Dynamics of microbiota and antimicrobial resistance in on-farm dairy processing plants using metagenomic and culture-dependent approaches. - Related Documents




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316301.0000Dynamics 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
316110.9999Longitudinal 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
316420.9998Variations 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
315630.9998Deciphering 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
337340.9998Evidence of Increased Antibiotic Resistance in Phylogenetically-Diverse Aeromonas Isolates from Semi-Intensive Fish Ponds Treated with Antibiotics. The genus Aeromonas is ubiquitous in aquatic environments encompassing a broad range of fish and human pathogens. Aeromonas strains are known for their enhanced capacity to acquire and exchange antibiotic resistance genes and therefore, are frequently targeted as indicator bacteria for monitoring antimicrobial resistance in aquatic environments. This study evaluated temporal trends in Aeromonas diversity and antibiotic resistance in two adjacent semi-intensive aquaculture facilities to ascertain the effects of antibiotic treatment on antimicrobial resistance. In the first facility, sulfadiazine-trimethoprim was added prophylactically to fingerling stocks and water column-associated Aeromonas were monitored periodically over an 11-month fish fattening cycle to assess temporal dynamics in taxonomy and antibiotic resistance. In the second facility, Aeromonas were isolated from fish skin ulcers sampled over a 3-year period and from pond water samples to assess associations between pathogenic strains to those in the water column. A total of 1200 Aeromonas isolates were initially screened for sulfadiazine resistance and further screened against five additional antimicrobials. In both facilities, strong correlations were observed between sulfadiazine resistance and trimethoprim and tetracycline resistances, whereas correlations between sulfadiazine resistance and ceftriaxone, gentamicin, and chloramphenicol resistances were low. Multidrug resistant strains as well as sul1, tetA, and intI1 gene-harboring strains were significantly higher in profiles sampled during the fish cycle than those isolated prior to stocking and these genes were extremely abundant in the pathogenic strains. Five phylogenetically distinct Aeromonas clusters were identified using partial rpoD gene sequence analysis. Interestingly, prior to fingerling stocking the diversity of water column strains was high, and representatives from all five clusters were identified, including an A. salmonicida cluster that harbored all characterized fish skin ulcer samples. Subsequent to stocking, diversity was much lower and most water column isolates in both facilities segregated into an A. veronii-associated cluster. This study demonstrated a strong correlation between aquaculture, Aeromonas diversity and antibiotic resistance. It provides strong evidence for linkage between prophylactic and systemic use of antibiotics in aquaculture and the propagation of antibiotic resistance.201627965628
536650.9998Fresh produce as a reservoir of antimicrobial resistance genes: A case study of Switzerland. Antimicrobial resistance (AMR) can be transferred to humans through food and fresh produce can be an ideal vector as it is often consumed raw or minimally processed. The production environment of fresh produce and the agricultural practices and regulations can vary substantially worldwide, and consequently, the contamination sources of AMR. In this study, 75 imported and 75 non-imported fresh produce samples purchased from Swiss retailers were tested for the presence of antimicrobial resistant bacteria (ARB) and antimicrobial resistance genes (ARGs). Moreover, the plasmidome of 4 selected samples was sequenced to have an insight on the diversity of mobile resistome. In total, 91 ARB were isolated from fresh produce, mainly cephalosporin-resistant Enterobacterales (n = 64) and carbapenem-resistant P. aeruginosa (n = 13). All P. aeruginosa, as well as 16 Enterobacterales' isolates were multidrug-resistant. No differences between imported and Swiss fresh produce were found regarding the number of ARB. In 95 % of samples at least one ARG was detected, being the most frequent sul1, bla(TEM), and ermB. Abundance of sul1 and intI1 correlated strongly with the total amount of ARGs, suggesting they could be good indicators for AMR in fresh produce. Furthermore, sul1 correlated with the fecal marker yccT, indicating that fecal contamination could be one of the sources of AMR. The gene sulI was significantly higher in most imported samples, suggesting higher anthropogenic contamination in the food production chain of imported produce. The analyses of the plasmidome of coriander and carrot samples revealed the presence of several ARGs as well as genes conferring resistance to antiseptics and disinfectants in mobile genetic elements. Overall, this study demonstrated that fresh produce contributes to the dissemination of ARGs and ARB.202437813266
316260.9998Metagenomic 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
344470.9998Multidrug resistance in bacteria associated with leafy greens and soil in urban agriculture systems. Urban farms and community gardens support local food production, though these agroecosystems can contain emerging environmental contaminants that may contribute to the dissemination of antimicrobial resistance (AMR). Our previous research enumerated AMR bacteria associated with leafy vegetable production environments in the greater Washington, D.C. area, identifying >100 isolates with multidrug-resistant (MDR) phenotypes. Here, we performed whole genome sequencing analysis of 87 of these strains recovered from leafy greens (n=29), root zone soil (n=42), and bulk soil (n=16) to comprehensively characterize their MDR genotypes, including taxonomy and any encoded ARGs, stress response genes, and mobile genetic elements (MGEs; e.g., plasmids, phages, conjugative elements). The MDR isolates spanned 4 phyla and 14 genera, with the majority identified as Pseudomonas (n = 29), Serratia (n = 22), Providencia (n = 11), and Bacillus (n = 11). Most of the ARGs were linked to multidrug efflux, while other abundant ARG classes reflected resistance to beta-lactams and tetracyclines. While the genotypes were often conserved within respective species and even genera, the observed phenotypes within taxonomic groups slightly varied, suggesting the potential roles of uncharacterized genetic elements in MDR function. Moreover, all of the MDR isolates encoded at least one gene annotated as a MGE, and there were 19 distinct ARGs located within 5,000 bp upstream or downstream of these sequences, suggesting potential implications for mobilization. Overall, our results indicate that the MDR bacteria in urban agriculture systems, including on fresh produce, are dominated by general soil-associated taxa that carry diverse ARGs and MGEs.202541059364
710780.9998A 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
710990.9998Explore 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
3467100.9998Epidemiological characteristics of antibiotic resistance genes in various bacteria worldwide. OBJECTIVES: This study aims to investigate the epidemiological characteristics of various bacteria carrying ARGs on a global scale over extended time periods. METHODS: A total of 25,285 globally isolated bacteria's genomes were analyzed to explore ARGs. The analysis focused on temporal, geographic, and species distribution, including pathogenic and non-pathogenic bacteria, intracellular parasitic states, ARG types, and their association with MGEs. Multiple linear regression was employed to identify ARG risk factors in bacteria. RESULTS: The overall prevalence of bacteria with ARGs was 64.2%, indicating that at least one ARG was present in 64.2% (16,243/25,285) of the included bacterial, with an average of 14.4 ARGs per bacterium. ARGs have been increasing globally, averaging one additional ARG every three years, closely linked to rising antibiotic consumption. Pathogenic bacteria harbored more ARGs than non-pathogenic ones. Intracellular parasitic bacteria still carry specific types of ARGs despite being less likely to generate ARGs. Clinical and human-associated bacteria showed higher ARG counts, and bacteria isolated from humans had the highest number of disinfectant-resistant genes. The average number of ARGs in bacteria isolated from high-middle-income and lower-middle-income countries is higher. Factors like motility, non-sporulation, Gram-positive staining, extracellular parasitism, and human pathogenicity are linked to higher ARGs levels. CONCLUSIONS: An increasing number of bacteria carrying ARGs pose a significant challenge to the control of antibiotics-resistant pathogens worldwide. The issue of bacteria carrying more ARGs requires greater global attention.202540147137
3238110.9998Extensive metagenomic analysis of the porcine gut resistome to identify indicators reflecting antimicrobial resistance. BACKGROUND: Antimicrobial resistance (AMR) has been regarded as a major threat to global health. Pigs are considered an important source of antimicrobial resistance genes (ARGs). However, there is still a lack of large-scale quantitative data on the distribution of ARGs in the pig production industry. The bacterial species integrated ARGs in the gut microbiome have not been clarified. RESULTS: In the present study, we used deep metagenomic sequencing data of 451 samples from 425 pigs including wild boars, Tibetan pigs, and commercial or cross-bred experimental pigs under different rearing modes, to comprehensively survey the diversity and distribution of ARGs and detect the bacteria integrated in these ARGs. We identified a total of 1295 open reading frames (ORFs) recognized as antimicrobial resistance protein-coding genes. The ORFs were clustered into 349 unique types of ARGs, and these could be further classified into 69 drug resistance classes. Tetracycline resistance was most enriched in pig feces. Pigs raised on commercial farms had a significantly higher AMR level than pigs under semi-free ranging conditions or wild boars. We tracked the changes in the composition of ARGs at different growth stages and gut locations. There were 30 drug resistance classes showing significantly different abundances in pigs between 25 and 240 days of age. The richness of ARGs and 41 drug resistance classes were significantly different between cecum lumen and feces in pigs from commercial farms, but not in wild boars. We identified 24 bacterial species that existed in almost all tested samples (core bacteria) and were integrated 128 ARGs in their genomes. However, only nine ARGs of these 128 ARGs were core ARGs, suggesting that most of the ARGs in these bacterial species might be acquired rather than constitutive. We selected three subsets of ARGs as indicators for evaluating the pollution level of ARGs in samples with high accuracy (r = 0.73~0.89). CONCLUSIONS: This study provides a primary overview of ARG profiles in various farms under different rearing modes, and the data serve as a reference for optimizing the use of antimicrobials and evaluating the risk of pollution by ARGs in pig farms. Video abstract.202235246246
3126120.9998Assessing Class 1 Integron Presence in Poultry Litter Amended with Wood Biochar and Wood Vinegar. Class 1 integrons are mobile genetic elements that facilitate the spread of antibiotic resistance genes among bacteria. The use of prophylactic antibiotics has resulted in the rise of antibiotic resistance genes accumulating in a wide range of settings, including poultry houses and the agricultural fields where poultry litter is applied as a fertilizer. Biochar and wood vinegar are forest products wastes that have generated increasing attention as additives to agricultural soils. The objectives of this study were to observe the prevalence of class 1 integrons in poultry litter blended with biochar and wood vinegar over time and to verify a modified class 1 integron screening assay. Poultry litter blends were sampled and screened for class 1 integrons using polymerase chain reaction, and 80 products, 79 of which showed positive, were sent for DNA sequencing. The GenBank® BLAST database was used to verify the presence of the class 1 integron-integrase gene (intI1). There was no change in prevalence over time in poultry litter blends. Out of 79 PCR products that were intI1 positive, 78 showed at least 95% sequence identity to intI1 encoding bacteria and 64 showed at least 97% sequence identity. This indicates that this method was effective for conducting baseline surveillance of class 1 integrons in poultry litter and poultry litter-blended biochar and/or wood vinegar. Most significantly, class 1 integron prevalence did not decrease over time, further supporting the recalcitrance of these elements and the need for improved monitoring systems.202134459936
3157130.9998Reservoirs 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
5360140.9998Diversity, 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
3245150.9998From Metagenomes to Functional Expression of Resistance: floR Gene Diversity in Bacteria from Salmon Farms. Background. The increase in antibiotic resistance in human-impacted environments, such as coastal waters with aquaculture activity, is related to the widespread use of antibiotics, even at sub-lethal concentrations. In Chile, the world's second largest producer of salmon, aquaculture is considered the main source of antibiotics in coastal waters. In this work, we aimed to characterize the genetic and phenotypic profiles of antibiotic resistance in bacterial communities from salmon farms. Methods. Bacterial metagenomes from an intensive aquaculture zone in southern Chile were sequenced, and the composition, abundance and sequence of antibiotic resistance genes (ARGs) were analyzed using assembled and raw read data. Total DNA from bacterial communities was used as a template to recover floR gene variants, which were tested by heterologous expression and functional characterization of phenicol resistance. Results. Prediction of ARGs in salmon farm metagenomes using more permissive parameters yielded significantly more results than the default Resistance Gene Identifier (RGI) software. ARGs grouped into drug classes showed similar abundance profiles to global ocean bacteria. The floR gene was the most abundant phenicol-resistance gene with the lowest gene counts, showing a conserved sequence although with variations from the reference floR. These differences were recovered by RGI prediction and, in greater depth, by mapping reads to the floR sequence using SNP base-calling. These variants were analyzed by heterologous expression, revealing the co-existence of high- and low-resistance sequences in the environmental bacteria. Conclusions. This study highlights the importance of combining metagenomic and phenotypic approaches to study the genetic variability in and evolution of antibiotic-resistant bacteria associated with salmon farms.202540001366
3137160.9998Evaluation 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
3159170.9998Longitudinal 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
3370180.9998Microbiological contamination and resistance genes in biofilms occurring during the drinking water treatment process. Biofilms are the predominant mode of microbial growth in drinking water systems. A dynamic exchange of individuals occurs between the attached and planktonic populations, while lateral gene transfer mediates genetic exchange in these bacterial communities. Integrons are important vectors for the spread of antimicrobial resistance. The presence of class 1 integrons (intI1, qac and sul genes) was assessed in biofilms occurring throughout the drinking water treatment process. Isolates from general and specific culture media, covering a wide range of environmental bacteria, fecal indicators and opportunistic pathogens were tested. From 96 isolates tested, 9.37% were found to possess genetic determinants of putative antimicrobial resistance, and these occurred in both Gram-positive and Gram-negative bacteria. Class 1 integron integrase gene was present in 8.33% of bacteria, all positive for the qacEΔ1 gene. The sul1 gene was present in 3.12% of total isolates, representing 37.5% of the class 1 integron positive cells. The present study shows that biofilm communities in a drinking water treatment plant are a reservoir of class 1 integrons, mainly in bacteria that may be associated with microbiological contamination. Eight out of nine integron bearing strains (88.8%) were identified based on 16S rRNA gene sequencing as either enteric bacteria or species that may be connected to animal and anthropogenic disturbance.201323247295
7110190.9998The "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