Microbial ecology, bacterial pathogens, and antibiotic resistant genes in swine manure wastewater as influenced by three swine management systems. - Related Documents




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710301.0000Microbial 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
710110.9998Tetracycline residues and tetracycline resistance genes in groundwater impacted by swine production facilities. Antibiotics are used at therapeutic levels to treat disease; at slightly lower levels as prophylactics; and at low, subtherapeutic levels for growth promotion and improvement of feed efficiency. Over 88% of swine producers in the United States gave antimicrobials to grower/finisher pigs in feed as a growth promoter in 2000. It is estimated that ca. 75% of antibiotics are not absorbed by animals and are excreted in urine and feces. The extensive use of antibiotics in swine production has resulted in antibiotic resistance in many intestinal bacteria, which are also excreted in swine feces, resulting in dissemination of resistance genes into the environment. To assess the impact of manure management on groundwater quality, groundwater samples have been collected near two swine confinement facilities that use lagoons for manure storage and treatment. Several key contaminant indicators - including inorganic ions, antibiotics, and antibiotic resistance genes - were analyzed in groundwater collected from the monitoring wells. Chloride, ammonium, potassium, and sodium were predominant inorganic constituents in the manure samples and served as indicators of groundwater contamination. Based on these analyses, shallow groundwater has been impacted by lagoon seepage at both sites. Liquid chromatography-mass spectroscopy (LC-MS) was used to measure the dissolved concentrations of tetracycline, chlortetracycline, and oxytetracycline in groundwater and manure. Although tetracyclines were regularly used at both facilities, they were infrequently detected in manure samples and then at relatively trace concentrations. Concentrations of all tetracyclines and their breakdown products in the groundwater sampled were generally less than 0.5 microg/L. Bacterial tetracycline resistance genes served as distinct genotypic markers to indicate the dissemination and mobility of antibiotic resistance genes that originated from the lagoons. Applying PCR to genomic DNA extracted from the lagoon and groundwater samples, four commonly occurring tetracycline (tet) resistance genes - tet(M), tet(O), tet(Q), and tet(W) - were detected. The detection frequency of tet genes was much higher in wells located closer to and down-gradient from the lagoons than in wells more distant from the lagoons. These results suggested that in the groundwater underlying both facilities tetracycline resistance genes exist and are somewhat persistent, but that the distribution and potentially the flux for each tet gene varied throughout the study period.200617127527
712620.9998Longitudinal characterization of antimicrobial resistance genes in feces shed from cattle fed different subtherapeutic antibiotics. BACKGROUND: Environmental transmission of antimicrobial-resistant bacteria and resistance gene determinants originating from livestock is affected by their persistence in agricultural-related matrices. This study investigated the effects of administering subtherapeutic concentrations of antimicrobials to beef cattle on the abundance and persistence of resistance genes within the microbial community of fecal deposits. Cattle (three pens per treatment, 10 steers per pen) were administered chlortetracycline, chlortetracycline plus sulfamethazine, tylosin, or no antimicrobials (control). Model fecal deposits (n = 3) were prepared by mixing fresh feces from each pen into a single composite sample. Real-time PCR was used to measure concentrations of tet, sul and erm resistance genes in DNA extracted from composites over 175 days of environmental exposure in the field. The microbial communities were analyzed by quantification and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S-rRNA. RESULTS: The concentrations of 16S-rRNA in feces were similar across treatments and increased by day 56, declining thereafter. DGGE profiles of 16S-rRNA differed amongst treatments and with time, illustrating temporal shifts in microbial communities. All measured resistance gene determinants were quantifiable in feces after 175 days. Antimicrobial treatment differentially affected the abundance of certain resistance genes but generally not their persistence. In the first 56 days, concentrations of tet(B), tet(C), sul1, sul2, erm(A) tended to increase, and decline thereafter, whereas tet(M) and tet(W) gradually declined over 175 days. At day 7, the concentration of erm(X) was greatest in feces from cattle fed tylosin, compared to all other treatments. CONCLUSION: The abundance of genes coding for antimicrobial resistance in bovine feces can be affected by inclusion of antibiotics in the feed. Resistance genes can persist in feces from cattle beyond 175 days with concentrations of some genes increasing with time. Management practices that accelerate DNA degradation such as frequent land application or composting of manure may reduce the extent to which bovine feces serves as a reservoir of antimicrobial resistance.201121261985
710230.9998Can probiotics improve the environmental microbiome and resistome of commercial poultry production? Food animal production systems have become more consolidated and integrated, producing large, concentrated animal populations and significant amounts of fecal waste. Increasing use of manure and litter as a more "natural" and affordable source of fertilizer may be contributing to contamination of fruits and vegetables with foodborne pathogens. In addition, human and animal manure have been identified as a significant source of antibiotic resistance genes thereby serving as a disseminator of resistance to soil and waterways. Therefore, identifying methods to remediate human and animal waste is critical in developing strategies to improve food safety and minimize the dissemination of antibiotic resistant bacteria. In this study, we sought to determine whether withdrawing antibiotic growth promoters or using alternatives to antibiotics would reduce the abundance of antibiotic resistance genes or prevalence of pathogens in poultry litter. Terminal restriction fragment length polymorphism (T-RFLP) paired with high throughput sequencing was used to evaluate the bacterial community composition of litter from broiler chickens that were treated with streptogramin growth-promoting antibiotics, probiotics, or prebiotics. The prevalence of resistance genes and pathogens was determined from sequencing results or PCR screens of litter community DNA. Streptogramin antibiotic usage did not elicit statistically significant differences in Shannon diversity indices or correlation coefficients among the flocks. However, T-RFLP revealed that there were inter-farm differences in the litter composition that was independent of antibiotic usage. The litter from all farms, regardless of antibiotic usage, contained streptogramin resistance genes (vatA, vatB, and vatE), macrolide-lincosamide-streptogramin B resistance genes (ermA and ermB), the tetracycline resistance gene tetM and class 1 integrons. There was inter-farm variability in the distribution of vatA and vatE with no statistically significant differences with regards to usage. Bacterial diversity was higher in litter when probiotics or prebiotics were administered to flocks but as the litter aged, diversity decreased. No statistically significant differences were detected in the abundance of class 1 integrons where 3%-5% of the community was estimated to harbor a copy. Abundance of pathogenic Clostridium species increased in aging litter despite the treatment while the abundance of tetracycline-resistant coliforms was unaffected by treatment. However some treatments decreased the prevalence of Salmonella. These findings suggest that withdrawing antibiotics or administering alternatives to antibiotics can change the litter bacterial community and reduce the prevalence of some pathogenic bacteria, but may not immediately impact the prevalence of antibiotic resistance.201324071920
710940.9998Explore 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
711450.9998Antibiotic Resistance Genes in Freshwater Trout Farms in a Watershed in Chile. Point sources such as wastewater treatment plants, terrestrial agriculture, and aquaculture may release antibiotic residues, antibiotic resistant bacteria, and antibiotic resistance genes (ARGs) into aquatic ecosystems. However, there is a lack of quantitative studies attributing environmental ARG abundance to specific sources. The goal of this study was to evaluate the role of freshwater trout farms in the release and dissemination of ARGs into the environment. Sediment samples upstream and downstream from five rainbow trout farms were collected over time in southern Chile. A microfluidic quantitative polymerase chain reaction approach was used to quantify an ARG array covering different mechanisms of resistance, and data were analyzed using principal component analysis (PCA) and linear mixed regression models. Surveys were also conducted to obtain information about management practices, including antibiotic use, at the farms. Florfenicol and oxytetracycline were used at these farms, although at different rates. A total of 93 samples were analyzed. In the PCA, , , , , (A), (B), (C), (W), and grouped together. A statistically significant increase in abundance of , , , and several genes was found downstream from the farms compared with upstream sites, and retention ponds had the highest ARG abundance at each site. Antibiotic resistance gene levels returned to baseline at an average distance of 132.7 m downstream from the farms. Although results from this study indicate an influence of trout farms on the presence of ARGs in the immediate environment, the extent of their contribution to ARG dissemination is unknown and deserves further investigation.201931589726
712560.9998Persistence of resistance to erythromycin and tetracycline in swine manure during simulated composting and lagoon treatments. The use of antimicrobials in food animal production leads to the development of antimicrobial resistance (AMR), and animal manure constitutes the largest reservoir of such AMR. In previous studies, composted swine manure was found to contain substantially lower abundance of AMR genes that encode resistance to tetracyclines (tet genes) and macrolide-lincosamide-streptogramin B (MLS(B)) superfamily (erm genes), than manures that were treated by lagoons or biofilters. In this study, temporal changes in AMR carried by both cultivated and uncultivated bacteria present in swine manure during simulated composting and lagoon storage were analyzed. Treatments were designed to simulate the environmental conditions of composting (55°C with modest aeration) and lagoon storage (ambient temperature with modest aeration). As determined by selective plate counting, over a 48-day period, cultivated aerobic heterotrophic erythromycin-resistant bacteria and tetracycline-resistant bacteria decreased by more than 4 and 7 logs, respectively, in the simulated composting treatment while only 1 to 2 logs for both resistant bacterial groups in the simulated lagoon treatment. Among six classes each of erm and tet genes quantified by class-specific real-time PCR assays, the abundance of erm(A), erm(C), erm(F), erm(T), erm(X), tet(G), tet(M), tet(O), tet(T), and tet(W) declined marginally during the first 17 days, but dramatically thereafter within 31 days of the composting treatment. No appreciable reduction of any of the erm or tet genes analyzed was observed during the simulated lagoon treatment. Correlation analysis showed that most of the AMR gene classes had similar persistence pattern over the course of the treatments, though not all AMR genes were destructed at the same rate during the treatments.201221811793
281470.9998Fate 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
710080.9998Spread of tetracycline resistance genes at a conventional dairy farm. The use of antibiotics in animal husbandry contributes to the worldwide problem of increasing antibiotic resistance in animal and human pathogens. Intensive animal production is considered an important source of antibiotic resistance genes released to the environment, while the contribution of smaller farms remains to be evaluated. Here we monitor the spread of tetracycline resistance (TC-r) genes at a middle-size conventional dairy farm, where chlortetracycline (CTC, as intrauterine suppository) is prophylactically used after each calving. Our study has shown that animals at the farm acquired the TC-r genes in their early age (1-2 weeks), likely due to colonization with TC-resistant bacteria from their mothers and/or the farm environment. The relative abundance of the TC-r genes tet(W), tet(Q), and tet(M) in fresh excrements of calves was about 1-2 orders of magnitude higher compared to heifers and dairy cows, possibly due to the presence of antibiotic residues in milk fed to calves. The occurrence and abundance of TC-r genes in fresh excrements of heifers and adult cows remained unaffected by intrauterine CTC applications, with tet(O), tet(Q), and tet(W) representing a "core TC-resistome" of the farm, and tet(A), tet(M), tet(Y), and tet(X) occurring occasionally. The genes tet(A), tet(M), tet(Y), and tet(X) were shown to be respectively harbored by Shigella, Lactobacillus and Clostridium, Acinetobacter, and Wautersiella. Soil in the farm proximity, as well as field soil to which manure from the farm was applied, was contaminated with TC-r genes occurring in the farm, and some of the TC-r genes persisted in the field over 3 months following the manure application. Concluding, our study shows that antibiotic resistance genes may be a stable part of the intestinal metagenome of cattle even if antibiotics are not used for growth stimulation, and that smaller dairy farms may also contribute to environmental pollution with antibiotic resistance genes.201526074912
314690.9998Resistomes 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
7104100.9998Antibiotic resistance genes load in an antibiotic free organic broiler farm. Antibiotic resistance is a serious concern for public health. Farm environments are relevant reservoirs of antibiotic resistant bacteria and antibiotic resistance genes (ARGs), thus strategies to limit the spread of ARGs from farms to the environment are needed. In this study a broiler farm, where antibiotics have never been used for any purpose, was selected to evaluate if this measure is effective in reducing the ARGs load in farm environment (FE) and in meat processing environment (MPE). Faecal samples from FE and MPE were processed for DNA extraction. Detection and quantification of the 16S rRNA gene and selected ARGs (bla(TEM), qnrS, sul2, and tetA) were carried out by PCR and digital droplet PCR (ddPCR), respectively. Generally, the relative abundance of the quantified ARGs in FE was similar or higher than that measured in intensive farms. Furthermore, apart for tetA, no differences in relative abundances of the other ARGs between FE and MPE were determined. These results suggest that the choice to not use antibiotics in broiler farming is not so effective to limit the ARGs spread in MPE and that further sources of ARGs should be considered including the preceding production phase with particular reference to the breeding stage.202235091251
3114110.9998Spatial 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
3137120.9998Evaluation 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
7123130.9998Presence and fate of antibiotic residues, antibiotic resistance genes and zoonotic bacteria during biological swine manure treatment. The presence and dissemination of antibiotic residues, antibiotic resistance genes and zoonotic bacteria in the environment is of growing concern worldwide. Manure management practices, such as biological removal of nitrogen from swine manure, may help to decrease levels of antibiotic residues, antibiotic resistance genes and zoonotic bacteria present in manure before fertilization, thereby reducing environmental contamination. Therefore, the aim of this study was to monitor the presence and fate of seven antibiotic residues (colistin, sulfadiazine, trimethoprim, doxycycline, oxytetracycline, ceftiofur and tylosin A), nine antibiotic resistance genes (tet(B), tet(L), tet(M), tet(O), tet(Q), tet(W), erm(B), erm(F) and sul2) and two zoonotic bacteria (Salmonella Typhimurium and Campylobacter coli) during biological nitrogen removal from swine manure over time. Samples from the raw manure, the solid fraction, the liquid fraction and the storage lagoon were analyzed on two farms at six time points with an interval of two weeks. Only the antibiotics which were used during the three months preceding the first sampling could be detected before and after biological nitrogen removal from swine manure. Of all the antibiotics studied, doxycycline was recovered in all of the samples and sulfadiazine was recovered in most samples on both farms. For both antibiotics, there appears to be a reduction of the amount of residues present in the storage lagoon compared to the liquid fraction, however, this reduction was not statistically significant. A significant reduction of the relative abundances of most of the antibiotic resistance genes studied was observed when comparing the liquid fraction and the storage lagoon. For tet(L), no differences were observed between the fractions sampled and for sul2 and erm(F), a significant increase in relative abundances was observed on the second farm sampled. For the zoonotic bacteria, a reduction of at least 1 log was observed after biological nitrogen removal from swine manure. The results indicate that the concentration of certain antibiotic residues and several antibiotic resistance genes and the amount of zoonotic bacteria present in the manure may be reduced in the end product of the biological nitrogen removal from swine manure.201930878661
7121140.9998Fate of fluoroquinolones in field soil environment after incorporation of poultry litter from a farm with enrofloxacin administration via drinking water. The practice of incorporating animal manure into soil is supported within the European Circular economy as a possible substitute for mineral fertilizers and will become crucial for the sustainability of agriculture. However, this practice may indirectly contribute to the dissemination of antibiotics, resistance bacteria, and resistance genes. In this study, medicated drinking water and poultry litter samples were obtained from a broiler-chick farm. The obtained poultry litter was incorporated into the soil at the experimental field site. The objectives of this research project were first to develop analytical methods able to quantify fluoroquinolones (FQs) in medicated drinking water, poultry litter, and soil samples by LC-MS; second to study the fate of these FQs in the soil environment after incorporation of poultry litter from flock medicated by enrofloxacin (ENR); and third to screen the occurrence of selected fluoroquinolone resistance encoding genes in poultry litter and soil samples (PCR analysis). FQs were quantified in the broiler farm's medicated drinking water (41.0 ± 0.3 mg∙L(-1) of ENR) and poultry litter (up to 70 mg∙kg(-1) of FQs). The persistence of FQs in the soil environment over 112 days was monitored and evaluated (ENR concentrations ranged from 36 μg∙kg(-1) to 9 μg∙kg(-1) after 100 days). The presence of resistance genes was confirmed in both poultry litter and soil samples, in agreement with the risk assessment for the selection of AMR in soil based on ENR concentrations. This work provides a new, comprehensive perspective on the entry and long-term fate of antimicrobials in the terrestrial environment and their consequences after the incorporation of poultry litter into agricultural fields.202438367114
3676150.9998Diversity of antibiotic resistance genes and encoding ribosomal protection proteins gene in livestock waste polluted environment. The rapid development and increase of antibiotic resistance are global phenomena resulting from the extensive use of antibiotics in human clinics and animal feeding operations. Antibiotics can promote the occurrence of antibiotic resistance genes (ARGs), which can be transferred horizontally to humans and animals through water and the food chain. In this study, the presence and abundance of ARGs in livestock waste was monitored by quantitative PCR. A diverse set of bacteria and tetracycline resistance genes encoding ribosomal protection proteins (RPPs) from three livestock farms and a river were analyzed through denaturing gradient gel electrophoresis (DGGE). The abundance of sul(I) was 10(3) to 10(5) orders of magnitude higher than that of sul(II). Among 11 tet-ARGs, the most abundant was tet(O). The results regarding bacterial diversity indicated that the presence of antibiotics might have an evident impact on bacterial diversity at every site, particularly at the investigated swine producer. The effect of livestock waste on the bacterial diversity of soil was stronger than that of water. Furthermore, a sequencing analysis showed that tet(M) exhibited two genotypes, while the other RPPs-encoding genes exhibited at least three genotypes. This study showed that various ARGs and RPPs-encoding genes are particularly widespread among livestock.201829469609
7124160.9998Changes in diversity of cultured bacteria resistant to erythromycin and tetracycline in swine manure during simulated composting and lagoon storage. This study investigated the impact of composting and lagoon storage on survival and change in diversity of tetracycline-resistant (Tc(r) ) and erythromycin-resistant (Em(r) ) bacteria and the resistance genes they carry in swine manure. Treatments were arranged as a 2 × 2 factorial design: composting vs lagoon storage and 0 vs 1% Surround WP Crop Protectant (a clay product) in three replicates. After 48 days of treatments, resistant bacteria were enumerated by selective plating and identified by 16S rRNA gene sequencing. The erm and the tet gene(s) carried by the resistant isolates were screened using class-specific PCR assays. The plate counts of Tc(r) and Em(r) bacteria decreased by 4-7 logs by composting, but only by 1-2 logs by the lagoon treatment. During the treatments, Acinetobacter gave way to Pseudomonas and Providencia as the largest resistant genera. The clay product had little effect on survival or diversity of resistant bacteria. Of six classes of erm and seven classes of tet genes tested, changes in prevalence were also noted. The results indicate that composting can dramatically shift Tc(r) and Em(r) bacterial populations, and composting can be an effective and practical approach to decrease dissemination of antibiotic resistance from swine farms to the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The presented research provided evidence that composting is much more effective than lagoon storage in dramatically decreasing culturable bacteria resistant to erythromycin and tetracycline in swine manure. Considerable diversity changes of resistant bacteria were also demonstrated during composting or lagoon storage. Overall, Acinetobacter was the major resistant genus in untreated swine manure, but pseudomonads and Providencia became the major resistant genera after the treatments. This is the first study that investigated diversity changes of cultured bacteria resistant to these two antibiotics during composting and lagoon storage of swine manure. New genes encoding resistance to the two antibiotics were also implied in the cultured isolates.201526031793
3164170.9998Variations 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
3431180.9998Correlation between Bacterial Cell Density and Abundance of Antibiotic Resistance on Milking Machine Surfaces Assessed by Cultivation and Direct qPCR Methods. The relative abundance of antibiotic-resistant bacteria and antibiotic-resistance genes was surveyed for different parts of a milking machine. A cultivation approach based on swab samples showed a highly diverse microbiota, harboring resistances against cloxacillin, ampicillin, penicillin, and tetracycline. This approach demonstrated a substantial cloxacillin resistance of numerous taxa within milking machine microbiota coming along with regular use of cloxacillin for dry-off therapy of dairy cows. For the less abundant tetracycline-resistant bacteria we found a positive correlation between microbial cell density and relative abundance of tetracycline-resistant microorganisms (R(2) = 0.73). This indicated an accelerated dispersion of resistant cells for sampling locations with high cell density. However, the direct quantification of the tetM gene from the swap samples by qPCR showed the reverse relation to bacterial density if normalized against the abundance of 16S rRNA genes (R(2) = 0.88). The abundance of 16S rRNA genes was analyzed by qPCR combined with a propidium monoazide treatment, which eliminates 16S rRNA gene signals in negative controls.202337166501
3214190.9998Characteristics of the antibiotic resistance genes in the soil of medical waste disposal sites. The inappropriate disposal of medical waste allows bacteria to acquire antibiotic resistance, which results in a threat to public health. Antibiotic resistance gene (ARG) profiles were determined for 45 different soil samples containing medical waste and 15 nearby soil samples as controls. Besides physical and chemical analyses (i.e., dry matter content, pH value, and metal content), the genomes of microorganisms from the soil samples were extracted for high-throughput sequencing. ARG abundances of these samples were obtained by searching the metagenomic sequences against the antibiotic resistance gene database and the copies of ARGs per copy of the 16S rRNA gene at different levels were assessed. The results showed medical waste accumulation significantly enriched the contents of Cu, Cr, Pb, and As in the tested soil samples. Compared to the controls, the samples collected from areas containing medical waste were significantly enriched (p < 0.05, t-test) with ARGs annotated as sulfonamide and multidrug resistance genes, and in particular, the subtypes sul1 and sul2 (sulfonamide resistance genes), and multidrug_transporter (multidrug resistance gene). Moreover, the ARGs of the samples from the polluted areas were more diverse than those of the control samples (p < 0.05, t-test). The comparatively higher abundance and diversity of ARGs in contaminated soil pose a potential risk to human health.202032402966