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315701.0000Reservoirs of antimicrobial resistance genes in retail raw milk. BACKGROUND: It has been estimated that at least 3% of the USA population consumes unpasteurized (raw) milk from animal sources, and the demand to legalize raw milk sales continues to increase. However, consumption of raw milk can cause foodborne illness and be a source of bacteria containing transferrable antimicrobial resistance genes (ARGs). To obtain a comprehensive understanding of the microbiome and antibiotic resistome in both raw and processed milk, we systematically analyzed 2034 retail milk samples including unpasteurized milk and pasteurized milk via vat pasteurization, high-temperature-short-time pasteurization, and ultra-pasteurization from the United States using complementary culture-based, 16S rRNA gene, and metagenomic sequencing techniques. RESULTS: Raw milk samples had the highest prevalence of viable bacteria which were measured as all aerobic bacteria, coliform, and Escherichia coli counts, and their microbiota was distinct from other types of milk. 16S rRNA gene sequencing revealed that Pseudomonadaceae dominated raw milk with limited levels of lactic acid bacteria. Among all milk samples, the microbiota remained stable with constant bacterial populations when stored at 4 °C. In contrast, storage at room temperature dramatically enriched the bacterial populations present in raw milk samples and, in parallel, significantly increased the richness and abundance of ARGs. Metagenomic sequencing indicated raw milk possessed dramatically more ARGs than pasteurized milk, and a conjugation assay documented the active transfer of bla(CMY-2), one ceftazidime resistance gene present in raw milk-borne E. coli, across bacterial species. The room temperature-enriched resistome differed in raw milk from distinct geographic locations, a difference likely associated with regionally distinct milk microbiota. CONCLUSION: Despite advertised "probiotic" effects, our results indicate that raw milk microbiota has minimal lactic acid bacteria. In addition, retail raw milk serves as a reservoir of ARGs, populations of which are readily amplified by spontaneous fermentation. There is an increased need to understand potential food safety risks from improper transportation and storage of raw milk with regard to ARGs. Video Abstract.202032591006
316210.9998Metagenomic Characterization of the Microbiome and Resistome of Retail Ground Beef Products. Ground beef can be a reservoir for a variety of bacteria, including spoilage organisms, and pathogenic foodborne bacteria. These bacteria can exhibit antimicrobial resistance (AMR) which is a public health concern if resistance in pathogens leads to treatment failure in humans. Culture-dependent techniques are commonly used to study individual bacterial species, but these techniques are unable to describe the whole community of microbial species (microbiome) and the profile of AMR genes they carry (resistome), which is critical for getting a holistic perspective of AMR. The objective of this study was to characterize the microbiome and resistome of retail ground beef products labeled as coming from conventional or raised without antibiotics (RWA) production systems. Sixteen ground beef products were purchased from 6 retail grocery outlets in Fort Collins, CO, half of which were labeled as produced from cattle raised conventionally and half of products were from RWA production. Total DNA was extracted and isolated from each sample and subjected to 16S rRNA amplicon sequencing for microbiome characterization and target-enriched shotgun sequencing to characterize the resistome. Differences in the microbiome and resistome of RWA and conventional ground beef were analyzed using the R programming software. Our results suggest that the resistome and microbiome of retail ground beef products with RWA packaging labels do not differ from products that do not carry claims regarding antimicrobial drug exposures during cattle production. The resistome predominantly consisted of tetracycline resistance making up more than 90% of reads mapped to resistance gene accessions in our samples. Firmicutes and Proteobacteria predominated in the microbiome of all samples (69.6% and 29.0%, respectively), but Proteobacteria composed a higher proportion in ground beef from conventionally raised cattle. In addition, our results suggest that product management, such as packaging type, could exert a stronger influence on the microbiome than the resistome in consumer-ready products. Metagenomic analyses of ground beef is a promising tool to investigate community-wide shifts in retail ground beef. Importantly, however, results from metagenomic sequencing must be carefully considered in parallel with traditional methods to better characterize the risk of AMR in retail products.202033240224
316320.9998Dynamics of microbiota and antimicrobial resistance in on-farm dairy processing plants using metagenomic and culture-dependent approaches. On-farm dairy processing plants, which are situated close to farms and larger dairy processing facilities, face unique challenges in maintaining environmental hygiene. This can impact various stages of dairy processing. These plants operate on smaller scales and use Low-Temperature-Long-Time (LTLT) pasteurization, making them more susceptible to microbial contamination through direct and indirect contact. Antimicrobial-resistant bacteria found on dairy farms pose risks to human health by potentially transferring resistance via dairy products. Our study aimed to investigate microbial distribution and antimicrobial resistance at four key stages: the farm, pre-pasteurization, post-pasteurization, and processing environments. We assessed microbial distribution by quantifying indicator bacteria and conducting metagenomic analysis. Antimicrobial resistance was examined by identifying resistance phenotypes and detecting resistance genes in bacterial isolates and metagenomes. Our results showed that the indicator bacteria were detected at all stages of on-farm dairy processing. We observed a significant reduction in aerobic microbes and coliforms post-pasteurization. However, contamination of the final dairy products increased, suggesting potential cross-contamination during post-pasteurization. Metagenomic analysis revealed that Pseudomonas, a representative psychrotrophic bacterium, was predominant in both the farm (24.1 %) and pre-pasteurization (65.9 %) stages, indicating microbial transfer from the farms to the processing plants. Post-pasteurization, Pseudomonas and other psychrotrophs like Acinetobacter and Enterobacteriaceae remained dominant. Core microbiota analysis identified 74 genera in total, including 13 psychrotrophic bacteria, across all stages. Of the 59 strains isolated from these plants, 49 were psychrotrophic. Antimicrobial resistance analysis showed that 74.6 % (44/59) of isolates were resistant to at least one antibiotic, with cefoxitin-, ampicillin-, amoxicillin-, and ticarcillin-resistant bacteria present at all stages. Identical antimicrobial resistance patterns were observed in isolates from serial stages of the same farm and season, suggesting bacterial transmission across stages. Additionally, 27.1 % (16/59) of isolates carried plasmid-mediated resistance genes, which were also detected in the metagenomes of non-isolated samples, indicating potential antimicrobial resistance gene transmission and their presence in uncultured bacteria. These findings reveal the persistence of antimicrobial-resistant psychrotrophic bacteria in on-farm dairy processing plants, which pose potential health risks via dairy consumption. Our study underscores the importance of both culture-dependent and culture-independent methods to fully understand their distribution and impact.202438640816
315830.9998Microbiological risk assessment and resistome analysis from shotgun metagenomics of bovine colostrum microbiome. Colostrum is known for its nutraceutical qualities, probiotic attributes, and health benefits. The aim of this study was to profile colostrum microbiome from bovine in rural sites of a developing country. The focus was on microbiological safety assessments and antimicrobial resistance, taking into account the risks linked with the consumption of raw colostrum. Shotgun sequencing was employed to analyze microbiome in raw buffalo and cow colostrum. Alpha and beta diversity analyses revealed increased inter and intra-variability within colostrum samples' microbiome from both livestock species. The colostrum microbiome was mainly comprised of bacteria, with over 90% abundance, whereas fungi and viruses were found in minor abundance. Known probiotic species, such as Leuconostoc mesenteroides, Lactococcus lactis, Streptococcus thermophilus, and Lactobacillus paracasei, were found in the colostrum samples. A relatively higher number of pathogenic and opportunistic pathogenic bacteria were identified in colostrum from both animals, including clinically significant bacteria like Clostridium botulinum, Pseudomonas aeruginosa, Escherichia coli, and Listeria monocytogenes. Binning retrieved 11 high-quality metagenome-assembled genomes (MAGs), with three MAGs potentially representing novel species from the genera Psychrobacter and Pantoea. Notably, 175 antimicrobial resistance genes (ARGs) and variants were detected, with 55 of them common to both buffalo and cow colostrum metagenomes. These ARGs confer resistance against aminoglycoside, fluoroquinolone, tetracycline, sulfonamide, and peptide antibiotics. In conclusion, this study describes a thorough overview of microbial communities in buffalo and cow colostrum samples. It emphasizes the importance of hygienic processing and pasteurization in minimizing the potential transmission of harmful microorganisms linked to the consumption of colostrum.202438404539
316440.9997Variations in Bacterial Communities and Antibiotic Resistance Genes Across Diverse Recycled and Surface Water Irrigation Sources in the Mid-Atlantic and Southwest United States: A CONSERVE Two-Year Field Study. Reduced availability of agricultural water has spurred increased interest in using recycled irrigation water for U.S. food crop production. However, there are significant knowledge gaps concerning the microbiological quality of these water sources. To address these gaps, we used 16S rRNA gene and metagenomic sequencing to characterize taxonomic and functional variations (e.g., antimicrobial resistance) in bacterial communities across diverse recycled and surface water irrigation sources. We collected 1 L water samples (n = 410) between 2016 and 2018 from the Mid-Atlantic (12 sites) and Southwest (10 sites) U.S. Samples were filtered, and DNA was extracted. The V3-V4 regions of the 16S rRNA gene were then PCR amplified and sequenced. Metagenomic sequencing was also performed to characterize antibiotic, metal, and biocide resistance genes. Bacterial alpha and beta diversities were significantly different (p < 0.001) across water types and seasons. Pathogenic bacteria, such as Salmonella enterica, Staphylococcus aureus, and Aeromonas hydrophilia were observed across sample types. The most common antibiotic resistance genes identified coded against macrolides/lincosamides/streptogramins, aminoglycosides, rifampin and elfamycins, and their read counts fluctuated across seasons. We also observed multi-metal and multi-biocide resistance across all water types. To our knowledge, this is the most comprehensive longitudinal study to date of U.S. recycled water and surface water used for irrigation. Our findings improve understanding of the potential differences in the risk of exposure to bacterial pathogens and antibiotic resistance genes originating from diverse irrigation water sources across seasons and U.S. regions.202236194536
316150.9997Longitudinal study on the effects of growth-promoting and therapeutic antibiotics on the dynamics of chicken cloacal and litter microbiomes and resistomes. BACKGROUND: Therapeutic and growth-promoting antibiotics are frequently used in broiler production. Indirect evidence indicates that these practices are linked to the proliferation of antimicrobial resistance (AMR), the spread of antibiotic-resistant bacteria from food animals to humans, and the environment, but there is a lack of comprehensive experimental data supporting this. We investigated the effects of growth promotor (bacitracin) and therapeutic (enrofloxacin) antibiotic administration on AMR in broilers for the duration of a production cycle, using a holistic approach that integrated both culture-dependent and culture-independent methods. We specifically focused on pathogen-harboring families (Enterobacteriaceae, Enterococcaceae, and Staphylococcaceae). RESULTS: Antibiotic-resistant bacteria and antibiotic resistance genes were ubiquitous in chicken cloaca and litter regardless of antibiotic administration. Environment (cloaca vs. litter) and growth stage were the primary drivers of variation in the microbiomes and resistomes, with increased bacterial diversity and a general decrease in abundance of the pathogen-harboring families with age. Bacitracin-fed groups had higher levels of bacitracin resistance genes and of vancomycin-resistant Enterococcaceae (total Enterococcaceae counts were not higher). Although metagenomic analyses classified 28-76% of the Enterococcaceae as the commensal human pathogens E. faecalis and E. faecium, culture-based analysis suggested that approximately 98% of the vancomycin-resistant Enterococcaceae were avian and not human-associated, suggesting differences in the taxonomic profiles of the resistant and non-resistant strains. Enrofloxacin treatments had varying effects, but generally facilitated increased relative abundance of multidrug-resistant Enterobacteriaceae strains, which were primarily E. coli. Metagenomic approaches revealed a diverse array of Staphylococcus spp., but the opportunistic pathogen S. aureus and methicillin resistance genes were not detected in culture-based or metagenomic analyses. Camphylobacteriaceae were significantly more abundant in the cloacal samples, especially in enrofloxacin-treated chickens, where a metagenome-assembled C. jejuni genome harboring fluoroquinolone and β-lactam resistance genes was identified. CONCLUSIONS: Within a "farm-to-fork, one health" perspective, considering the evidence that bacitracin and enrofloxacin used in poultry production can select for resistance, we recommend their use be regulated. Furthermore, we suggest routine surveillance of ESBL E. coli, vancomycin-resistant E. faecalis and E. faecium, and fluoroquinolone-resistant C. jejuni strains considering their pathogenic nature and capacity to disseminate AMR to the environment. Video Abstract.202134454634
314660.9997Resistomes from oxytetracycline-treated pigs are readily transferred to untreated pen mates. Pork is currently a major part of Danish food export and is also a key dietary source of protein across the world. Industrial pork production, however, comes with high antibiotic usage in many countries, including Denmark. This has created consumer demand for meat Raised Without Antibiotics (RWA). Previous work has demonstrated that levels of antibiotic resistance genes (ARGs) are indeed increased in antibiotically treated animals, but also suggest that these ARGs are transferred to untreated pen-mates. In a Danish commercial farm, we studied four groups of physically separated pigs: one group of only antibiotic treated pigs (n = 20), one group of only untreated pigs (n = 30 total, n = 15 analysed), and one group combining treated (n = 15) and untreated pigs (n = 15). These groups were followed for 16 weeks during which all pigs were profiled for both their faecal microbiome (through 16 S rRNA gene sequencing) and resistome (by use of a high-throughput qPCR platform targeting 82 ARGs and their variants). We found that the resistome of treated pigs was substantially enriched in resistance genes compared to untreated pigs but, importantly, observed that untreated pigs co-reared with treated pigs had levels of resistance genes approaching their treated pen mates, suggesting that the treated enterotype is readily transferred to the untreated animal. From this, we conclude that mixing of treated and untreated pigs causes spill-over of antibiotic resistant bacteria and/or resistance genes from treated pigs when these are co-reared. To optimize RWA production, treated and untreated pigs should be physically separated to limit the proliferation of ARGs.202439578929
313770.9997Evaluation of pre-treated healthcare wastes during COVID-19 pandemic reveals pathogenic microbiota, antibiotics residues, and antibiotic resistance genes against beta-lactams. The disposal of healthcare waste without prior elimination of pathogens and hazardous contaminants has negative effects on the environment and public health. This study aimed to profile the complete microbial community and correlate it with the antibiotic compounds identified in microwave pre-treated healthcare wastes collected from three different waste operators in Peninsular Malaysia. The bacterial and fungal compositions were determined via amplicon sequencing by targeting the full-length 16S rRNA gene and partial 18S with full-length ITS1-ITS2 regions, respectively. The antibiotic compounds were characterized using high-throughput spectrometry. There was significant variation in bacterial and fungal composition in three groups of samples, with alpha- (p-value = 0.04) and beta-diversity (p-values <0.006 and < 0.002), respectively. FC samples were found to acquire more pathogenic microorganisms than FA and FV samples. Paenibacillus and unclassified Bacilli genera were shared among three groups of samples, meanwhile, antibiotic-resistant bacteria Proteus mirabilis, Enterococcus faecium, and Enterococcus faecalis were found in modest quantities. A total of 19 antibiotic compounds were discovered and linked with the microbial abundance detected in the healthcare waste samples. The principal component analysis demonstrated a positive antibiotic-bacteria correlation for genera Pseudomonas, Aerococcus, Comamonas, and Vagococcus, while the other bacteria were negatively linked with antibiotics. Nevertheless, deep bioinformatic analysis confirmed the presence of bla(TEM-1) and penP which are associated with the production of class A beta-lactamase and beta-lactam resistance pathways. Microorganisms and contaminants, which serve as putative indicators in healthcare waste treatment evaluation revealed the ineffectiveness of microbial inactivation using the microwave sterilization method. Our findings suggested that the occurrence of clinically relevant microorganisms, antibiotic contaminants, and associated antibiotic resistance genes (ARGs) represent environmental and human health hazards when released into landfills via ARGs transmission.202336565841
315980.9997Longitudinal development of the dust microbiome in a newly opened Norwegian kindergarten. BACKGROUND: In Norway, 91% of children aged 1-5 attend kindergarten where they are exposed to indoor microbiomes which can have relevance for development and health. In order to gain a better understanding of the composition of the indoor microbiome and how it is affected by occupancy over time, floor dust samples from a newly opened kindergarten were investigated. Samples were collected during an 11-month period. Samples were analyzed for bacterial composition using 16S rRNA gene sequencing. Samples were also screened for four clinically relevant antibiotic resistance genes. In addition, Petrifilm analyses were used to evaluate surface hygiene. RESULTS: Significant changes in the microbial community composition were observed over time (PERMANOVA, P < 0.05). Particularly, changes in the abundance and the proportions of human associated bacteria were found. A decrease in the prevalence of Propionibacterium from over 16% abundance to less than 1% and an increase in Streptococcus from 10 to 16% were the most significant findings. Four classes of clinically relevant antibiotic resistance genes were tested for; three were detected in the dust, indicating the presence of resistant bacteria and a potential for resistance spread. Petrifilm analysis showed that some surfaces in the kindergarten were of consistent poor hygienic quality, and new hygienic routines are required. CONCLUSIONS: This study, which is the first of its kind performed at a newly opened kindergarten, reveals changes in the microbiome over time as well as the presence of antibiotic resistance genes and hygiene issues which are of relevance for occupant health.201830219104
311290.9997Farm-to-fork changes in poultry microbiomes and resistomes in Maputo City, Mozambique. Increasing demand for poultry has spurred poultry production in low- and middle-income countries like Mozambique. Poultry may be an important source of foodborne, antimicrobial-resistant bacteria to consumers in settings with limited water, sanitation, and hygiene infrastructure. The Chicken Exposures and Enteric Pathogens in Children Exposed through Environmental Pathways (ChEEP ChEEP) study was conducted in Maputo City, Mozambique from 2019 to 2021 to quantify enteric pathogen exposures along the supply chain for commercial and local (i.e., scavenger) chicken breeds. Here, we performed metagenomic sequencing of total DNA from banked ChEEP ChEEP samples to characterize fecal and carcass microbiomes and resistome diversity between chicken breeds and along the supply chain. Fecal samples (n = 26) were collected from commercial and local chickens at production sites and markets and carcass (n = 49) and rinse bucket samples (n = 26) from markets. We conducted taxonomic profiling and identified antimicrobial resistance genes (ARGs) from metagenomic sequence data, focusing especially on potential human pathogens and "high-risk" ARGs. We estimated alpha diversity for each sample and compared by site and breed. We estimated Bray-Curtis dissimilarity between samples and examined clustering. We found that commercial and local chickens harbored distinct fecal potential pathogens and resistomes at production and market sites. Many potentially pathogenic bacteria and ARGs present in chicken fecal samples are also present on carcasses sold to consumers. Finally, commercial chicken carcasses contain high-risk ARGs that are not necessarily introduced from chicken feces. These results indicate markets are an important site of exposure to potentially pathogenic bacteria and high-risk ARGs. IMPORTANCE: While chicken eggs and meat are a critical protein source in low-income settings, antibiotics are routinely fed to chickens with consequences for selection of antimicrobial resistance. Evaluating how poultry gut bacterial communities, including potential human pathogens and high-risk antimicrobial resistance genes, differ from farm to market could help identify where to target interventions to minimize transmission risks to human populations. In this study in Maputo City, Mozambique, we found compositional differences between commercial and local chicken breeds at production and market sites. We also found that while all potentially pathogenic bacteria and many high-risk antimicrobial resistance genes persisted from production and market through processing, some resistance genes were detected on carcass samples only after processing, suggesting human or environmental contamination is occurring within markets. Overall, our findings indicate that open-air markets may represent a critical juncture for human exposures to pathogens and antimicrobial resistance genes from poultry and poultry products.202539699181
3114100.9997Spatial and temporal dynamics of microbiomes and resistomes in broiler litter stockpiles. Farmers apply broiler chicken litter to soils to enrich organic matter and provide crops with nutrients, following varying periods of stockpiling. However, litter frequently harbors fecal-derived microbial pathogens and associated antibiotic resistance genes (ARGs), and may be a source of microbial contamination of produce. We coupled a cutting-edge Loop Genomics long-read 16S rRNA amplicon-sequencing platform with high-throughput qPCR that targeted a suite of ARGs, to assess temporal (five time points over a 60-day period) and spatial (top, middle and bottom layers) microbiome and resistome dynamics in a broiler litter stockpile. We focused on potentially pathogenic species from the Enterobacteriaceae, Enterococcaceae and Staphylococcaceae families associated with food-borne disease. Bacterial diversity was significantly lower in the middle of the stockpile, where targeted pathogens were lowest and Bacillaceae were abundant. E. coli was the most abundant Enterobacteriaceae species, and high levels of the opportunistic pathogen Enterococcus faecium were detected. Correlation analyses revealed that the latter was significantly associated with aminoglycoside (aac(6')-Ib(aka aacA4), aadA5), tetracycline (tetG), vancomycin (vanC), phenicol (floR) and MLSB (mphB) resistance genes. Staphylococcaceae were primarily non-pathogenic, but extremely low levels of the opportunistic pathogen S. aureus were detected, as was the opportunistic pathogen S. saprophyticus, which was linked to vancomycin (vanSA, vanC1), MLSB (vatE, ermB) and tetracycline (tetK) resistance genes. Collectively, we found that stockpile microbiomes and resistomes are strongly dictated by temporal fluctuations and spatial heterogeneity. Insights from this study can be exploited to improve stockpile management practice to support sustainable antimicrobial resistance mitigation policies in the future.202134900133
3931110.9997Commercial farmed swine harbour a variety of pathogenic bacteria and antimicrobial resistance genes. Introduction. The northern region of Thailand serves as a crucial area for swine production, contributing to the Thai community food supply. Previous studies have highlighted the presence of foodborne bacterial pathogens originating from swine farms in this region, posing a threat to both human and animal health.Gap statement. Multiple swine bacterial pathogens have been studied at a species level, but the distribution and co-occurrence of bacterial pathogens in agricultural swine has not been well established.Aim. Our study employed the intestinal scraping technique to directly examine the bacterial micro-organisms interacting with the swine host.Methodology. We used shotgun metagenomic sequencing to analyse the bacterial pathogens inhabiting the caecal microbiome of swine from five commercial farms in northern Thailand.Results. A variety of pathogenic and opportunistic bacteria were identified, including Escherichia coli, Clostridium botulinum, Staphylococcus aureus and the Corynebacterium genus. From a One Health perspective, these species are important foodborne and opportunistic pathogens in both humans and agricultural animals, making swine a critical pathogen reservoir that can cause illness in humans, especially farm workers. Additionally, the swine caecal microbiome contains commensal bacteria such as Bifidobacterium, Lactobacillus and Faecalibacterium, which are associated with normal physiology and feed utilization in healthy swine. Antimicrobial resistance genes were also detected in all samples, specifically conferring resistance to tetracycline and aminoglycosides, which have historically been used extensively in swine farming.Conclusion. The findings further support the need for improved sanitation standards in swine farms, and additional monitoring of agricultural animals and farm workers to reduce contamination and improved produce safety for human consumption.202438230911
7107120.9997A Comprehensive Study of the Microbiome, Resistome, and Physical and Chemical Characteristics of Chicken Waste from Intensive Farms. The application of chicken waste to farmland could be detrimental to public health. It may contribute to the dissemination of antibiotic-resistance genes (ARGs) and antibiotic-resistant bacteria (ARB) from feces and their subsequent entry into the food chain. The present study analyzes the metagenome and resistome of chicken manure and litter obtained from a commercial chicken farm in Poland. ARB were isolated, identified, and screened for antibiogram fingerprints using standard microbiological and molecular methods. The physicochemical properties of the chicken waste were also determined. ARGs, integrons, and mobile genetic elements (MGE) in chicken waste were analyzed using high-throughput SmartChip qPCR. The results confirm the presence of many ARGs, probably located in MGE, which can be transferred to other bacteria. Potentially pathogenic or opportunistic microorganisms and phytopathogens were isolated. More than 50% of the isolated strains were classified as being multi-drug resistant, and the remainder were resistant to at least one antibiotic class; these pose a real risk of entering the groundwater and contaminating the surrounding environment. Our results indicate that while chicken manure can be sufficient sources of the nutrients essential for plant growth, its microbiological aspects make this material highly dangerous to the environment.202236009027
7110130.9997The "best practices for farming" successfully contributed to decrease the antibiotic resistance gene abundances within dairy farms. INTRODUCTION: Farms are significant hotspots for the dissemination of antibiotic-resistant bacteria and genes (ARGs) into the environment and directly to humans. The prevalence of ARGs on farms underscores the need for effective strategies to reduce their spread. This study aimed to evaluate the impact of a guideline on "best practices for farming" aimed at reducing the dissemination of antibiotic resistance. METHODS: A guideline focused on prudent antibiotic use, selective therapy, and hygienic and immune-prophylactic practices was developed and provided to the owners of 10 selected dairy farms and their veterinarians. Fecal samples were collected from lactating cows, dry cows, and calves both before and after the implementation of the guideline. ARGs (bla (TEM), ermB, sul2, and tetA) were initially screened by end-point PCR, followed by quantification using digital droplet PCR. ARG abundance was expressed in relative terms by dividing the copy number of ARGs by the copy number of the 16S rRNA gene. RESULTS: The ARG abundances were higher in lactating cows compared to other categories. Despite similar levels of antibiotic administration (based on veterinary prescription data from the sampled farms) in both sampling campaigns, the total abundance of selected ARGs, particularly bla (TEM) and tetA, significantly decreased after the adoption of the farming guidelines. DISCUSSION: This study highlights the positive impact of prudent antibiotic use and the implementation of farming best practices in reducing the abundance of ARGs. The lactating cow category emerged as a crucial point of intervention for reducing the spread of antibiotic resistance. These findings contribute to ongoing efforts to address antibiotic resistance in farm environments and strengthen the evidence supporting the adoption of good farming practices.202439840338
7109140.9997Explore the Contamination of Antibiotic Resistance Genes (ARGs) and Antibiotic-Resistant Bacteria (ARB) of the Processing Lines at Typical Broiler Slaughterhouse in China. Farms are a major source of antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB), and previous research mainly focuses on polluted soils and breeding environments. However, slaughtering is an important link in the transmission of ARGs and ARB from farmland to dining table. In this study, we aim to reveal the pollution of ARGs and ARB in the slaughter process of broilers. First, by qualitative and quantitative analysis of ARGs in samples collected from the broiler slaughtering and processing production chain, the contamination level of ARGs was reflected; secondly, potential hosts for ARGs and microbial community were analyzed to reflect the possible transmission rules; thirdly, through the antibiotic susceptibility spectrum analysis of four typical food-borne pathogens, the distribution of ARB was revealed. The results showed that 24 types of ARGs were detected positive on the broiler slaughter production line, and tetracycline-resistance genes (20.45%) were the most frequently detected. The types of ARGs vary with sampling process, and all sampling links contain high levels of sul2 and intI1. The most abundant ARGs were detected in chicken surface in the scalding stage and entrails surface in the evisceration stage. There was a significant correlation between intI1 and tetM, suggesting that tetM might be able to enter the human food chain through class-1 integrons. The host range of the oqxB gene is the most extensive, including Sphingobacterium, Bacteroidia unclassified, Rothia, Microbacterium, Algoriella, etc. In the relevant links of the slaughter production line, the microbial community structure is similar. Removing viscera may cause diffusion of ARGs carried by intestinal microorganisms and contaminate chicken and following processing production. The four food-borne pathogens we tested are widely present in all aspects of the slaughter process, and most of them have multi-drug resistance and even have a high degree of resistance to some veterinary drugs banned by the Ministry of Agriculture. Our study preliminarily revealed the pollution of ARGs and ARB in the slaughter process of broilers, and these results are helpful to carry out food safety risk assessment and formulate corresponding control measures.202540232101
7103150.9997Microbial ecology, bacterial pathogens, and antibiotic resistant genes in swine manure wastewater as influenced by three swine management systems. The environmental influence of farm management in concentrated animal feeding operations (CAFO) can yield vast changes to the microbial biota and ecological structure of both the pig and waste manure lagoon wastewater. While some of these changes may not be negative, it is possible that CAFOs can enrich antibiotic resistant bacteria or pathogens based on farm type, thereby influencing the impact imparted by the land application of its respective wastewater. The purpose of this study was to measure the microbial constituents of swine-sow, -nursery, and -finisher farm manure lagoon wastewater and determine the changes induced by farm management. A total of 37 farms were visited in the Mid-South USA and analyzed for the genes 16S rRNA, spaQ (Salmonella spp.), Camp-16S (Campylobacter spp.), tetA, tetB, ermF, ermA, mecA, and intI using quantitative PCR. Additionally, 16S rRNA sequence libraries were created. Overall, it appeared that finisher farms were significantly different from nursery and sow farms in nearly all genes measured and in 16S rRNA clone libraries. Nearly all antibiotic resistance genes were detected in all farms. Interestingly, the mecA resistance gene (e.g. methicillin resistant Staphylococcus aureus) was below detection limits on most farms, and decreased as the pigs aged. Finisher farms generally had fewer antibiotic resistance genes, which corroborated previous phenotypic data; additionally, finisher farms produced a less diverse 16S rRNA sequence library. Comparisons of Camp-16S and spaQ GU (genomic unit) values to previous culture data demonstrated ratios from 10 to 10,000:1 depending on farm type, indicating viable but not cultivatable bacteria were dominant. The current study indicated that swine farm management schemes positively and negatively affect microbial and antibiotic resistant populations in CAFO wastewater which has future "downstream" implications from both an environmental and public health perspective.201424704907
3156160.9997Deciphering Resistome and Virulome Diversity in a Porcine Slaughterhouse and Pork Products Through Its Production Chain. We aimed to better understand resistome and virulome patterns on animal and process-area surfaces through a pig slaughterhouse to track possible contamination within the food production chain. Culture-dependent methods revealed high levels of microbial contamination, corresponding to mesophilic and pathogenic bacteria on both the animal and process-area surfaces mainly in the anesthesia (AA and AS) zone followed by "scorching and whip" (FA and FS) zone and also in the end products. To evaluate the potential risk of antibiotic resistance and virulence determinants, shotgun metagenomic DNA-sequencing of isolates from selected areas/products uncovered a high diversity and richness of antibiotic resistance genes (ARGs): 55-62 genes in the anesthesia area (AA and AS) and 35-40 in "animal-arrival zone" (MA and MS). The "scorching and whip" (FA and FS) area, however, exhibited lowered abundance of ARGs (1-6), indicating that the scalding and depilating process (an intermediate zone between "anesthesia" and "scorching and whip") significantly decreased bacterial load by 1-3 log(10) but also diminished the resistome. The high prevalence of antibiotic-inactivating enzyme genes in the "animal-arrival zone" (60-65%) and "anesthesia" area (56%) were mainly represented by those for aminoglycoside (46-51%) and lincosamide (14-19%) resistance, which did not reflect selective pressures by antibiotics most commonly used in pig therapy-tetracyclines and beta-lactams. Contrary to ARGs, greater number of virulence resistance genes were detected after evisceration in some products such as kidney, which reflected the poor hygienic practices. More than 19 general virulence features-mainly adherence, secretion system, chemotaxis and motility, invasion and motility were detected in some products. However, immune evasion determinants were detected in almost all samples analyzed from the beginning of the process, with highest amounts found from the anesthesia area. We conclude that there are two main sources of contamination in a pig slaughterhouse: the microorganisms carried on the animals' hide, and those from the evisceration step. As such, focussing control measures, e.g., enhanced disinfection procedures, on these contamination-source areas may reduce risks to food safety and consumer health, since the antibiotic and virulence determinants may spread to end products and the environment; further, ARG and virulence traits can exacerbate pathogen treatments.201830258416
3129170.9997Effect of therapeutic administration of β-lactam antibiotics on the bacterial community and antibiotic resistance patterns in milk. Dairy cows with mastitis are frequently treated with antibiotics. The potential effect of antibiotics on the milk microbiome is still not clear. Therefore, the objective of this research was to investigate the effect of 2 commonly used cephalosporins on the milk microbiota of dairy cows and the antibiotic resistance genes in the milk. The milk samples were collected from 7 dairy cows at the period before medication (d 0), medication (d 1, 2, 3), withdrawal period (d 4, 6, 8), and the period after withdrawal (d 9, 11, 13, 15). We applied 16S rRNA sequencing to explore the microbiota changes, and antibiotic resistance patterns were investigated by quantitative PCR. The microbiota richness and diversity in each sample were calculated using the Chao 1 (richness), Shannon (diversity), and Simpson (diversity) indices. The cephalosporins treatment lowered the Simpson diversity value at the period of withdrawal. Members of the Enterobacter genera were the most affected bacteria associated with mastitis. Meanwhile, antibiotic resistance genes in the milk were also influenced by antibiotic treatment. The cephalosporins treatment raised the proportion of bla(TEM) in milk samples at the period of withdrawal. Therefore, the treatment of cephalosporins led to change in the milk microbiota and increase of β-lactam resistance gene in the milk at the time of withdrawal period.202133741154
2814180.9997Fate of antimicrobial-resistant enterococci and staphylococci and resistance determinants in stored poultry litter. The use of antimicrobials in commercial broiler poultry production results in the presence of drug-resistant bacteria shed in the excreta of these birds. Because these wastes are largely land-disposed these pathogens can affect the surrounding environment and population. In this analysis, we characterized the survival of antimicrobial-resistant enterococci and staphylococci and resistance genes in poultry litter. Temperature, moisture, and pH were measured in the litter over a 120-day period from storage sheds at three conventional US broiler chicken farms, as well as colony-forming units of Enterococcus spp. and Staphylococcus spp. Selected isolates from each sampling event were tested for resistance to eight antimicrobials used in poultry feeds as well as the presence of resistance genes and mobile genetic elements. Temperatures greater than 60 degrees C were only intermittently observed in the core of the litter piles. Both antimicrobial-resistant enterococci and staphylococci, as well as resistance genes persisted throughout the 120-day study period. Resistance genes identified in the study include: erm(A), erm(B), erm (C), msr(A/B), msr(C), and vat(E). This study indicates that typical storage practices of poultry litter are insufficient for eliminating drug-resistant enterococci and staphylococci, which may then be released into the environment through land disposal.200919541298
5360190.9996Diversity, distribution and quantification of antibiotic resistance genes in goat and lamb slaughterhouse surfaces and meat products. The distribution and quantification of tetracycline, sulfonamide and beta-lactam resistance genes were assessed in slaughterhouse zones throughout meat chain production and the meat products; this study represents the first to report quantitatively monitor antibiotic resistance genes (ARG) in goat and lamb slaughterhouse using a culture independent approach, since most studies focused on individual bacterial species and their specific resistance types. Quantitative PCR (qPCR) revealed a high prevalence of tetracycline resistance genes tetA and tetB in almost all slaughterhouse zones. Sulfonamide resistance genes were largely distributed, while beta-lactam resistance genes were less predominant. Statistical analysis revealed that resistant bacteria, in most cases, were spread by the same route in almost all slaughterhouse zones, except for tetB, blaCTX and blaTEM genes, which occurred in few zones as isolated 'hot spots.' The sum of all analyzed ARG indicated that slaughterhouse surfaces and end products act as reservoirs of ARG, mainly tet genes, which were more prevalent in slaughtering room (SR), cutting room (CR) and commercial meat products (MP). Resistance gene patterns suggest they were disseminated throughout slaughterhouse zones being also detected in commercial meat products, with significant correlations between different sampling zones/end products and total resistance in SR, CR and white room (WR) zones, and also refrigerator 4 (F4) and MP were observed. Strategically controlling key zones in slaughterhouse (SR, CR and WR) by adequate disinfection methods could strategically reduce the risks of ARG transmission and minimize the issues of food safety and environment contamination.201425479100