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 (MLS_B), 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 MLS_B 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 MLS_B 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.     

Modulations of the chicken cecal microbiome and metagenome in response to anticoccidial and growth promoter treatment

With increasing pressures to reduce or eliminate the use of antimicrobials for growth promotion purposes in production animals, there is a growing need to better understand the effects elicited by these agents in order to identify alternative approaches that might be used to maintain animal health. Antibiotic usage at subtherapeutic levels is postulated to confer a number of modulations in the microbes within the gut that ultimately result in growth promotion and reduced occurrence of disease. This study examined the effects of the coccidiostat monensin and the growth promoters virginiamycin and tylosin on the broiler chicken cecal microbiome and metagenome. Using a longitudinal design, cecal contents of commercial chickens were extracted and examined using 16S rRNA and total DNA shotgun metagenomic pyrosequencing. A number of genus-level enrichments and depletions were observed in response to monensin alone, or monensin in combination with virginiamycin or tylosin. Of note, monensin effects included depletions of Roseburia, Lactobacillus and Enterococcus, and enrichments in Coprococcus and Anaerofilum. The most notable effect observed in the monensin/virginiamycin and monensin/tylosin treatments, but not in the monensin-alone treatments, was enrichments in Escherichia coli. Analysis of the metagenomic dataset identified enrichments in transport system genes, type I fimbrial genes, and type IV conjugative secretion system genes. No significant differences were observed with regard to antimicrobial resistance gene counts. Overall, this study provides a more comprehensive glimpse of the chicken cecum microbial community, the modulations of this community in response to growth promoters, and targets for future efforts to mimic these effects using alternative approaches.     

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.     

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.     

Induction of ermAMR from a Clinical Strain of Enterococcus faecalis by 16-Membered-Ring Macrolide Antibiotics

We cloned the MLS_B resistance determinant by PCR from a clinical isolate of Enterococcus faecalis 373, which is induced more strongly by a 16-membered-ring macrolide, tylosin, than by erythromycin. To elucidate the molecular basis of resistance of E. faecalis 373, we analyzed the cloned gene, designated ermAMR, by site-directed mutagenesis and reporter gene assay. Our results showed that an arginine-to-cysteine change in the seventh codon of the putative leader peptide endowed tylosin with resistance inducibility and that TAAA duplication enabled the control region to express the downstream methylase gene at a drastically increased level.     

Specific Inhibition of 50S Ribosomal Subunit Formation in Staphylococcus aureus Cells by 16-Membered Macrolide, Lincosamide, and Streptogramin B Antibiotics

The translational functions of the bacterial ribosome are the target for a large number of antimicrobial agents. The 14- and 16-membered macrolides, the lincosamides, and the streptogramin B type antibiotics are thought to share certain inhibitory properties, based on both biochemical and genetic studies. We have shown previously that the 14-membered macrolides, like erythromycin, have an equivalent inhibitory effect on translation and the formation of the 50S ribosomal subunit in growing bacterial cells. To extend this work, we have now tested the 16-membered macrolides spiramycin and tylosin, the lincosamides lincomycin and clindamycin, and 3 streptogramin B compounds pristinamycin I_A, virginiamycin S, and CP37277. Each of these was a specific inhibitor of 50S subunit formation, in addition to having an inhibitory effect on translation. By contrast, two streptogramin A compounds, virginiamycin M1 and CP36926, as well as chloramphenicol, were effective inhibitors of translation without showing a specific effect on the assembly of the large ribosomal subunit. A combination of an A and B type streptogramin (virginiamycin M1 and pristinamycin I_A) demonstrated a synergistic inhibition of protein synthesis without exhibiting a specific inhibition of 50S subunit formation. These results extend our observations on 50S assembly inhibition to the entire class of MLS_B antibiotics and reinforce other suggestions concerning their common ribosome-binding site and inhibitory functions. Received: 13 January 2000 / Accepted 2 March 2000     

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.     

Comparison of Agar Dilution and Broth Microdilution Methods of Anaerobic Antimicrobial Susceptibility Testing using Several Veterinary Antibiotics against Clostridium perfringens Strains Originating from Porcine and Avian Sources

A broth microdilution and a reference agar dilution method was used to determine minimal inhibitory concentrations (MICs) of five veterinary antimicrobials when tested against 96 animal-derived and six American Type Culture Collection (ATCC) strains of Clostridium perfringens. These antimicrobials [bacitracin methylenedisalicylate (bacitracin-MD), tylosin, virginiamycin, erthromycin and tetracycline] are approved for use in animal feed at different levels for growth enhancement, control, and treatment of a variety of enteric diseases. For bacitracin-MD, MICs were higher using the broth microdilution method when compared to the agar dilution method. The two methods had the lowest agreement when using bacitracin-MD compared to the method agreements of other antimicrobials tested (only 34.3% of the C. perfringens tested within ±1 doubling dilution). Tylosin MICs were lower by the broth microdilution method but had better agreement between methods with 75.5% of the C. perfringens tested within ±1 doubling dilution. Good correlation between methods was found for virginiamycin, tetracycline, and erythromycin with 85.3, 76.5, 81.4% of the C. perfringens tested within ±1 doubling dilution, respectively. Differences in susceptibility to individual antimicrobials were found among the avian and porcine strains by both methods. For avian strains, bacitracin-MD, tylosin, and erthromycin MIC₉₀values had differences of at least four doubling dilutions between methods. There were biases toward higher bacitracin-MD and lower tylosin and erythromycin MIC₉₀values using the broth microdilution method. MIC₉₀values against porcine and ATCC strains were more comparable between methods for all five antimicrobials than those generated against avian strains but the biases were still present. Most animal-derived strains were inhibited by approved livestock feed levels of the antimicrobials. Caution should be used when evaluating the potential effectiveness of feed-based antimicrobials against C. perfringens when results are generated using an in vitro test that may not be in agreement with the reference agar dilution method.     

The effects of dietary additives on faecal levels of Lactobacillus spp., coliforms,and Escherichia coli,and faecal prevalence of Salmonella spp. and Campylobacter spp. in US production nursery swine1

Aims: In the United States, carbadox and copper sulfate are growth promoters commonly used in combination in nursery swine diets. Our aim was to determine how selected dietary additives affect selected bacterial populations and pathogens in nursery swine, and compare to larch extract, which contains potential antibacterial activities. Methods and Results: Piglets were weaned and sorted into one of the four treatments: (i) basal diet without antimicrobials; (ii) basal diet with carbadox + copper sulfate; (iii) basal diet + 1000 ppm larch extract; or (iv) basal diet + 2000 ppm larch extract. Diets were fed for a 4‐week period after weaning. In both trials, the carbadox + copper sulfate group consumed more feed over the 4‐week period relative to the other three diet groups (P < 0·05), but did not gain significantly more weight. Faecal shedding of Salmonella spp. was not affected by dietary supplement in either trial, but faecal shedding of Campylobacter spp. was the lowest for the carbadox + copper sulfate diet. In faecal samples collected at the end of each trial, Lactobacillus spp. cell counts for the basal and larch extract diets were nearly 1·0 log₁₀ g^?1 faeces greater (P < 0·05) than the carbadox + copper sulfate group, whereas the coliforms and Escherichia coli were nearly 1·0 log₁₀ g^?1 faeces lower (P < 0·05). Conclusions: Compared to basal fed animals, supplementation with carbadox + copper sulfate significantly altered faecal E. coli, coliform bacteria and Lactobacillus spp. Larch extract has no benefit up to 0·2% of diet in regard to pathogen shedding, whereas carbadox + copper sulfate decreased faecal shedding of Campylobacter spp. Significance and Impact of the Study: Current swine management practices in the United States may be beneficial to managing Campylobacter spp. shedding in nursery swine, but also result in significant changes in the resident gastrointestinal microflora.     

Antimicrobial susceptibilities of enterococci isolated from faeces of broiler and layer chickens

Enterococci were isolated from faecal droppings of chickens in broiler and layer farms and the susceptibilities to nine therapeutic antimicrobial agents and six growth-promoting antibiotics were determined by the agar dilution method. Resistance to therapeutic antimicrobial agents such as ampicillin, clindamycin, erythromycin, streptomycin, tetracycline or tylosin was more frequent in enterococcal isolates from broiler farms than in those from layer farms. Resistance to ofloxacin was rare, occurring in only one (0.7%) of the Enterococcus faecium isolates from broiler farms. Resistance to growth-promoting antibiotics such as avilamycin, salinomycin and virginiamycin was common among isolates from broiler farms. Of the E. faecium isolates from broiler farms, 12.4% were resistant to avilamycin and 27.4% were resistant to virginiamycin. Resistance to salinomycin was detected in all enterococcal species, ranging from 12.4% of E. faecium isolates to 50% of E. hirae isolates.     

Effects of the antimicrobial tylosin on the microbial community structure of an anaerobic sequencing batch reactor

The effects of the antimicrobial tylosin on a methanogenic microbial community were studied in a glucose‐fed laboratory‐scale anaerobic sequencing batch reactor (ASBR) exposed to stepwise increases of tylosin (0, 1.67, and 167 mg/L). The microbial community structure was determined using quantitative fluorescence in situ hybridization (FISH) and phylogenetic analyses of bacterial 16S ribosomal RNA (rRNA) gene clone libraries of biomass samples. During the periods without tylosin addition and with an influent tylosin concentration of 1.67 mg/L, 16S rRNA gene sequences related to Syntrophobacter were detected and the relative abundance of Methanosaeta species was high. During the highest tylosin dose of 167 mg/L, 16S rRNA gene sequences related to Syntrophobacter species were not detected and the relative abundance of Methanosaeta decreased considerably. Throughout the experimental period, Propionibacteriaceae and high GC Gram‐positive bacteria were present, based on 16S rRNA gene sequences and FISH analyses, respectively. The accumulation of propionate and subsequent reactor failure after long‐term exposure to tylosin are attributed to the direct inhibition of propionate‐oxidizing syntrophic bacteria closely related to Syntrophobacter and the indirect inhibition of Methanosaeta by high propionate concentrations and low pH. Biotechnol. Bioeng. 2011;108: 296–305. © 2010 Wiley Periodicals, Inc.     

Susceptibility and resistance of ruminal bacteria to antimicrobial feed additives

Susceptibility and resistance of ruminal bacterial species to avoparcin, narasin, salinomycin, thiopeptin, tylosin, virginiamycin, and two new ionophore antibiotics, RO22-6924/004 and RO21-6447/009, were determined. Generally, antimicrobial compounds were inhibitory to gram-positive bacteria and those bacteria that have gram-positive-like cell wall structure. MICs ranged from 0.09 to 24.0 micrograms/ml. Gram-negative bacteria were resistant at the highest concentration tested (48.0 micrograms/ml). On the basis of their fermentation products, ruminal bacteria that produce lactic acid, butyric acid, formic acid, or hydrogen were susceptible and bacteria that produce succinic acid or ferment lactic acid were resistant to the antimicrobial compounds. Selenomonas ruminantium was the only major lactic acid-producing bacteria resistant to all the antimicrobial compounds tested. Avoparcin and tylosin appeared to be less inhibitory (MIC greater than 6.0 micrograms/ml) than the other compounds to the two major lactic acid-producing bacteria, Streptococcus bovis and Lactobacillus sp. Ionophore compounds seemed to be more inhibitory (MIC, 0.09 to 1.50 micrograms/ml) than nonionophore compounds (MIC, 0.75 to 12.0 micrograms/ml) to the major butyric acid-producing bacteria. Treponema bryantii, an anaerobic rumen spirochete, was less sensitive to virginiamycin than to the other antimicrobial compounds. Ionophore compounds were generally bacteriostatic, and nonionophore compounds were bactericidal. The specific growth rate of Bacteroides ruminicola was reduced by all the antimicrobial compounds except avoparcin. The antibacterial spectra of the feed additives were remarkably similar, and it appears that MICs may not be good indicators of the potency of the compounds in altering ruminal fermentation characteristics.     

Susceptibility and resistance of ruminal bacteria to antimicrobial feed additives.

Susceptibility and resistance of ruminal bacterial species to avoparcin, narasin, salinomycin, thiopeptin, tylosin, virginiamycin, and two new ionophore antibiotics, RO22-6924/004 and RO21-6447/009, were determined. Generally, antimicrobial compounds were inhibitory to gram-positive bacteria and those bacteria that have gram-positive-like cell wall structure. MICs ranged from 0.09 to 24.0 micrograms/ml. Gram-negative bacteria were resistant at the highest concentration tested (48.0 micrograms/ml). On the basis of their fermentation products, ruminal bacteria that produce lactic acid, butyric acid, formic acid, or hydrogen were susceptible and bacteria that produce succinic acid or ferment lactic acid were resistant to the antimicrobial compounds. Selenomonas ruminantium was the only major lactic acid-producing bacteria resistant to all the antimicrobial compounds tested. Avoparcin and tylosin appeared to be less inhibitory (MIC greater than 6.0 micrograms/ml) than the other compounds to the two major lactic acid-producing bacteria, Streptococcus bovis and Lactobacillus sp. Ionophore compounds seemed to be more inhibitory (MIC, 0.09 to 1.50 micrograms/ml) than nonionophore compounds (MIC, 0.75 to 12.0 micrograms/ml) to the major butyric acid-producing bacteria. Treponema bryantii, an anaerobic rumen spirochete, was less sensitive to virginiamycin than to the other antimicrobial compounds. Ionophore compounds were generally bacteriostatic, and nonionophore compounds were bactericidal. The specific growth rate of Bacteroides ruminicola was reduced by all the antimicrobial compounds except avoparcin. The antibacterial spectra of the feed additives were remarkably similar, and it appears that MICs may not be good indicators of the potency of the compounds in altering ruminal fermentation characteristics.     

THE COMBINED USE OF WHOLE CUPHEA SEEDS CONTAINING MEDIUM CHAIN FATTY ACIDS AND AN EXOGENOUS LIPASE IN PIGLET NUTRITION

In search for an alternative for nutritional antimicrobials in piglet feeding, the effects of adding whole Cuphea seeds, as a natural source of medium chain fatty acids (MCFA), with known antimicrobial effects, and an exogenous lipase to a weaner diet were studied. The foregut flora, the gut morphology, some digestive parameters and the zootechnical performance of weaned piglets were investigated. Thirty newly weaned piglets, initial weight 7.0 ± 0.4 kg, were divided according to litter, sex and weight in two groups (control diet; Cuphea+lipase diet). The Cuphea seeds (lanceolata and ignea) (50 g kg^?1) were substituted for soybean oil (15 g kg^?1), Alphacell (25 g kg^?1) and soy protein isolate (10 g kg^?1) in the control diet. Also 500 mg kg^?1 microbial lipase was added to the Cuphea diet. The piglets were weighted individually on days 0, 3, 7, 14 and 16. Feed intake was recorded per pen during days 0 to 3, 3 to 7, 7 to 14 and 14 to 16. On day 7 five piglets of each experimental group were euthanized for counting the gastric and small intestinal gut flora and for gut morphology at two sites of the small intestine (proximal, distal). The results indicate a trend towards improved performances parameters by feeding Cuphea + lipase. The enzymic released MCFA (1.7 g kg^?1 fresh gastric contents) tended to decrease the number of Coliforms in the proximal small intestine, but increased the number in the stomach and distal small intestine. With Cuphea, the number of Streptococci was significantly lower in small intestine, but not in the stomach, while the number of Lactobacilli was significantly lower in the distal small intestine and tended to be lower in the stomach and proximal small intestine. No differences between the diets were noted for the total anaerobic microbial load in the stomach or in the gut. Feeding Cuphea+lipase resulted in a significantly greater villus height (distal small intestine) and a lesser crypt depth (proximal and distal small intestine) and greater villus/crypt ratio depth (proximal and distal small intestine). The intra-epithelial lymphocyte (IEL) counts per 100 enterocytes were significantly decreased in the proximal small intestine and tended to decrease in the distal small intestine by feeding the Cuphea+lipase diet. Both phenomena are indicative for a more healthy and better functional state of the mucosa. Present results are in line with foregoing research, showing that manipulation of the gut ecosystem by the enzymic in situ released MCFA in the stomach and foregut can result in improved performances of the piglets, which makes the concept a potential alternative for in-feed nutritional antibiotics.     

Changes in Antimicrobial Susceptibility of Native Enterococcus faecium in Chickens Fed Virginiamycin

The extent of transfer of antimicrobial resistance from agricultural environments to humans is controversial. To assess the potential hazard posed by streptogramin use in food animals, this study evaluated the effect of virginiamycin exposure on antimicrobial resistance in Enterococcus faecium recovered from treated broilers. Four consecutive broiler feeding trials were conducted using animals raised on common litter. In the first three trials, one group of birds was fed virginiamycin continuously in feed at 20 g/ton, and a second group served as the nontreated control. In the fourth trial, antimicrobial-free feed was given to both groups. Fecal samples were cultured 1 day after chickens hatched and then at 1, 3, 5, and 7 weeks of age. Isolates from each time point were tested for susceptibility to a panel of different antimicrobials. Quinupristin/dalfopristin-resistant E. faecium appeared after 5 weeks of treatment in trial 1 and within 7 days of trials 2 to 4. Following removal of virginiamycin in trial 4, no resistant isolates were detected after 5 weeks. PCR failed to detect vat, vgb, or erm(B) in any of the streptogramin-resistant E. faecium isolates, whereas the msr(C) gene was detected in 97% of resistant isolates. In an experimental setting using broiler chickens, continuous virginiamycin exposure was required to maintain a stable streptogramin-resistant population of E. faecium in the animals. The bases of resistance could not be explained by known genetic determinants.     

Use of virginiamycin to control the growth of lactic acid bacteria during alcohol fermentation

The antibiotic virginiamycin was investigated for its effects on growth and lactic acid production by seven strains of lactobacilli during the alcoholic fermentation of wheat mash by yeast. The lowest concentration of virginiamycin tested (0.5 mg Lactrol ^TMkg⁻¹ mash), was effective against most of the lactic acid bacteria under study, but Lactobacillus plantarum was not significantly inhibited at this concentration. The use of virginiamycin prevented or reduced potential yield losses of up to 11% of the produced ethanol due to the growth and metabolism of lactobacilli. However, when the same concentration of virginiamycin was added to mash not inoculated with yeast, Lactobacillus rhamnosus and L. paracasei grew after an extensive lag of 48 h and L. plantarum grew after a similar lag even in the presence of 2 mg virginiamycin kg⁻¹ mash. Results showed a variation in sensitivity to virginiamycin between the different strains tested and also a possible reduction in effectiveness of virginiamycin over prolonged incubation in wheat mash, especially in the absence of yeast. Received 05 August 1996/ Accepted in revised form 18 December 1996     

Large extents of intensive land use limit community reorganization during climate warming

Climate change is increasingly altering the composition of ecological communities, in combination with other environmental pressures such as high‐intensity land use. Pressures are expected to interact in their effects, but the extent to which intensive human land use constrains community responses to climate change is currently unclear. A generic indicator of climate change impact, the community temperature index (CTI), has previously been used to suggest that both bird and butterflies are successfully ‘tracking’ climate change. Here, we assessed community changes at over 600 English bird or butterfly monitoring sites over three decades and tested how the surrounding land has influenced these changes. We partitioned community changes into warm‐ and cold‐associated assemblages and found that English bird communities have not reorganized successfully in response to climate change. CTI increases for birds are primarily attributable to the loss of cold‐associated species, whilst for butterflies, warm‐associated species have tended to increase. Importantly, the area of intensively managed land use around monitoring sites appears to influence these community changes, with large extents of intensively managed land limiting ‘adaptive’ community reorganization in response to climate change. Specifically, high‐intensity land use appears to exacerbate declines in cold‐adapted bird and butterfly species, and prevent increases in warm‐associated birds. This has broad implications for managing landscapes to promote climate change adaptation.     

Intracolonial differences in gut bacterial community between worker and soldier castes of Coptotermes formosanus

Abstract The establishment of symbiotic relationships with intestinal microorganisms enables termites to thrive on recalcitrant substrates such as cellulose and wood. A termite colony is composed of several different castes which have distinct feeding habits. The soldiers, for example, cannot feed by themselves and depend on workers, who feed them with digested or semi‐digested foods. To investigate the influence of feeding habits on the bacterial symbionts, a comparative study of gut bacteria between worker and soldier castes of the termite Coptotermes formosanus was conducted. The bacterial communities of both castes were investigated using denaturing gradient gel electrophoresis (DGGE) and clonal analysis of 16S ribosomal DNA (rDNA). Both methods indicated Bacteroidetes was the common predominant group; the common dominant phylotype was affiliated with a reported uncultured Bacteroidetes phylotype (BCf1–03). There were significant differences in Bacteroidetes and Spirochaetes between two castes. Compared to the gut bacteria of workers, those of soldiers were lower in abundance and diversity of Bacteroidetes and slightly higher in Spirochaetes. Two phylotypes (W8, W11) affiliated to Bacteroidetes and two (W26, W29) affiliated to Spirochaetes were exclusively found in the DGGE profile of the worker caste. Bacteroidetes are assumed to be involved in fermentation of sugars and nitrogenous compounds as well as degradation of uric acid. Spirochaetes are supposed to aid in the functions of acetogenesis and N₂‐fixation. The different feeding habits between workers and soldiers of C. formosanus may explain the observed differences in the gut bacterial community.     

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.     

Antimicrobial susceptibility of Clostridium difficile isolated from neonatal pigs with enteritis

The minimum inhibitory concentration (MIC) of eight antimicrobial agents was determined by the agar dilution method for 80 isolates of Clostridium difficile from neonatal pigs with enteritis. MICs(50) for erythromycin, tilmicosin, and tylosin were relatively low (0.25-0.50 microg/mL), but MICs(90) (64 or > or =256 microg/mL) suggest in vivo resistance of a proportion of isolates. Susceptibility to tetracycline varied widely, with MIC(50) and MIC(90) of 8 and 32 microg/mL, respectively. The MICs(90) for tiamulin (8 microg/mL) and virginiamycin (16 microg/mL) suggest moderate susceptibility. Bacitracin and ceftiofur (MICs(90) > or =256 microg/mL) have little activity against C. difficile. Tiamulin and virginiamycin may decrease fecal shedding of C. difficile by sows, and erythromycin, tetracycline, and tylosin may be useful for treatment of infected piglets.