The phenotypic and genotypic antimicrobial resistance patterns of Salmonella isolated from chickens and meat at poultry slaughterhouses in Japan and Thailand.

Background and Aim: Poultry meat is a popular food consumed globally. However, poultry farming is a source of Salmonella contamination which causes human salmonellosis. This study aimed to estimate the prevalence and antimicrobial resistance (AMR) of Salmonella among chickens and meat at poultry slaughterhouses in province study areas in Thailand and Japan. Materials and Methods: Chicken meat and feces samples were collected in Thailand and Japan. In Nakhon Ratchasima Province, Thailand, 558 samples were obtained from slaughterhouses from January 2021 to March 2022. In Gifu Prefecture, Japan, 140 samples (70 each of intestinal contents and meat) were purchased from slaughterhouses from June to October 2022. For Salmonella detection, the samples were cultivated according to the International Organization for Standardization 6579:2002/AMD 1:2007 method and confirmed using polymerase chain reaction (PCR) and agglutination tests for serotyping. Isolated Salmonella were tested for AMR to nine antibiotics using the disk diffusion method. Selected phenotypic multidrug-resistant (MDR) isolates were evaluated for AMR genes (AMRGs) using PCR. Results: Salmonella prevalence from chickens and meat at slaughterhouses in Thailand and Japan was 41.2% and 40.7%, respectively. All the Salmonella isolates in Japan were serotyped as Schwarzengrund, and no Salmonella isolates were resistant to the nine antibiotics tested. In contrast, most of the Thai Salmonella isolates from chicken cloacal swabs and meat were resistant to doxycycline (78.3%) and colistin (63.5%). The prevalence of MDR Salmonella (MDRS) in chickens and meat from Thailand and Japan was 29.1% (67/230) and 0% (0/57), respectively. However, the most frequent AMRGs found in MDRS in Thailand were extended-spectrum beta-lactamase-Temoneira (ESBL-TEM) (45.1%). All isolated Salmonella from Japan revealed a class 1 integron gene (Int-1). Conclusion: Phenotypic MDRS isolates from Thailand showed the greatest correlation to AMRG and ESBL-TEM. Although there were no phenotypic AMR Salmonella isolates found in Japan, they can be found associated with Int-1, which may carry other AMRGs within the gene cassettes.

Risk factors associated with Salmonella prevalence, its antibiotic resistance, and egg antibiotic residues in the layer farming environment.

Background and Aim: Human salmonellosis with non-typhoidal Salmonella remains a global public health concern related to the consumption of contaminated eggs and egg-based products. This study aimed to examine the prevalence of Salmonella, antimicrobial-resistant Salmonella, and egg antibiotic residues concerning risk factors associated with Salmonella contamination in eggs, the layer farming environment, and laying hens kept in battery-cage closed-housing systems. Materials and Methods: This study used a repeated cross-sectional design to collect 488 samples from eggs, laying hens, and the farm environment on one laying farm for Salmonella detection according to ISO 6579:2002/AMD 1:2007. Salmonella-positive samples were further tested for serotype and antimicrobial susceptibility using the disk diffusion test. The layer farm contact person was interviewed at the sampling time to evaluate the risk factors associated with Salmonella contamination using logistic regression analysis. For each month, 24 eggs (144 eggs in total) were also randomly sampled from the collection egg area at the farm for antibiotic residue detection using the European Four Plate Test. Results: The highest Salmonella prevalence rates were in the samples from the layer pen floors, followed by the egg sizing machine (ESM) and eggshells at 65.5%, 52.5%, and 15%, respectively. Salmonella enterica serovar Corvallis was the dominant serovar (48.38%), followed by Mbandaka (37.76%), Braenderup (14.29%), and Typhimurium (4.08%). Rodent presence at the farm and the frequency of changing the disinfectant foot dip were significant factors related to Salmonella contamination on the pen floors (odds ratio [OR]=22.5, 95% confidence interval [CI]=2.11-240.48, p=0.01; OR=24, 95% CI=2.78-206.96, p=0.004, respectively). Hand-washing before sorting and cleaning the ESM were the significant factors (OR=13, 95% CI=1.2-140.73, p=0.04). The most resistant Salmonella isolates were resistant to oxytetracycline. One isolate of S. enterica Typhimurium was resistant to cefotaxime, enrofloxacin, and oxytetracycline. The antibiotic residues in the egg yolks were streptomycin, enrofloxacin, and tetracycline at prevalence rates of 36.11%, 11.81%, and 7.64%, respectively. Streptomycin was the most abundant residue in the albumen and yolk, followed by tetracycline. Conclusion: Salmonella prevalence in layer farming with a closed-housing system is related to effective biosecurity and hygiene issues, such as rodent control, clean farm equipment, and good worker hygiene. In addition, eggs' antibiotic residues may be related to treating antimicrobial-resistant Salmonella isolates and medicated feed with inappropriate antibiotic withdrawal time.

Prevalence and multidrug resistance of Enterococcus species isolated from chickens at slaughterhouses in Nakhon Ratchasima Province, Thailand.

Background and Aim: Enterococcus is a commensal bacteria found in humans and animals, which can cause human nosocomial infections. One of the most contaminated enterococcal sources is poultry meat. Therefore, this study estimated the prevalence and antimicrobial resistance (AMR) profile of Enterococcus from chickens and their meat products at local slaughterhouses in Nakhon Ratchasima Province, Thailand. Materials and Methods: From January 2021 to March 2022, 558 samples from 279 cloacal swabs and breast meat were collected from 31 local slaughterhouses in the area. Then, the samples were screened for Enterococcus using modified de Man, Rogosa, and Sharpe agar. Next, selected Gram-positive, catalase-negative, and cocci-shaped colonies were investigated for enterococcal confirmation using Enterococcosel Agar (EA). We also cultivated the samples directly on EA. However, the disk diffusion method was used to investigate positive Enterococcus resistance profiles to 16 antimicrobial agents. Finally, selected phenotypic multidrug-resistant (MDR) Enterococcus isolates were further assessed to identify AMR genes by polymerase chain reaction. Results: Investigations showed that the prevalence of Enterococcus isolates from the chicken cloacal swabs and meat samples were 29.75% (83/279) and 28.32% (78/279), respectively. Most Enterococcus positive isolates were resistant to colistin, followed by cefoxitin, cephalexin, and streptomycin. These isolates also showed a prevalence of MDR species (65.22%; 105/161) and 66 patterns. Furthermore, selected MDR Enterococcus (MDRE) from cloacal swabs and breast meat were positive for the resistant extended-spectrum beta-lactamase TEM genes at 71.43% (20/28) and 78.26% (18/23), respectively, whereas other AMR genes detected in the selected MDR enterococci from the cloacal swabs and breast meat were beta-lactamase TEM (bla TEM [0%, 1.96%]), Class 1 integrase (intI1 [14.28%, 0%]), colistin (mrc-1 [3.57%, 0%]), and vancomycin (vanA [14.28%, 0%]). Conclusion: This study indicated that phenotypic MDRE correlated with extended-spectrum beta-lactamase TEM gene presence, leading to an AMR reservoir that can be transferred to other bacteria.

Encapsulating Viability of Multi-strain Lactobacilli as Potential Probiotic in Pigs.

Important aspects of the selection of probiotics to be used for mixing in animal feed include host species specificity and probiotic cell survival during production and storage of their products. The research was to screen and investigate some probiotic properties of lactic acid bacteria (LAB) isolated from pig fecal samples. One hundred and thirty-eight representative LAB isolates, which were isolated from 51 pig fecal samples, were tested for acid and bile tolerance, antimicrobial susceptibility, antibacterial activity, potential adhesion to the cell surface, and survival rates when stored in varied microencapsulation forms: freeze-dried, spray-dried, and micro-beads. The antibacterial activity results of the ten LAB isolates, which were acid- (pH 2, 3 h) and bile- (50% (v/v) fresh pig bile, 8 h) tolerant and suitable for resisting the five antibiotics commonly used for treating pig infections with pathogenic indicator strains, showed that three isolates (L21, L80, L103) had strong inhibition to Escherichia coli, Salmonella group B, and Salmonella group D using co-culturing and agar spot assays. The three isolates had high hydrophobicity (65-73%) and did not show antagonistic growth against each other. All three selected isolates had greater than 80% survival in freeze-dried and micro-bead forms at 25-30 °C after 2 days of storage (80.4-86.75%, 7.31-7.89 log CFU/ml). Sequence analysis of the 16S rRNA genes demonstrated that the three isolates belong to Lactobacillus plantarum (strain L21 and strain L80) and L. paraplantarum (strain L103). The single and multiple strains of these bacteria may have potential use as probiotics in pig diets.

Effect of direct-fed microbials on culturable gut microbiotas in broiler chickens: a meta-analysis of controlled trials.

OBJECTIVE: This meta-analysis was conducted to evaluate the overall effect of direct-fed microbial (DFM) or probiotic supplementation on the log concentrations of culturable gut microbiota in broiler chickens. METHODS: Relevant studies were collected from PubMed, SCOPUS, Poultry Science Journal, and Google Scholar. The studies included controlled trials using DFM supplementation in broiler chickens and reporting log concentrations of the culturable gut microbiota. The overall effect of DFM supplementation was determined using standardized mean difference (SMD) with a random-effects model. Subgroups were analyzed to identify pre-specified characteristics possibly associated with the heterogeneity of the results. Risk of bias and publication bias were assessed. RESULTS: Eighteen taxa of the culturable gut microbiota were identified from 42 studies. The overall effect of DFM supplementation on the log concentrations of all 18 taxa did not differ significantly from the controls (SMD = -0.06, 95% confidence interval [-0.16, 0.04], p = 0.228, I2 = 85%, n = 699 comparisons), but the 18 taxa could be further classified into three categories by the direction of the effect size: taxa whose log concentrations did not differ significantly from the controls (category 1), taxa whose log concentrations increased significantly with DFM supplementation (category 2), and taxa whose log concentrations decreased significantly with DFM supplementation (category 3). Category 1 comprised nine taxa, including total bacterial counts. Category 2 comprised four taxa: Bacillus, Bifidobacterium, Clostridium butyricum, and Lactobacillus. Category 3 comprised five taxa: Clostridium perfringens, coliforms, Escherichia coli, Enterococcus, and Salmonella. Some characteristics identified by the subgroup analysis were associated with result heterogeneity. Most studies, however, were present with unclear risk of bias. Publication bias was also identified. CONCLUSION: DFM supplementation increased the concentrations of some beneficial bacteria (e.g. Bifidobacterium and Lactobacillus) and decreased those of some detrimental bacteria (e.g. Clostridium perfringens and Salmonella) in the guts of broiler chickens.

Antimicrobial susceptibility of lactic acid bacteria isolated from human and food-producing animal feces in Khon Kaen Province, Thailand.

The aim of this study was to investigate the susceptibility of 93 Lactobacillus strains to seven antimicrobial agents, i.e., penicillin G, amoxicillin-clavulanic acid, vancomycin, tetracycline, streptomycin, ciprofloxacin, and sulfamethoxazole-trimethoprim, by disk diffusion test. The Lactobacillus strains were isolated from fecal samples taken from 90 healthy, food-producing animals (fattening pigs, free-grazing ducks, and beef cattle) and 30 healthy human subjects (1- to 6-year-olds) in Khon Kaen. The minimum inhibitory concentration (MIC) values of tetracycline and ciprofloxacin against all strains were determined using the E-test. All 93 Lactobacillus isolates were identified at the species level using 16S rRNA gene sequencing. The most common species of Lactobacillus isolated from fattening pigs, free-grazing ducks, beef cattle, and humans were L. reuteri (30 %), L. salivarius (46.7 %), L. acetotolerans (20 %), and L. gasseri (33.3 %), respectively. A total of 83 Lactobacillus strains were resistant to the examined antibiotics. Some strains were resistant to two to six types of antibiotics. More than 50 % of Lactobacillus species were intrinsically resistant to vancomycin, streptomycin, ciprofloxacin, and sulfamethoxazole-trimethoprim. The prevalence of acquired resistance to tetracycline was observed for Lactobacillus isolates from fattening pigs, humans, free-grazing ducks, and beef cattle at 92.3, 85.7, 77.8, and 68.4 %, respectively. These results demonstrate the impact of antibiotic use in human and veterinary medicine on antibiotic treatment efficacy and may support the spread of transferable antibiotic resistant genes to other bacteria via the food chain.

Probiotic isolates from unconventional sources: a review.

The use of probiotics for human and animal health is continuously increasing. The probiotics used in humans commonly come from dairy foods, whereas the sources of probiotics used in animals are often the animals' own digestive tracts. Increasingly, probiotics from sources other than milk products are being selected for use in people who are lactose intolerant. These sources are non-dairy fermented foods and beverages, non-dairy and non-fermented foods such as fresh fruits and vegetables, feces of breast-fed infants and human breast milk. The probiotics that are used in both humans and animals are selected in stages; after the initial isolation of the appropriate culture medium, the probiotics must meet important qualifications, including being non-pathogenic acid and bile-tolerant strains that possess the ability to act against pathogens in the gastrointestinal tract and the safety-enhancing property of not being able to transfer any antibiotic resistance genes to other bacteria. The final stages of selection involve the accurate identification of the probiotic species.

Heterophil Phagocytic Activity Stimulated by Lactobacillus salivarius L61 and L55 Supplementation in Broilers with Salmonella Infection.

Newborn chicks are susceptible to Salmonella enterica serovar Enteritidis (SE). The objective of this study was to evaluate the effect of Lactobacillus probiotic isolated from chicken feces on heterophil phagocytosis in broiler chicks. A total of 150 newborn broiler chicks were divided into 5 groups (30 chicks per group) as follows: group 1 (normal control), given feed and water only, group 2 (positive control) given feed, water and SE infection, group 3 (L61 treated) given feed, water, SE infection followed by Lactobacillus salivarius L61 treatment, group 4 (L55 treated) given feed, water, SE infection followed by L. salivarius L55 treatment, and group 5 given feed, water, SE infection followed by L. salivarius L61 + L55 combination treatment. After SE infection, L. salivarius treatment lasted for 7 days. The results showed that L. salivarius L61 and L. salivarius L55 treatment, either alone or combination of both, increased the survival rate after SE infection, and upregulated heterophil phagocytosis and phagocytic index (PI). Conversely, chick groups treated with Lactobacillus showed lower SE recovery rate from cecal tonsils than that of the positive control group. The PI values of the chicken group with SE infection, followed by the combination of L. salivarius L61 and L. salivarius L55 were the highest as compared to either positive control or normal control group. Two Lactobacillus strains supplementation group showed significantly (p<0.05) higher PI value at 48 h than 24 h after treatment.