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.     

Antimicrobial Resistance and Virulence Genes of Escherichia coli Isolates from Swine in Ontario

A total of 318 Escherichia coli isolates obtained from diarrheic and healthy pigs in Ontario from 2001 to 2003 were examined for their susceptibility to 19 antimicrobial agents. They were tested by PCR for the presence of resistance genes for tetracycline, streptomycin, sulfonamides, and apramycin and of 12 common virulence genes of porcine E. coli. Antimicrobial resistance frequency among E. coli isolates from swine in Ontario was moderate in comparison with other countries and was higher in isolates from pigs with diarrhea than in isolates from healthy finisher pigs. Resistance profiles suggest that cephamycinases may be produced by ≥8% of enterotoxigenic E. coli (ETEC). Resistance to quinolones was detected only in enterotoxigenic E. coli (≤3%). The presence of sul3 was demonstrated for the first time in Canada in porcine E. coli isolates. Associations were observed among tetA, sul1, aadA, and aac(3)IV and among tetB, sul2, and strA/strB, with a strong negative association between tetA and tetB. The paa and sepA genes were detected in 92% of porcine ETEC, and strong statistical associations due to colocation on a large plasmid were observed between tetA, estA, paa, and sepA. Due at least in part to gene linkages, the distribution of resistance genes was very different between ETEC isolates and other porcine E. coli isolates. This demonstrates that antimicrobial resistance epidemiology differs significantly between pathogenic and commensal E. coli isolates. These results may have important implications with regards to the spread and persistence of resistance and virulence genes in bacterial populations and to the prudent use of antimicrobial agents.     

Antimicrobial Resistance in Escherichia coli Recovered from Feedlot Cattle and Associations with Antimicrobial Use

The objectives of this study were to estimate the prevalence of antimicrobial resistance (AMR) and to investigate the associations between exposures to antimicrobial drugs (AMDs) and AMR in fecal non-type specific Escherichia coli (NTSEC) recovered from a large population of feedlot cattle. Two-stage random sampling was used to select individually identified cattle for enrollment, which were sampled at arrival and then a second time later in the feeding period. Advanced regression techniques were used to estimate resistance prevalences, and to investigate associations between AMD exposures in enrolled cattle and penmates and AMR identified in NTSEC recovered from the second sample set. Resistance was most commonly detected to tetracycline, streptomycin, and sulfisoxazole, and was rarely identified for critically important AMDs. All cattle were exposed to AMDs in feed, and 45% were treated parenterally. While resistance prevalence generally increased during the feeding period, most AMD exposures were not significantly associated with AMR outcomes. Exposures of enrolled cattle to tetracycline were associated with increased resistance to tetracycline and trimethoprim sulfa, while beta-lactam exposures were associated with decreased likelihood of detecting streptomycin resistance. Pen-level AMD exposure measures were not associated with resistance outcomes. These findings suggest that tetracycline treatment of feedlot cattle can be associated with modest increases in risk for recovery of resistant NTSEC, but the numerous treatments with an advanced macrolide (tulathromycin) were not associated with detectable increases in resistance in NTSEC. All cattle were exposed to in-feed treatments of tetracycline and this could limit the ability to identify the full impact of these exposures, but these exposures varied for enrolled cattle varied, providing an opportunity to evaluate a dose response. While AMD exposures were not associated with detectably increased risks for resistance to critically important AMDs, rare resistance outcomes and infrequent exposure to other important AMDs (e.g., cephalosporins) limited our ability to rigorously investigate questions regarding factors that can influence resistance to these important AMDs.     

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.     

Effects of Tylosin Use on Erythromycin Resistance in Enterococci Isolated from Swine

The effect of tylosin on erythromycin-resistant enterococci was examined on three farms; farm A used tylosin for growth promotion, farm B used tylosin for treatment of disease, and farm C did not use tylosin for either growth promotion or disease treatment. A total of 1,187 enterococci were isolated from gestation, farrowing, suckling, nursery, and finishing swine from the farms. From a subset of those isolates (n = 662), 59% (124 out of 208), 28% (80 out of 281), and 2% (4 out of 170) were resistant to erythromycin (MIC ≥ 8 μg/ml) from farms A, B, and C, respectively. PCR analysis and Southern blotting revealed that 95% (65 out of 68) of isolates chosen from all three farms for further study were positive for ermB, but all were negative for ermA and ermC. By using Southern blotting, ermB was localized to the chromosome in 56 of the isolates while 9 isolates from farms A and B contained ermB on two similar-sized plasmid bands (12 to 16 kb). Pulsed-field gel electrophoresis revealed that the isolates were genetically diverse and represented a heterogeneous population of enterococci. This study suggests that although there was resistance to a greater number of enterococcal isolates on a farm where tylosin was used as a growth promotant, resistant enterococci also existed on a farm where no antimicrobial agents were used.     

Antimicrobial Resistance in Escherichia coli Isolates from Swine and Wild Small Mammals in the Proximity of Swine Farms and in Natural Environments in Ontario, Canada

Wild animals not normally exposed to antimicrobial agents can acquire antimicrobial agent-resistant bacteria through contact with humans and domestic animals and through the environment. In this study we assessed the frequency of antimicrobial resistance in generic Escherichia coli isolates from wild small mammals (mice, voles, and shrews) and the effect of their habitat (farm or natural area) on antimicrobial resistance. Additionally, we compared the types and frequency of antimicrobial resistance in E. coli isolates from swine on the same farms from which wild small mammals were collected. Animals residing in the vicinity of farms were five times more likely to carry E. coli isolates with tetracycline resistance determinants than animals living in natural areas; resistance to tetracycline was also the most frequently observed resistance in isolates recovered from swine (83%). Our results suggest that E. coli isolates from wild small mammals living on farms have higher rates of resistance and are more frequently multiresistant than E. coli isolates from environments, such as natural areas, that are less impacted by human and agricultural activities. No Salmonella isolates were recovered from any of the wild small mammal feces. This study suggests that close proximity to food animal agriculture increases the likelihood that E. coli isolates from wild animals are resistant to some antimicrobials, possibly due to exposure to resistant E. coli isolates from livestock, to the resistance genes of these isolates, or to antimicrobials through contact with animal feed.     

Phylogenetic Analysis Reveals Common Antimicrobial Resistant Campylobacter coli Population in Antimicrobial-Free (ABF) and Commercial Swine Systems

The objective of this study was to compare the population biology of antimicrobial resistant (AR) Campylobacter coli isolated from swine reared in the conventional and antimicrobial-free (ABF) swine production systems at farm, slaughter and environment. A total of 200 C. coli isolates selected from fecal, environmental, and carcass samples of ABF (n = 100) and conventional (n = 100) swine production systems were typed by multilocus sequence typing (MLST). Sequence data from seven housekeeping genes was analyzed for the identification of allelic profiles, sequence types (STs) and clonal complex determination. Phylogenetic trees were generated to establish the relationships between the genotyped isolates. A total of 51 STs were detected including two novel alleles (glnA 424 and glyA 464) and 14 novel STs reported for the first time. The majority of the C. coli isolates belonged to ST-854 (ABF: 31, conventional: 17), and were grouped in clonal complex ST-828 (ABF: 68%, conventional: 66%). The mean genetic diversity (H) for the ABF (0.3963+/−0.0806) and conventional (0.4655+/−0.0714) systems were similar. The index of association () for the ABF ( = 0.1513) and conventional ( = 0.0991) C. coli populations were close to linkage equilibrium, indicative of a freely recombining population. Identical STs were detected between the pigs and their environment both at farm and slaughter. A minimum spanning tree revealed the close clustering of C. coli STs that originated from swine and carcass with those from the environment. In conclusion, our study reveals a genotypic diverse C. coli population that shares a common ancestry in the conventional and ABF swine production systems. This could potentially explain the high prevalence of antimicrobial resistant C. coli in the ABF system in the absence of antimicrobial selection pressure.     

Antimicrobial Resistance of Escherichia coli O157 Isolated from Humans, Cattle, Swine, and Food

A total of 361 Escherichia coli O157 isolates, recovered from humans, cattle, swine, and food during the years 1985 to 2000, were examined to better understand the prevalence of antimicrobial resistance among these organisms. Based on broth microdilution results, 220 (61%) of the isolates were susceptible to all 13 antimicrobials tested. Ninety-nine (27%) of the isolates, however, were resistant to tetracycline, 93 (26%) were resistant to sulfamethoxazole, 61 (17%) were resistant to cephalothin, and 48 (13%) were resistant to ampicillin. Highest frequencies of resistance occurred among swine isolates (n = 70), where 52 (74%) were resistant to sulfamethoxazole, 50 (71%) were resistant to tetracycline, 38 (54%) were resistant to cephalothin, and 17 (24%) were resistant to ampicillin. Based on the presence of Shiga toxin genes as determined by PCR, 210 (58%) of the isolates were identified as Shiga toxin-producing E. coli (STEC). Among these, resistance was generally low, yet 21 (10%) were resistant to sulfamethoxazole and 19 (9%) were resistant to tetracycline. Based on latex agglutination, 189 (52%) of the isolates were identified as E. coli O157:H7, among which 19 (10%) were resistant to sulfamethoxazole and 16 (8%) were resistant to tetracycline. The data suggest that selection pressure imposed by the use of tetracycline derivatives, sulfa drugs, cephalosporins, and penicillins, whether therapeutically in human and veterinary medicine or as prophylaxis in the animal production environment, is a key driving force in the selection of antimicrobial resistance in STEC and non-STEC O157.     

Prevalence of Antibiotic Resistance in Drinking Water Treatment and Distribution Systems

The occurrence and spread of antibiotic-resistant bacteria (ARB) are pressing public health problems worldwide, and aquatic ecosystems are a recognized reservoir for ARB. We used culture-dependent methods and quantitative molecular techniques to detect and quantify ARB and antibiotic resistance genes (ARGs) in source waters, drinking water treatment plants, and tap water from several cities in Michigan and Ohio. We found ARGs and heterotrophic ARB in all finished water and tap water tested, although the amounts were small. The quantities of most ARGs were greater in tap water than in finished water and source water. In general, the levels of bacteria were higher in source water than in tap water, and the levels of ARB were higher in tap water than in finished water, indicating that there was regrowth of bacteria in drinking water distribution systems. Elevated resistance to some antibiotics was observed during water treatment and in tap water. Water treatment might increase the antibiotic resistance of surviving bacteria, and water distribution systems may serve as an important reservoir for the spread of antibiotic resistance to opportunistic pathogens.The occurrence and spread of antibiotic-resistant bacteria (ARB) are pressing public health problems worldwide, and aquatic ecosystems are a recognized reservoir for ARB and antibiotic resistance genes (ARGs) (4, 6, 8, 11, 12, 15, 39). Naturally occurring ARB and ARGs in the aquatic environment are selected for and enriched for by antibiotics found in sewage and agricultural runoff, which result from the widespread and increased use of antibiotics (4, 11, 12, 15, 38). Historically, concerns about the microbial quality of drinking water have focused on the occurrence of pathogens in drinking water distribution systems (5, 34). However, the presence of trace levels of antibiotics and ARB in source water and finished drinking water may also greatly affect public health and is an emerging issue for the general public and the drinking water industry (3, 30). Although several studies have detected ARB in drinking water systems (2, 3, 20, 30, 38), most previous studies focused on cultivable bacteria and/or indicator organisms. Little is known about the fate of ARGs in drinking water systems, and it was recently proposed that ARGs are emerging contaminants (24).We used culture-dependent methods and molecular techniques to investigate the prevalence and dynamics of heterotrophic ARB and ARGs in a drinking water source (source RW-P) and treated drinking water (source DW-P) (see Materials and Methods in the supplemental material). We tested water from a drinking water plant located in Michigan and tap water from several small cities located in Michigan and Ohio (sources TW-1, TW-2, TW-3, and TW-4). Two independent samples were collected each time at each collection site at three different times, and we used four replicates from each sample for tests. We tested bacterial resistance to the following antibiotics: amoxicillin (amoxicilline), chloramphenicol, ciprofloxacin, gentamicin, rifampin (rifampicin), sulfisoxazole, and tetracycline. We also examined the presence of eight ARGs, including beta-lactam resistance genes (bla_TEM and bla_SHV), chloramphenicol resistance genes (cat and cmr), sulfonamide resistance genes (sulI and sulII), and tetracycline resistance genes (tetO and tetW).Total heterotrophic plate counts (HPC) were determined using R₂A agar without added antibiotics. The water treatment process reduced the total HPC from 9.9 × 10⁶ CFU/100 ml in source water to 68 CFU/100 ml in treated drinking water, indicating that there was efficient removal and/or deactivation of total HPC (Table ​(Table1).1). In contrast, the total 16S rRNA gene copy number decreased from 3.4 × 10⁷ copies/100 ml in source water to 1.6 × 10⁶ copies/100 ml in treated drinking water (Fig. ​(Fig.1).1). The discrepancy between the reduction in the HPC and the reduction in the total 16S rRNA gene copy number suggests that the final disinfection step effectively inactivated bacteria but most of the dead or damaged cells were still present in finished drinking water. The number of HPC in tap water ranged from 3.44 × 10² to 6.1 × 10⁴ CFU/100 ml water, values that are lower than those for source water but significantly higher than those for treated drinking water, indicating that there is regrowth of bacteria in drinking water distribution systems. The copy numbers of total 16S rRNA genes in tap water ranged from 2.45 × 10⁵ to 1.02 × 10⁷ copies/100 ml water. The higher levels suggested by the 16S rRNA data are consistent with results of previous studies demonstrating that only 5 to 10% and 1% of bacteria in wastewater and soil, respectively, can be cultivated or identified by culture-based methods (9, 37). A significant correlation (P < 0.05, R² = 0.78) was found between the 16S rRNA gene copy number and the total HPC if treated drinking water (DW-P) data were not included (Fig. ​(Fig.1).1). This suggests that cultivable bacteria in drinking water represent only a small portion of the total bacterial biomass. Including treated drinking water (DW-P) data resulted in a distorted correlation, suggesting that a large proportion of the 16S rRNA genes present came from dead and/or damaged cells. The levels of total heterotrophic bacteria were significantly higher in tap water (TW-1) than in treated drinking water (DW-P), indicating that there was bacterial regrowth in the water distribution system.Open in a separate windowFIG. 1.Heterotrophic bacteria and the 16S rRNA gene in different water samples. (A) Copy numbers of the 16S rRNA gene and numbers of heterotrophic bacteria (CFU) in 100 ml water. (B) Correlation (P < 0.05, R² = 0.78) between the copy number of the 16S rRNA gene and the number of heterotrophic bacteria in different water samples (without the data for DW-P). RW-P, source water from the drinking water treatment plant; DW-P, finished drinking water from the drinking water treatment plant; TW-1, tap water from the city where the drinking water treatment plant is located; TW-2, TW-3, and TW-4, tap water from three towns in Michigan and Ohio close to the city where the TW-1 drinking water treatment plant is located. The statistical analysis was done using six samples for each type of water sample. Lg, log₁₀.

TABLE 1.

Prevalence of ARB HPC in source water, finished drinking water, and tap water from four towns^a

Escherichia coli and Klebsiella pneumoniae Producing CTX-M Cephalosporinase from Swine Finishing Barns and Their Association with Antimicrobial Use

We report the recovery of Escherichia coli or Klebsiella pneumoniae containing the extended-spectrum β-lactamase gene bla_CTX-M from 24 of 1,495 (1.6%) swine fecal samples in 8 of 50 (16%) finishing barns located in 5 U.S. states. We did not detect an association between antimicrobial use and recovery of bla_CTX-M.     

Genetics of Antimicrobial Resistance

Antimicrobial resistant strains of bacteria are an increasing threat to animal and human health. Resistance mechanisms to circumvent the toxic action of antimicrobials have been identified and described for all known antimicrobials currently available for clinical use in human and veterinary medicine. Acquired bacterial antibiotic resistance can result from the mutation of normal cellular genes, the acquisition of foreign resistance genes, or a combination of these two mechanisms. The most common resistance mechanisms employed by bacteria include enzymatic degradation or alteration of the antimicrobial, mutation in the antimicrobial target site, decreased cell wall permeability to antimicrobials, and active efflux of the antimicrobial across the cell membrane. The spread of mobile genetic elements such as plasmids, transposons, and integrons has greatly contributed to the rapid dissemination of antimicrobial resistance among several bacterial genera of human and veterinary importance. Antimicrobial resistance genes have been shown to accumulate on mobile elements, leading to a situation where multidrug resistance phenotypes can be transferred to a susceptible recipient via a single genetic event. The increasing prevalence of antimicrobial resistant bacterial pathogens has severe implications for the future treatment and prevention of infectious diseases in both animals and humans. The versatility with which bacteria adapt to their environment and exchange DNA between different genera highlights the need to implement effective antimicrobial stewardship and infection control programs in both human and veterinary medicine.     

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.     

Antimicrobial Resistance in Scandinavia after a Ban of Antimicrobial Growth Promoters

The banned use of antimicrobial growth promoters resulted in a considerably decreased use of antimicrobials in food animal production in Sweden (65%), Denmark (47%), Norway (40%) and Finland (27%). The current prevalence of antimicrobial resistance in animal bacterial populations is also considerably lower than in some other countries in the EU. In the swine production, no or limited effect was found in the finisher production (>25 to 30 kg). Temporary negative effects occurred during the post weaning period (7–30 kg). In Denmark, the cost of production from birth to slaughter per pig produced increased by approximately 1.0 [euro]with a high variability between pig producers. In the broiler production the termination had no significant negative effect on animal health and welfare or on production economy.     

Focus: Antimicrobial Resistance: Antibiotics and Antimicrobial Resistance in Acne: Epidemiological Trends and Clinical Practice Considerations

Antimicrobial resistance is an increasing public health problem worldwide. The interest of a focus on antimicrobial resistance in acne lies on the facts that acne vulgaris (acne) is the most common skin disease worldwide, that the bacterium Cutibacterium acnes (C. acnes, formerly Propionibacterium acnes) plays a key role in the pathogenesis of acne, while at the same time being part of the skin flora, and that antibiotics are commonly recommended for acne treatment. The overuse of topical and/or systemic antibiotics, the long treatment courses used for acne, and the availability of over-the-counter antibiotic preparations, have led to the worldwide emergence of resistant strains in acne patients. In this review, we discuss the epidemiological trends of antimicrobial resistance in acne, the need to avoid the perturbation of the skin microbiome caused by anti-acne antibiotics, and the clinical practice considerations related to the emergence of resistant strains in acne patients. In light of the increasing risk of antimicrobial resistance, raising concerns over the misuse of antibiotics, prescribing patterns can be a critical target for antibiotic stewardship efforts. Also, the selection of non-antibiotic therapies for acne, whenever possible, may offer significant advantages.