Acquired Resistance to Macrolide–Lincosamide–Streptogramin Antibiotics in Lactic Acid Bacteria of Food Origin

Antibiotic resistance is a growing problem in clinical settings as well as in food industry. Lactic acid bacteria (LAB) commercially used as starter cultures and probiotic supplements are considered as reservoirs of several antibiotic resistance genes. Macrolide–lincosamide–streptogramin (MLS) antibiotics have a proven record of excellence in clinical settings. However, the intensive use of tylosin, lincomysin and virginamycin antibiotics of this group as growth promoters in animal husbandry and poultry has resulted in development of resistance in LAB of animal origin. Among the three different mechanisms of MLS resistance, the most commonly observed in LAB are the methylase and efflux mediated resistance. This review summarizes the updated information on MLS resistance genes detected and how resistance to these antibiotics poses a threat when present in food grade LAB.     

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     

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.     

Phenotypic and Genetic Characteristics of Macrolide and Lincosamide Resistant Ureaplasma urealyticum Isolated in Guangzhou, China

The phenotypic and genetic characteristics of resistance to macrolides and lincosamides among 72 Ureaplasma urealyticum clinical strains isolated in Guangzhou, China were investigated in this study. Strains were studied by resistance phenotyping, detection of resistance genes (ermB, msrA, msrB, msrC, and msrD), and determining the significance of an association between the presence of resistance genes and the int-Tn gene (a genetic marker of transposon). The ermB, msrA, msrB, msrC, and msrD genes were obtained in 21, 1, 12, 0, and 24 strains, respectively. The msrB and msrD genes were detected in strains of macrolides (M) or macrolides–streptogramin B (MS) resistance phenotype, and the ermB gene was detected in strains of M or macrolide–lincosamide (ML) phenotype in U. urealyticum. Statistical analysis revealed that only the ermB gene was closely associated with the int-Tn gene. It was concluded that U. urealyticum harbored the ermB, msrA, msrB, and msrD genes which confer resistance to macrolides and (or) lincosamides. The ermB gene may be located in the U. urealyticum transposon.     

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.     

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.     

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.     

Persistence of Resistance to Erythromycin and Tetracycline in Swine Manure During Simulated Composting and Lagoon Treatments

The use of antimicrobials in food animal production leads to the development of antimicrobial resistance (AMR), and animal manure constitutes the largest reservoir of such AMR. In previous studies, composted swine manure was found to contain substantially lower abundance of AMR genes that encode resistance to tetracyclines (tet genes) and macrolide–lincosamide–streptogramin B (MLS_B) superfamily (erm genes), than manures that were treated by lagoons or biofilters. In this study, temporal changes in AMR carried by both cultivated and uncultivated bacteria present in swine manure during simulated composting and lagoon storage were analyzed. Treatments were designed to simulate the environmental conditions of composting (55°C with modest aeration) and lagoon storage (ambient temperature with modest aeration). As determined by selective plate counting, over a 48-day period, cultivated aerobic heterotrophic erythromycin-resistant bacteria and tetracycline-resistant bacteria decreased by more than 4 and 7 logs, respectively, in the simulated composting treatment while only 1 to 2 logs for both resistant bacterial groups in the simulated lagoon treatment. Among six classes each of erm and tet genes quantified by class-specific real-time PCR assays, the abundance of erm(A), erm(C), erm(F), erm(T), erm(X), tet(G), tet(M), tet(O), tet(T), and tet(W) declined marginally during the first 17 days, but dramatically thereafter within 31 days of the composting treatment. No appreciable reduction of any of the erm or tet genes analyzed was observed during the simulated lagoon treatment. Correlation analysis showed that most of the AMR gene classes had similar persistence pattern over the course of the treatments, though not all AMR genes were destructed at the same rate during the treatments.     

Housefly Larva Vermicomposting Efficiently Attenuates Antibiotic Resistance Genes in Swine Manure,with Concomitant Bacterial Population Changes

Manure from swine treated with antimicrobials as feed additives is a major source for the expansion of the antibiotic resistance gene (ARG) reservoir in the environment. Vermicomposting via housefly larvae (Musca domestica) can be efficiently used to treat manure and regenerate biofertilizer, but few studies have investigated its effect on ARG attenuation. Here, we tracked the abundances of 9 ARGs and the composition and structure of the bacterial communities in manure samples across 6 days of full-scale manure vermicomposting. On day 6, the abundances of genes encoding tetracycline resistance [tet(M), tet(O), tet(Q), and tet(W)] were reduced (P < 0.05), while those of genes encoding sulfonamide resistance (sul1 and sul2) were increased (P < 0.05) when normalized to 16S rRNA. The abundances of tetracycline resistance genes were correlated (P < 0.05) with the changing concentrations of tetracyclines in the manure. The overall diversity and richness of the bacteria significantly decreased during vermicomposting, accompanied by a 100 times increase in the relative abundance of Flavobacteriaceae spp. Variations in the abundances of ARGs were correlated with the changing microbial community structure and the relative abundances of the family Ruminococcaceae, class Bacilli, or phylum Proteobacteria. Vermicomposting, as a waste management practice, can reduce the overall abundance of ARGs. More research is warranted to assess the use of this waste management practice as a measure to attenuate the dissemination of antimicrobial residues and ARGs from livestock production before vermicompost can be safely used as biofertilizer in agroecosystems.     

Effect of Dissolved Organic Matter from Wheat Straw or Swine Manure on the Cu Adsorption in Three Chinese Soils

Batch equilibration experiments were conducted to evaluate the effects of dissolved organic matter (DOM) from wheat straw (DOMw) and swine manure (DOMs) on copper (Cu) adsorption and behavior in Haplic Phaeozems, Haplic Acrisol, and Eutric Fluvisol in China. Results showed that the Cu adsorption isotherms were well fitted with both Langmuir and Freundlich equations. The Cu maximum potential adsorption capacity of the three soils followed the order of Eutric Fluvisol > Haplic Phaeozems > Haplic Acrisol. DOMw and DOMs increased the Cu adsorption capacity in Haplic Phaeozems and Haplic Acrisol, and the promoting role of DOMs on Cu adsorption was obviously higher than that of DOMw. Increasing DOM concentration of DOMw and DOMs promoted the Cu adsorption in Haplic Phaeozems and Haplic Acrisol. However, this promoting effect weakened with increasing DOM concentration. Moreover, DOMw and DOMs inhibited the Cu adsorption in Eutric Fluvisol, and this inhibitory effect significantly increased with increasing DOM concentration. The results may be used to assess the potential environmental contamination of the studied soils and to control the application of organic fertilizers.     

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.     

彩绒革盖菌在猪粪堆肥中应用的初步研究

在以猪粪为原料的静态条垛堆肥的堆体试验中添加了彩绒革盖菌,研究其对堆肥发酵的影响。研究表明彩绒革盖菌在堆肥二次发酵时期有利于堆体温度的提升和保温,说明了在进入堆肥后期彩绒革盖菌对其中剩余的木质素等成分有很好的分解能力,有利于堆肥的腐熟和养分的释放,初步表明彩绒革盖菌是一株理想的堆肥发酵菌株。     

Bacterial Resistance to the Synergistic Antibiotics of the PA 114, Streptogramin, and Vernamycin Complexes

A mutant of Bacillus subtilis was isolated that was resistant to the growth inhibitory activity of the synergistic antibiotics of the PA 114, streptogramin, and vernamycin complexes. Escherichia coli is naturally resistant to the action of these antibiotics. In both cases, it was shown that resistance was due to an inability of the bacteria to transport the antibiotics into the cell.     

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.     

Effect of Liquid Swine Manure on Hatch and Viability of Heterodera glycines

Experiments were conducted in the laboratory and greenhouse to determine the effect of raw and anaerobically digested liquid swine manures on the hatch and viability of Heterodera glycines, the soybean cyst nematode. Anaerobic digestion was performed for 15 and 35 days to enrich volatile fatty acids (VFA) and ammonium (NH₄ ⁺), respectively. All filtrates of the raw, VFA-enriched, and NH₄ ⁺-enriched manures at 10⁻¹ to 250⁻¹ dilutions inhibited H. glycines hatch, and the reduction of hatch was increased with increasing concentration of the manure. Cumulative hatch at day 21 was only 2.1% to 3.7% in the 10⁻¹ dilution manures, while the hatch in water was 21% to 27.3%. The high concentrations appeared to be lethal to some eggs. Most second-stage juveniles (J2) of H. glycines were killed when incubated for 8 hours in the manure filtrate at the original concentration (>90% mortality) or for 48 hours at the 64⁻¹ dilution (> 82% mortality). When J2 were treated with the manures at 10⁻¹ to 250⁻¹ dilutions for 4 hours, only the 10⁻¹ dilution of VFA-enriched and raw manures resulted in a lower number of J2 that penetrated soybean roots as compared with lower concentrations. The VFA-enriched manure was the best, raw manure intermediate, and NH₄ ⁺-enriched manure the least effective in inhibiting H. glycines hatch and killing eggs and J2.     

Product and Economic Analysis of Direct Liquefaction of Swine Manure

Direct hydrothermal liquefaction of biomass is a technology that has shown promising results in treating waste and producing oil. A batch hydrothermal liquefaction system was used to treat swine manure, and it successfully converted up to 70% of swine manure volatile solids into oil and reduced manure chemical oxygen demand by up to 75% (He et al., Trans ASAE 43(6):1827?C1833, 2000). A continuous-flow reactor was developed and resulted in similar conversion rates to the batch process, indicating the potential for scale-up (Ocfemia, 2005). This study investigates the hydrothermal process in relation to a livestock system to determine the impact on oil yields and fertilizer values that might be realized in a farm-scale application. Oil products from the hydrothermal process are maximized using manure containing around 20% solids, but typical swine confinement facilities contain wetter manure slurries. A preliminary investigation of liquid?Csolid separation methods was conducted to determine the resultant oil yields and the effects of the hydrothermal process on fertilizer values in the wastewater as compared to the unprocessed manure. Energy and economic analyses of the liquid?Csolid separation and hydrothermal liquefaction processes were also conducted. The hydrothermal process results in an oil product as well as a fertilizer product that retains the majority of its nitrogen value with a reduced level of phosphorus (as compared to the unprocessed swine manure). The economic analyses indicate feasibility for several different liquid?Csolid separation methods, dependent on the equipment and maintenance costs assumed for each method. Conveyor-belt manure collection systems in conjunction with hydrothermal liquefaction are especially promising.     

Temporal Dynamics and Impact of Manure Storage on Antibiotic Resistance Patterns and Population Structure of Escherichia coli Isolates from a Commercial Swine Farm

Many confined-livestock farms store their wastes for several months prior to use as a fertilizer. Storing manure for extended periods could significantly bias the composition of enteric bacterial populations subsequently released into the environment. Here, we compared populations of Escherichia coli isolated from fresh feces and from the manure-holding tank (stored manure) of a commercial swine farm, each sampled monthly for 6 months. The 4,668 confirmed E. coli isolates were evaluated for resistance to amikacin, ampicillin, cephalothin, chloramphenicol, kanamycin, nalidixic acid, streptomycin, sulfamethoxazole, tetracycline, trimethoprim, and trimethoprim plus sulfamethoxazole. A subset of 1,687 isolates was fingerprinted by repetitive extragenic palindromic PCR (rep-PCR) with the BOXA1R primer to evaluate the diversity and the population structure of the collection. The population in the stored manure was generally more diverse than that in the fresh feces. Half of the genotypes detected in the stored manure were never detected in the fresh fecal material, and only 16% were detected only in the fresh feces. But the majority of the isolates (84%) were assigned to the 34% of genotypes shared between the two environments. The structure of the E. coli population showed important monthly variations both in the extent and distribution of the diversity of the observed genotypes. The frequency of detection of resistance to specific antibiotics was not significantly different between the two collections and varied importantly between monthly samples. Resistance to multiple antibiotics was much more temporally dynamic in the fresh feces than in the stored manure. There was no relationship between the distribution of rep-PCR fingerprints and the distribution of antibiotic resistance profiles, suggesting that specific antibiotic resistance determinants were dynamically distributed within the population.     

Survival of Salmonella enterica Serovar Newport in Manure and Manure-Amended Soils

Salmonella enterica serovar Newport has undergone a rapid epidemic spread in dairy cattle. This provides an efficient mechanism for pathogen amplification and dissemination into the environment through manure spreading on agricultural land. The objective of this study was to determine the survival characteristics of Salmonella serovar Newport in manure and manure-amended soils where the pathogen may be amplified. A multidrug-resistant (MDR) Salmonella serovar Newport strain and a drug-susceptible (DS) strain, both bovine isolates, were inoculated into dairy manure that was incubated under constant temperature and moisture conditions alone or after being mixed with sterilized or nonsterilized soil. Salmonella serovar Newport concentrations increased by up to 400% in the first 1 to 3 days following inoculation, and a trend of steady decline followed. With manure treatment, a sharp decline in cell concentration occurred after day 35, possibly due to microbial antagonism. For all treatments, decreases in Salmonella serovar Newport concentrations over time fit a first-order kinetic model. Log reduction time was 14 to 32 days for 1 log₁₀, 28 to 64 days for 2 log₁₀, and 42 to 96 days for 3 log₁₀ declines in the organisms' populations from initially inoculated concentrations. Most-probable-number monitoring data indicated that the organisms persisted for 184, 332, and 405 days in manure, manure-amended nonsterilized soil, and manure-amended sterilized soil, respectively. The MDR strain and the DS strain had similar survival patterns.