Antimicrobial use and resistance in swine waste treatment systems

Chlortetracycline and the macrolide tylosin were identified as commonly used antimicrobials for growth promotion and prophylaxis in swine production. Resistance to these antimicrobials was measured throughout the waste treatment processes at five swine farms by culture-based and molecular methods. Conventional farm samples had the highest levels of resistance with both culture-based and molecular methods and had similar levels of resistance despite differences in antimicrobial usage. The levels of resistance in organic farm samples, where no antimicrobials were used, were very low by a culture-based method targeting fecal streptococci. However, when the same samples were analyzed with a molecular method detecting methylation of a specific nucleotide in the 23S rRNA that results in resistance to macrolides, lincosamides, and streptogramin B (MLSB), an unexpectedly high level of resistant rRNA (approximately 50%) was observed, suggesting that the fecal streptococci were not an appropriate target group to evaluate resistance in the overall microbial community and that background levels of MLSB resistance may be substantial. All of the feed samples tested, including those from the organic farm, contained tetracycline resistance genes. Generally, the same tetracycline resistance genes and frequency of detection were found in the manure and lagoon samples for each commercial farm. The levels of tetracycline and MLSB resistance remained high throughout the waste treatment systems, suggesting that the potential impact of land application of treated wastes and waste treatment by-products on environmental levels of resistance should be investigated further.     

Macrolide Resistance in Microorganisms at Antimicrobial-Free Swine Farms

To investigate the relationship between agricultural antimicrobial use and resistance, a variety of methods for quantification of macrolide-lincosamide-streptogramin B (MLS_B) resistance were applied to organic swine farm manure samples. Fluorescence in situ hybridization was used to indirectly quantify the specific rRNA methylation resulting in MLS_B resistance. Using this method, an unexpectedly high prevalence of ribosomal methylation and, hence, predicted MLS_B resistance was observed in manure samples from two swine finisher farms that reported no antimicrobial use (37.6% ± 6.3% and 40.5% ± 5.4%, respectively). A culture-based method targeting relatively abundant clostridia showed a lower but still unexpectedly high prevalence of resistance at both farms (27.7% ± 11.3% and 11.7% ± 8.6%, respectively), while the prevalence of resistance in cultured fecal streptococci was low at both farms (4.0%). These differences in the prevalence of resistance across microorganisms suggest the need for caution when extrapolating from data obtained with indicator organisms. A third antimicrobial-free swine farm, a breeder-to-finisher operation, had low levels of MLS_B resistance in manure samples with all methods used (<9%). Tetracycline antimicrobials were detected in manure samples from one of the finisher farms and may provide a partial explanation for the high level of MLS_B resistance. Taken together, these findings highlight the need for a more fundamental understanding of the relationship between antimicrobial use and the prevalence of antimicrobial resistance.Clinical data have documented a substantial rise in the levels of antimicrobial resistance (reviewed in reference 22). In response to this alarming rise, national and international initiatives have been developed to limit the use of antimicrobials in both human and veterinary medicine, with some successes. However, some of the data suggest a more complicated relationship between the patterns of antimicrobial use and the resulting prevalence of resistance. For both avoparcin and chloramphenicol, a ban was not effective in reducing the prevalence of resistance to the respective antimicrobial in pig isolates (2, 9). This may be due to coselection by the continued use of other types of antimicrobials (1, 15, 16, 33). Coselection by other antimicrobials, however, cannot explain the persistence of antimicrobial resistance for years after all use of antimicrobials was stopped, as documented in other studies of swine (13, 25). A better understanding of this complex relationship is needed to provide a basis for developing more-effective measures to control the prevalence of antimicrobial resistance. One means for investigating the factors influencing the prevalence of resistance is through comparisons between conventional farms and organic, antimicrobial-free farms (12, 13, 18, 25) or the wilderness (14, 19).The current study focused on macrolide antimicrobials, for which the most clinically relevant resistance mechanisms are efflux and target site modification (20). Resistance via modification of the target site on the ribosome may be achieved either through point mutations in rRNA or proteins or through acquisition of an erm gene catalyzing a site-specific mono- or dimethylation of the 23S rRNA (37). The point mutations confer various levels of resistance and degrees of cross-resistance (35), and their known distribution is currently limited, although this may simply reflect the historical experimental focus (20, 35). Dimethylation of A2058 (Escherichia coli numbering), on the other hand, consistently results in high-level resistance (for antimicrobial concentrations above 1 mg/ml) for three structurally unrelated classes of antimicrobials, macrolides, lincosamides, and streptogramin Bs, or macrolide-lincosamide-streptogramin B (MLS_B) antimicrobials, because of their shared target site (37). Constitutive expression of an erm dimethylase can also confer resistance to the newer ketolides, which are erythromycin (macrolide) derivatives developed for use on macrolide-resistant pathogens, and the degree of resistance correlates with the degree of methylation (11). The ribosomal methylation resistance mechanism is of particular concern for this work for the following three reasons. (i) It confers a high level of resistance. (ii) It can be acquired through horizontal gene transfer and thus has the potential for rapid spread. (iii) It is relevant to swine production environments in the United States because all three classes of MLS_B antimicrobials are used there. A variety of methods have been used to quantify macrolide resistance, including traditional culture-based methods (for an example, see reference 9), PCR (for examples, see references 27 and 32), or fluorescence in situ hybridization (FISH) (for an example, see reference 34) detection of specific point mutations known to result in resistance in the targeted microorganisms, using PCR to detect erm and mef (efflux) genes (for examples, see references 6 and 31) and using membrane hybridizations to detect the degree of methylation at A2058 (5, 18).In our previous study of swine production, a discrepancy was observed between culture-based measurements of resistance to the macrolide tylosin and membrane hybridizations quantifying the ribosomal methylation leading to MLS_B resistance (18). Cultured fecal streptococci showed a low prevalence of tylosin resistance (4.0%) in manure samples from an organic farm, as expected in the absence of the selective pressure imposed by the use of antimicrobials. However, membrane hybridizations quantifying the ribosomal methylation leading to MLS_B resistance in all bacteria in the swine waste samples suggested the presence of a much higher level of resistance (approximately 50%). One explanation for this discrepancy is that the prevalence of resistance in the fecal streptococci was not representative of the overall prevalence of resistance in this community. However, the high level of resistance measured with the molecular method was surprising in the absence of antimicrobial use and could also be explained as an artifact of the membrane hybridization methodology.The primary objectives of this paper were to resolve this discrepancy between culture-based and molecular methods and, if the unexpectedly high prevalence of antimicrobial resistance was confirmed, to investigate possible explanations. To accomplish the first objective, we developed a variation of FISH to indirectly quantify the specific rRNA methylation resulting in MLS_B resistance and provide insight into the identity of the putative resistant microorganisms. The major group identified, Clostridium cluster XIVa, was targeted with a culture-based method to provide an independent quantification of resistance. The results presented here have confirmed an unexpectedly high prevalence of MLS_B resistance at two organic farms. They also support the hypothesis that the prior discrepancy resulted from differences in the prevalence of resistance across groups of microorganisms.     

Effects of Swine Manure on Macrolide,Lincosamide, and Streptogramin B Antimicrobial Resistance in Soils

Current agricultural practices involve inclusion of antimicrobials in animal feed and result in manure containing antimicrobials and antimicrobial-resistant microorganisms. This work evaluated the effects of land application of swine manure on the levels of tetracycline, macrolide, and lincosamide antimicrobials and on macrolide, lincosamide, and streptogramin B (MLS_B) resistance in field soil samples and laboratory soil batch tests. MLS_B and tetracycline antimicrobials were quantified after solid-phase extraction using liquid chromatography-tandem mass spectrometry. The prevalence of the ribosomal modification responsible for MLS_B resistance in the same samples was quantified using fluorescence in situ hybridization. Macrolide antimicrobials were not detected in soil samples, while tetracyclines were detected, suggesting that the latter compounds persist in soil. No significant differences in ribosomal methylation or presumed MLS_B resistance were observed when amended and unamended field soils were compared, although a transient (<20-day) increase was observed in most batch tests. Clostridium cluster XIVa accounted for the largest fraction of resistant bacteria identified in amended soils. Overall, this study did not detect a persistent increase in the prevalence of MLS_B resistance due to land application of treated swine manure.Treated swine manure contains substantial levels of both antimicrobial-resistant microorganisms (10, 26) and antimicrobials (7, 18, 33). Land application of manure could therefore contribute to public health risks associated with the increasing prevalence of antimicrobial resistance in pathogens both directly, through the dissemination of antimicrobial-resistant pathogens, and indirectly, through the introduction of and selection for antimicrobial resistance genes. Because limited data are available, this connection is largely a theoretical connection, particularly for the indirect effects. However, a recent retrospective study of antimicrobial resistance in soil did support the hypothesis that there is an environmental connection by documenting that there was an increase in the abundance of antibiotic resistance genes in samples collected from 1940 to 2008, during which time antimicrobial production increased dramatically (12).The fate of antimicrobials in amended soils is a function of their sorptive properties, the soil characteristics, and the potential for abiotic and biotic degradation of the antimicrobials. Tetracyclines tend to adsorb to soil (21, 23), which leads to persistence in amended soils (3, 7, 11), although they are also susceptible to degradation (3, 4). The macrolide tylosin frequently is not detected (3, 4, 7, 11, 33) and is likely rapidly degraded in manure and soils (8, 16, 24). However, persistence of tylosin for several months in amended soil has also been reported (6). The differences in degradation rates may be caused by differences in soil characteristics, manure-to-soil ratios, and/or microbial communities (15, 16, 21).Addition of both antimicrobials and antimicrobial-resistant microorganisms might be expected to result in an increase in the levels of resistance. However, most studies have not shown that there is a long-term increase in antimicrobial resistance due to land application of manure at agronomically prescribed rates (5, 9, 26). Transient (i.e., <45-day) increases have been reported (9, 26), as have elevated levels of resistance at sites near manure piles (5). In contrast, another report showed that there were significantly higher levels of tylosin resistance in soils that received animal manure from operations that used subtherapeutic levels of antimicrobials than in soils at sites where there was no use of subtherapeutic levels of antimicrobials (19). One limitation of these studies was their use of culture-based methods to quantify resistance; the results may not be representative of the entire microbial community. The molecular methods that have been used to quantify resistance also have limitations, and the most serious limitation is the inability of these methods to examine the full diversity of known and unknown resistance genes. The previous molecular studies of the impact of land application on resistance were largely restricted to qualitative analyses (10, 25), although quantitative PCR methods for analysis of tetracycline resistance genes have recently been used for cattle and swine lagoons (14, 20). In a retrospective soil study, Knapp et al. (12), who also used quantitative PCR, found multiple site differences, which made it difficult to evaluate the impact of manure application. However, the site with the highest manure application rate did not show the highest levels of antimicrobial resistance, suggesting that there are other factors that have a greater influence on the prevalence of resistance.In the present study, a variation of the fluorescence in situ hybridization (FISH) technique was used to assess the impact of land application of swine manure on the levels of macrolide-lincosamide-streptogramin B (MLS_B) resistance. Although the MLS_B antimicrobials are chemically distinct, methylation or mutation of a single base of the 23S rRNA prevents binding and results in cross-resistance to all three classes (29). The prevalence of MLS_B antimicrobial resistance in the microbial community can therefore be quantified indirectly by hybridization of an oligonucleotide probe to unmethylated, MLS_B-sensitive ribosomes, using either membrane hybridization (1, 10) or FISH (31). These methods do not require culturing or a comprehensive knowledge of the diversity of resistance gene sequences, but they do not detect resistance to specific antimicrobials that results from other mechanisms, such as macrolide efflux.This study focused on evaluating the impact of land application of swine manure on the levels of antimicrobials and the prevalence of antimicrobial resistance in the soil environment. The concentrations of tetracycline, macrolide, and lincosamide antimicrobials and the prevalence of MLS_B resistance were compared for field soils that received no manure, swine manure from farms that did not use antimicrobials (referred to below as organic farms), and swine manure from conventional farms to determine whether land application affects the levels of antimicrobials and MLS_B resistance. The effects of addition of manure, antimicrobials (lincomycin and chlortetracycline), and MLS_B-resistant microorganisms on the prevalence of MLS_B resistance were also compared using soil batch tests.     

Continuous feeding of antimicrobial growth promoters to commercial swine during the growing/finishing phase does not modify faecal community erythromycin resistance or community structure

Aims: To investigate the effect of continuous feeding of antimicrobial growth promoters (tylosin or virginiamycin) on the swine faecal community. Methods and Results: The study consisted of two separate on‐farm feeding trials. Swine were fed rations containing tylosin (44 or 88 mg kg^?1 of feed) or virginiamycin (11 or 22 mg kg^?1 of feed) continuously over the growing/finishing phases. The temporal impact of continuous antimicrobial feeding on the faecal community was assessed and compared to nondosed control animals through anaerobic cultivation, the analysis of community 16S rRNA gene libraries and faecal volatile fatty acid content. Feeding either antimicrobial had no detectable effect on the faecal community. Conclusions: Erythromycin methylase genes encoding resistance to the macrolide–lincosamide–streptogramin B (MLS_B) antimicrobials are present at a high level within the faecal community of intensively raised swine. Continuous antimicrobial feeding over the entire growing/finishing phase had no effect on community erm‐methylase gene copy numbers or faecal community structure. Significance and Impact of the Study: Antimicrobial growth promoters are believed to function by altering gut bacterial communities. However, widespread MLS_B resistance within the faecal community of intensively raised swine likely negates any potential effects that these antimicrobials might have on altering the faecal community. These findings suggest that if AGP‐mediated alterations to gut communities are an important mechanism for growth promotion, it is unlikely that these would be associated with the colonic community.     

Molecular Ecology Of Macrolide–Lincosamide–Streptogramin B Methylases in Waste Lagoons and Subsurface Waters Associated with Swine Production

RNA methylase genes are common antibiotic resistance determinants for multiple drugs of the macrolide, lincosamide, and streptogramin B (MLS_B) families. We used molecular methods to investigate the diversity, distribution, and abundance of MLS_B methylases in waste lagoons and groundwater wells at two swine farms with a history of tylosin (a macrolide antibiotic structurally related to erythromycin) and tetracycline usage. Phylogenetic analysis guided primer design for quantification of MLS_B resistance genes found in tylosin-producing Streptomyces (tlr(B), tlr(D)) and commensal/pathogenic bacteria (erm(A), erm(B), erm(C), erm(F), erm(G), erm(Q)). The near absence of tlr genes at these sites suggested a lack of native antibiotic-producing organisms. The gene combination erm(ABCF) was found in all lagoon samples analyzed. These four genes were also detected with high frequency in wells previously found to be contaminated by lagoon leakage. A weak correlation was found between the distribution of erm genes and previously reported patterns of tetracycline resistance determinants, suggesting that dissemination of these genes into the environment is not necessarily linked. Considerations of gene origins in history (i.e., phylogeny) and gene distributions in the landscape provide a useful “molecular ecology” framework for studying environmental spread of antibiotic resistance.     

Patterns of Antimicrobial Resistance Observed in Escherichia coli Isolates Obtained from Domestic- and Wild-Animal Fecal Samples, Human Septage, and Surface Water

A repeated cross-sectional study was conducted to determine the patterns of antimicrobial resistance in 1,286 Escherichia coli strains isolated from human septage, wildlife, domestic animals, farm environments, and surface water in the Red Cedar watershed in Michigan. Isolation and identification of E. coli were done by using enrichment media, selective media, and biochemical tests. Antimicrobial susceptibility testing by the disk diffusion method was conducted for neomycin, gentamicin, streptomycin, chloramphenicol, ofloxacin, trimethoprim-sulfamethoxazole, tetracycline, ampicillin, nalidixic acid, nitrofurantoin, cephalothin, and sulfisoxazole. Resistance to at least one antimicrobial agent was demonstrated in isolates from livestock, companion animals, human septage, wildlife, and surface water. In general, E. coli isolates from domestic species showed resistance to the largest number of antimicrobial agents compared to isolates from human septage, wildlife, and surface water. The agents to which resistance was demonstrated most frequently were tetracycline, cephalothin, sulfisoxazole, and streptomycin. There were similarities in the patterns of resistance in fecal samples and farm environment samples by animal, and the levels of cephalothin-resistant isolates were higher in farm environment samples than in fecal samples. Multidrug resistance was seen in a variety of sources, and the highest levels of multidrug-resistant E. coli were observed for swine fecal samples. The fact that water sample isolates were resistant only to cephalothin may suggest that the resistance patterns for farm environment samples may be more representative of the risk of contamination of surface waters with antimicrobial agent-resistant bacteria.     

Patterns of antimicrobial resistance observed in Escherichia coli isolates obtained from domestic- and wild-animal fecal samples, human septage, and surface water

A repeated cross-sectional study was conducted to determine the patterns of antimicrobial resistance in 1,286 Escherichia coli strains isolated from human septage, wildlife, domestic animals, farm environments, and surface water in the Red Cedar watershed in Michigan. Isolation and identification of E. coli were done by using enrichment media, selective media, and biochemical tests. Antimicrobial susceptibility testing by the disk diffusion method was conducted for neomycin, gentamicin, streptomycin, chloramphenicol, ofloxacin, trimethoprim-sulfamethoxazole, tetracycline, ampicillin, nalidixic acid, nitrofurantoin, cephalothin, and sulfisoxazole. Resistance to at least one antimicrobial agent was demonstrated in isolates from livestock, companion animals, human septage, wildlife, and surface water. In general, E. coli isolates from domestic species showed resistance to the largest number of antimicrobial agents compared to isolates from human septage, wildlife, and surface water. The agents to which resistance was demonstrated most frequently were tetracycline, cephalothin, sulfisoxazole, and streptomycin. There were similarities in the patterns of resistance in fecal samples and farm environment samples by animal, and the levels of cephalothin-resistant isolates were higher in farm environment samples than in fecal samples. Multidrug resistance was seen in a variety of sources, and the highest levels of multidrug-resistant E. coli were observed for swine fecal samples. The fact that water sample isolates were resistant only to cephalothin may suggest that the resistance patterns for farm environment samples may be more representative of the risk of contamination of surface waters with antimicrobial agent-resistant bacteria.     

Antimicrobial resistance in generic Escherichia coli isolates from wild small mammals living in swine farm, residential, landfill, and natural environments in southern Ontario, Canada

To assess the impacts of different types of human activity on the development of resistant bacteria in the feces of wild small mammals, we compared the prevalences and patterns of antimicrobial resistance and resistance genes in generic Escherichia coli and Salmonella enterica isolates from fecal samples collected from wild small mammals living in four environments: swine farms, residential areas, landfills, and natural habitats. Resistance to antimicrobials was observed in E. coli isolates from animals in all environments: 25/52 (48%) animals trapped at swine farms, 6/69 (9%) animals trapped in residential areas, 3/20 (15%) animals trapped at landfills, and 1/22 (5%) animals trapped in natural habitats. Animals trapped on farms were significantly more likely to carry E. coli isolates with resistance to tetracycline, ampicillin, sulfisoxazole, and streptomycin than animals trapped in residential areas. The resistance genes sul2, aadA, and tet(A) were significantly more likely to be detected in E. coli isolates from animals trapped on farms than from those trapped in residential areas. Three S. enterica serotypes (Give, Typhimurium, and Newport) were recovered from the feces of 4/302 (1%) wild small mammals. All Salmonella isolates were pansusceptible. Our results show that swine farm origin is significantly associated with the presence of resistant bacteria and resistance genes in wild small mammals in southern Ontario, Canada. However, resistant fecal bacteria were found in small mammals living in all environments studied, indicating that environmental exposure to antimicrobials, antimicrobial residues, resistant bacteria, or resistance genes is widespread.     

Incidence of salmonellae in fecal samples of production swine and swine at slaughter plants in the United States in 1978.

Nine swine slaughter plants and 19 swine production units were randomly selected for sampling from the six highest swine-producing states representing a total of 64% of the United States swine production. Three composites of 10 fresh swine fecal samples were obtained from each slaughter plant, representing three different farm sources of swine. Two composite fecal samples were collected from two different production pens from each production unit. Samples were analyzed for salmonellae. Isolated salmonellae were biochemically and serologically identified and tested for antibiotic susceptibility and resistance transfer ability. Salmonellae were recovered from swine at seven of the nine slaughter plants and 16 of the 27 composites of slaughter swine. Of the 19 production units, 3 had swine shedding salmonellae. Resistances found included streptomycin, tetracycline, and sulfadiazine. Of the 52 total isolates tested, 71% had some level of antibiotic resistance. Only 3 of 37 resistant isolated could transfer resistance under the conditions used.     

Effects of heat stress on the antimicrobial drug resistance of Escherichia coli of the intestinal flora of swine

The effects of heat stress on the antimicrobial drug resistance of Escherichia coli of the intestinal tract of swine were studied in animals from a farm that had not been supplementing antimicrobials in feed for the past 10 years. In one study, 10 finisher hogs were heat stressed (34 degrees C) for 24 h. Antimicrobial resistance levels after stress were significantly higher (P < 0.05) when compared with pre-stress levels for amikacin, ampicillin, cephalothin, neomycin and tetracycline from faecal samples. This high level of resistance persisted to slaughter that occurred at 10 days post-stress for most of the antimicrobials mentioned. In a second study, samples of different sections of the gastrointestinal tract were collected after heat stress and compared with control, non-stressed animals. Results indicated that E. coli which colonized the ileum and caecum had a higher level of resistance to ampicillin and tetracycline than the E. coli which colonized the colon and rectum. When animals were exposed to heat stress, resistance to ampicillin and tetracycline of E. coli in the lower digestive tract increased (P < 0.05) to a level similar to that observed in the ileum and caecum. Based on these findings, an investigation was made to test the hypothesis that (a) an increase in intestinal motility increases shedding of resistant E. coli and (b) heat stress induces a reduction in intestinal transit time in swine. For each study, two groups of three, randomly selected finisher hogs each were formed (treated and control groups). In study (a), induction of increased motility and peristalsis was obtained using an intramuscular injection of the cholinergic drug neostigmine methylsulphate. Escherichia coli isolates were obtained from the ileum, caecum, colon and rectum after animals were slaughtered. A higher level of ampicillin-resistant E. coli was found in the caecum (40%) than in other segments of the intestinal tract. In treated animals, level of resistance increased for organisms from the colon and rectum. Similar results were obtained for tetracycline resistance. In study (b), intestinal transit time was measured using chromium-EDTA as a marker. Swine were euthanized and samples were collected throughout the intestinal tract (duodenum to rectum) 8 h after administration of the marker to control and heat-stressed animals. Results indicated a reduced transit time for the stressed group. These findings corroborate the initial hypothesis that an outflow of resistant organisms moves from the upper tract (ileum and caecum) to the lower tract (colon and rectum).     

Occurrence and Persistence of Erythromycin Resistance Genes (erm) and Tetracycline Resistance Genes (tet) in Waste Treatment Systems on Swine Farms

Animal manure from modern animal agriculture constitutes the single largest source of antibiotic resistance (AR) owing to the use of large quantities of antibiotics. After animal manure enters the environment, the AR disseminates into the environment and can pose a potentially serious threat to the health and well-being of both humans and animals. In this study, we evaluated the efficiency of three different on-farm waste treatment systems in reducing AR. Three classes of erythromycin resistance genes (erm) genes (B, F, and X) conferring resistances to macrolide–lincosamides–streptogramin B (MLS_B) and one class of tetracycline resistance genes (tet) gene (G) conferring resistance to tetracyclines were used as models. Real-time polymerase chain reaction assays were used to determine the reservoir sizes of these AR genes present in the entire microbiome. These classes of AR genes varied considerably in abundance, with erm(B) being more predominant than erm(F), erm(X), and tet(G). These AR genes also varied in persistence in different waste treatment systems. Aerobic biofiltration reduced erm(X) more effectively than other AR genes, while mesophilic anaerobic digestion and lagoon storage did not appreciably reduce any of these AR genes. Unlike chemical pollutants, some AR genes could increase after reduction in a preceding stage of the treatment processes. Season might also affect the persistence of AR. These results indicate that AR arising from swine-feeding operations can survive typical swine waste treatment processes and thus treatments that are more effective in destructing AR on farms are required.     

Sampling 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.     

Tetracycline 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.     

Epidemiology of Antimicrobial Resistance in Escherichia coli Isolates from Raccoons (Procyon lotor) and the Environment on Swine Farms and Conservation Areas in Southern Ontario

Antimicrobial resistance is a global threat to livestock, human and environmental health. Although resistant bacteria have been detected in wildlife, their role in the epidemiology of antimicrobial resistance is not clear. Our objective was to investigate demographic, temporal and climatic factors associated with carriage of antimicrobial resistant Escherichia coli in raccoons and the environment. We collected samples from raccoon paws and feces and from soil, manure pit and dumpsters on five swine farms and five conservation areas in Ontario, Canada once every five weeks from May to November, 2011–2013 and tested them for E. coli and susceptibility to 15 antimicrobials. Of samples testing positive for E. coli, resistance to ≥ 1 antimicrobials was detected in 7.4% (77/1044; 95% CI, 5.9–9.1) of raccoon fecal samples, 6.3% (23/365; 95% CI, 4.0–9.3) of paw samples, 9.6% (121/1260; 8.0–11.4) of soil samples, 57.4% (31/54; 95% CI, 43.2–70.8) of manure pit samples, and 13.8% (4/29; 95% CI, 3.9–31.7) of dumpster samples. Using univariable logistic regression, there was no significant difference in the occurrence of resistant E. coli in raccoon feces on conservation areas versus farms; however, E. coli isolates resistant to ≥ 1 antimicrobials were significantly less likely to be detected from raccoon paw samples on swine farms than conservation areas and significantly more likely to be detected in soil samples from swine farms than conservation areas. Resistant phenotypes and genotypes that were absent from the swine farm environment were detected in raccoons from conservation areas, suggesting that conservation areas and swine farms may have different exposures to resistant bacteria. However, the similar resistance patterns and genes in E. coli from raccoon fecal and environmental samples from the same location types suggest that resistant bacteria may be exchanged between raccoons and their environment.     

Isolation of Tn916-like conjugal elements from swine lot effluent

Isolates of Enterococcus faecalis obtained from a swine farrowing house outflow were examined for genetic elements similar to Tn916. Of the enterococci isolated, 71% were resistant to tetracycline. Among the tetracycline-resistant enterococci isolated from the outflow samples, approximately 34% were able to transfer the tetracycline resistance phenotype to Bacillus thuringiensis in cross-genus matings. The frequencies of transfer for 10 random isolates were comparable to those for transfer of Tn916 from E. faecalis to B. thuringiensis. In addition, these elements were shown to mobilize plasmid pC194 between Bacillus species, as did Tn916. Southern blot and polymerase chain reaction (PCR) analysis showed these elements share extensive structural homology with Tn916. The selected conjugal elements were capable of transfer to a Bacillus recipient in a soil environment. When the swine waste was introduced into the soil, the tetracycline resistant fecal enterococci levels rose from essentially undetectable levels to approximately 4 x 10(4) and remained at this level for 4 weeks. After six months, including one winter, levels had decreased to 5 x 10(3).     

Field Trial Evaluating the Influence of Prophylactic and Therapeutic Antimicrobial Administration on Antimicrobial Resistance of Fecal Escherichia coli in Dairy Calves

The objective of this study was to describe the influence of in-feed and therapeutic antimicrobials on resistance in commensal fecal Escherichia coli isolated from preweaned calves. Four groups of 30, day-old calf-ranch calves were enrolled and raised until 4 weeks of age. Groups 1 to 3 were raised without antimicrobials in the feed. Group 1 was isolated from the other groups and received no antimicrobial therapy. Group 2 was housed on the calf ranch and did not receive antimicrobial therapy, whereas groups 3 and 4 could be treated with antimicrobials. Group 4 was fed neomycin and tetracycline HCl in the milk replacer. Fecal samples were collected from calves on days 1, 14, and 28. Three E. coli isolates per sample were evaluated for susceptibility to 12 antimicrobials. Cluster analysis was used to group isolates having similar susceptibility patterns. Cumulative logistic regression was used to evaluate factors associated with increasing levels of multiple antimicrobial resistance. In-feed antimicrobials were associated with higher levels of multiple antimicrobial resistance in fecal E. coli.f In calves not receiving in-feed antimicrobials, older calves had higher levels of resistance compared to day-old calves. Individual antimicrobial therapy increased resistance in these calves but appeared to be transient. There was no environmental influence on resistance in E. coli populations among study groups.     

Field trial evaluating the influence of prophylactic and therapeutic antimicrobial administration on antimicrobial resistance of fecal Escherichia coli in dairy calves

The objective of this study was to describe the influence of in-feed and therapeutic antimicrobials on resistance in commensal fecal Escherichia coli isolated from preweaned calves. Four groups of 30, day-old calf-ranch calves were enrolled and raised until 4 weeks of age. Groups 1 to 3 were raised without antimicrobials in the feed. Group 1 was isolated from the other groups and received no antimicrobial therapy. Group 2 was housed on the calf ranch and did not receive antimicrobial therapy, whereas groups 3 and 4 could be treated with antimicrobials. Group 4 was fed neomycin and tetracycline HCl in the milk replacer. Fecal samples were collected from calves on days 1, 14, and 28. Three E. coli isolates per sample were evaluated for susceptibility to 12 antimicrobials. Cluster analysis was used to group isolates having similar susceptibility patterns. Cumulative logistic regression was used to evaluate factors associated with increasing levels of multiple antimicrobial resistance. In-feed antimicrobials were associated with higher levels of multiple antimicrobial resistance in fecal E. coli.f In calves not receiving in-feed antimicrobials, older calves had higher levels of resistance compared to day-old calves. Individual antimicrobial therapy increased resistance in these calves but appeared to be transient. There was no environmental influence on resistance in E. coli populations among study groups.     

Longitudinal study of antimicrobial resistance among Escherichia coli isolates from integrated multisite cohorts of humans and swine

In a 3-year longitudinal study, we examined the relationship between the seasonal prevalence of antimicrobial-resistant (AR) Escherichia coli isolates from human wastewater and swine fecal samples and the following risk factors: the host species, the production type (swine), the vocation (human swine workers, non-swine workers, and slaughter plant workers), and the season, in a multisite, vertically integrated swine and human population representative of a closed agri-food system. Human and swine E. coli (n = 4,048 and 3,429, respectively) isolates from wastewater and fecal samples were tested for antimicrobial susceptibility, using the Sensititre broth microdilution system. There were significant (P < 0.05) differences among AR E. coli prevalence levels of (i) the host species, in which swine isolates were at higher risk for resistance to tetracycline, kanamycin, ceftiofur, gentamicin, streptomycin, chloramphenicol, sulfisoxazole, and ampicillin; (ii) the swine production group, in which purchased boars, nursery piglets, and breeding boars isolates had a higher risk of resistance to streptomycin and tetracycline; and iii) the vocation cohorts, in which swine worker cohort isolates exhibited lower sulfisoxazole and cefoxitin prevalence than the non-swine worker cohorts, while the slaughter plant worker cohort isolates exhibited elevated cefoxitin prevalence compared to that of non-swine workers. While a high variability was observed among seasonal samples over the 3-year period, no significant temporal trends were apparent. There were significant differences in the prevalence levels of multidrug-resistant isolates between host species, with swine at a higher risk of carrying multidrug-resistant strains than humans. Considering vocation, slaughter plant workers were at higher risk of exhibiting multidrug-resistant E. coli than non-swine workers.     

Evaluation of Swine-Specific PCR Assays Used for Fecal Source Tracking and Analysis of Molecular Diversity of Swine-Specific “Bacteroidales” Populations

In this study, we evaluated the specificity, distribution, and sensitivity of Prevotella strain-based (PF163 and PigBac1) and methanogen-based (P23-2) PCR assays proposed to detect swine fecal pollution in environmental waters. The assays were tested against 222 fecal DNA extracts derived from target and nontarget animal hosts and against 34 groundwater and 15 surface water samples from five different sites. We also investigated the phylogenetic diversity of 1,340 “Bacteroidales” 16S rRNA gene sequences derived from swine feces, swine waste lagoons, swine manure pits, and waters adjacent to swine operations. Most swine fecal samples were positive for the host-specific Prevotella-based PCR assays (80 to 87%), while fewer were positive with the methanogen-targeted PCR assay (53%). Similarly, the Prevotella markers were detected more frequently than the methanogen-targeted assay markers in waters historically impacted with swine fecal contamination. However, the PF163 PCR assay cross-reacted with 23% of nontarget fecal DNA extracts, although Bayesian statistics suggested that it yielded the highest probability of detecting pig fecal contamination in a given water sample. Phylogenetic analyses revealed previously unknown swine-associated clades comprised of clones from geographically diverse swine sources and from water samples adjacent to swine operations that are not targeted by the Prevotella assays. While deeper sequencing coverage might be necessary to better understand the molecular diversity of fecal Bacteroidales species, results of sequence analyses supported the presence of swine fecal pollution in the studied watersheds. Overall, due to nontarget cross amplification and poor geographic stability of currently available host-specific PCR assays, development of additional assays is necessary to accurately detect sources of swine fecal pollution.The size of swine farming operations has increased significantly during the last few decades as a result of the high demand for pork products. In fact, pork is now considered the most popular meat worldwide (15). In the United States, the number of large confined swine animal units increased by 3 orders of magnitude from 1982 to 1997 (18), making the swine industry among the top three producers of domesticated animal feces. A direct consequence of this trend is the increase in swine fecal waste, which in turn has raised environmental concerns. When introduced to water, swine fecal waste can present a risk to human health because this waste can harbor a variety of human pathogens (5, 13, 15, 21, 36). The diversity and relatively high frequency of human pathogens in swine feces make swine important reservoirs of zoonotic pathogens. Moreover, the marked increase in the number of large operations has resulted in increased manure production and application in small geographic areas, creating an imbalance between the assimilative capacity of manure-treated farmland and the amount of manure nutrients produced on each farm. This imbalance is evidenced by the 20% increase (from 1982 to 1997) in nitrogen and phosphorus produced in swine operations, thus potentially contributing to the detrimental eutrophication of aquatic ecosystems (18). Swine manure spills and leaks are commonplace in the top hog production states, such as Iowa and North Carolina, due to failure or overflow of manure storage, uncontrolled runoff from open feedlots, improper manure application on cropland, deliberate pumping of manure onto the ground, and intentional breaches in storage lagoons (28, 37).Recently, swine-associated PCR-based methods targeting members of the “Bacteroidales” order (i.e., Prevotella species) and methanogen populations (12, 29, 35) have been proposed to discriminate swine fecal pollution events from other potential fecal contributions (i.e., human, bovine, and wildlife) to environmental waters. Nevertheless, the value of these assays in reliably detecting fecal pollution sources in watershed-based studies has not been thoroughly investigated. The main goals of this study were to determine host specificity, frequency of detection, and detection limits of currently available swine-associated PCR-based, microbial source tracking assays. To achieve these objectives, assays were tested against swine and nontarget fecal samples, samples from swine manure pits and swine waste lagoons, and water samples presumed to be impacted by swine fecal sources. Furthermore, we investigated the phylogenetic diversity of Bacteroidales 16S rRNA gene sequences derived from some of the aforementioned samples to resolve the level of specificity, relative abundance, and environmental occurrence of Bacteroidales-specific 16S rRNA gene sequences.     

Molecular ecology of tetracycline resistance: development and validation of primers for detection of tetracycline resistance genes encoding ribosomal protection proteins

Phylogenetic analysis of tetracycline resistance genes encoding the ribosomal protection proteins (RPPs) revealed the monophyletic origin of these genes. The most deeply branching class, exemplified by tet and otrA, consisted of genes from the antibiotic-producing organisms Streptomyces rimosus and Streptomyces lividans. With a high degree of confidence, the corresponding genes of the other seven classes (Tet M, Tet S, Tet O, Tet W, Tet Q, Tet T, and TetB P) formed phylogenetically distinct separate clusters. Based on this phylogenetic analysis, a set of PCR primers for detection, retrieval, and sequence analysis of the corresponding gene fragments from a variety of bacterial and environmental sources was developed and characterized. A pair of degenerate primers targeted all tetracycline resistance genes encoding RPPs except otrA and tet, and seven other primer pairs were designed to target the specific classes. The primers were used to detect the circulation of these genes in the rumina of cows, in swine feed and feces, and in swine fecal streptococci. Classes Tet O and Tet W were found in the intestinal contents of both animals, while Tet M was confined to pigs and Tet Q was confined to the rumen. The tet(O) and tet(W) genes circulating in the microbiota of the rumen and the gastrointestinal tract of pigs were identical despite the differences in animal hosts and antibiotic use regimens. Swine fecal streptococci uniformly possessed the tet(O) gene, and 22% of them also carried tet(M). This population could be considered one of the main reservoirs of these two resistance genes in the pig gastrointestinal tract. All classes of RPPs except Tet T and TetB P were found in the commercial components of swine feed. This is the first demonstration of the applicability of molecular ecology techniques to estimation of the gene pool and the flux of antibiotic resistance genes in production animals.