Real-time PCR methods for quantitative monitoring of streptomycin and tetracycline resistance genes in agricultural ecosystems. - Related Documents




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341601.0000Real-time PCR methods for quantitative monitoring of streptomycin and tetracycline resistance genes in agricultural ecosystems. Antibiotic application in plant agriculture is primarily used to control fire blight caused by Erwinia amylovora in pome fruit orchards. In order to facilitate environmental impact assessment for antibiotic applications, we developed and validated culture-independent quantitative real-time PCR multiplex assays for streptomycin (strA, strB, aadA and insertion sequence IS1133) and tetracycline (tetB, tetM and tetW) resistance elements in plant and soil samples. The qPCR were reproducible and consistent whether the DNA was extracted directly from bacteria, plant and soil samples inoculated with bacteria or soil samples prior to and after manure slurry treatment. The genes most frequently identified in soils pre- and post-slurry treatment were strB, aadA, tetB and tetM. All genes tested were detected in soils pre-slurry treatment, and a decrease in relative concentrations of tetB and the streptomycin resistance genes was observed in samples taken post-slurry treatment. These multiplex qPCR assays offer a cost-effective, reliable method for simultaneous quantification of antibiotic resistance genes in complex, environmental sample matrices.201121549164
532510.9998Genes encoding tetracycline resistance in a full-scale municipal wastewater treatment plant investigated during one year. Tetracycline-resistant bacteria and genes encoding tetracycline resistance are common in anthropogenic environments. We studied how wastewater treatment affects the prevalence and concentration of two genes, tetA and tetB, that encode resistance to tetracycline. Using real-time polymerase chain reaction (PCR) we analysed wastewater samples collected monthly for one year at eight key-sites in a full-scale municipal wastewater treatment plant (WWTP). We detected tetA and tetB at each sampling site and the concentration of both genes, expressed per wastewater volume or per total-DNA, decreased over the treatment process. The reduction of tetA and tetB was partly the result of the sedimentation process. The ratio of tetA and tetB, respectively, to total DNA was lower in or after the biological processes. Taken together our data show that tetracycline resistance genes occur throughout the WWTP, and that the concentrations are reduced under conventional operational strategies.201020154388
343120.9998Correlation 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
528930.9998Examination of the Aerobic Microflora of Swine Feces and Stored Swine Manure. Understanding antibiotic resistance in agricultural ecosystems is critical for determining the effects of subtherapeutic and therapeutic uses of antibiotics for domestic animals. This study was conducted to ascertain the relative levels of antibiotic resistance in the aerobic bacterial population to tetracycline, tylosin, and erythromycin. Swine feces and manure samples were plated onto various agar media with and without antibiotics and incubated at 37°C. Colonies were counted daily. Randomly selected colonies were isolated and characterized by 16S rRNA sequence analyses and additional antibiotic resistance and biochemical analyses. Colonies were recovered at levels of 10 to 10 CFU mL for swine slurry and 10 to 10 CFU g swine feces, approximately 100-fold lower than numbers obtained under anaerobic conditions. Addition of antibiotics to the media resulted in counts that were 60 to 80% of those in control media without added antibiotics. Polymerase chain reaction analyses for antibiotic resistance genes demonstrated the presence of a number of different resistance genes from the isolates. The recoverable aerobic microflora of swine feces and manure contain high percentages of antibiotic-resistant bacteria, which include both known and novel genera and species, and a variety of antibiotic resistance genes. Further analyses of these and additional isolates should provide additional information on these organisms as potential reservoirs of antibiotic resistance genes in these ecosystems.201627065407
529240.9998Antibiotic-Resistant Bacteria in Hydroponic Lettuce in Retail: A Comparative Survey. Hydroponic produce is gaining popularity due to its suitability for urban agriculture. The general public also considers that hydroponic produce is free from microbiological contamination. In this study, we compared the frequency and abundance of tetracycline-resistant and sulphadiazine-resistant bacteria and the minimal inhibitory concentration (MIC) of these isolates in conventional, organic, and hydroponic lettuce sold in retail. We also determined the frequency of samples carrying tetB, tetX, sul1, sul2, and int1 genes by PCR and further quantified the copy number of tetX, sul1, and int1 genes in samples positive for these genes using qPCR. As expected, the number of resistant bacteria and the MICs of these isolates were lowest in hydroponic lettuce and highest in organic lettuce. All tested resistant genes, except int1, were detected in samples of all three production methods, but no significant difference was observed between the three groups in the frequency of samples carrying the resistance genes examined or in their copy number. To the best of our knowledge, it is the first study directly reporting the existence of antibiotic-resistant bacteria and resistance genes in hydroponic vegetables sold in retail. The result highlights that the risk of antibiotic-resistant bacteria contamination in hydroponic produce should be further investigated.202032967196
341750.9998tet genes as indicators of changes in the water environment: relationships between culture-dependent and culture-independent approaches. The aim of this study was to identify tetracycline resistance determinants that could be used as molecular indicators of anthropogenic changes in aquatic environments. Two parallel approaches were used to examine the prevalence of tet genes: a culture-based method involving standard PCR and a method relying on quantitative PCR. The studied site was the Łyna River in Olsztyn (Poland). The culture-dependent method revealed that the concentrations of doxycycline-resistant bacteria harboring the tet(B) gene were higher in wastewater and downstream river samples than in upstream water samples. The tet(B) gene was transferred from environmental bacteria to Escherichia coli. The results generated by the culture-independent method validated statistically significant differences in tet(B) concentrations between upstream and downstream river sections, and revealed that tet(B) levels were correlated with the presence of other tetracycline resistance genes, dissolved oxygen concentrations, temperature and doxycycline concentrations in water. Our findings indicate that doxycycline-resistant bacteria, in particular E. coli harboring tet(B) or increased concentrations of tet(B), are potentially robust indicators of changes in water environments.201525461073
712460.9998Changes 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
312570.9998Development of a rapid method for direct detection of tet(M) genes in soil from Danish farmland. A method for direct detection of antibiotic resistance genes in soil samples has been developed. The tetracycline resistance gene, tet(M), was used as a model. The method was validated on Danish farmland soil that had repeatedly been treated with pig manure slurry containing resistant bacteria. The tet(M) gene was directly detected in 10-80% of the samples from the various farmland soils and could be detected in all samples tested after selective enrichment. To validate the obtained results, the method was applied to garden soil samples where lower prevalence of resistance was found. RESULT: A detection limit of 10(2)-10(3) copies of the tet(M) gene per gram of soil (in a Bacillus cereus group bacterium) was achieved. tet(M) gene was detected in soil samples with the highest prevalence on farmland treated with pig manure slurry.200414664871
351980.9998Fate of chlortetracycline- and tylosin-resistant bacteria in an aerobic thermophilic sequencing batch reactor treating swine waste. Antibiotics have been added to animal feed for decades. Consequently, food animals and their wastes constitute a reservoir of antibiotic-resistant bacteria. The objective of this work was to characterize the impact of an aerobic thermophilic biotreatment on aerobic, antibiotic-resistant bacteria in swine waste. The proportion of tylosin- and chlortetracycline-resistant bacteria grown at 25 degrees C, 37 degrees C, and 60 degrees C decreased after treatment, but they were still abundant (10(2) to 10(8) most probable number ml(-1)) in the treated swine waste. The presence of 14 genes conferring resistance to tylosin and chlortetracycline was assessed by polymerase chain reaction in bacterial populations grown at 25 degrees C, 37 degrees C, and 60 degrees C, with or without antibiotics. In 22 cases, genes were detected before but not after treatment. The overall gene diversity was wider before [tet(BLMOSY), erm(AB)] than after [tet(LMOS), erm(B)] treatment. Analysis by denaturing gradient gel electrophoresis of amplified 16S ribosomal DNA (rDNA) fragments generally showed a reduction of the bacterial diversity, except for total populations grown at 60 degrees C and for tylosin-resistant populations grown at 37 degrees C. The latter were further investigated by cloning and sequencing their 16S rDNA. Phylotypes found before treatment were all closely related to Enterococcus hirae, whereas six different phylotypes, related to Pseudomonas, Alcaligenes, and Pusillimonas, were found after treatment. This work demonstrated that the aerobic thermophilic biotreatment cannot be considered as a means for preventing the dissemination of aerobic antibiotic-resistant bacteria and their resistance genes to the environment. However, since pathogens do not survive the biotreatment, the effluent does not represent an immediate threat to animal or human health.200919125305
712690.9998Longitudinal 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
3676100.9998Diversity 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
7123110.9998Presence 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
2796120.9998Distribution of specific tetracycline and erythromycin resistance genes in environmental samples assessed by macroarray detection. A macroarray system was developed to screen environmental samples for the presence of specific tetracycline (Tc(R)) and erythromycin (erm(R)) resistance genes. The macroarray was loaded with polymerase chain reaction (PCR) amplicons of 23 Tc(R) genes and 10 erm(R) genes. Total bacterial genomic DNA was extracted from soil and animal faecal samples collected from different European countries. Macroarray hybridization was performed under stringent conditions and the results were analysed by fluorescence scanning. Pig herds in Norway, reared without antibiotic use, had a significantly lower incidence of antibiotic resistant bacteria than those reared in other European countries, and organic herds contained lower numbers of resistant bacteria than intensively farmed animals. The relative proportions of the different genes were constant across the different countries. Ribosome protection type Tc(R) genes were the most common resistance genes in animal faecal samples, with the tet(W) gene the most abundant, followed by tet(O) and tet(Q). Different resistance genes were present in soil samples, where erm(V) and erm(E) were the most prevalent followed by the efflux type Tc(R) genes. The macroarray proved a powerful tool to screen DNA extracted from environmental samples to identify the most abundant Tc(R) and erm(R) genes within those tested, avoiding the need for culturing and biased PCR amplification steps.200717298370
3696130.9998Assessment of Tetracyclines Residues and Tetracycline Resistant Bacteria in Conventional and Organic Baby Foods. Children are very vulnerable to bacterial infections and they are sometimes subject to antimicrobials for healing. The presence of resistance genes may counteract effects of antimicrobials. This work has thereby compared the amount of tetracycline resistance genes, tet(A) and tet(B), between conventional and organic meat-based or vegetable-based baby foods and used the quantification of these genes to assess the presence of tetracycline residues in these samples. Counts of bacteria harboring the tet(A) gene were higher than those containing tet(B), and there was no difference between the organic and the conventional samples. Samples with detectable amounts of tetracycline residues were also positive for the presence of tet genes, and when the presence of the genes was not detected, the samples were also negative for the presence of residues. The percentages of tetracycline residues were higher in organic samples than in conventional ones. It cannot be concluded that organic formulas are safer than conventional ones for the studied parameters.201528231206
3699140.9998Investigation on gene transfer from genetically modified corn (Zea mays L.) plants to soil bacteria. Knowledge about the prevalence and diversity of antibiotic resistance genes in soil bacteria communities is required to evaluate the possibility and ecological consequences of the transfer of these genes carried by genetically modified (GM) plants to soil bacteria. The neomycin phosphotransferase gene (nptII) conferring resistance to kanamycin and neomycin is one of the antibiotic resistance genes commonly present in GM plants. In this study, we investigated kanamycin-resistant (Km(R)) and neomycin-resistant (Nm(R)) soil bacterial populations in a 3-year field trial using a commercial GM corn (Zea mays L.) carrying the nptII gene and its near isogenic line. The results showed that a portion (2.3 - 15.6 %) of cultivable soil bacteria was naturally resistant to kanamycin or neomycin. However, no significant difference in the population level of Km(R) or Nm(R) soil bacteria was observed between the GM and non-GM corn fields. The nptII gene was not detected in any of the total 3000 Km(R) or Nm(R) isolates screened by PCR. Further, total soil bacterial cells were collected through Nycodenz gradient centrifugation and bacterial community DNA was subjected to PCR. Detection limit was about 500 cells per gram of fresh soil. Our study suggests that the nptII gene was relatively rare in the soil bacterial populations and there was no evidence of gene transfer from a GM corn plant to soil bacteria based on the data from total soil bacterial communities.201121722080
3415150.9998Sampling and Pooling Methods for Capturing Herd Level Antibiotic Resistance in Swine Feces using qPCR and CFU Approaches. The aim of this article was to define the sampling level and method combination that captures antibiotic resistance at pig herd level utilizing qPCR antibiotic resistance gene quantification and culture-based quantification of antibiotic resistant coliform indicator bacteria. Fourteen qPCR assays for commonly detected antibiotic resistance genes were developed, and used to quantify antibiotic resistance genes in total DNA from swine fecal samples that were obtained using different sampling and pooling methods. In parallel, the number of antibiotic resistant coliform indicator bacteria was determined in the same swine fecal samples. The results showed that the qPCR assays were capable of detecting differences in antibiotic resistance levels in individual animals that the coliform bacteria colony forming units (CFU) could not. Also, the qPCR assays more accurately quantified antibiotic resistance genes when comparing individual sampling and pooling methods. qPCR on pooled samples was found to be a good representative for the general resistance level in a pig herd compared to the coliform CFU counts. It had significantly reduced relative standard deviations compared to coliform CFU counts in the same samples, and therefore differences in antibiotic resistance levels between samples were more readily detected. To our knowledge, this is the first study to describe sampling and pooling methods for qPCR quantification of antibiotic resistance genes in total DNA extracted from swine feces.201526114765
3520160.9998Influence of tetracycline on tetracycline-resistant heterotrophs and tet genes in activated sludge process. The concentrations of tetracycline-intermediate resistant, tetracycline-resistant heterotrophic bacteria, and total heterotrophic bacteria were examined to assess the influence of tetracycline on tetracycline-resistant heterotrophs by the R2A agar cultivation method in the tetracycline fortified activated sludge process and in the natural background. Results showed that the percentages of both tetracycline-intermediate resistant and tetracycline-resistant heterotrophic bacteria in total heterotrophic bacteria were significantly increased, after tetracycline was fed to activated sludge for a 3 months period under four different operating conditions, as compared with the background. In order to investigate the mechanism of activated sludge resistance to tetracycline, polymerase chain reaction experiments were carried out to analyze the existence and evolution of tet genes in the presence of tetracycline. Results revealed that only tet A and tet B genes out of the 11 target tet genes were observed in tetracycline treated activated sludge while no tet gene was detected in background. This indicated that tet A gene could accumulate in activated sludge with slower and continuous influent, while the accumulation of tet B gene could be attributed to shorter hydraulic retention time. Therefore, it was proposed in this study that tetracycline-resistant genes created by efflux pumps spread earlier and quicker to encode resistance to tetracycline, which facilitated the increase in tetracycline-resistance.201525424345
7125170.9998Persistence 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
7122180.9997Bacterial antibiotic resistance levels in Danish farmland as a result of treatment with pig manure slurry. Resistance to tetracycline, macrolides and streptomycin was measured for a period of 8 months in soil bacteria obtained from farmland treated with pig manure slurry. This was done by spread plating bacteria on selective media (Luria Bertani (LB) medium supplemented with antibiotics). To account for seasonal variations in numbers of soil bacteria, ratios of resistant bacteria divided by total count on nonselective plates were calculated. Soil samples were collected from four different farms and from a control soil on a fifth farm. The control soil was not amended with animal manure. The occurrence of tetracycline-resistant bacteria was elevated after spread of pig manure slurry but declined throughout the sampling period to a level corresponding to the control soil. Higher load of pig manure slurry yielded higher occurrence of tetracycline resistance after spreading; however, the tetracycline resistance declined to normal occurrence defined by the tetracycline resistance occurrence in the control soil. Concentrations of tetracycline in soil and in pig manure slurry were measured using HPLC. No tetracycline exceeding the detection limit was found in soil samples. Manure slurry concentrations of tetracycline for three of the farms were 42, 81 and 698 microg/l, respectively. For streptomycin and macrolides, only minor variations in resistance levels were detected. Results obtained in this study thus indicate that tetracycline resistance levels in soil are temporarily influenced by the addition of pig manure slurry. The results indicate also that increased amount of pig manure slurry amendment may result in increased levels of tetracycline resistance in the soil.200312504155
5291190.9997Low-Concentration Ciprofloxacin Selects Plasmid-Mediated Quinolone Resistance Encoding Genes and Affects Bacterial Taxa in Soil Containing Manure. The spread of antimicrobial resistance in environment is promoted at least in part by the inappropriate use of antibiotics in animals and humans. The present study was designed to investigate the impact of different concentrations of ciprofloxacin in soil containing manure on the development of plasmid-mediated quinolone resistance (PMQR) - encoding genes and the abundance of soil bacterial communities. For these studies, high-throughput next-generation sequencing of 16S rRNA, real-time polymerase chain reaction and standard microbiologic culture methods were utilized. We demonstrated that the dissipate rate of relative abundances of some of PMQR-encoding genes, such as qnrS, oqxA and aac(6('))-Ib-cr, were significantly lower with ciprofloxacin 0.04 and 0.4 mg/kg exposure as compared to no-ciprofloxacin control and ciprofloxacin 4 mg/kg exposure during 2 month. Also, the number of ciprofloxacin resistant bacteria was significantly greater in ciprofloxacin 0.04 and 0.4 mg/kg exposure as compared with no-ciprofloxacin control and the ciprofloxacin 4 mg/kg exposure. In addition, lower ciprofloxacin concentration provided a selective advantage for the populations of Xanthomonadales and Bacillales in orders while Agrobacterium, Bacillus, Enterococcus, and Burkholderia in genera. These findings suggest that lower concentration of ciprofloxacin resulted in a slower rate of PMQR-encoding genes dissipation and selected development of ciprofloxacin-resistant bacteria in soil amended with manure.201627847506