Isolation and encapsulation of bacteriophage with chitosan nanoparticles for biocontrol of multidrug-resistant methicillin-resistant Staphylococcus aureus isolated from broiler poultry farms.

This study was divided into two parts. The first part, the determination of methicillin-resistant Staphylococcus aureus (MRSA) prevalence in 25 broiler chicken farms, with the detection of multidrug resistant MRSA strains. The prevalence of MRSA was 31.8% (159 out of 500 samples) at the level of birds and it was 27% (27 out of 100) in the environmental samples. The highest antimicrobial resistance of the recovered MRSA strains was recorded to streptomycin (96%). All isolates (100%) had multidrug resistance (MDR) to four or more antibiotics with 16 distinct antibiotic resistant patterns, and multiple antibiotic resistance index (MARI) of 0.4-1. The second part, implementing novel biocontrol method for the isolated multidrug resistant MRSA strains through the isolation of its specific phage and detection of its survival rate at different pH and temperature degrees and lytic activity with and without encapsulation by chitosan nanoparticles (CS-NPs). Encapsulated and non-encapsulated MRSA phages were characterized using transmission electron microscope (TEM). Encapsulation of MRSA phage with CS-NPs increasing its lytic activity and its resistance to adverse conditions from pH and temperature. The findings of this study suggested that CS-NPs act as a protective barrier for MRSA phage for the control of multidrug resistant MRSA in broiler chicken farms.

Multidrug resistant and multivirulent avian bacterial pathogens: tackling experimental leg disorders using phytobiotics and antibiotics alone or in combination.

Locomotor disorders caused by multidrug-resistant (MDR) bacterial pathogens denote one of the most detrimental issues that collectively threaten the poultry industry leading to pronounced economic losses across the world. Hence, searching for effective alternatives, especially those extracted from plant origins became of great priority targeting a partial or complete replacement of chemical antimicrobials to tackle their developing resistance. Therefore, we aimed to determine the prevalence and antimicrobial resistance of Staphylococcus aureus (S. aureus), Salmonella species, Mycoplasma synoviae (M. synoviae), and Escherichia coli (E. coli) recovered from 500 broilers and ducks (250 each) with locomotor disorders in various farms in Dakahlia and Sharkia Governorates, Egypt. Additionally, we assessed, for the first time, the in vitro antimicrobial effectiveness of marjoram, garlic, ginger and cinnamon essential oils (EOs) against MDR and multivirulent bacterial isolates as well as the in vivo efficiency of the most effective antibiotics and EOs either separately or in combination in the treatment of experimentally induced poultry leg disorders. The overall prevalence rates of S. aureus, E. coli, Salmonella species, and M. synoviae were 54, 48, 36, and 2%, respectively. Salmonella species and S. aureus prevailed among ducks and broilers (36 and 76%, respectively). Notably, MDR was observed in 100, 91.7, 81.1, and 78.5% of M. synoviae, E. coli, Salmonella, and S. aureus isolates, respectively. Our in vitro results displayed that marjoram was the most forceful EO against MDR and multivirulent chicken vancomycin-resistant S. aureus (VRSA) and duck S. Typhimurium isolates. The current in vivo results declared that marjoram in combination with florfenicol or amoxicillin/clavulanic acid succeeded in relieving the induced duck and chicken leg disorders caused by S. Typhimurium and VRSA, respectively. This was evidenced by improvement in the clinical and histopathological pictures with a reduction of bacterial loads in the experimental birds. Our encountered successful in vitro and in vivo synergistic effectiveness of marjoram combined with florfenicol or amoxicillin/clavulanic acid recommends their therapeutic application for leg disorders and offers opportunities for reducing the antibiotics usage in the poultry industry.

Synergistic influence of probiotic and florfenicol on embryonic viability, performance, and multidrug-resistant Salmonella Enteritidis in broiler chickens.

This study was conducted during the period of August 2021 to April 2022 and divided into two parts. The first part involved the isolation and characterization of Salmonella from 200 diseased broiler chickens collected from farms in Dakahlia Governorate, Egypt, with the detection of its antimicrobial susceptibility. The second experimental part involved in ovo inoculation of probiotics and florfenicol to evaluate their effects on hatchability, embryonic viability, growth performance traits and the control of multidrug-resistant Salmonella Enteritidis infections post hatching. The point prevalence of Salmonella in the internal organs of diseased chickens was 13% (26/200), including 6 serotypes: S. Enteritidis, S. Typhimurium, S. Santiago, S. Colindale, S. Takoradi and S. Daula. Multidrug resistance was found in 92% (24/26) of the isolated strains with a multiantibiotic resistance index of 0.33-0.88 and 24 antibiotic resistance patterns. The in ovo inoculation of probiotic with florfenicol showed significant improvement in the growth performance parameters compared with other groups and had the ability to prevent colonization of multidrug resistant S. Enteritidis in the majority of the experimental chicks, and the remaining chicks showed very low colonization, as detected by RT‒PCR. These findings suggested the application of in ovo inoculation techniques with both probiotics and florfenicol as a promising tool to control multidrug-resistant S. Enteritidis in poultry farms.

Effects of dietary nano-selenium supplementation on Riemerella anatipestifer vaccinated and challenged Pekin ducklings (Anas platyrhynchos).

Riemerella anatipestifer (RA) is a common disease causing economic losses to duck farms worldwide. Novel supplements are crucially needed to control this bacterium, enhance poultry performance, and produce synergistic effects with vaccines in stimulating the immune system. This study investigated the effect of nano-selenium (Nano-Se) on the vaccinated (VAC) and challenged (Ch) Pekin ducklings (Anas platyrhynchos) with RA. Five experimental groups (G1-G5) were included in this study: G1 was the control group, G2 was the RA-challenged group, G3 was the Nano-Se+Ch group, G4 was the VAC+Ch group, and G5 was the Nano-Se+VAC+Ch group. The Nano-Se (0.3 mg/kg diet) was supplemented for 5 weeks post-vaccination (PV). The ducklings were vaccinated subcutaneously with the RA vaccine at 7 days of age and challenged with RA at the 3rd week PV. Blood, pharyngeal swabs and tissue samples were collected at the 3rd week PV and at different times post-challenge (PC). The growth performance (weight gain and feed conversion ratio), clinical signs, gross lesions, mortality, bacterial shedding, haematological, immunological, and biochemical parameters, cytokines production, and histopathological lesion scores showed significant differences (P < 0.05) between the challenged (G2) group and the supplemented (G3 & G5) groups. G5 showed the highest (P < 0.05) growth performance, phagocytic activity, IgM and IgG, splenic interleukin-2 (IL-2), IL-10, and interferon-gamma (IFN-γ) gene expressions, and the lowest mortality, bacterial shedding, hepatic and renal damage, heterophil/lymphocyte ratio and lesion scores compared to the other groups. In conclusion, the supplementation of nano-selenium for five weeks in the diet can improve the growth performance, immune status, and cytokines production in ducklings vaccinated and challenged with RA.

Modulatory Effect of Competitive Exclusion on the Transmission of ESBL E. coli in Chickens.

The extensive use of antimicrobial agents in broiler farms causes the emergence of antimicrobial resistance of E. coli producing severe economic losses to the poultry industry; therefore, monitoring the transmission of ESBL E. coli is of great significance throughout broiler farms. For this reason, we investigated the efficiency of competitive exclusion (CE) products to control the excretion and transmission of ESBL-producing E. coli in broiler chickens. Three hundred samples from 100 broiler chickens were screened for the incidence of E. coli by standard microbiological techniques. The overall isolation percentage was 39% and differentiated serologically into ten different serotypes: O158, O128, O125, O124, O91, O78, O55, O44, O2, and O1. The isolates represented absolute resistance to ampicillin, cefotaxime, and cephalexin. The effectiveness of CE (commercial probiotic product; Gro2MAX) on ESBL-producing E. coli (O78) isolate transmission and excretion was studied in vivo. The results showed that the CE product has interesting properties, making it an excellent candidate for targeted drug delivery by inhibiting bacterial growth and downregulating biofilm, adhesins, and toxin-associated genes loci. The histopathological findings demonstrated the capability of CE in repairing internal organ tissues. Our outcomes suggested that the administration of CE (probiotic products) in broiler farms could be a safe and alternative approach to control the transmission of ESBL-producing virulent E. coli in broiler chickens.

The potential role of migratory birds in the transmission of pathogenic Campylobacter species to broiler chickens in broiler poultry farms and live bird markets.

BACKGROUND: Campylobacter species (spp.) are one of the most important zoonotic bacteria possessing potential hazards for animal and human health worldwide. Migratory birds are implicated as significant carriers for microbes and a play very important role in the dissemination of Campylobacter to broiler chickens and their environment. The purpose of this investigation was to detect the prevalence, antibiotic resistant patterns, virulence and diversity of pathogenic Campylobacter spp. in 7 migratory bird species (Northern shoveler, Common pochard, Common teal, Northern pintail, Eared Grebe, Great Crested Grebe and Garganey) and broiler chickens that were collected from broiler poultry farms and live bird markets. RESULTS: The prevalence of Campylobacter was 12.5% (25/200), of which 15% (15/100) was recovered from 5 migratory bird species only and 10% (10/100) from broiler chickens. At the level of migratory birds, eight isolates (53.3%) were Campylobacter jejuni (C. jejuni) and 7 isolates (46.7%) were Campylobacter coli (C. coli) meanwhile, in broiler chickens C. jejuni and C. coli were 50% (5/10) for each. All isolated strains had phenotypic resistance to doxycycline, while all of the isolates were susceptible to amikacin. The multidrug resistance to three, four or five antimicrobial classes was found in 72% (18/25) of the isolated strains. The multiantibiotic resistance index between the examined isolates was 0.22-0.77, with 10 antibiotic resistance patterns. The virulence of isolated Campylobacter strains (from both migratory birds and broiler chicken birds) was detected by targeting the VirB11, ciaB and iam genes which were recorded at 16%, 52% and 100%, respectively. Additionally, 100% and 84% of the antibiotic resistance genes were identified as tetA and BlaOXA-61, respectively. CONCLUSIONS: The results of this study revealed the diversity between all the isolated strains from migratory birds and their similarity to broiler chicken isolates. The findings of the present study highlight the impact of migratory birds visiting Egypt and other countries on pathogenic Campylobacter spp. carrying pathogenic virulence and resistance genes, necessitating the application of biosecurity measures to prevent migratory birds from entering farms during their migration period.

Effects of the prebiotic lactoferrin on multidrug-resistant Escherichia coli infections in broiler chickens.

BACKGROUND AND AIM: Increased multidrug resistance in Escherichia coli has created challenges for the poultry industry. Consequently, new antimicrobial agents should preferentially be utilized for the prevention and treatment of E. coli outbreaks. This study aimed to evaluate the effects of lactoferrin (LF) as a prebiotic on broiler chicks challenged with multidrug-resistant E. coli in comparison with antibiotics. MATERIALS AND METHODS: A total of 70 diseased flocks from Egypt were collected for E. coli isolation and identification, serotyping, and antimicrobial susceptibility pattern determination. E. coli was isolated and characterized phenotypically and one isolate that showed multidrug-resistance was selected. A challenge trial was performed to evaluate the effectiveness of LF as a prebiotic on the isolated multidrug-resistant E. coli. Liver samples were collected from the experimental chicks and subjected to E. coli enumeration to illustrate the effectiveness of LF on the liver cells and bacteria using an electron microscope. Serum samples were also collected to estimate lysozyme and nitric oxide (NO) concentrations. RESULTS: After isolation of E. coli with a percentage of 54.3% from the diseased broilers, the strain was serotyped (identified serotypes: O2, O18, O55, O78, O86a, O111, O125, O126, O127, O157, O159, and O166). Multi-antibiotic resistance was found to be harbored in a high percentage among 11 antibiotic discs. The LF in the prophylactic and treated groups was found to have a significant effect in comparison with the group treated with the drug of choice (ciprofloxacin). Furthermore, a significant difference in the NO (one of non-specific immune response) and a non-significant difference in lysozyme concentrations were reported in the group fed on rations with LF in comparison with the non-fed group. CONCLUSION: LF was thus identified as an effective prebiotic that can improve chick performance, help them to overcome multidrug-resistant E. coli and stimulate immunity.

Wild Birds in Live Birds Markets: Potential Reservoirs of Enzootic Avian Influenza Viruses and Antimicrobial Resistant Enterobacteriaceae in Northern Egypt.

Wild migratory birds are often implicated in the introduction, maintenance, and global dissemination of different pathogens, such as influenza A viruses (IAV) and antimicrobial-resistant (AMR) bacteria. Trapping of migratory birds during their resting periods at the northern coast of Egypt is a common and ancient practice performed mainly for selling in live bird markets (LBM). In the present study, samples were collected from 148 wild birds, representing 14 species, which were being offered for sale in LBM. All birds were tested for the presence of AIV and enterobacteriaceae. Ten samples collected from Northern Shoveler birds (Spatula clypeata) were positive for IAV and PCR sub-typing and pan HA/NA sequencing assays detected H5N8, H9N2, and H6N2 viruses in four, four, and one birds, respectively. Sequencing of the full haemagglutinin (HA) gene revealed a high similarity with currently circulating IAV in Egypt. From all the birds, E.coli was recovered from 37.2% and Salmonella from 20.2%, with 66%-96% and 23%-43% isolates being resistant to at least one of seven selected critically important antimicrobials (CIA), respectively. The presence of enzootic IAV and the wide prevalence of AMR enterobacteriaceae in wild birds highlight the potential role of LBM in the spread of different pathogens from and to wild birds. Continued surveillance of both AIV and antimicrobial-resistant enterobacteriaceae in wild birds' habitats is urgently needed.

Evaluation of bacteriophage efficacy in reducing the impact of single and mixed infections with Escherichia coli and infectious bronchitis in chickens.

Infectious bronchitis virus (IBV) represents a major threat to poultry production worldwide particularly when complicated with bacterial infection. In the present study samples were collected from forty broiler farms with respiratory manifestations to characterize IBV and E. coli. Bacteriophages were isolated and enriched from sampled farms to study its efficacy to control single and mixed infections with E. coli and IBV in vivo. Twelve out of forty farms were positive for IBV. Phylogenetic analysis of partial spike protein revealed that all positive cases clustered within the GI-23 genotype. Eight out of forty farms were positive for E. coli serogroups O26, O78, O86, O114, O119, with O125 found on three farms. Bacteriophage treatment delayed the onset and reduced the severity of clinical signs, and prevented the mortality associated with single and mixed infection with IBV and E. coli. Furthermore, in mixed infections, bacteriophage treatment significantly reduced E. coli as well as IBV shedding. Groups treated with bacteriophages showed a significant reduction of E. coli shedding that gradually decreased over time, in contrast to higher and gradually increasing shedding without bacteriophage treatment. In conclusion, bacteriophage treatment significantly reduced the pathogenicity and shedding of IBVand E. coli in mixed infections.

Assessing the impact of bacteriophages in the treatment of Salmonella in broiler chickens.

Salmonellosis is one of the main bacterial infections affecting commercial poultry, causing losses to poultry production, and posing a public health concern. Samples from internal organs (liver, cecum and spleen) of one hundred diseased broiler chickens were collected and subjected to Salmonella isolation, identification and serotyping. S. typhimurium and S. enteritidis were selected from the isolated Salmonella to prepare bacteriophages from sewage water taken at broiler farms. An experimental infection of one day old specific pathogen free (SPF) chicks followed by treatment with the prepared bacteriophages isolated from both Salmonella was performed. Caecal samples from infected chicks were subjected at intervals to bacteriophage isolation and Salmonella quantitation. The effectiveness of bacteriophage treatments on Salmonella colonization in cecum of infected chicks increased after five successive doses. At 3 day post infection (dpi), cecal contents showed a marginal decrease in Salmonella loads with more reduction at 5 dpi. From 7 dpi to the end of the experiment at 15 dpi, all the chicks were cleared for both Salmonella. The findings of this study demonstrate that bacteriophage treatment is efficacious in reducing S. typhimurium and S. enteritidis colonization in broiler chickens within a short period and could be used as an alternative to antibiotics.