Survival of highly related ESBL- and pAmpC- producing Escherichia coli in broiler farms identified before and after cleaning and disinfection using cgMLST.

BACKGROUND: Broiler chickens are frequently colonized with Extended-Spectrum Beta-Lactamase- (ESBL-) and plasmid mediated AmpC Beta-Lactamase- (pAmpC-) producing Enterobacterales, and we are confronted with the potential spread of these resistant bacteria in the food chain, in the environment, and to humans. Research focused on identifying of transmission routes and investigating potential intervention measures against ESBL- and pAmpC- producing bacteria in the broiler production chain. However, few data are available on the effects of cleaning and disinfection (C&D) procedures in broiler stables on ESBL- and pAmpC- producing bacteria. RESULTS: We systematically investigated five broiler stables before and after C&D and identified potential ESBL- and pAmpC- colonization sites after C&D in the broiler stables, including the anteroom and the nearby surrounding environment of the broiler stables. Phenotypically resistant E. coli isolates grown on MacConkey agar with cefotaxime were further analyzed for their beta-lactam resistance genes and phylogenetic groups, as well as the relation of isolates from the investigated stables before and after C&D by whole genome sequencing. Survival of ESBL- and pAmpC- producing E. coli is highly likely at sites where C&D was not performed or where insufficient cleaning was performed prior to disinfection. For the first time, we showed highly related ESBL-/pAmpC- producing E. coli isolates detected before and after C&D in four of five broiler stables examined with cgMLST. Survival of resistant isolates in investigated broiler stables as well as transmission of resistant isolates from broiler stables to the anteroom and surrounding environment and between broiler farms was shown. In addition, enterococci (frequently utilized to detect fecal contamination and for C&D control) can be used as an indicator bacterium for the detection of ESBL-/pAmpC- E. coli after C&D. CONCLUSION: We conclude that C&D can reduce ESBL-/pAmpC- producing E. coli in conventional broiler stables, but complete ESBL- and pAmpC- elimination does not seem to be possible in practice as several factors influence the C&D outcome (e.g. broiler stable condition, ESBL-/pAmpC- status prior to C&D, C&D procedures used, and biosecurity measures on the farm). A multifactorial approach, combining various hygiene- and management measures, is needed to reduce ESBL-/pAmpC- E. coli in broiler farms.

Efficacy evaluation of hydrogen peroxide disinfectant based zinc oxide nanoparticles against diarrhea causing Escherichia coli in ruminant animals and broiler chickens.

Different strains of Escherichia coli that exhibit genetic characteristics linked to diarrhea pose a major threat to both human and animal health. The purpose of this study was to determine the prevalence of pathogenic Escherichia coli (E. coli), the genetic linkages and routes of transmission between E. coli isolates from different animal species. The efficiency of disinfectants such as hydrogen peroxide (H2O2), Virkon®S, TH4+, nano zinc oxide (ZnO NPs), and H2O2-based zinc oxide nanoparticles (H2O2/ZnO NPs) against isolated strains of E. coli was evaluated. Using 100 fecal samples from different diarrheal species (cow n = 30, sheep n = 40, and broiler chicken n = 30) for E. coli isolation and identification using the entero-bacterial repetitive intergenic consensus (ERIC-PCR) fingerprinting technique. The E. coli properties isolated from several diarrheal species were examined for their pathogenicity in vitro. Scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HR-TEM), Fourier-transform infrared spectrum (FT-IR), X-ray diffraction (XRD), zeta potential, and particle size distribution were used for the synthesis and characterization of ZnO NPs and H2O2/ZnO NPs. The broth macro-dilution method was used to assess the effectiveness of disinfectants and disinfectant-based nanoparticles against E. coli strains. Regarding the results, the hemolytic activity and Congo red binding assays of pathogenic E. coli isolates were 55.3 and 44.7%, respectively. Eleven virulent E. coli isolates were typed into five ERIC-types (A1, A2, B1, B2, and B3) using the ERIC-PCR method. These types clustered into two main clusters (A and B) with 75% similarity. In conclusion, there was 90% similarity between the sheep samples' ERIC types A1 and A2. On the other hand, 89% of the ERIC types B1, B2, and B3 of cows and poultry samples were comparable. The H2O2/ZnO NPs composite exhibits potential antibacterial action against E. coli isolates at 0.04 mg/ml after 120 min of exposure.

Endotoxin Translocation Is Increased in Broiler Chickens Fed a Fusarium Mycotoxin-Contaminated Diet.

Broiler chickens in livestock production face numerous challenges that can impact their health and welfare, including mycotoxin contamination and heat stress. In this study, we aimed to investigate the combined effects of two mycotoxins, deoxynivalenol (DON) and fumonisins (FBs), along with short-term heat stress conditions, on broiler gut health and endotoxin translocation. An experiment was conducted to assess the impacts of mycotoxin exposure on broilers, focusing on intestinal endotoxin activity, gene expression related to gut barrier function and inflammation, and the plasma concentration of the endotoxin marker 3-OH C14:0 either at thermoneutral conditions or short-term heat stress conditions. Independently of heat stress, broilers fed DON-contaminated diets exhibited reduced body weight gain during the starter phase (Day 1-12) compared to the control group, while broilers fed FB-contaminated diets experienced decreased body weight gain throughout the entire trial period (Day 1-24). Furthermore, under thermoneutral conditions, broilers fed DON-contaminated diets showed an increase in 3-OH C14:0 concentration in the plasma. Moreover, under heat stress conditions, the expression of genes related to gut barrier function (Claudin 5, Zonulin 1 and 2) and inflammation (Toll-like receptor 4, Interleukin-1 beta, Interleukin-6) was significantly affected by diets contaminated with mycotoxins, depending on the gut segment. This effect was particularly prominent in broilers fed diets contaminated with FBs. Notably, the plasma concentration of 3-OH C14:0 increased in broilers exposed to both DON- and FB-contaminated diets under heat stress conditions. These findings shed light on the intricate interactions between mycotoxins, heat stress, gut health, and endotoxin translocation in broiler chickens, highlighting the importance of understanding these interactions for the development of effective management strategies in livestock production to enhance broiler health and welfare.

Intestinal microbiome profiles in broiler chickens raised without antibiotics exhibit altered microbiome dynamics relative to conventionally raised chickens.

The present study was undertaken to profile and compare the cecal microbial communities in conventionally (CONV) grown and raised without antibiotics (RWA) broiler chickens. Three hundred chickens were collected from five CONV and five RWA chicken farms on days 10, 24, and 35 of age. Microbial genomic DNA was extracted from cecal contents, and the V4-V5 hypervariable regions of the 16S rRNA gene were amplified and sequenced. Analysis of 16S rRNA sequence data indicated significant differences in the cecal microbial diversity and composition between CONV and RWA chickens on days 10, 24, and 35 days of age. On days 10 and 24, CONV chickens had higher richness and diversity of the cecal microbiome relative to RWA chickens. However, on day 35, this pattern reversed such that RWA chickens had higher richness and diversity of the cecal microbiome than the CONV groups. On days 10 and 24, the microbiomes of both CONV and RWA chickens were dominated by members of the phylum Firmicutes. On day 35, while Firmicutes remained dominant in the RWA chickens, the microbiome of CONV chickens exhibited am abundance of Bacteroidetes. The cecal microbiome of CONV chickens was enriched with the genus Faecalibacterium, Pseudoflavonifractor, unclassified Clostridium_IV, Bacteroides, Alistipes, and Butyricimonas, whereas the cecal microbiome of RWA chickens was enriched with genus Anaerofilum, Butyricicoccu, Clostridium_XlVb and unclassified Lachnospiraceae. Overall, the cecal microbiome richness, diversity, and composition were greatly influenced by the management program applied in these farms. These findings provide a foundation for further research on tailoring feed formulation or developing a consortium to modify the gut microbiome composition of RWA chickens.

Occurrence, antibiotic resistance profiles and associated risk factors of Klebsiella pneumoniae in poultry farms in selected districts of Somalia Reginal State, Ethiopia.

BACKGROUND: Klebsiella pneumoniae is an opportunistic infection that causes production losses and death in the chicken industry. A cross-sectional study was conducted on exotic chicken breeds reared at the Jigjiga poultry farm from November 2022 to May 2023 to estimate the occurrence, associated risk factors, and antimicrobial susceptibility profiles of Klebsiella pneumoniae. The chickens were selected using systematic random sampling techniques. A total of 384 cloacal swabs were collected aseptically and transported to the laboratory for analysis. For statistical analysis, STATA® version 14.0 statistical software was used. RESULTS: From 384 examined faecal samples, 258 (67.2%) prevalences of Klebsiella pneumoniae were found. Furthermore, the association of the study's risk factors with the prevalence of Klebsiella pneumoniae was explored, and no statistically significant association was identified between sex and age. Nonetheless, relative prevalence at the age level was higher in chickens aged 12 months (67.6%) and Sasso breeds (90.0%). Similarly, male chickens and those raised for meat and egg production had a high prevalence rate of 72.5 and 75.8%, respectively. A total of 30 isolated Klebsiella pneumoniae colonies were tested in vitro for antibiotic sensitivity for six drugs, and it was shown that Klebsiella pneumoniae is moderately sensitive to Penicillin G (43.3%) while having higher resistance to Oxytetracycline (80.0%). CONCLUSIONS: The current findings revealed that the research area had the highest prevalence of Klebsiella pneumoniae, and the isolates were resistant to commonly used drugs in the study area. Thus, a long-term intervention plan, thorough research to determine a nationwide status, as well as further multi-drug resistance patterns and molecular characterization, were urged.

A comprehensive study of colisepticaemia progression in layer chickens applying novel tools elucidates pathogenesis and transmission of Escherichia coli into eggs.

Colisepticaemia caused by avian pathogenic Escherichia coli (APEC) is a challenging disease due to its high economic importance in poultry, dubious pathogenesis and potential link with zoonosis and food safety. The existing in vitro studies can't define hallmark traits of APEC isolates, suggesting a paradigm shift towards host response to understand pathogenesis. This study investigated the comprehensive pathological and microbial progression of colisepticaemia, and transmission of E. coli into eggs using novel tools. In total 48 hens were allocated into three groups and were inoculated intratracheally with ilux2-E. coli PA14/17480/5-/ovary (bioluminescent strain), E. coli PA14/17480/5-/ovary or phosphate buffered saline. Infection with both strains led to typical clinical signs and lesions of colibacillosis as in field outbreaks. Based on lung histopathology, colisepticaemia progression was divided into four disease stages as: stage I (1-3 days post infection (dpi)), stage II (6 dpi), stage III (9 dpi) and stage IV (16 dpi) that were histologically characterized by predominance of heterophils, mixed cells, pyogranuloma, and convalescence, respectively. As disease progressed, bacterial colonization in host organs also decreased, revealed by the quantification of bacterial bioluminescence, bacteriology, and quantitative immunohistochemistry. Furthermore, immunofluorescence, immunohistochemistry, and bacteria re-isolation showed that E. coli colonized the reproductive tract of infected hens and reached to egg yolk and albumen. In conclusion, the study provides novel insights into the pathogenesis of colisepticemia by characterizing microbial and pathological changes at different disease stages, and of the bacteria transmission to table eggs, which have serious consequences on poultry health and food safety.

Effects of different supplemental levels of protease DE200 on the production performance, egg quality, and cecum microflora of laying hens.

This study aimed to investigate the effects of different levels of the protease DE200 on the performance, egg quality, organ index, and cecum microflora of Hy-line W36 laying hens. In this experiment, a total of 180 laying hens aged 300 d were randomly divided into three treatment groups and fed diets containing 0, 100, or 200 g/t DE200. The experimental period was 8 wk, including 2 wk of prefeeding and 6 wk of the formal experiment. Regular feeding was performed thrice a day and eggs were collected twice daily, and the feed intake and the egg quality were recorded. The results showed that in terms of production performance, dietary supplementation with different levels of DE200 significantly increased egg production (EP; P < 0.05) and significantly decreased the feed conversion ratio (FCR; P < 0.05) and average daily feed intake (ADFI; P < 0.05) without affecting egg weight (EW). In addition, the addition of DE200 significantly reduced the egg breakage rate (P < 0.05) and tended to increase the Haugh unit and decrease the water content of the yolk (P > 0.05). In the cecal microflora, the addition of DE200 increased the proportions of Bacteroidetes and Firmicutes at the phylum level while reducing the proportion of Fusobacteria. Furthermore, at the genus level, the addition of DE200 increased the proportions of Bacteroides and Faecalibacterium and reduced the proportion of Megamonas. This study suggested that the protease DE200 can be used as a feed supplement to improve the production performance of laying hens.

In the production of laying hens, improving the efficiency of dietary protein utilization is important. The aim of this study was to investigate the effects of the protease DE200 on the performance, egg quality and cecal microflora of Hyline white laying hens. A total of 180 laying hens aged 300 d were randomly divided into three treatment groups and fed diets containing 0, 100, or 200 g/t DE200 for 56 d. The results showed that supplementation with 100 or 200 g/t DE200 in the basal diet improved the production performance and egg quality of laying hens. DE200 (100 g/t) improved the balance of the cecal microflora, and DE200 (200 g/t) increased the richness and diversity of the cecal microflora of laying hens. Dietary supplementation with DE200 can improve the intestinal health and nutrient utilization efficiency of laying hens by improving the intestinal flora.

Modeling the influence of propionic acid concentration and pH on the kinetics of Salmonella Typhimurium.

Salmonella Typhimurium is a foodborne pathogen often found in the poultry production chain. Antibiotics have been used to reduce S. Typhimurium contamination in poultry aviaries and improve chicken growth. However, antibiotics were banned in several countries. Alternatively, organic acids, such as propionic acid (PA), can control pathogens. This study determined the PA minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and mathematically modeled S. Typhimurium growth/inactivation kinetics under the influence of PA at different pH values (4.5, 5.5, and 6.5) which are within the pH range of the chicken gastrointestinal tract. The PA MIC against S. Typhimurium was pH-dependent, resulting in 5.0, 3.5 and 9.0 mM undissociated PA at pH 4.5, 5.5, and 6.5, respectively. The Baranyi and Roberts and the Weibull model fit growth and inactivation data well, respectively. Secondary models were proposed. The validated model predicted 3-log reduction of S. Typhimurium in 3 h at 68.2 mM of undissociated PA and pH 4.5. The models presented a good capacity to describe the kinetics of S. Typhimurium subjected to PA, representing a useful tool to predict PA antibacterial action depending on the pH.

Phenotypic and genotypic characterization of resistance and virulence in Pseudomonas aeruginosa isolated from poultry farms in Egypt using whole genome sequencing.

Pseudomonas aeruginosa (P. aeruginosa) is an ESKAPE pathogen that can quickly develop resistance to most antibiotics. This bacterium is a zoonotic pathogen that can be found in humans, animals, foods, and environmental samples, making it a One-Health concern. P. aeruginosa threatens the poultry industry in Egypt, leading to significant economic losses. However, the investigation of this bacterium using NGS technology is nearly non-existent in Egypt. In this study, 38 isolates obtained from broiler farms of the Delta region were phenotypically investigated, and their genomes were characterized using whole genome sequencing (WGS). The study found that 100% of the isolates were resistant to fosfomycin and harbored the fosA gene. They were also resistant to trimethoprim/sulfamethoxazole, although only one isolate harbored the sul1 gene. Non-susceptibility (resistant, susceptible with increased dose) of colistin was observed in all isolates. WGS analysis revealed a high level of diversity between isolates, and MLST analysis allocated the 38 P. aeruginosa isolates into 11 distinct sequence types. The most predominant sequence type was ST267, found in 13 isolates, followed by ST1395 in 8 isolates. The isolates were susceptible to almost all tested antibiotics carrying only few different antimicrobial resistance (AMR) genes. Various AMR genes that confer resistance mainly to ß-lactam, aminoglycoside, sulfonamide, and phenicol compounds were identified. Additionally, several virulence associated genes were found without any significant differences in number and distribution among isolates. The majority of the virulence genes was identified in almost all isolates. The fact that P. aeruginosa, which harbors several AMR and virulence-associated factors, is present in poultry farms is alarming and threatens public health. The misuse of antimicrobial compounds in poultry farms plays a significant role in resistance development. Thus, increasing awareness and implementing strict veterinary regulations to guide the use of veterinary antibiotics is required to reduce health and environmental risks. Further studies from a One-Health perspective using WGS are necessary to trace the potential transmission routes of resistance between animals and humans and clarify resistance mechanisms.

The spontaneously produced lysogenic prophage phi456 promotes bacterial resistance to adverse environments and enhances the colonization ability of avian pathogenic Escherichia coli strain DE456.

In the last decade, prophages that possess the ability of lysogenic transformation have become increasingly significant. Their transfer and subsequent activity in the host have a significant impact on the evolution of bacteria. Here, we investigate the role of prophage phi456 with high spontaneous induction in the bacterial genome of Avian pathogenic Escherichia coli (APEC) DE456. The phage particles, phi456, that were released from DE456 were isolated, purified, and sequenced. Additionally, phage particles were no longer observed either during normal growth or induced by nalidixic acid in DE456Δphi456. This indicated that the released phage particles from DE456 were only phi456. We demonstrated that phi456 contributed to biofilm formation through spontaneous induction of the accompanying increase in the eDNA content. The survival ability of DE456Δphi456 was decreased in avian macrophage HD11 under oxidative stress and acidic conditions. This is likely due to a decrease in the transcription levels of three crucial genes-rpoS, katE, and oxyR-which are needed to help the bacteria adapt to and survive in adverse environments. It has been observed through animal experiments that the presence of phi456 in the DE456 genome enhances colonization ability in vivo. Additionally, the number of type I fimbriae in DE456Δphi456 was observed to be reduced under transmission electron microscopy when compared to the wild-type strain. The qRT-PCR results indicated that the expression levels of the subunit of I fimbriae (fimA) and its apical adhesin (fimH) were significantly lower in DE456Δphi456. Therefore, it can be concluded that phi456 plays a crucial role in helping bacterial hosts survive in unfavorable conditions and enhancing the colonization ability in DE456.

The European livestock resistome.

Metagenomic sequencing has proven to be a powerful tool in the monitoring of antimicrobial resistance (AMR). Here, we provide a comparative analysis of the resistome from pigs, poultry, veal calves, turkey, and rainbow trout, for a total of 538 herds across nine European countries. We calculated the effects of per-farm management practices and antimicrobial usage (AMU) on the resistome in pigs, broilers, and veal calves. We also provide an in-depth study of the associations between bacterial diversity, resistome diversity, and AMR abundances as well as co-occurrence analysis of bacterial taxa and antimicrobial resistance genes (ARGs) and the universality of the latter. The resistomes of veal calves and pigs clustered together, as did those of avian origin, while the rainbow trout resistome was different. Moreover, we identified clear core resistomes for each specific food-producing animal species. We identified positive associations between bacterial alpha diversity and both resistome alpha diversity and abundance. Network analyses revealed very few taxa-ARG associations in pigs but a large number for the avian species. Using updated reference databases and optimized bioinformatics, previously reported significant associations between AMU, biosecurity, and AMR in pig and poultry farms were validated. AMU is an important driver for AMR; however, our integrated analyses suggest that factors contributing to increased bacterial diversity might also be associated with higher AMR load. We also found that dispersal limitations of ARGs are shaping livestock resistomes, and future efforts to fight AMR should continue to emphasize biosecurity measures.IMPORTANCEUnderstanding the occurrence, diversity, and drivers for antimicrobial resistance (AMR) is important to focus future control efforts. So far, almost all attempts to limit AMR in livestock have addressed antimicrobial consumption. We here performed an integrated analysis of the resistomes of five important farmed animal populations across Europe finding that the resistome and AMR levels are also shaped by factors related to bacterial diversity, as well as dispersal limitations. Thus, future studies and interventions aimed at reducing AMR should not only address antimicrobial usage but also consider other epidemiological and ecological factors.

Predictive modeling of Salmonella spp. growth behavior in cooked and raw chicken samples: Real-time PCR quantification approach and model assessment in different handling scenarios.

The increasing prevalence of Salmonella contamination in poultry meat emphasizes the importance of suitable predictive microbiological models for estimating Salmonella growth behavior. This study was conducted to evaluate the potential of chicken juice as a model system to predict the behavior of Salmonella spp. in cooked and raw chicken products and to assess its ability to predict cross-contamination scenarios. A cocktail of four Salmonella serovars was inoculated into chicken juice, sliced chicken, ground chicken, and chicken patties, with subsequent incubation at 10, 15, 20, and 25°C for 39 h. The number of Salmonella spp. in each sample was determined using real-time polymerase chain reaction. Growth curves were fitted into the primary Baranyi and Roberts model to obtain growth parameters. Interactions between temperature and growth parameters were described using the secondary Ratkowsky's square root model. The predictive results generated by the chicken juice model were compared with those obtained from other chicken meat models. Furthermore, the parameters of the chicken juice model were used to predict Salmonella spp. numbers in six worst-case cross-contamination scenarios. Performance of the chicken juice model was evaluated using the acceptable prediction zone from -1.0 (fail-safe) to 0.5 (fail-dangerous) log. Chicken juice model accurately predicted all observed data points within the acceptable range, with the distribution of residuals being wider near the fail-safe zone (75%) than near the fail-dangerous zone (25%). This study offers valuable insights into a novel approach for modeling Salmonella growth in chicken meat products, with implications for food safety through the development of strategic interventions. PRACTICAL APPLICATION: The findings of this study have important implications in the food industry, as chicken juice could be a useful tool for predicting Salmonella behavior in different chicken products and thus reducing the risk of foodborne illnesses through the development of strategic interventions. However, it is important to recognize that some modifications to the chicken juice model will be necessary to accurately mimic all real-life conditions, as multiple factors particularly those related to food processing can vary between different products.

Potential for the development of Taraxacum mongolicum aqueous extract as a phytogenic feed additive for poultry.

Introduction: Taraxacum mongolicum (TM) is a kind of medicinal and edible homologous plant which is included in the catalogue of feed raw materials in China. It is rich in polyphenols, flavonoids, polysaccharides and other active substances, and shows many benefits to livestock, poultry and aquatic products. The study aimed to assess the potential of TM aqueous extract (TMAE) as a substitute for poultry AGPs. Methods: A total of 240 one-day-old Arbor Acker broilers were randomly assigned to four groups and fed a basal diet (Con) supplemented with 500, 1000, and 2000 mg/kg TMAE (Low, Medium, and High groups). The growth performance of the broilers was measured on day 21 and day 42. At the end of the trial, the researchers measured slaughter performance and collected serum, liver, spleen, ileum, and intestinal contents to investigate the effects of TMAE on serum biochemistry, antioxidant capacity, immune function, organ coefficient, intestinal morphology, flora composition, and short-chain fatty acids (SCFAs). Results: The results showed that broilers treated with TMAE had a significantly higher average daily gain from 22 to 42 days old compared to the Con group. Various doses of TMAE resulted in different levels of improvement in serum chemistry. High doses increased serum alkaline phosphatase and decreased creatinine. TMAE also increased the antioxidant capacity of serum, liver, and ileum in broilers. Additionally, middle and high doses of TMAE enhanced the innate immune function of the liver (IL-10) and ileum (Occludin) in broilers. Compared to the control group, the TMAE treatment group exhibited an increase in the ratio of villi length to villi crypt in the duodenum. TMAE increased the abundance of beneficial bacteria, such as Alistipes and Lactobacillus, while reducing the accumulation of harmful bacteria, such as Colidextracter and Sellimonas. The cecum's SCFAs content increased with a medium dose of TMAE. Supplementing broiler diets with TMAE at varying doses enhanced growth performance and overall health. The most significant benefits were observed at a dose of 1000 mg/kg, including improved serum biochemical parameters, intestinal morphology, antioxidant capacity of the liver and ileum, immune function of the liver and ileum, and increased SCFAs content. Lactobacillus aviarius, norank_f_norank_o__Clostridia_UCG-014, and Flavonifractor are potentially dominant members of the intestinal microflora. Conclusion: In conclusion, TMAE is a promising poultry feed additive and 1000 mg/kg is an effective reference dose.

Feeding strategy and prebiotic supplementation: Effects on immune responses and gut health in the early life stage of broiler chickens.

This study aimed to investigate the effects of early or late feeding strategies and prebiotic, on immune responses and gut health during the early life stage of broiler chickens. A total of 240 day-old male broiler chicks were used in a 2 × 3 factorial arrangement of treatments that comprised 2 feeding strategies (early or late) and 3 levels of prebiotic (0, recommended dosage or three times the recommended dosage) in a completely randomized design with 4 pen replicates and 10 broilers per each. Compared to broiler chickens that had early access to feed, delayed access to feed resulted in an increased population of Escherichia coli and a decreased population of Lactobacillus spp. and Bifidobacterium spp. in the ileum (P < 0.05). Additionally, delayed access to feed led to a decrease in villus height, crypt depth, villus height: villus width ratio, goblet cell density, and mucin 2 gene expression in the ileum (P < 0.05). The supplementation of prebiotics in both the late and early feeding strategy groups resulted in increased villus height, crypt depth, goblet cell density, mucin 2 gene expression, and antibodies against Infectious Bursal Disease (IBD). Additionally, it led to an improvement in the foot web thickness index (P < 0.05). Furthermore, it resulted in a significant decrease in the population of Escherichia coli, while the populations of Lactobacillus spp. and Bifidobacterium spp. in the ileum were significantly increased (P < 0.05). Therefore, this study suggests that incorporating prebiotics in the starter diet can effectively enhance immune responses and promote gut health, regardless of the feeding strategy (early or late). In conclusion, this study demonstrates the potential benefits of incorporating prebiotics into poultry diets to alleviate the detrimental effects of delayed access to feed and improve gut health during the early life stage of broiler chickens.

A broad host phage, CP6, for combating multidrug-resistant Campylobacter prevalent in poultry meat.

Campylobacter is a major cause of bacterial foodborne diarrhea worldwide. Consumption of raw or undercooked chicken meat contaminated with Campylobacter is the most common causative agent of human infections. Given the high prevalence of contamination in poultry meat and the recent rise of multi-drug-resistant (MDR) Campylobacter strains, an effective intervention method of reducing bird colonization is needed. In this study, the Campylobacter-specific lytic phage CP6 was isolated from chicken feces. Phage CP6 exhibited a broad host range against different MDR Campylobacter isolates (97.4% of strains were infected). Some biological characteristics were observed, such as a good pH (3-9) stability and moderate temperature tolerance (<50 ℃). The complete genome sequence revealed a linear double-stranded DNA (178,350 bp, group II Campylobacter phage) with 27.51% GC content, including 209 predicted open reading frames, among which only 54 were annotated with known functions. Phylogenetic analysis of the phage major capsid protein demonstrated that phage CP6 was closely related to Campylobacter phage CPt10, CP21, CP20, IBB35, and CP220. CP6 phage exerted good antimicrobial effects on MDR Campylobacter in vitro culture and reduced CFUs of the host cells by up to 1-log compared with the control in artificially contaminated chicken breast meat. Our findings suggested the potential of CP6 phage as a promising antimicrobial agent for combating MDR Campylobacter in food processing.

Diet replacement with whole insect larvae affects intestinal morphology and microbiota of broiler chickens.

Insect-based diets are gaining interest as potential ingredients in improving poultry gut health. This study assessed the dietary treatment with whole dried Tenebrio molitor larvae (TM) on broiler chickens' gut microbiota and morphology. 120 Ross-308 broilers received treated diets with 5% (TM5) and 10% (TM10) replacement ratio in a 35-day trial. Intestinal histomorphometry was assessed, as well as claudin-3 expression pattern and ileal and caecal digesta for microbial community diversity. Null hypothesis was tested with two-way ANOVA considering the intestinal segment and diet as main factors. The TM5 group presented higher villi in the duodenum and ileum compared to the other two (P < 0.001), while treated groups showed shallower crypts in the duodenum (P < 0.001) and deeper in the jejunum and ileum than the control (P < 0.001). Treatments increased the caecal Firmicutes/Bacteroidetes ratio and led to significant changes at the genus level. While Lactobacilli survived in the caecum, a significant reduction was evident in the ileum of both groups, mainly owed to L. aviarius. Staphylococci and Methanobrevibacter significantly increased in the ileum of the TM5 group. Results suggest that dietary supplementation with whole dried TM larvae has no adverse effect on the intestinal epithelium formation and positively affects bacterial population richness and diversity.

In-vitro selection of lactic acid bacteria to combat Salmonella enterica and Campylobacter jejuni in broiler chickens.

Campylobacter and Salmonella are the two most prominent foodborne zoonotic pathogens reported in the European Union. As poultry is one of the major sources of these pathogens, it is imperative to mitigate the colonization of these pathogens in poultry. Many strains of lactic acid bacteria (LAB) have demonstrated anti-Salmonella and anti-Campylobacter characteristics to varying degrees and spectrums which are attributed to the production of various metabolites. However, the production of these compounds and consequent antimicrobial properties are highly strain dependent. Therefore, the current study was performed to select a potent LAB and determine its causal attribute in inhibiting Salmonella enterica and Campylobacter jejuni, in-vitro. Six LAB (Lactiplantibacillus plantarum (LP), Lacticaseibacillus casei (LC), Limosilactobacillus reuteri (LR), Lacticaseibacillus rhamnosus (LRh), Leuconostoc mesenteroides (LM) and Pediococcus pentosaceus (PP)) and three serovars of Salmonella enterica (Typhimurium, Enterica and Braenderup) and Campylobacter jejuni were used in the current study. Spot overlays, well diffusion, co-culture and co-aggregation assays against Salmonella and well diffusion assays against Campylobacter jejuni were performed. Organic acid profiling of culture supernatants was performed using HPLC. The results indicated that LRh, LM and PP had the most significant anti-Salmonella effects while LP, LC, LM and PP displayed the most significant anti-Campylobacter effects. Lactic acid and formic acid detected in the culture supernatants seem the most likely source of the anti-Salmonella and anti-Campylobacter effects exhibited by these LAB. In conclusion, Leuconostoc mesenteroides displayed the most significant overall anti-pathogenic effects when compared to the other LAB strains studied, indicating its potential application in-vivo.

Recommendation of a standardized broth microdilution method for antimicrobial susceptibility testing of Avibacterium paragallinarum and resistance monitoring.

This study aimed to develop a method for standardized broth microdilution antimicrobial susceptibility testing (AST) of Avibacterium (Av.) paragallinarum, the causative agent of infectious coryza in chickens. For this, a total of 83 Av. paragallinarum isolates and strains were collected from 15 countries. To select unrelated isolates for method validation steps, macrorestriction analyses were performed with 15 Av. paragallinarum. The visible growth of Av. paragallinarum was examined in six broth media and growth curves were compiled. In Veterinary Fastidious Medium and cation-adjusted Mueller-Hinton broth (CAMHB) + 1% chicken serum + 0.0025% NADH (CAMHB + CS + NADH), visible growth of all isolates was detected and both media allowed adequate bacterial growth. Due to the better readability of Av. paragallinarum growth in microtiter plates, CAMHB + CS + NADH was chosen for AST. Repetitions of MIC testing with five epidemiologically unrelated isolates using a panel of 24 antimicrobial agents resulted in high essential MIC agreements of 96%-100% after 48-h incubation at 35 ± 2°C. Hence, the remaining 78 Av. paragallinarum were tested and demonstrated easily readable MICs with the proposed method. Differences in MICs were detected between isolates from different continents, with isolates from Africa showing lower MICs compared to isolates from America and Europe, which more often showed elevated MICs of aminoglycosides, quinolones, tetracyclines, and/or trimethoprim/sulfamethoxazole. PCR analyses of isolates used for method development revealed that isolates with elevated MICs of tetracyclines harbored the tetracycline resistance gene tet(B) but none of the other tested resistance genes were detected. Therefore, whole-genome sequencing data from 62 Av. paragallinarum were analyzed and revealed the presence of sequences showing nucleotide sequence identity to the genes aph(6)-Id, aph(3″)-Ib, blaTEM-1B, catA2, sul2, tet(B), tet(H), and mcr-like. Overall, the proposed method using CAMHB + CS + NADH for susceptibility testing with 48-h incubation time at 35 ± 2°C in ambient air was shown to be suitable for Av. paragallinarum. Due to a variety of resistance genes detected, the development of clinical breakpoints is highly recommended. IMPORTANCE: Avibacterium paragallinarum is an important pathogen in veterinary medicine that causes infectious coryza in chickens. Since antibiotics are often used for treatment and resistance of the pathogen is known, targeted therapy should be given after resistance testing of the pathogen. Unfortunately, there is currently no accepted method in standards that allows susceptibility testing of this fastidious pathogen. Therefore, we have worked out a method that allows harmonized susceptibility testing of the pathogen. The method meets the requirements of the CLSI and could be used by diagnostic laboratories.

Transcriptome analysis reveals the inhibitory mechanism of phloretin on virulence expression of Staphylococcus aureus and its application in cooked chicken.

Staphylococcus aureus (S. aureus) enterotoxins have aroused great concern to food safety owing to its increased risk of food poisoning. The current research aimed to investigate the anti-virulence mechanisms of phloretin against S. aureus in terms of toxin activity and gene expression. The results indicated that phloretin could effectively inhibit the production of hemolysins and enterotoxins, and its anti-virulence effect was exerted in a concentration-dependent manner. Transcriptome results indicated that phloretin could downregulate the transcription level of majority virulence factors related genes (68 %) of S. aureus, including the quorum sensing-related genes (agrB, agrC, agrA, sspA, splF, splD and others) and bacterial secretion system-related genes (secDF, secY2, and yidC). In addition, it was speculated that phloretin was most likely to bind to the AgrA DNA binding domain, thereby affecting the expression of downstream virulence genes (hla, seb, spa, rot, geh, etc) based on molecular docking. Finally, the application in cooked chicken indicated that phloretin could effectively decrease the content of enterotoxins and improve the storage quality of cooked chicken. These findings not only evidenced the feasible anti-virulence activity of phloretin, but also provided a new strategy to prevent S. aureus food poisoning in cooked meat preservation.

Status quo: Levels of Campylobacter spp. and hygiene indicators in German slaughterhouses for broiler and turkey.

Poultry is a common reservoir for Campylobacter and a main source for human campylobacteriosis. With broiler being the predominant poultry for food production, most food safety related research is conducted for this species, for turkey, few studies are available. Although animals are typically colonized at the farm level, the slaughtering process is considered an important factor in re- and cross-contamination. We examined the development of Campylobacter, E. coli and total colony counts (TCC) after several processing steps in three broiler and one turkey slaughterhouses. Whole carcass rinsing and neck skin sampling was applied for broilers resulting in 486 samples in total, while 126 neck skin samples were collected for turkeys. A decrease in the loads of the different bacterial groups along the broiler slaughtering process was observed. Campylobacter mean counts dropped from 4.5 ± 1.7 log10 CFU/ml after killing to 1.6 ± 0.4 log10 CFU/ml after chilling. However, an increase in Campylobacter counts was evident after evisceration before the values again decreased by the final processing step. Although the Campylobacter prevalence in the turkey samples showed a similar development, the bacterial loads were much lower with 1.7 ± 0.3 log10 CFU/g after killing and 1.7 ± 0.4 log10 CFU/g after chilling compared to those of broilers. The loads of E. coli and total colony count of turkey were higher after killing, were reduced by scalding and remained stable until after chilling. This study highlights trends during the slaughtering process in reducing the levels of Campylobacter, E. coli, and total colony counts for broiler and turkey carcasses, from the initial step to after chilling. These results contribute to our understanding of microbial dynamics during meat processing.