High frequency of multidrug-resistant Escherichia coli from cattle in the Cerrado and Pantanal biomes of Brazil.

The indiscriminate use of antimicrobials has led to the emergence of resistant bacteria, especially pathogenic strains of Escherichia coli, which are associated with diseases in animals and humans. The aim of the present study was to characterize E. coli isolates in calves with regards to the presence of virulence genes and investigate the resistance of the isolates to different antimicrobials. Between 2021 and 2023, 456 fecal samples were collected from calves in the Pantanal and Cerrado biomes of the state of Mato Grosso do Sul, Brazil. All samples were subjected to microbiological analysis and disc diffusion antibiogram testing. The polymerase chain reaction method was used to detect virulence genes. Bacterial growth was found in 451 of the 456 samples and biochemically identified as Escherichia coli. All 451 isolates (100 %) exhibited some phenotypic resistance to antimicrobials and 67.62 % exhibited multidrug resistance. The frequency of multidrug-resistant isolates in the Cerrado biome was significantly higher than that in the Pantanal biome (p = 0.0001). In the Cerrado, the most common pathotype was Shiga toxin-producing Escherichia coli (STEC) (28 %), followed by toxigenic Escherichia coli (ETEC) (11 %), enterohemorrhagic Escherichia coli (EHEC) (8 %) and enteropathogenic Escherichia coli (EPEC) (2 %). In most cases, the concomitant occurrence of pathotypes was more common, the most frequent of which were ETEC + STEC (33 %), ETEC + EHEC (15 %) and ETEC + EPEC (3 %). The STEC pathotype (30 %) was also found more frequently in the Pantanal, followed by EHEC (12 %), ETEC (9 %) and EPEC (6 %). The STEC pathotype had a significantly higher frequency of multidrug resistance (p = 0.0486) compared to the other pathotypes identified. The frequency of resistance was lower in strains from the Pantanal biome compared to those from the Cerrado biome. Although some factors are discussed in this paper, it is necessary to clarify the reasons for this difference and the possible impacts of these findings on both animal and human health in the region.

In vitro evaluation of two novel Escherichia bacteriophages against multiple drug resistant avian pathogenic Escherichia coli.

BACKGROUND: In recent years, there has been a growing interest in phage therapy as an effective therapeutic tool against colibacillosis caused by avian pathogenic Escherichia coli (APEC) which resulted from the increasing number of multidrug resistant (MDR) APEC strains. METHODS: In the present study, we reported the characterization of a new lytic bacteriophage (Escherichia phage AG- MK-2022. Basu) isolated from poultry slaughterhouse wastewater. In addition, the in vitro bacteriolytic activity of the newly isolated phage (Escherichia phage AG- MK-2022. Basu) and the Escherichia phage VaT-2019a isolate PE17 (GenBank: MK353636.1) were assessed against MDR- APEC strains (n = 100) isolated from broiler chickens with clinical signs of colibacillosis. RESULTS: Escherichia phage AG- MK-2022. Basu belongs to the Myoviridae family and exhibits a broad host range. Furthermore, the phage showed stability under a wide range of temperatures, pH values and different concentrations of NaCl. Genome analysis of the Escherichia phage AG- MK-2022. Basu revealed that the phage possesses no antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and any E. coli virulence associated genes. In vitro bacterial challenge tests demonstrated that two phages, the Escherichia phage VaT-2019a isolate PE17 and the Escherichia phage AG- MK-2022. Basu exhibited high bactericidal activity against APEC strains and lysed 95% of the tested APEC strains. CONCLUSIONS: The current study findings indicate that both phages could be suggested as safe biocontrol agents and alternatives to antibiotics for controlling MDR-APEC strains isolated from broilers.

A study on selected responses and immune structures of broiler chickens with experimental colibacillosis with or without florfenicol administration.

BACKGROUND: Colibacillosis in broiler chickens is associated with economic loss and localized or systemic infection. Usually, the last resort is antibacterial therapy. Insight into the disease pathogenesis, host responses and plausible immunomodulatory effects of the antibacterials is important in choosing antibacterial agent and optimization of the treatment. Selected responses of broiler chickens experimentally infected with Escherichia coli (E. coli) and also those treated with florfenicol are evaluated in this study. Chickens (n = 70, 5 weeks old) were randomly assigned to four groups. The control groups included normal control (NC) and intratracheal infection control (ITC) (received sterile bacterial medium). The experimental groups consisted of intratracheal infection (IT) that received bacterial suspension and intratracheal infection with florfenicol administration (ITF) group. RESULTS: Florfenicol reversed the decreased albumin/globulin ratio to the level of control groups (p > 0.05). Serum interleukin 10 (IL-10) and interferon-gamma (IFN-γ) concentrations decreased in IT birds as compared to NC group. Florfenicol decreased the serum interleukin 6 (IL-6) concentration as compared to IT group. Milder signs of inflammation, septicemia, and left shift were observed in the leukogram of the ITF group. Florfenicol decreased the severity of histopathological lesions in lungs and liver. Depletion of lymphoid tissue was detected in spleen, thymus and bursa of IT group but was absent in ITF birds. The number of colony forming units of E. coli in liver samples of ITF group was only slightly lower than IT birds. CONCLUSIONS: Experimental E. coli infection of chickens by intratracheal route is associated with remarkable inflammatory responses as shown by changes in biochemical and hematological parameters. Histopathological lesions in lymphoid organs (especially in the spleen) were also prominent. Florfenicol has positive immunomodulatory effects and improves many of the lesions before the full manifestation of its antibacterial effects. These effects of florfenicol should be considered in pharmacotherapy decision-making process.

Effect of phage vB_EcoM_FJ1 on the reduction of ETEC O9:H9 infection in a neonatal pig cell line.

Enterotoxigenic Escherichia coli (ETEC) colonizes the intestine of young pigs causing severe diarrhoea and consequently bringing high production costs. The rise of antibiotic selective pressure together with ongoing limitations on their use, demands new strategies to tackle this pathology. The pertinence of using bacteriophages as an alternative is being explored, and in this work, the efficacy of phage vB_EcoM_FJ1 (FJ1) in reducing the load of ETEC EC43-Ph (serotype O9:H9 expressing the enterotoxin STa and two adhesins F5 and F41) was assessed. Foreseeing the oral application on piglets, FJ1 was encapsulated on calcium carbonate and alginate microparticles, thus preventing phage release under adverse conditions of the simulated gastric fluid (pH 3.0) and allowing phage availability in simulated intestinal fluid (pH 6.5). A single dose of encapsulated FJ1, provided to IPEC-1 cultured cells (from intestinal epithelium of piglets) previously infected by EC43, provided bacterial reductions of about 99.9% after 6 h. Although bacteriophage-insensitive mutants (BIMs) have emerged from treatment, the consequent fitness costs associated with this new phenotype were demonstrated, comparatively to the originating strain. The higher competence of the pig complement system to decrease BIMs' viability, the lower level of colonization of IPEC-1 cells observed with these mutants, and the increased survival rates and health index recorded in infected Galleria mellonella larvae supported this observation. Most of all, FJ1 established a proof-of-concept of the efficiency of phages to fight against ETEC in piglet intestinal cells.

A molecular epidemiological study on Escherichia coli in young chicks with colibacillosis identified two possible outbreaks across farms.

Avian pathogenic Escherichia coli (APEC) is the cause of colibacillosis outbreaks in young poultry chicks, resulting in acute to peracute death. The high morbidity and mortality caused by colibacillosis results in poor animal welfare, reduced sustainability and economical loss worldwide. To advance the understanding of the molecular epidemiology, genomic relatedness and virulence traits of APEC, we performed systematic sampling from 45 confirmed colibacillosis broiler flocks with high first week mortality (FWM) during 2018-2021. From these flocks, 219 APEC isolates were whole genome sequenced (WGS) and bioinformatic analyses were performed. The bioinformatic analyses included sequence typing (ST), serotyping, detection of virulence-associated genes (VAGs) and phylogenetic analysis. Our results showed a high prevalence of ST23, ST429 and ST95 among APEC isolates from Norwegian broiler flocks, and identified ST23, ST429, ST117 and ST371 to cause disease more often alone, compared to ST95, ST69 and ST10. Phylogenetic analyses, together with associated metadata, identified two distinct outbreaks of colibacillosis across farms caused by ST429 and ST23 and gave insight into expected SNP distances within and between flocks identified with the same ST. Further, our results highlighted the need for combining two typing methods, such as serotyping and sequence typing, to better discriminate strains of APEC. Ultimately, systematic sampling of APEC from multiple birds in a flock, together with WGS as a diagnostic tool is important to identify the disease-causing APEC within a flock and to detect outbreaks of colibacillosis across farms.

Molecular characteristics of fluoroquinolone-resistant Escherichia coli isolated from suckling piglets with colibacillosis.

OBJECTIVES: Colibacillosis is a frequent enteric disease in the pig industry that causes significant economic losses. The objective of this study was to investigate the molecular characteristics of fluoroquinolone (FQ)-resistant E. coli isolates from suckling piglets with colibacillosis. RESULTS: A total of 43 FQ-resistant E. coli isolates were tested in this study and all isolates showed multi-drug resistance (MDR) and mutations in quinolone resistance determining regions (gyrA or parC). Especially, FQ-resistant E. coli isolates with double mutations in both gyrA and parC were shown a high FQs minimum inhibitory concentration (≥ 64 mg/L for ciprofloxacin, ≥ 128 mg/L for enrofloxacin, and ≥ 256 mg/L for norfloxacin). Among 43 FQ-resistant E. coli isolates, 12 (27.9%) were showed plasmid-mediated quinolone resistance (PMQR) positive E. coli. Prevalence of PMQR gene, aac(6')-Ib-cr, qnrS, and qepA, were identified in 7, 3, and 2 E. coli isolates, respectively. We identified the following in PMQR-positive E. coli isolates: the tetracycline resistance genes tetD (12 isolates, 100.0%), tetE (12 isolates, 100.0%), tetA (11 isolates, 91.7%), and tetB (1 isolate, 8.3%); ß-lactamases-encoding blaCMY-2 (10 isolates, 83.3%), blaTEM-1 (7 isolates, 58.3%), blaOXA-1 (7 isolates, 58.3%), blaSHV-1 (3 isolates, 16.7%), and blaAAC-2 (1 isolate, 8.3%); and the chloramphenicol resistance genes (10 isolates, 83.3%); the sulfonamide resistance genes sul1 (9 isolates, 75.0%) and sul2 (10 isolates, 83.3%); the aminoglycoside modifying enzyme gene aac(3)-II (2 isolates, 16.7%). The F4 (7 isolates, 58.3%), LT:STb:EAST1 (5 isolates, 41.7%), and paa (3 isolates, 25.0%) were most common fimbrial antigen, combinations of toxin genes, and non-fimbrial adhesins genes, respectively. All PMQR-positive E. coli carried class I integrons but only 4 isolates carried the gene cassette. The most prevalent plasmid replicon was FIB (9 isolates, 75.0%), followed by FIC, HI1, and N (7 isolates, 58.3%), respectively. CONCLUSIONS: Because FQ-resistant E. coli can serve as a reservoir of FQ resistant genetic determinants that can be transferred to pathogenic bacteria in humans or pigs, this represents a public health hazard.

Longitudinal study on background lesions in broiler breeder flocks and their progeny, and genomic characterisation of Escherichia coli.

In broiler breeders, background mortality is rarely addressed, however, it represents the death of a vast number of birds, a constant productivity loss, welfare concerns and it might affect chick quality. The study aimed to unveil lesions leading to mortality in a study population perceived as healthy, combined with whole-genome sequencing (WGS) of Escherichia coli, a well-known contributor to disease problems in poultry. Broiler breeders (n = 340) originating from three distinct, putative healthy flocks and their progeny (n = 154) were subjected to a comprehensive post-mortem examination, bacteriological sampling, and sequencing of 77 E. coli isolates. Productivity data confirmed an exemplary health status of the enrolled flocks, and post-mortem examination further verified the absence of general disease problems. Among the submitted broiler breeders, exudative peritonitis (31.2%) was the most frequent lesion linked to infectious disease, whereas airsacculitis, pericarditis, perihepatitis, and salpingitis occurred in 18.5%, 3.5%, 3.8% and 17%, respectively. Yolksacculitis occurred in 15.6% of the broilers, whilst pericarditis, perihepatitis and peritonitis were diagnosed in 9.7%, 7.1% and 9.1%, respectively. WGS revealed a diverse population where ST95 dominated the population retrieved from broiler breeders, whereas ST10 was highly prevalent among broilers. Both lineages could be isolated from extraintestinal sites of birds without lesions indicative of infection. In general, the genetic diversity within flocks was comparable to the diversity between farms, and the overall occurrence of resistance markers was low. In conclusion, a comprehensive insight into lesions associated with background mortality is presented, together with a vast diversity of E. coli isolated from extraintestinal sites during a non-outbreak situation.

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 10 years, from 2009 to 2018, were used to evaluate the phenotypic AMR of 264 APEC isolates obtained from 158 broiler breeder flocks of a large company in Portugal. The APEC isolates were tested against eleven antimicrobials by the Kirby-Bauer disc diffusion test. The annual proportion of AMR was calculated by dividing the number of APEC isolates 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%, enrofloxacin 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 seven (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. RESEARCH HIGHLIGHTSDecreasing trend of antimicrobial resistance in avian pathogenic E. coli over time.Over 50% of isolates still resistant to amoxicillin, tetracycline and doxycycline.Multidrug resistance decreased from 100% in 2009 to 48% in 2018.Further reduction of antimicrobial resistance in broiler breeders desirable.

Nebulization as a more efficient method than atomizer for experimental reproduction of avian colibacillosis in young chickens.

Infection and immunity studies involving genetically modified organisms (GMOs), such as gene knockout bacterial mutants, require stringent physical containment to prevent the accidental spread of these organisms into the environment. Experimental respiratory tract infection models often require the animals, for example birds, to be transported several times between a negative pressure housing isolator and a bespoke aerosol exposure chamber under positive pressure. While the exposure chamber is sealed and fitted with HEPA filters, the repeated movements of infected animals and opening of the chamber can still pose a serious risk of breaching containment of the organism in the experimental facility. In the current study, the ability of two aerosol infection protocols that expose birds to avian pathogenic E. coli (APEC) aerosols directly within the housing isolator was evaluated. Young chicks were exposed to APEC E956 within the negative pressure housing isolators using either a nebulizer or an atomizer. Birds exposed twice (days 1 and 4) to aerosols of APEC E956 produced by the nebulizer developed a rapidly progressing disease mimicking field cases of avian colibacillosis. However, birds exposed to aerosols of APEC E956 produced by an atomizer did not develop colibacillosis even after three exposures to APEC E956 on days 1, 4 and 7. Consequently, the current study reports the nebulizer was more efficacious in producing avian colibacillosis under stricter bacterial containment settings.RESEARCH HIGHLIGHTS Two aerosol exposure methods were evaluated to develop avian colibacillosis.Nebulizer method found to be more efficient in reproducing avian colibacillosis.Refined infection method can be used to study genetically modified organisms (GMOs).

Antimicrobial susceptibility, plasmid replicon typing, phylogenetic grouping, and virulence potential of avian pathogenic and faecal Escherichia coli isolated from meat chickens in Australia.

Globally, avian colibacillosis is a leading cause of morbidity and mortality in poultry, associated with economic losses and welfare problems. Here, clinical avian pathogenic E. coli isolates (CEC; n = 50) and faecal E. coli isolates from healthy (FEC; n = 187) Australian meat chickens collected between 2006 and 2014 were subjected to antimicrobial susceptibility testing, phylogenetic grouping, plasmid replicon (PR) typing, multilocus sequence typing, and virulence gene (VG) profiling. Extended-spectrum cephalosporin (ESC)- and fluoroquinolone (FQ)-resistant E. coli isolates underwent further genetic characterization. Significant proportions of CEC and FEC were, respectively, susceptible (13/50; 48/187) or MDR (9/50; 26/187) to 20 tested antimicrobials. Phylogenetic groups A and C, and PR types IncFIB and IncFrep were most represented. Five tested CEC-associated VGs were more prevalent in CEC (≥ 90%) than FEC (≤ 58%). Some isolates (CEC n = 3; FEC n = 7) were resistant to ESCs and/or FQs and possessed signature mutations in chromosomal FQ target genes and plasmid-mediated qnrS, blaCMY-2, and blaDHA-1 genes. Sequence type 354 (n = 4), associated with extraintestinal infections in a broad range of hosts, was prevalent among ESC- and/or FQ-resistant FEC. This study confirmed existence of a small reservoir of ESC- and FQ-resistant E. coli in Australian commercial meat chickens despite absence of use in the industry of these drugs. Otherwise, diversity of VGs and PR types in both FEC and CEC populations was identified. We hypothesize that the source of ESC- and FQ-resistant E. coli is external to poultry production facilities.RESEARCH HIGHLIGHTSLow-level resistance to older and newer generation antimicrobial drugs detected.The most common sequence type (ST) associated with FQ resistance was ST354 (4/10).A small proportion of CEC (n = 3) and FEC (n = 7) were resistant to ESCs and/or FQs.

New strategies to prevent and control avian pathogenic Escherichia coli (APEC).

Avian pathogenic Escherichia coli (APEC) infections are associated with major economical losses and decreased animal welfare. In broiler production, APEC infections have traditionally been controlled by antibiotics, resulting in an increased prevalence of antibiotic-resistant E. coli. Concerns have been raised that transfer of antibiotic-resistant APEC via the food chain may result in risks for extra-intestinal infection of humans related to zoonotic transfer and increased difficulties in the treatment of human infections caused APEC-related E. coli types. In this review, the risks associated with APEC are presented based on new knowledge on transmission, virulence and antibiotic resistance of APEC. A major new change in our understanding of APEC is the high degree of genuine vertical transfer of APEC from parents to offspring. A new strategy for controlling APEC, including control of antibiotic-resistant APEC, has to focus on limiting vertical transfer from parents to offspring, and subsequent horizontal transmission within and between flocks and farms, by using all-in-all-out production systems and implementing a high level of biosecurity. Vaccination and the use of competitive exclusion are important tools to be considered. A specific reduction of antibiotic-resistant APEC can be obtained by implementing culling strategies, only allowing the use of antibiotics in cases where animal welfare is threatened. Strategies to reduce APEC, including antibiotic-resistant APEC, need to be implemented in the whole production pyramid, but it has to start at the very top of the production pyramid.

The Avian Pathogenic Escherichia coli (APEC) pathotype is comprised of multiple distinct, independent genotypes.

Avian Pathogenic E. coli (APEC) is the causative agent of avian colibacillosis, resulting in economic losses to the poultry industry through morbidity, mortality and carcass condemnation, and impacts the welfare of poultry. Colibacillosis remains a complex disease to manage, hampered by diagnostic and classification strategies for E. coli that are inadequate for defining APEC. However, increased accessibility of whole genome sequencing (WGS) technology has enabled phylogenetic approaches to be applied to the classification of E. coli and genomic characterization of the most common APEC serotypes associated with colibacillosis O1, O2 and O78. These approaches have demonstrated that the O78 serotype is representative of two distinct APEC lineages, ST-23 in phylogroup C and ST-117 in phylogroup G. The O1 and O2 serotypes belong to a third lineage comprised of three sub-populations in phylogroup B2; ST-95, ST-140 and ST-428/ST-429. The frequency with which these genotypes are associated with colibacillosis implicates them as the predominant APEC populations and distinct from those causing incidental or opportunistic infections. The fact that these are disparate clusters from multiple phylogroups suggests that these lineages may have become adapted to the poultry niche independently. WGS studies have highlighted the limitations of traditional APEC classification and can now provide a path towards a robust and more meaningful definition of the APEC pathotype. Future studies should focus on characterizing individual APEC populations in detail and using this information to develop improved diagnostics and interventions.

Aerosol is the optimal route of respiratory tract infection to induce pathological lesions of colibacillosis by a lux-tagged avian pathogenic Escherichia coli in chickens.

Pathogenesis of colibacillosis caused by avian pathogenic Escherichia coli (APEC) in poultry is unclear and experimental studies reveal substantial inconsistency. In this study, the impact of three infection routes differing in the site of deposition of inoculum in the respiratory tract, were investigated. Two-weeks-old chickens were infected with a lux-tagged APEC strain via aerosol, intranasally or intratracheally, and sequentially sampled along with uninfected birds. At 1 and 3 days post infection (dpi), liver or spleen to body-weight ratios in all infected groups were significantly higher than in negative control, while at 7 dpi, such differences were significant in both organs in the aerosol-infected group. The infection-strain colonized tracheas and lungs in infected birds at 1 dpi and persisted until 7 dpi. Among infected groups, in lungs, bacterial load at 1 dpi was significantly lower in intranasally-inoculated birds. Histology revealed that, independent of infection route, lesions were mostly seen in the lower respiratory organs (lungs and air sacs) characterized by bronchitis/pneumonia and airsacculitis. Birds infected via aerosol showed the highest mean lesion score in lungs while intranasal application caused the mildest pathological changes, and difference between the two groups was significant at 1 dpi. In spleen, heterophilic infiltrations were prominent in affected birds. Interestingly, tracheas were pathologically unaffected. Altogether, the results demonstrated the importance of infection route, with aerosol being the most suitable to induce pathological lesions of colibacillosis without predisposing factors. Furthermore, the lux-tagged APEC strain was discriminated from native isolates enabling exact differentiation and enumeration.RESEARCH HIGHLIGHTS Lux-tagged APEC strain was used for infection to differentiate from native E. coli.Pathologically, lungs, air sacs and spleen but not trachea were affected.The route of infection strongly impacts the pathological outcome with APEC.The infection with APEC via aerosol caused the most severe lesions in chickens.

Impact of Escherichia coli infection in broiler breeder chicks: The effect of oxidative stress on weight gain.

The aim of this study was to determine whether oxidative stress occurs in Escherichia coli-infected broiler breeder chicks, as well as the impact of this infection on bird growth. Twenty birds, 25-day-old female birds were divided into two groups (n = 10 per group): an intraperitoneally-infected group (1 mL containing 1.5 × 108 CFU of E. coli) and a control group that received 1 mL of culture medium (uninfected birds). Birds were weighed individually at the beginning and at the end of the experiment, and samples were collected on days 0, 5 and 10 post-infection (PI). No clinical signs were observed throughout the experimental period; nevertheless, on day 10 PI, there was lower growth and weight gain in infected birds than in the control group. The infected birds showed pericarditis and liver congestion, as well as moderate periportal inflammatory infiltrates with predominance of neutrophils. Significantly higher numbers of total leukocytes, lymphocytes, heterophils and monocytes were observed in the infected group on days 5 and 10 PI, as well as significantly higher total protein and globulin levels; albumin values significantly decreased over the same period. Levels of serum oxidative biomarkers (lipid peroxidation (TBARS) and free radicals (ROS)) were significantly higher at 10 PI, as was glutathione S-transferase (GST) activity during the same period. Hepatic ROS and protein thiol levels were significantly higher in E. coli-infected birds, as well as activities of the antioxidant enzymes catalase, superoxide dismutase. In the spleen, only GST activity was significantly higher for the infected group, unlike the brain, where SOD activity, ROS and non-protein thiol levels were significantly higher in infected birds than in the control group. These data suggested that colibacillosis causes oxidative stress in broiler breeder chicks, negatively affecting their weight gain.

Design, development, and evaluation of the efficacy of a nucleic acid-free version of a bacterial ghost candidate vaccine against avian pathogenic E. coli (APEC) O78:K80 serotype.

One of the major bacterial infectious diseases in the poultry industry is avian pathogenic Escherichia coli (APEC), which causes colibacillosis in chickens. To develop a novel nucleic acid-free bacterial ghost (BG) vaccine against the O78:K80 serotype of APEC, in this study we constructed a plasmid that harbored E-lysis and S nuclease (SNUC). Following the expression, the O78:K80 bacteria lost all of their cytoplasmic content and nucleic acids by enzymatic digestion. The functionality of these two proteins in the production procedure of bacterial ghosts was confirmed by monitoring the number of colonies, scanning electron microscopy imaging, gel electrophoresis of genomic DNA, and qPCR on the plasmid content of bacterial ghosts. The protective efficacy of the ghost vaccine generated from O78:K80 serotype of APEC was tested in chickens by injection and inhalation routes and compared with that in chickens that received the injection of a killed vaccine. The O78:K80 BG vaccine candidate, used as injection and inhalation, in comparison with the killed vaccine, triggered higher proinflammatory cytokine expression including IL-6, IL-1ß, and TNFSF15; a higher level of antibody-dependent humoral (IgY and IgA) and cellular immune responses (IFNγ and lymphocyte proliferation); and lower lesion scores. According to the results of this study, we suggest that the bacterial ghost technology has the potential to be applied for the development of novel vaccines against avian colibacillosis. This technology provides an effective and reliable approach to make multivalent vaccines for more prevalent APEC strains involved in the establishment of this infectious disease in the poultry industry.

The role of Enterococcus faecalis during co-infection with avian pathogenic Escherichia coli in avian colibacillosis.

Enterococcus spp. (ENT) are frequently co-isolated with avian pathogenic E. coli (APEC) from poultry with colibacillosis, a leading cause of flock mortality. Although largely overlooked, ENT may play an active role in these infections. To assess the frequency of ENT co-isolation in colibacillosis, cultures were collected from birds with gross lesions of omphalitis, polyserositis, and septicaemia over a 3-year period from three turkey flocks and three broiler flocks. In birds diagnosed with colibacillosis based on gross findings and isolation of E. coli, ENT were co-isolated with APEC in 35.7% (n = 41/115) of colibacillosis mortality and 3.7% of total mortality (n = 41/1122). Co-isolated APEC and ENT pairs (n = 41) were further characterized using antimicrobial resistance phenotyping and in vitro co-culture assays. E. faecalis (EF) was the most commonly co-isolated species (68% n = 28/41) and tetracycline resistance was the resistance phenotype most commonly found among APEC (51% n = 21/41) and ENT (93% n = 38/41). Under iron-restricted conditions, EF enhanced APEC growth in a proximity-dependent manner and APEC grown in mixed culture with EF exhibited a significant growth and survival advantage (P ≤ 0.01). In an embryo lethality assay, APEC co-infection with EF resulted in decreased survival of broiler embryos compared to mono-infections (P ≤ 0.05). These data demonstrate that EF augmented APEC survival and growth under iron limiting conditions, possibly translating to the increased virulence of APEC in broiler embryos. Thus, ENT co-infections may be a previously unrecognized contributor to colibacillosis-related mortality. Further investigations into the mechanism of this interaction are warranted. RESEARCH HIGHLIGHTS Enterococcus is frequently co-isolated with avian pathogenic E. coli (APEC). Enterococcus faecalis (EF) enhances survival of APEC in iron restricted conditions. EF co-infection increases APEC virulence in broiler embryos.

A comparison of virulence genes, antimicrobial resistance profiles and genetic diversity of avian pathogenic Escherichia coli (APEC) isolates from broilers and broiler breeders in Thailand and Australia.

ABSTRACT Avian pathogenic Escherichia coli (APEC) is the causative agent of colibacillosis resulting in economic losses in the poultry industry worldwide. A total of 168 APEC isolates, equal numbers from Australian and Thai broilers/broiler breeders, were identified and tested for their susceptibility to ten antimicrobial agents. Most of the Thai APEC isolates were multidrug-resistant (MDR) (60.7%) whilst Australian APEC isolates showed a MDR rate of just 10.7%. The Thai APEC isolates exhibited high resistance to tetracycline (TET) (84.5%), amoxicillin (AMX) (70.2%) and trimethoprim-sulfamethoxazole (SXT) (51.2%) whilst the Australian APEC isolates showed lower levels of resistance (TET 36.9%, AMX 29.8%, SXT 17.86%). The 34 Thai APEC and four Australian APEC isolates which were resistant to nalidixic acid were characterized for their carriage of mutations in the quinolone resistance determining region of gyrA, gyrB, parC and parE. While no mutations were detected in gyrB in the Thai isolates, the Ser83Leu and Asp87Asn substitutions in gyrA and Ser80Ile in parC were common (n = 9/34). In regard to the Australian isolates, the Ser83Leu and Asp678Glu substitution in gyrA, Pro385Ala and Ser492Asn in gyrB and Met241Ile and Asp475Glu in parC were identified (n = 3/4). Rep-PCR analysis of the 84 Thai and 84 Australian APEC isolates showed 16 main clusters that mostly contained isolates from both countries. Our results suggest that the emergence of MDR is a major concern for the Thai APEC isolates and that more prudent use of antimicrobial agents in Thai poultry production is required.

Risks and disease aetiologies of compromised performance in commercial broilers kept at lower stocking density and limited antimicrobial use.

The health status of broilers raised at lower stocking density and limited antimicrobial use (but routine anticoccidials) was assessed in order to identify prevalent causes of disease, mortality and reduced performance. "Dead-on-farm"(DOF) broilers from 145 commercial flocks were investigated at two different time points (TP1:7-14 and TP2:28-35 days of age); per sampling, 6-10 DOF broilers were selected for post-mortem investigation and gross pathomorphological changes were assessed from 2717 birds in total. Post-mortem findings were substantiated by bacteriological, virological and parasitological investigations. Furthermore, production data of all flocks were collected and used to perform comprehensive statistical analysis. Overall, colibacillosis was found most important with a significant negative impact on flock health, productivity and profitability through all ages of broiler production. At TP1, primary reasons for mortality comprised yolk sac infections, generally found together with fibrinous polyserositis due to E. coli. Furthermore, femoral lesions, which correlated with increased flock mortality, were associated with detection of E. coli. At TP2, ascites was detected frequently in DOF broilers, correlating with increased production losses in the fourth and fifth weeks of life. No aetiological link between the presence of ascites and the detection of the investigated pathogens was observed, instead a positive correlation was noticed with altitude above sea level of the farm, and with the sex of the birds. Disease conditions could not be linked with the housing system. Presence of infectious bronchitis virus, avian reovirus and fowl adenovirus did not correlate with macroscopic lesions or a specific disease. RESEARCH HIGHLIGHTS In young broilers lesions of visceral organs due to bacterial infections dominated. Colibacillosis impacts broiler health, productivity and profitability independent of the age of birds. Disorders of the locomotor system were frequently observed throughout production. Older broilers frequently showed pathologic changes due to metabolic disorders. Overall, a shift from infectious towards metabolic disease conditions was noticed.

Antimicrobial resistance and prevalence of tetracycline resistance genes in Escherichia coli isolated from lesions of colibacillosis in broiler chickens in Sistan, Iran.

BACKGROUND: Antibiotics have long been the first line of defense to prevent Escherichia coli infections, but they have lost their potency since bacteria have grown increasingly resistant to treatment. The present research aimed to study the drug resistance and the prevalence of tetracycline resistance genes in E. coli isolated from broilers with colibacillosis. RESULTS: The results showed that the most prevalent type of drug resistance was to tetracycline at 95.0%, and the least was to gentamicin at 21.7%. The prevalences of antimicrobial resistance among the tested antibiotics were significantly different (p < 0.001). A statistically significant difference was observed between the prevalence of the tet genes (p < 0.001). The tetD positive isolates and antibiotic sensitivity to tetracycline showed statistical significant differences (p = 0.017). CONCLUSIONS: Considering the results, tetA is the most common tetracycline resistance gene, and the presence of tetD and antibiotic sensitivity to tetracycline had a significant relationship in E. coli isolated from colibacillosis infections.

Genomic analysis of Escherichia coli strains isolated from diseased chicken in the Czech Republic.

BACKGROUND: Avian pathogenic Escherichia coli (APEC) can cause various extraintestinal infections in poultry, resulting in massive economic losses in poultry industry. In addition, some avian E. coli strains may have zoonotic potential, making poultry a possible source of infection for humans. Due to its extreme genetic diversity, this pathotype remains poorly defined. This study aimed to investigate the diversity of colibacillosis-associated E. coli isolates from Central European countries with a focus on the Czech Republic. RESULTS: Of 95 clinical isolates subjected to preliminary characterization, 32 were selected for whole-genome sequencing. A multi resistant phenotype was detected in a majority of the sequenced strains with the predominant resistance to ß-lactams and quinolones being associated with TEM-type beta-lactamase genes and chromosomal gyrA mutations respectively. The phylogenetic analysis confirmed a great diversity of isolates, that were derived from nearly all phylogenetic groups, with predominace of B2, B1 and C phylogroups. Clusters of closely related isolates within ST23 (phylogroup C) and ST429 (phylogroup B2) indicated a possible local spread of these clones. Besides, the ST429 cluster carried blaCMY-2, - 59 genes for AmpC beta-lactamase and isolates of both clusters were generally well-equipped with virulence-associated genes, with considerable differences in distribution of certain virulence-associated genes between phylogenetically distant lineages. Other important and potentially zoonotic APEC STs were detected, incl. ST117, ST354 and ST95, showing several molecular features typical for human ExPEC. CONCLUSIONS: The results support the concept of local spread of virulent APEC clones, as well as of zoonotic potential of specific poultry-associated lineages, and highlight the need to investigate the possible source of these pathogenic strains.