Among the 77 phenotypically resistant isolates, 32.4% were positive for mcr-1. The average abundance of mcr-1 in the first interval was 66.6%, which decreased to 25% in the last interval. To the best of our knowledge, this is the first study reporting the presence of the mcr-1 gene among the APEC isolates from turkeys in the Gaza Strip. Banned veterinary use of colistin significantly decreased the percentage of resistant APEC isolates from turkeys in Gaza Strip. Further studies are needed to investigate other colistin resistance genes and track the emergence of AMR.

Bird immunobiological parameters in the dissemination of the biofilm-forming bacteria Escherichia coli.

With the development of industrial maintenance technology, a group of pathogens called avian pathogenic Escherichia coli (APEC) became very common. The initiation, development, and outcome of the infectious process mediated by virulent APEC strains occur through a decrease in the colonization resistance of the intestine, an immunobiological marker of homeostasis stability in susceptible species. This study focused on the pathogenetic features of colibacillosis and the morphological features of E. coli. Clinical, immunological, bacteriological, and histological studies were conducted on 15-day-old white Leghorn birds (n = 20). The birds were divided into two groups: Control group (Group I; n = 10) with birds intranasally inoculated with 0.5 mL of 0.9% NaCl solution and experimental group (Group II; n = 10) with birds intranasally inoculated with 0.5 mL of an E. coli suspension at 1 billion/mL. During the biofilm formation, clusters of microcolonies were formed as a gel-like intercellular matrix that accumulated due to cell coagulation. The intercellular matrix "glues" heteromorphic cells together and forms a structure of densely packed heteromorphic cells arranged in an orderly manner and growing in different directions. During the experimental reproduction of E. coli, excessive growth was observed in material isolated from poultry. Pathogenic E. coli strains implementing virulence factors adhered to the receptors of erythrocytes, alveolocytes, and enterocytes. Multicellular heterogeneous biofilms, united by an intercellular matrix, were located at the apical poles of the respiratory tract alveolocytes and enterocytes of the terminal ileum villi. Many bacteria exudate containing desquamated epithelial cells with an admixture of mucus, and polymorphonuclear leukocytes were detected in the lumen of the birds' abdominal organs. Invasive bacteria damaged the epithelial layer, violated the endothelial layer of blood vessels, and developed inflammatory hyperemia of the lamina propria of the respiratory and digestive systems' mucous membrane. A correlative dependence of changes developed by the type of delayed hypersensitivity reaction was established. Signs of accidental transformation of the thymus, atrophy of the bursa of Fabricius, disseminated thrombosis, and septic spleen developed. Moreover, toxic cardiomyocyte dystrophy, signs of congestive vascular hyperemia, massive disintegration of lymphocytes, macrophage reactions, perivascular edema resulting from the release of plasma, and shaped blood elements were detected. The development and outcome of the infectious process in escherichiosis primarily depend on the homeostasis stability of susceptible species and virulence factors of the pathogenic microorganisms. One of the selected strains, E. coli O78:K80 displayed the highest ability to form biofilms. Its strong adhesion ability to bird erythrocytes was demonstrated. Deepening the scientific knowledge of the interaction between eukaryotes and prokaryotes will contribute to a better understanding of the pathogenetic aspects of avian escherichiosis and eventually find promising anti-adhesive drugs that could reduce primary bacterial contamination in vivo and in vitro.

Characterization of a lytic Escherichia coli phage CE1 and its potential use in therapy against avian pathogenic Escherichia coli infections.

The high incidence of Avian pathogenic Escherichia coli (APEC) in poultry has resulted in significant economic losses. It has become necessary to find alternatives to antibiotics due to the alarming rise in antibiotic resistance. Phage therapy has shown promising results in numerous studies. In the current study, a lytic phage vB_EcoM_CE1 (short for CE1) against Escherichia coli (E. coli) was isolated from broiler feces, showing a relatively wide host range and lysing 56.9% (33/58) of high pathogenic strains of APEC. According to morphological observations and phylogenetic analysis, phage CE1 belongs to the Tequatrovirus genus, Straboviridae family, containing an icosahedral capsid (80 ~ 100 nm in diameter) and a retractable tail (120 nm in length). This phage was stable below 60°C for 1 h over the pH range of 4 to 10. Whole-genome sequencing revealed that phage CE1 contained a linear double-stranded DNA genome spanning 167,955 bp with a GC content of 35.4%. A total of 271 ORFs and 8 tRNAs were identified. There was no evidence of virulence genes, drug-resistance genes, or lysogeny genes in the genome. The in vitro test showed high bactericidal activity of phage CE1 against E. coli at a wide range of MOIs, and good air and water disinfectant properties. Phage CE1 showed perfect protection against broilers challenged with APEC strain in vivo. This study provides some basic information for further research into treating colibacillosis, or killing E. coli in breeding environments.

Characterization of Avian Pathogenic Escherichia coli Isolated from Broiler Breeders with Colibacillosis in Mississippi

Avian pathogenic Escherichia coli (APEC) causes colibacillosis in poultry, a leading cause of poultry mortality worldwide. It is crucial to control APEC in broiler breeders as it is vertically transferred to progeny via eggs. However, there is only limited knowledge on the current APEC population in breeders. This study characterized 28 APEC strains isolated from broiler breeders with colibacillosis. The genotypic-virulence characteristics as well as antimicrobial and heavy-metal resistance patterns of the isolates were determined. Results showed that O88 is the most prevalent serogroup and B2 is the predominant phylogenetic group. Among virulence genes, genes for iron acquisition (iroN and iutA), protectins (iss and ompT), and toxin production (hlyF) exhibited the highest prevalence. Further, 93% of the isolates carried at least one antimicrobial resistance gene with highest prevalence for tetracycline gene tetA. Among the isolates, 10.71% exhibited multidrug resistance. All isolates carried at least one heavy-metal resistance gene with the highest prevalence for arsenic gene arsC and the highest resistance towards silver. Our findings provide insight into the characteristics of current APEC populations in broiler breeders in Mississippi. This will help future research on the pathogenesis of APEC and the development of effective prevention and control strategies against APEC in broiler breeders.

Evaluation of immunogenicity and efficacy of the enterobactin conjugate vaccine in protecting chickens from colibacillosis.

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 (100μg per bird) at 7days of age, followed by booster immunization at 21days of age. The chickens were intratracheally challenged with an APEC strain (108 CFU/bird) or PBS at 28days of age. At 5days 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.

Prevalensi Strain Avian Pathogenik Escherichia coli (APEC) Penyebab Kolibasilosis pada Burung Puyuh

PREVALENCE OF AVIAN PATHOGENIC Escherichia coli (APEC) STRAINS ON COLIBACILLOSIS IN QUAIL Wahyu Prihtiyantoro1), Khusnan1), Mitra Slipranata2)† dan Imron Rosyidi2) 1 Akademi Peternakan Brahmaputra, Jl. Ki Ageng Pemanahan, Nitikan, Sorosutan Umbulharjo Yogyakarta,Tlp. 0274-384370, E-mail: wahyumatra@gmail.com 2 Fakultas Kedokteran Hewan, Universitas Gadjah Mada, Jl. Fauna 2, Karangmalang, Yogyakarta 55281 ABSTRACTAvian Pathogenic Escherichia coli (APEC) is a pathogen that causes colibacillosis in poultry, including salpingitis, omphalitis, cellulitis, swollen head syndrome, coligranuloma yolk sac inflammation, and air sacs inflammation. APEC is a zoonotic strain which spread through raw meat and processed meat products of animals and birds. In this research, the isolation and the identification of Escherichia coli were done by using selective media MacConkey, Kligger Iron Agar, and Gram staining. To analyse genetopically, polymerase chain reaction (PCR) was used to detect 16SrRNA genes, vt1 genes, and vt2 genes. Thirty one (55,36%) isolates of 56 specimens collected from quail were detected as Escherichia coli. The detection of APEC strains towards 31 Escherichia coli isolates were done by using polymerase chain reaction (PCR) with vt1 and vt2 specific primer. From the results showed that 32,26% (10/31) was APEC strains and 67.74% was non-APEC strains. From 10 isolates, 90% had vt1 gene and 10% had vt2 gene. Escherichia coli isolates were found in eyes (32,26%), infraorbital sinus fluid (32,26%), nasal fluid (16,20%), also in lungs, air sacs, ascites, and heart for 3,2% each. The isolates could not be found in the specimens from the skull. These isolates are zoonotic and have an impact on human health.Keywords: APEC, colibacillosis, quail, vt1 and vt2

The effect of a spontaneous induction prophage, phi458, on biofilm formation and virulence in avian pathogenic Escherichia coli.

Prophage sequences are present in most bacterial genomes and account for up to 20% of its host genome. Integration of temperate phages may have an impact on the expression of host genes, while some prophages could turn into the lytic cycle and affect bacterial host biological characteristics. We investigated the role of spontaneous induction prophages in avian pathogenic Escherichia coli (APEC), which is the causative agent of avian colibacillosis in poultry, and considered a potential zoonotic bacterium related to the fact it serves as an armory of extraintestinal pathogenic E. coli. We found that APEC strain DE458 had a high spontaneous induction rate in vivo and in vitro. The released phage particles, phi458, were isolated, purified, and sequenced, and the deletion mutant, DE458Δphi458, was constructed and characterized. Biofilm formation of DE458Δphi458 was strongly decreased compared to that of the wild-type strain (p < 0.01). In addition, while the addition of DNase (100 μg/ml) did not affect prophage release but could digest eDNA, it significantly reduced the biofilm production of DE458 biofilm to a level close to that of DE458Δphi458. Compared to DE458, the adhesion and invasion abilities of DE458Δphi458 increased by approximately 6-20 times (p < 0.05). The virulence of DE458Δphi458 was enhanced by approximately 10-fold in chickens based on a 50% lethal dose. Furthermore, avian infection assays showed that the bacterial loads of DE458Δphi458 in the lung and liver were increased by 16.5- and 10-fold (p < 0.05), respectively, compared with those of the WT strain. The qRT-PCR revealed that deletion of phi458 led to upregulation of type I fimbriate-related gene fimH and curli-related gene csgC by 3- and 2.8-fold, respectively (p < 0.01). Our study revealed that phi458 promoted biofilm formation by spontaneously inducing and decreasing virulence by repressing virulence genes.

Occurrence, antimicrobial resistance, and potential zoonosis risk of avian pathogenic Escherichia coli in Indonesia: A review

Avian pathogenic Escherichia coli (APEC) causes avian colibacillosis or colibacillosis and is a major endemic disease of poultry worldwide, including in Indonesia. It is characterized by a black proventriculus and can damage other organs, leading to pericarditis, perihepatitis, water sacculitis, mesenteritis, and omphalitis. The APEC strain is one of the six main sub-pathotypes of the extraintestinal pathogenic E. coli (ExPEC) pathotype. The relationship between APEC and infection in humans is questionable. The previous studies have suggested poultry products, including meat and eggs, as a potential source of infection for the transmission of ExPEC disease to humans. Due to the absence of reporting of disease incidents and the lack of literature updates on this disease, it seems as if APEC does not exist in Indonesia. Since bacterial resistance is a growing problem in Indonesia, and globally, the World Health Organization issued a statement regarding the importance of assessing related factors and their control strategies. Antimicrobial resistance, especially multidrug resistance, presents a challenge when treating infectious diseases. In Indonesia, the incidence of resistance to several antimicrobials in cases of avian colibacillosis is high. In addition, avian and human extraintestinal E. coli infections present a potential zoonotic risk. Furthermore, a relationship exists between antibiotic resistance to foodborne bacteria and the occurrence of antibiotic resistance in humans, so the use of antibiotics in the poultry industry must be controlled. Therefore, the One Health strategy should be implemented to prevent the overuse or misuse of antibiotics in the poultry industry. This review aimed to increase awareness of people who are at risk of getting Avian pathogenic Escherichia coli (APEC) from poultry by controlling the spread of APEC by maintaining a clean environment and hygienic personnel in poultry farms.

Modification of avian pathogenic Escherichia coli χ7122 lipopolysaccharide increases accessibility to glycoconjugate antigens

BackgroundWorldwide, an estimated 70.7 billion broilers were produced in 2020. With the reduction in use of prophylactic antibiotics as a result of consumer pressure and regulatory oversight alternative approaches, such as vaccination, are required to control bacterial infections. A potential way to produce a multivalent vaccine is via the generation of a glycoconjugate vaccine which consists of an antigenic protein covalently linked to an immunogenic carbohydrate. Protein-glycan coupling technology (PGCT) is an approach to generate glycoconjugates using enzymes that can couple proteins and glycan when produced in bacterial cells. Previous studies have used PGCT to generate a live-attenuated avian pathogenic Escherichia coli (APEC) strain capable of N-glycosylation of target proteins using a chromosomally integrated Campylobacter jejuni pgl locus. However, this proved ineffective against C. jejuni challenge.ResultsIn this study we demonstrate the lack of surface exposure of glycosylated protein in APEC strain χ7122 carrying the pgl locus. Furthermore, we hypothesise that this may be due to the complex cell-surface architecture of E. coli. To this end, we removed the lipopolysaccharide O-antigen of APEC χ7122 pgl+ via deletion of the wecA gene and demonstrate increased surface exposure of glycosylated antigens (NetB and FlpA) in this strain. We hypothesise that increasing the surface expression of the glycosylated protein would increase the chance of host immune cells being exposed to the glycoconjugate, and therefore the generation of an efficacious immune response would be more likely.ConclusionsOur results demonstrate an increase in cell surface exposure and therefore accessibility of glycosylated antigens upon removal of lipopolysaccharide antigen from the APEC cell surface.

Efficacy of passive immunization in broiler chicks via an inactivated Escherichia coli autogenous vaccine administered to broiler breeder hens

ABSTRACT Avian pathogenic Escherichia coli (APEC) cause extra-intestinal infections called colibacillosis, which is the dominant bacterial disease in broilers. To date, given the diversity of APEC strains and the need for an acceptable level of protection in day-old chicks, no satisfactory commercial vaccine is available. As part of a French nationwide project, we selected three representative strains among several hundred APEC that cause colibacillosis disease. We first performed experiments to develop colibacillosis in vivo models, using an inoculum of 3 × 107 CFU of each E. coli strain per chick. Two APEC strains (19-381 and 19-383-M1) were found to be highly virulent for day-old chicks, whereas the third strain (19-385-M1) induced no mortality nor morbidity. We then produced an autogenous vaccine using the (Llyod, 1982; MaCQueen, 1967) 19–381 and 19-383-M1 APEC strains and a passive immunization trial was undertaken. Specific-pathogen-free Leghorn hens were vaccinated twice 2 weeks apart, the control group receiving a saline solution. The vaccinated and control hens exhibited no clinical signs, and egg production and fertility of both groups were similar. Fertile eggs were collected for 2 weeks after the second vaccination and chicks were obtained. After challenge with each APEC (19-381 and 19-383-M1), chicks appeared to be partially protected from infection with the 19-383-M1 strain, with 40% mortality compared with 80% for the non-vaccinated chicks. No protection was found when the chicks were challenged with the 19–381 strain. Now, further work is needed to consider some aspects: severity of the pathogen challenge model, persistence of the protection, number of APEC strains in the autogenous vaccine, choice of adjuvants, and heterologous protection by the vaccine made from strain 19-383-M1. RESEARCH HIGHLIGHTS Three APEC strains were characterized and selected to develop in vivo models of colibacillosis. A bivalent autogenous vaccine was produced and a passive immunization trial was carried out. Protection of chicks was demonstrated when challenged with the 19-383-M1 APEC strain (homologous challenge). Further work is needed in particular to evaluate the protection against heterologous challenge.

Virulence Factors and Antimicrobial Resistance Profile of Escherichia Coli Isolated from Laying Hens in Italy.

Simple SummaryColibacillosis is a disease of great importance in the poultry industry, but many of its features and characteristics still need to be identified. This survey on avian Escherichia coli investigated the correlation between the presence of specific virulence genes, antimicrobial resistance features and serogroups. The results highlighted that half of the tested strains were avian pathogenic Escherichia coli (APEC). Moreover, a high prevalence of two specific serogroups was detected, namely, O2 and O88. Finally, antimicrobial resistance was lower than in other studies. Further investigations of APEC strains’ antimicrobial resistance features would support farmers, veterinarians and local authorities in planning actions for a better control of colibacillosis in poultry production.Colibacillosis is the most common bacterial disease in the poultry industry. The isolation of Escherichia coli (E. coli) strains with multiple resistance to various classes of antimicrobials has been increasing in recent years. In this study, antimicrobial resistance features, serotyping and the presence of avian pathogenic Escherichia coli (APEC) virulence genes were investigated on a total of 71 E. coli strains isolated during outbreaks of colibacillosis in laying hens. The correlation between these features was evaluated. The most frequently isolated serogroups were O2 and O88. Resistance was often detected with nalidixic acid (49%) and ampicillin (38%), while all strains were sensitive to ceftiofur and florfenicol. Overall, 25% of the isolates showed resistance to at least three or more antimicrobial classes (multidrug-resistant strains), and 56% of the isolates were defined as APEC strains (due to the presence of at least five virulence genes). Correlation between the different parameters (virulence genes, serogroup and antimicrobial resistance) did not reveal relevant associations. The comparison of the obtained results with those of similar studies highlighted the importance of continuous monitoring in order to have a better understanding of colibacillosis. An evaluation of the national epidemiological situation would allow, especially with regard to antimicrobial resistance, to focus on the right measures in order to prioritize the available resources for effective disease control.

Identification of novel biofilm genes in avian pathogenic Escherichia coli by Tn5 transposon mutant library.

Avian pathogenic Escherichia coli (APEC) is the main pathogens that inflict the poultry industry. Biofilm as the pathogenic factors of APEC, which can enhance the anti-host immune system of APEC and improve its survival in the environment. In order to screen for new genes related to APEC biofilm. The APEC strain APEC81 was used to construct a mutant library by Tn5 insertion mutagenesis. Moreover the 28 mutant strains with severely weakened biofilm were successfully screened from 1500 mutant strains by crystal violet staining, in which 17 genes were obtained by high-efficiency thermal asymmetric interlaced PCR. The reported genes include 3 flagella genes (fliS, fliD, andfliR), 4 curli fimbriae genes (csgD, csgA, csgF, andcsgG) and 3 type 1 fimbriae genes (fimA, fimD, andfimC). The novel genes include 3 coenzyme genes (gltA, bglX, and mltF) and 4 putative protein genes (yehE, 07045, 11735, 11255). To investigate whether these 17 genes co-regulate the biofilm, the 17 identified genes were deleted from APEC strain APEC81. The results showed that except for the 11735 and 11255 genes, the deletion of 15 genes significantly reduced the biofilm formation ability of APEC81 (P < 0.05). The result of rdar (red, dry and rough) colony morphology showed that curli fimbriae genes (csgD, csgA, csgF, andcsgG) and other functional genes (fimC, glxK, yehE, 07045, and11255) affected the colony morphology. In particular, the hypothetical protein YehE had the greatest influence on the biofilm. It was predicted to have the same structure as the type 1 fimbria protein. When yehE was deleted, the fimE transcription was up-regulated, and the fimA and fimB transcription were down-regulated, resulting in a decrease in type 1 fimbriae. Hence, the yehE mutant significantly reduced the biofilm and the adhesion and invasion ability to cells (P < 0.05). This study identified 5 novel genes (gltA, bglX, mltF, yehE, and07045) related to biofilm formation and confirmed that yehE affects biofilm formation by type 1 fimbriae, which will benefit further study of the mechanism of biofilm regulation in APEC.

Genomic characteristics, virulence, and antimicrobial resistance in avian pathogenic Escherichia coli MTR_BAU02 strain isolated from layer farm in Bangladesh

Colibacillosis, caused by avian pathogenic Escherichia coli (APEC), is one of the most significant infectious diseases affecting poultry worldwide. This study aimed to determine the genomic diversity, virulence factor genes (VFGs), and antimicrobial resistance genes (ARGs) in the APEC MTR_BAU02 strain isolated from a layer chicken using whole-genome sequencing (WGS). Paired-end (2×250) WGS was performed using Illumina MiSeq sequencer (Illumina, San Diego, CA) and de novo assembly was performed using SPAdes. Core genome multilocus sequence typing (cgMLST) analysis between APEC MTR_BAU02 and all of the ST1196 E. coli strains retrieved from the National Center for Biotechnology Information (NCBI) GenBank database was performed using the BacWGSTdb 2.0 server. We utilized different databases to detect ARGs, VFGs, and genomic functional features of the APEC MTR_BAU02 strain. The complete genome of APEC MTR_BAU02 consists of 94 contigs comprising 4,924,680 bp (51.1% guanine-cytosine [GC] content), including 4681 protein-coding sequences, one chromosome, and one plasmid, and was assigned to ST1196. The closest relatives of APEC MTR_BAU02 were four isolates originating from human clinical specimens (diarrhetic stool) in Bangladesh and two clinical isolates originating from chicken in India, which differed by 694 core genome multilocus sequence typing (cgMLST) alleles. One hundred and twenty-two ARGs and 92 VFGs were identified in the APEC MTR_BAU02 genome. Metabolic functional annotations detected 380 SEED subsystems including genes coding for carbohydrate metabolism, protein metabolism, cofactors, vitamins, prosthetic groups and pigments, respiration, membrane transport, stress response, motility and chemotaxis, and virulence, disease, and defense. This study reports the genome sequence of a multidrug-resistant APEC strain isolated from layer birds in Bangladesh. The ARGs and VFGs, widespread in APEC MTR_BAU02, are similar to those found in human isolates, and highlight the growing threat of antimicrobial resistance in both poultry and humans.

Effect of nutritional and environmental conditions on biofilm formation of avian pathogenic Escherichia coli.

To study the effects of environmental stress and nutrient conditions on biofilm formation of avian pathogenic Escherichia coli (APEC). The APEC strain DE17 was used to study biofilm formation under various conditions of environmental stress (including different temperatures, pH, metal ions, and antibiotics) and nutrient conditions (Luria-Bertani [LB] and M9 media, with the addition of different carbohydrates, if necessary). The DE17 biofilm formation ability was strongest at 25°C in LB medium. Compared to incubation at 37°C, three biofilm-related genes (csgD, dgcC, and pfs) were significantly upregulated and two genes (flhC and flhD) were downregulated at 25°C, which resulted in decreased motility. However, biofilm formation was strongest in M9 medium supplemented with glucose at 37°C, and the number of live bacteria was the highest as determined by confocal laser scanning microscopy. The bacteria in the biofilm were surrounded by a thick extracellular matrix, and honeycomb-like or rough surfaces were observed by scanning electron microscopy. Moreover, biofilm formation of the DE17 strain was remarkably inhibited under acidic conditions, whereas neutral and alkaline conditions were more suitable for biofilm formation. Biofilm formation was also inhibited at specific concentrations of cations (Na+ , K+ , Ca2+ , and Mg2+ ) and antibiotics (ampicillin, chloramphenicol, kanamycin, and spectinomycin). The real-time quantitative reverse transcription PCR showed that the transcription levels of biofilm-related genes change under different environmental conditions. Nutritional and environmental factors played an important role in DE17 biofilm development. The transcription levels of biofilm-related genes changed under different environmental and nutrient conditions. The findings suggest that nutritional and environmental factors play an important role in APEC biofilm development. Depending on the different conditions involved in this study, it can serve as a guide to treating biofilm-related infections and to eliminating biofilms from the environment.

Investigation of the impact of food preservatives on avian pathogenicEscherichia coli(APEC) and their role in driving zoonotic disease

The excessive use of antibiotics in agriculture is routinely described as a major contributor to bacterial antimicrobial resistance. Globally, antibiotics are also widely used as growth supplements in livestock. This has led to concerns regarding use of human-use antibiotics in food and food-producing animals. In more recent times organic acids such as propionic acid (PA) and formic acid (FA) have been used as alternative antimicrobials or preservatives in place of antibiotics. PA is a short chain fatty acid naturally abundant in the human and animal intestine as a breakdown product of non-digestible carbohydrates. In the human intestine it plays important roles in regulating the immune response in the human body. Recently, a study has shown that exposure of a Crohn’s Disease associated bacterial pathotype, adherent-invasive Escherichia coli(AIEC),to PA significantly altered its phenotype resulting in increased adhesion and invasion of epithelial cells and increased persistence through biofilm-formation. AIEC are both evolutionarily and phylogenetically related to avian pathogenicEscherichia coli (APEC). PA and FA use is widespread in chickens a known source of zoonotic disease. Our results indicate that virulence of some APEC strains is increased by exposure to alternative antimicrobials such as PA and FA. This included increased adhesion of APEC strain E. coli 601 to human intestinal epithelial cells after exposure to FA which could be a potential risk of zoonotic disease. Further investigation of APEC strains is currently underway in a fermentation model of the poultry gut. This approach will improve our understanding of how commonly used.

Phenotypic antimicrobial resistance (AMR) of avian pathogenic Escherichia coli (APEC) from broiler breeder flocks between 2009 and 2018

Colibacillosis is one of the most important diseases in poultry production. The use of antimicrobials remains a therapeutic cornerstone for avian pathogenic E. coli (APEC), thereby contributing to the development of antimicrobial resistance (AMR). The aim of this study was to characterize AMR in broiler breeder flocks reared under commercial conditions. Data covering ten years, from 2009 to 2018, were used to evaluate the phenotypic AMR of 264 APEC obtained from 158 broiler breeder flocks of a large company in Portugal. The APEC isolates were tested against eleven antimicrobials by the Kirby-Bauer disk diffusion test. The annual proportion of AMR was calculated by dividing the number of APEC with phenotypic resistance by the total number of APEC isolated that year. Similarly, the overall AMR of the whole period was calculated. The relationship of antimicrobial resistance with time (years) was investigated with a generalized linear model using logistic regression. The overall AMR of the 10-year period was: amoxicillin 78%, ampicillin 73.5%, tetracycline 63.3%, doxycycline 56.4%, apramycin 34.5%, neomycin 68.2%, enrofloxacine 32.6%, flumequine 39.4%, co-trimoxazole 47.7%, florfenicol 46.6% and lincospectin 66.3%. Over time, a significant decrease in AMR was observed for amoxicillin and ampicillin, neomycin, flumequine, co-trimoxazole, florfenicol and lincospectin. Multidrug resistance (MDR) decreased from 100% in 2009 to 48% in 2018. Only 7 (2.7%) APEC strains were fully susceptible to all tested antimicrobials. The decrease over time of AMR in APEC likely reflects the efficacy of manifold improvements in broiler breeder production systems. A further reduction in AMR is still desirable.

Epidemiological Prevalence of Phenotypical Resistances and Mobilised Colistin Resistance in Avian Commensal and Pathogenic E. coli from Denmark, France, The Netherlands, and the UK.

Colistin has been used for the treatment of non-invasive gastrointestinal infections caused by avian pathogenic E. coli (APEC). The discovery of mobilised colistin resistance (mcr) in E. coli has instigated a One Health approach to minimise colistin use and the spread of resistance. The aim of this study was to compare colistin susceptibility of APECs (collected from Denmark n = 25 and France n = 39) versus commensal E. coli (collected from the Netherlands n = 51 and the UK n = 60), alongside genetic (mcr-1–5) and phenotypic resistance against six other antimicrobial classes (aminoglycosides, cephalosporins, fluoroquinolones, penicillins, sulphonamides/trimethoprim, tetracyclines). Minimum inhibitory concentration (MIC) values were determined using a broth microdilution method (EUCAST guidelines), and phenotypic resistance was determined using disk diffusion. Colistin MIC values of APEC were significantly lower than those for commensals by 1 dilution (p < 0.0001, Anderson-Darling test), and differences in distributions were observed between countries. No isolate carried mcr-1–5. Three phenotypically resistant isolates were identified in 2/62 APEC and 1/111 commensal isolates. Gentamicin or gentamicin–ceftriaxone co-resistance was observed in two of these isolates. This study showed a low prevalence of phenotypic colistin resistance, with no apparent difference in colistin resistance between commensal E. coli strains and APEC strains.

Molecular and Serological Characteristics of Avian Pathogenic Escherichia coli Isolated from Various Clinical Cases of Poultry Colibacillosis in Poland.

Simple SummaryInfections caused by avian pathogenic Escherichia coli (APEC) represent a serious threat for poultry production and economic losses. These bacteria have a number of mechanisms that enable them to colonize various ecosystems. We analyzed E. coli from chicken and turkey broilers, laying hens, and broiler parents. In this work, we characterized virulence factors in APEC isolated from poultry in Poland. We also attempted to show a correlation between lesion severity, the presence of virulence genes, and the E. coli serogroup. The most prevalent serotype among E. coli isolates from all types of chickens was serotype O78, in contrast to isolates from turkeys, in which the prevalence of four serotypes, O1, O2, O18, and O78, was similar. Once the serotype was distinguished, it was possible to assign certain virulence characteristics to the isolate and to associate it with the symptoms present, as well as to track the frequency of occurrence. All of the eight virulence genes analyzed were most common in cases with severe, generalized disease processes. The results also confirmed that there is a great variety of APEC strains in the poultry population.Escherichia coli infections are a major problem in modern poultry production. Avian pathogenic E. coli (APEC) strains have several mechanisms that enable them to colonize various ecosystems. In this study, 290 E. coli isolates were recovered from clinical cases of colibacillosis in chicken and turkey broilers and from laying and breeding hens. The samples were taken from organs with pathological changes suggesting colibacillosis. The lesions were assigned to three groups depending on their advancement, of which the largest (60% of the isolates) was group 3, with the most extensive changes. The most common serotype was shown to be O78 (14%). The most frequently detected gene among those tested was iss, while papC was the least prevalent. An analysis of the number of genes present per isolate revealed that the presence of four genes was the most common (22%), while only 1% of the strains tested had all eight genes. The most frequently detected genes for each serotype were iss and iucD for O78; irp2 and cvi/cva for O1; irp2, iucD, and iss for O2, and iss and iucD for O8, for which the least frequent was papC. All O18 serotype strains had the iss gene, while none had the vat gene.

Investigation of Serotype Prevalence of Escherichia coli Strains Isolated from Layer Poultry in Greece and Interactions with Other Infectious Agents.

Colibacillosis is the most common bacterial disease in poultry and it is caused by avian pathogenic Escherichia coli (APEC), which is assigned to various O-serogroups. Previous studies have shown that APEC strains are more often related to certain O-serogroups such asO78, O2 and O1. E. coli has been reported to act either as a primary or secondary agent in complicating other infections. The aim of this study was to investigate the occurrence of and characterize the O-serogroups of E. coli strains isolated from commercial layer and layer breeder flocks showing macroscopic lesions of colibacillosis and increased or normal mortality in Greece. Furthermore, we attempted to assess the interaction between infectious agents such as Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), infectious bronchitis (IBV) and infectious laryngotracheitis (ILT) with E. coli infections in layer flocks with increased mortality. Our study revealed that in addition to the common serogroups (O78, O2), many other, and less common serogroups were identified, including O111. The O78, O111 and O2 serogroups were frequently detected in flocks with lesions of colibacillosis and increased mortality whereas O2, O88 and O8 were reported more commonly in birds with colibacillosis lesions but normal mortality rates. These data provide important information for colibacillosis monitoring and define preventative measures, especially by using effective vaccination programs because E. coli vaccines are reported to mainly offer homologous protection. Finally, concerning the association of the four tested infectious agents with E. coli mortality, our study did not reveal a statistically significant effect of the above infectious agents tested with E. coli infection mortality.

Avian Pathogenic Escherichia coli through Pfs Affects the Tran-Scription of Membrane Proteins to Resist β-Lactam Antibiotics.

Avian pathogenic Escherichia coli (APEC) is a causative agent of colibacillosis, one of the principal causes of morbidity and mortality in poultry worldwide. Nowadays, antibiotics are mainly used to prevent and control poultry colibacillosis, but the situation of drug resistance is serious. 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (Pfs) is involved in methylation reactions, polyamine synthesis, vitamin synthesis, and quorum sensing (QS) pathways. In this study, compared with the APEC wild-type strain DE17, the pfs deletion strain DE17Δpfs was more susceptible to β-lactam antibiotics (amoxicillin, ceftazidime, cefuroxime) by drug sensitivity test and minimum inhibitory concentration (MIC), and the MIC of the DE17Δpfs was half that of the DE17. Quorum sensing signal molecule AI-2 is involved in antibiotic resistance. In the case of pfs inactivation, the DE17Δpfs cannot synthesize AI-2, so it is necessary to add AI-2 to study whether it affects APEC resistance. When the exogenous AI-2 was added, the MIC of all APEC did not change. Transcriptome sequencing indicated that the transcription levels of a lot of outer membrane protein genes and metabolic genes had changed due to the deletion of pfs. Moreover, the transcription levels of the efflux pump gene tolC and penicillin binding protein (fstI and mrcA) were significantly reduced (p < 0.05), while the transcription levels of the porin protein genes (ompF, ompC, and ompD) were significantly increased (p < 0.05). In addition, it was also found that the outer membrane permeability of the DE17Δpfs was significantly increased (p < 0.05). The results indicated that pfs does not affect APEC strain DE17 resistance to β-lactam antibiotics through AI-2, but pfs affects the sensitivity of APEC to β-lactam antibiotics by affecting antibiotic-related genes. This study can provide a reference for screening new drug targets.