Influence of antibiotic selection on genetic composition of Escherichia coli populations from conventional and organic dairy farms

The widespread agricultural use of antimicrobials has long been considered a crucial influence on the prevalence of resistant genes and bacterial strains. It has been suggested that antibiotic applications in agricultural settings are a driving force for the development of antimicrobial resistance, and epidemiologic evidence supports the view that there is a direct link between resistant human pathogens, retail produce, farm animals, and farm environments. Despite such concerns, little is understood about the population processes underlying the emergence and spread of antibiotic resistance and the reversibility of resistance when antibiotic selective pressure is removed. In this study, hierarchical log-linear modeling was used to assess the association between farm type (conventional versus organic), age of cattle (calf versus cow), bacterial phenotype (resistant versus susceptible), and the genetic composition of Escherichia coli populations (E. coli Reference Collection [ECOR] phylogroup A, B1, B2, or D) among 678 susceptible and resistant strains from a previously published study of 60 matched dairy farms (30 conventional and 30 organic) in Wisconsin. The analysis provides evidence for clonal resistance (ampicillin resistance) and genetic hitchhiking (tetracycline resistance [Tet(r)]), estimated the rate of compositional change from conventional farming to organic farming (mean, 8 years; range, 3 to 15 years), and discovered a significant association between low multidrug resistance, organic farms, and strains of the numerically dominant phylogroup B1. These data suggest that organic farming practices not only change the frequency of resistant strains but also impact the overall population genetic composition of the resident E. coli flora. In addition, the results support the hypothesis that the current prevalence of Tet(r) loci on dairy farms has little to do with the use of this antibiotic.     

Dairy Farm Age and Resistance to Antimicrobial Agents in <Emphasis Type="Italic">Escherichia coli</Emphasis> Isolated from Dairy Topsoil

Antimicrobial agent usage is common in animal agriculture for therapeutic and prophylactic purposes. Selective pressure exerted by these antimicrobials on soil bacteria could result in the selection of strains that are resistant due to chromosomal- or plasmid-derived genetic components. Multiple antimicrobial resistances in Escherichia coli and the direct relationship between antimicrobial agent use over time has been extensively studied, yet the relationship between the age of an animal agriculture environment such as a dairy farm and antibiotic resistance remains unclear. Therefore, we tested the hypothesis that antimicrobial-resistance profiles of E. coli isolated from dairy farm topsoil correlate with dairy farm age. E. coli isolated from eleven dairy farms of varying ages within Roosevelt County, NM were used for MIC determinations to chloramphenicol, nalidixic acid, penicillin, tetracycline, ampicillin, amoxicillin/clavulanic acid, gentamicin, trimethoprim/sulfamethoxazole, cefotaxime, and ciprofloxacin. The minimum inhibitory concentration values of four antibiotics ranged 0.75 to >256 μg/ml, 1 to >256 μg/ml, 12 to >256 μg/ml, and 0.75 to >256 μg/ml for chloramphenicol, nalidixic acid, penicillin, and tetracycline, respectively. The study did not show a direct relationship between antibiotic resistance and the age of dairy farms.     

Similarity of Tetracycline Resistance Genes Isolated from Fish Farm Bacteria to Those from Clinical Isolates

Tetracycline-resistant (Tet^r) bacteria were isolated from fishes collected at three different fish farms in the southern part of Japan in August and September 2000. Of the 66 Tet^r gram-negative strains, 29 were identified as carrying tetB only. Four carried tetY, and another four carried tetD. Three strains carried tetC, two strains carried tetB and tetY, and one strain carried tetC and tetG. Sequence analyses indicated the identity in Tet^r genes between the fish farm bacteria and clinical bacteria: 99.3 to 99.9% for tetB, 98.2 to 100% for tetC, 99.7 to 100% for tetD, 92.0 to 96.2% for tetG, and 97.1 to 100% for tetY. Eleven of the Tet^r strains transferred Tet^r genes by conjugation to Escherichia coli HB-101. All transconjugants were resistant to tetracycline, oxycycline, doxycycline, and minocycline. The donors included strains of Photobacterium, Vibrio, Pseudomonas, Alteromonas, Citrobacter, and Salmonella spp., and they transferred tetB, tetY, or tetD to the recipients. Because NaCl enhanced their growth, these Tet^r strains, except for the Pseudomonas, Citrobacter, and Salmonella strains, were recognized as marine bacteria. Our results suggest that tet genes from fish farm bacteria have the same origins as those from clinical strains.     

Virulence determinants and antimicrobial resistance of E. coli isolated from bovine clinical mastitis in some selected dairy farms of Bangladesh

E. coli is one of the major significant pathogens causing mastitis, the most complex and costly diseases in the dairy industry worldwide. Present study was undertaken to isolate, detect the virulence factors, phylogroup, antimicrobial susceptibility and antimicrobial resistance genes in E. coli from cows with clinical mastitis. A total of 68 milk samples comprising 53 from clinical mastitis and 15 from apparently healthy cattle were collected from four different established dairy farms in Bangladesh. E. coli was isolated from the milk samples and identified by PCR targeting malB gene and sequencing of 16S rRNA gene. E. coli isolates were screened by PCR for the detection of major virulence genes (stx, eae and cdt) of diarrheagenic E. coli followed by phylogenetic grouping. Antimicrobial susceptibility of the E. coli isolates was determined by disk diffusion test and E. coli showing resistance was further screened for the presence of antimicrobial resistance genes. E. coli was isolated from 35.8% of the mastitis milk samples but none from the apparently healthy cattle milk. All the E. coli isolates were negative for stx, eae and cdt genes and belonged to the phylogenetic groups A and B1 which comprising of commensal E. coli. Antibiotic sensitivity testing revealed 84.2% (16/19) of the isolates as multidrug resistant. Highest resistance was observed against amoxicillin (94.5%) followed by ampicillin (89.5%) and tetracycline (89.5%). E. coli were found resistant against all the classes of antimicrobials used at the farm level. Tetracycline resistance gene (tetA) was detected in 100% of the tetracycline resistant E. coli and blaTEM-1 was present in 38.9% of the E. coli isolates. Findings of this study indicate a potential threat of developing antimicrobial resistance in commensal E. coli and their association with clinical mastitis. Occurrence of multidrug resistant E. coli might be responsible for the failure of antibiotic therapies in clinical mastitis as well as pose potential threat of transmitting and development of antibiotic resistance in human.     

Comparison of Prevalence and Antimicrobial Susceptibilities of Campylobacter spp. Isolates from Organic and Conventional Dairy Herds in Wisconsin

The prevalence and antimicrobial susceptibilities of Campylobacter spp. isolates from bovine feces were compared between organic and conventional dairy herds. Thirty organic dairy herds, where antimicrobials are rarely used for calves and never used for cows, were compared with 30 neighboring conventional dairy farms, where antimicrobials were routinely used for animals for all ages. Fecal specimens from 10 cows and 10 calves on 120 farm visits yielded 332 Campylobacter isolates. The prevalence of Campylobacter spp. in organic and conventional farms was 26.7 and 29.1%, and the prevalence was not statistically different between the two types of farms. Campylobacter prevalence was significantly higher in March than in September, higher in calves than in cows, and higher in smaller farms than in large farms. The rates of retained placenta, pneumonia, mastitis, and abortion were associated with the proportion of Campylobacter isolation from fecal samples. The gradient disk diffusion MIC method (Etest) was used for testing susceptibility to four antimicrobial agents: ciprofloxacin, gentamicin, erythromycin, and tetracycline. Two isolates were resistant to ciprofloxacin, and none of isolates was resistant to gentamicin or erythromycin. Resistance to tetracycline was 45% (148 of 332 isolates). Tetracycline resistance was found more frequently in calves than in cows (P = 0.042), but no difference was observed between organic and conventional farms. When we used Campylobacter spp. as indicator bacteria, we saw no evidence that restriction of antimicrobial use on dairy farms was associated with prevalence of resistance to ciprofloxacin, gentamicin, erythromycin, and tetracycline.     

Escherichia coli Population Structure and Antibiotic Resistance at a Buffalo/Cattle Interface in Southern Africa

At a human/livestock/wildlife interface, Escherichia coli populations were used to assess the risk of bacterial and antibiotic resistance dissemination between hosts. We used phenotypic and genotypic characterization techniques to describe the structure and the level of antibiotic resistance of E. coli commensal populations and the resistant Enterobacteriaceae carriage of sympatric African buffalo (Syncerus caffer caffer) and cattle populations characterized by their contact patterns in the southern part of Hwange ecosystem in Zimbabwe. Our results (i) confirmed our assumption that buffalo and cattle share similar phylogroup profiles, dominated by B1 (44.5%) and E (29.0%) phylogroups, with some variability in A phylogroup presence (from 1.9 to 12%); (ii) identified a significant gradient of antibiotic resistance from isolated buffalo to buffalo in contact with cattle and cattle populations expressed as the Murray score among Enterobacteriaceae (0.146, 0.258, and 0.340, respectively) and as the presence of tetracycline-, trimethoprim-, and amoxicillin-resistant subdominant E. coli strains (0, 5.7, and 38%, respectively); (iii) evidenced the dissemination of tetracycline, trimethoprim, and amoxicillin resistance genes (tet, dfrA, and bla_TEM-1) in 26 isolated subdominant E. coli strains between nearby buffalo and cattle populations, that led us (iv) to hypothesize the role of the human/animal interface in the dissemination of genetic material from human to cattle and toward wildlife. The study of antibiotic resistance dissemination in multihost systems and at anthropized/natural interface is necessary to better understand and mitigate its multiple threats. These results also contribute to attempts aiming at using E. coli as a tool for the identification of pathogen transmission pathway in multihost systems.     

The Streptomycin-Sulfadiazine-Tetracycline Antimicrobial Resistance Element of Calf-Adapted Escherichia coli Is Widely Distributed among Isolates from Washington State Cattle

Association of specific antimicrobial resistance patterns with unrelated selective traits has long been implicated in the maintenance of antimicrobial resistance in a population. Previously we demonstrated that Escherichia coli strains with a specific resistance pattern (resistant to streptomycin, sulfadiazine, and tetracycline [SSuT]) have a selective advantage in dairy calf intestinal environments and in the presence of a milk supplement commonly fed to the calves. In the present study we identified the sequence of the genetic element that confers the SSuT phenotype and show that this element is present in a genetically diverse group of E. coli isolates, as assessed by macrorestriction digestion and pulsed-field gel electrophoresis. This element was also found in E. coli isolates from 18 different cattle farms in Washington State. Using in vitro competition experiments we further demonstrated that SSuT strains from 17 of 18 farms were able to outcompete pansusceptible strains. In a separate set of experiments, we were able to transfer the antimicrobial resistance phenotype by electroporation to a laboratory strain of E. coli (DH10B), making that new strain more competitive during in vitro competition with the parental DH10B strain. These data indicate that a relatively large genetic element conferring the SSuT phenotype is widely distributed in E. coli from cattle in Washington State. Furthermore, our results indicate that this element is responsible for maintenance of these traits owing to linkage to genetic traits that confer a selective advantage in the intestinal lumens of dairy calves.     

Whole-genome sequencing and gene sharing network analysis powered by machine learning identifies antibiotic resistance sharing between animals,humans and environment in livestock farming

Anthropogenic environments such as those created by intensive farming of livestock, have been proposed to provide ideal selection pressure for the emergence of antimicrobial-resistant Escherichia coli bacteria and antimicrobial resistance genes (ARGs) and spread to humans. Here, we performed a longitudinal study in a large-scale commercial poultry farm in China, collecting E. coli isolates from both farm and slaughterhouse; targeting animals, carcasses, workers and their households and environment. By using whole-genome phylogenetic analysis and network analysis based on single nucleotide polymorphisms (SNPs), we found highly interrelated non-pathogenic and pathogenic E. coli strains with phylogenetic intermixing, and a high prevalence of shared multidrug resistance profiles amongst livestock, human and environment. Through an original data processing pipeline which combines omics, machine learning, gene sharing network and mobile genetic elements analysis, we investigated the resistance to 26 different antimicrobials and identified 361 genes associated to antimicrobial resistance (AMR) phenotypes; 58 of these were known AMR-associated genes and 35 were associated to multidrug resistance. We uncovered an extensive network of genes, correlated to AMR phenotypes, shared among livestock, humans, farm and slaughterhouse environments. We also found several human, livestock and environmental isolates sharing closely related mobile genetic elements carrying ARGs across host species and environments. In a scenario where no consensus exists on how antibiotic use in the livestock may affect antibiotic resistance in the human population, our findings provide novel insights into the broader epidemiology of antimicrobial resistance in livestock farming. Moreover, our original data analysis method has the potential to uncover AMR transmission pathways when applied to the study of other pathogens active in other anthropogenic environments characterised by complex interconnections between host species.     

Effects of Therapeutic Ceftiofur Administration to Dairy Cattle on Escherichia coli Dynamics in the Intestinal Tract

The goal of this study was to follow ceftiofur-treated and untreated cattle in a normally functioning dairy to examine enteric Escherichia coli for changes in antibiotic resistance profiles and genetic diversity. Prior to treatment, all of the bacteria cultured from the cows were susceptible to ceftiofur. Ceftiofur-resistant E. coli was only isolated from treated cows during and immediately following the cessation of treatment, and the 12 bla_CMY-2-positive isolates clustered into two genetic groups. E. coli bacterial counts dropped significantly in the treated animals (P < 0.027), reflecting a disappearance of the antibiotic-susceptible strains. The resistant bacterial population, however, did not increase in quantity within the treated cows; levels stayed low and were overtaken by a returning susceptible population. There was no difference in the genetic diversities of the E. coli between the treated and untreated cows prior to ceftiofur administration or after the susceptible population of E. coli returned in the treated cows. A cluster analysis of antibiotic susceptibility profiles resulted in six clusters, two of which were multidrug resistant and were comprised solely of isolates from the treated cows immediately following treatment. The antibiotic treatment provided a window to detect the presence of ceftiofur-resistant E. coli but did not appear to cause its emergence or result in its amplification. The finding of resistant isolates following antibiotic treatment is not sufficient to estimate the strength of selection pressure nor is it sufficient to demonstrate a causal link between antibiotic use and the emergence or amplification of resistance.     

Effect of Conventional and Organic Production Practices on the Prevalence and Antimicrobial Resistance of Campylobacter spp. in Poultry

Intestinal tracts of broilers and turkeys from 10 conventional broiler farms and 10 conventional turkey farms, where antimicrobials were routinely used, and from 5 organic broiler farms and 5 organic turkey farms, where antimicrobials had never been used, were collected and cultured for Campylobacter species. A total of 694 Campylobacter isolates from the conventional and organic poultry operations were tested for antimicrobial resistance to nine antimicrobial agents by the agar dilution method. Although Campylobacter species were highly prevalent in both the conventional and organic poultry operations, the antimicrobial resistance rates were significantly different between the organic operations and the conventional operations. Less than 2% of Campylobacter strains isolated from organically raised poultry were resistant to fluoroquinolones, while 46% and 67% of Campylobacter isolates from conventionally raised broilers and conventionally raised turkeys, respectively, were resistant to these antimicrobials. In addition, a high frequency of resistance to erythromycin (80%), clindamycin (64%), kanamycin (76%), and ampicillin (31%) was observed among Campylobacter isolates from conventionally raised turkeys. None of the Campylobacter isolates obtained in this study was resistant to gentamicin, while a large number of the isolates from both conventional and organic poultry operations were resistant to tetracycline. Multidrug resistance was observed mainly among Campylobacter strains isolated from the conventional turkey operation (81%). Findings from this study clearly indicate the influence of conventional and organic poultry production practices on antimicrobial resistance of Campylobacter on poultry farms.     

Survey of Shiga toxigenic Escherichia coli O157 and drug-resistant coliform bacteria from in-line milk filters on dairy farms in the Czech Republic

Aims: To determine the occurrence of Shiga toxin‐producing Escherichia coli (STEC) O157 and coliform bacteria isolates resistant to antimicrobial agents in dairy herds by examining milk filters and to analyse the influence of management factors and antibiotic use on antimicrobial resistance. Methods and Results: A total of 192 in‐line milk filters were sampled on 192 dairy farms in the Czech Republic. Information on feeding, husbandry, production, and antibiotic therapy were obtained by questionnaire. The milk filters were cultured for STEC O157 and coliform bacteria. All recovered isolates were examined for antimicrobial susceptibility and presence of antimicrobial‐resistance genes. STEC O157 was detected in four (2%) of the filters. Resistant nonpathogenic E. coli and coliform bacteria isolates with specific genes were detected in 44 (23%) of the filters. Conclusions: The study demonstrated a high prevalence of resistant coliform bacteria in milk filters obtained on Czech dairy farms. Significance and Impact of the Study: The occurrence of resistant coliform bacteria in milk filters was significantly higher among isolates from farms where antibiotic therapy against mastitis was employed during the dry period (P < 0·05).     

Genetic Diversity and Antibiotic Resistance Patterns in a Campylobacter Population Isolated from Poultry Farms in Switzerland

The diversity and genetic interrelation of Campylobacter jejuni and C. coli isolated from Swiss poultry were assessed by three independent typing methods. Samples were derived prior to slaughter from 100 randomly selected flocks (five birds per flock) raised on three different farm types. The observed flock prevalence was 54% in total, with 50% for conventional and 69% for free-range farms. Birds held on farms with a confined roaming area had the lowest prevalence of 37%. Campylobacter isolates were characterized by amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism of flaA PCR fragments (flaA-RFLP), and disk diffusion testing for eight antimicrobial agents that are commonly used in veterinary or human medicine in Switzerland. Analysis of the genotypic results indicates that the Campylobacter population in Swiss poultry is genetically highly diverse. Nevertheless, occasionally, isolates with identical or nearly identical characteristics were isolated from different farms or farm types in different locations. Genetic typing by AFLP and flaA-RFLP was found to be complementary. The majority of isolates (67%) were susceptible to all tested antibiotics; however, single, double, and triple resistances were observed in 7%, 23%, and 2% of the strains, respectively. There was no correlation between genotype and antibiotic resistance. Surprisingly, sulfonamide resistance was frequently found together with streptomycin resistance. Our findings illustrate the results of common genetic exchange in the studied bacterial population.     

Prevalence of antibiotic resistant mastitis pathogens in dairy cows in Egypt and potential biological control agents produced from plant endophytic actinobacteria

Dairy production is threatened by antibiotic resistant pathogens worldwide, and alternative solutions to treat mastitis are not available. The prevalence of antibiotic resistant strains is not well known in less developed countries. The prevalence of pathogenic bacteria and their resistance to 21 commercial antibiotics were studied in milk samples taken from 122 dairy cows suffering from the symptoms of mastitis in Egypt. The bacterial species were identified with molecular methods, and antibiotic resistance was studied with disc diffusion method. The prevalence of Streptococcus aureus, Escherichia coli and Pseudomonas aeruginosa were 30%, 17% and 3.5%, respectively. Most (90%) of the S. aureus strains showed resistance to penicillin whereas only 10% of the strains were resistant to oxacillin. Nearly half (40%) of E. coli strains showed resistance to streptomycin. Six P. aeruginosa strains showed resistance to several antibiotics, including ceftriaxone, enrofloxacin and levofloxacin. This points out that despite P. aeruginosa was not common, it should be followed up carefully. Potential biocontrol agents against antibiotic resistant mastitis bacteria were searched among 30 endophytic actinobacterial strains derived from wild medicinal plants. Three plants, namely Mentha longifolia, Malva parviflora and Pulicaria undulata were chosen for a more detailed study; their endophytic actinobacteria were used to prepare metabolic extracts. The crude metabolites of the actinobacteria were extracted with ethyl acetate. All metabolic extracts inhibited the growth of S. aureus, methicillin-resistant Staphylococcus aureus (MRSA), E. coli and P. aeruginosa in vitro. The 16S rRNA sequence analysis revealed that the most efficient actinobacterial strains were two Micromonospora sp. and one Actinobacteria bacterium. We conclude that the combination of the metabolites of several endophytic actinobacteria derived from several medicinal plants would be the most efficient against pathogens. Different metabolite cocktails should be studied further in order to develop novel biocontrol agents to treat antibiotic resistant mastitis bacteria in dairy cows.     

Diversity and Distribution of Commensal Fecal Escherichia coli Bacteria in Beef Cattle Administered Selected Subtherapeutic Antimicrobials in a Feedlot Setting

Escherichia coli strains isolated from fecal samples were screened to examine changes in phenotypic and genotypic characteristics including antimicrobial susceptibility, clonal type, and carriage of resistance determinants. The goal of this 197-day study was to investigate the influence of administration of chlortetracycline alone (T) or in combination with sulfamethazine (TS) on the development of resistance, dissemination of defined strain types, and prevalence of resistance determinants in feedlot cattle. Inherent tetracycline resistance was detected in cattle with no prior antimicrobial exposure. Antimicrobial administration was not found to be essential for the maintenance of inherently ampicillin-resistant and tetracycline-resistant (Tet^r) E. coli in control animals; however, higher Tet^r E. coli shedding was observed in animals subjected to the two treatments. At day 0, high tetracycline (26.7%), lower sulfamethoxazole-tetracycline (19.2%), and several other resistances were detected, which by the finishing phase (day 197) were restricted to ampicillin-tetracycline (47.5%), tetracycline (31.7%), and ampicillin-tetracycline-sulfamethoxazole (20.8%) from both treated and untreated cattle. Among the determinants, bla_TEM1, tet(A), and sul2 were prevalent at days 0 and 197. Further, E. coli from day 0 showed diverse antibiogram profiles and strain types, which by the finishing phase were limited to up to three, irrespective of the treatment. Some genetically identical strains expressed different phenotypes and harbored diverse determinants, indicating that mobile genetic elements contribute to resistance dissemination. This was supported by an increased linked inheritance of ampicillin and tetracycline resistance genes and prevalence of specific strains at day 197. Animals in the cohort shed increasingly similar genotypes by the finishing phase due to animal-to-animal strain transmission. Thus, characterizing inherent resistance and propagation of cohort-specific strains is crucial for determining antimicrobial resistance in cattle.     

Comparison of prevalence and antimicrobial susceptibilities of Campylobacter spp. isolates from organic and conventional dairy herds in Wisconsin

The prevalence and antimicrobial susceptibilities of Campylobacter spp. isolates from bovine feces were compared between organic and conventional dairy herds. Thirty organic dairy herds, where antimicrobials are rarely used for calves and never used for cows, were compared with 30 neighboring conventional dairy farms, where antimicrobials were routinely used for animals for all ages. Fecal specimens from 10 cows and 10 calves on 120 farm visits yielded 332 Campylobacter isolates. The prevalence of Campylobacter spp. in organic and conventional farms was 26.7 and 29.1%, and the prevalence was not statistically different between the two types of farms. Campylobacter prevalence was significantly higher in March than in September, higher in calves than in cows, and higher in smaller farms than in large farms. The rates of retained placenta, pneumonia, mastitis, and abortion were associated with the proportion of Campylobacter isolation from fecal samples. The gradient disk diffusion MIC method (Etest) was used for testing susceptibility to four antimicrobial agents: ciprofloxacin, gentamicin, erythromycin, and tetracycline. Two isolates were resistant to ciprofloxacin, and none of isolates was resistant to gentamicin or erythromycin. Resistance to tetracycline was 45% (148 of 332 isolates). Tetracycline resistance was found more frequently in calves than in cows (P = 0.042), but no difference was observed between organic and conventional farms. When we used Campylobacter spp. as indicator bacteria, we saw no evidence that restriction of antimicrobial use on dairy farms was associated with prevalence of resistance to ciprofloxacin, gentamicin, erythromycin, and tetracycline.     

Use of a Nonmedicated Dietary Supplement Correlates with Increased Prevalence of Streptomycin-Sulfa-Tetracycline-Resistant Escherichia coli on a Dairy Farm

We examined how a dietary supplement affects the prevalence of antibiotic-resistant Escherichia coli on a dairy farm in Washington State. Between 2001 and 2004 the prevalence of fecal E. coli strains resistant to streptomycin, sulfadiazine, and tetracycline (SSuT strains) declined from 59.2% to 26.1% in the calf population. In 2003 the dairy discontinued use of a dietary supplement, and we hypothesized that the decline in prevalence of SSuT strains was related to this change in management. To test this we established three treatments in which calves received no supplement, the dietary supplement with oxytetracycline, or the dietary supplement without oxytetracycline. Calves receiving either dietary supplement had a significantly higher prevalence of SSuT E. coli than the no-supplement control group (≈37% versus 20%, respectively; P = 0.03). Importantly, there was no evidence that oxytetracycline contributed to an increased prevalence of fecal SSuT E. coli. We compared the growth characteristics of SSuT and non-SSuT E. coli in LB broth enriched with either the complete dietary supplement or its individual constituents. Both the complete dietary supplement and its vitamin D component supported a significantly higher cell density of SSuT strains (P = 0.003 and P = 0.001, respectively). The dry milk and vitamin A components of the dietary supplement did not support different cell densities. These results were consistent with selection and maintenance of SSuT E. coli due to environmental components independent of antibiotic selection.     

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

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

Longitudinal Study of Escherichia coli O157:H7 Dissemination on Four Dairy Farms in Wisconsin

A 14-month longitudinal study was conducted on four dairy farms (C, H, R, and X) in Wisconsin to ascertain the source(s) and dissemination of Escherichia coli O157:H7. A cohort of 15 heifer calves from each farm were sampled weekly by digital rectal retrieval from birth to a minimum of 7 months of age (range, 7 to 13 months). Over the 14 months of the study, the cohort heifers and other randomly selected cattle from farms C and H tested negative. Farm R had two separate periods of E. coli O157:H7 shedding lasting 4 months (November 1995 to February 1996) and 1 month (July to August 1996), while farm X had at least one positive cohort animal for a 5-month period (May to October 1996). Heifers shed O157:H7 strains in feces for 1 to 16 weeks at levels ranging from 2.0 × 10² to 8.7 × 10⁴ CFU per g. E. coli O157:H7 was also isolated from other noncohort cattle, feed, flies, a pigeon, and water associated with the cohort heifers on farms R and/or X. When present in animal drinking water, E. coli O157:H7 disseminated through the cohort cattle and other cattle that used the water source. E. coli O157:H7 was found in water at <1 to 23 CFU/ml. Genomic subtyping by pulsed-field gel electrophoresis demonstrated that a single O157:H7 strain comprised a majority of the isolates from cohort and noncohort cattle, water, and other positive samples (i.e., from feed, flies, and a pigeon, etc.) on a farm. The isolates from farm R displayed two predominant XbaI restriction endonuclease digestion profiles (REDP), REDP 3 and REDP 7, during the first and second periods of shedding, respectively. Six additional REDP that were ≥89% similar to REDP 3 or REDP 7 were identified among the farm R isolates. Additionally, the REDP of an O157:H7 isolate from a heifer on farm R in 1994 was indistinguishable from REDP 3. Farm X had one O157:H7 strain that predominated (96% of positive samples had strains with REDP 9), and the REDP of an isolate from a heifer in 1994 was indistinguishable from REDP 9. These results suggest that E. coli O157:H7 is disseminated from a common source on farms and that strains can persist in a herd for a 2-year period.     

Toxin Production and Antibiotic Resistances in Escherichia coli Isolated from Bathing Areas Along the Coastline of the Oslo Fjord

The presence of enterovirulent and/or antibiotic resistant strains of Escherichia coli in recreational bathing waters would represent a clear health issue. In total, 144 E. coli isolated from 26 beaches along the inner Oslo fjord were examined for virulence determinants and resistance to clinically important antibiotics. No isolates possessed the genetic determinants associated with enterotoxigenic strains and none showed the prototypic sorbitol negative, O157:H7 phenotype. A small number (～1 %) produced alpha-hemolysin. Occurrences and patterns of antibiotic resistances were similar to those of E. coli isolated previously from environmental samples. In total, 6 % of the strains showed one or more clinically relevant resistances and 1.4 % were multi-drug resistant. Microarray analyses suggested that the resistance determinants were generally associated with mobile genetic elements. Resistant strains were not clonally related, and were, furthermore not concentrated at one or a few beach sites. This suggests that these strains are entering the waters at a low rate but in a widespread manner. The study demonstrates that resistant E. coli are present in coastal bathing waters where they can come into contact with bathers, and that the resistance determinants are potentially transferable. Some of the resistances registered in the study are to important antibiotics used in human medicine such as fluoroquinolones. The spread of antibiotic resistant genes, from the clinical setting to the environment, has clear implications with respect to the current management of bacterial infections and the long term value of antimicrobial therapy. The present study is the first of its kind in Norway.     

Comparative study on the antibiotic susceptibility and plasmid profiles of Vibrio alginolyticus strains isolated from four Tunisian marine biotopes

The antibiotic resistance patterns and the plasmids profiles of the predominant etiological agent responsible for vibriosis in Tunisia, V. alginolyticus were studied to contribute to control their spread in some Mediterranean aquaculture farms and seawater. The sixty-nine V. alginolyticus strains isolated from different marine Tunisian biotopes (bathing waters, aquaculture and conchylicole farms and a river connected to the seawater during the cold seasons) were multi-drug resistant with high resistance rate to ampicillin, kanamycin, doxycyclin, erythromycin, imipinem, and nalidixic acid. The multiple resistance index ranged from 0.3 to 0.7 for the isolates of Khenis, from 0.5 to 0.8 for those of Menzel Jmil, from 0.5 to 0.75 (Hergla) and from 0.3 to 0.7 for the isolates of Oued Soltane. The high value of antibiotic resistance index was recorded for the V. alginolyticus population isolated from the fish farm in Hergla (ARI?=?0.672) followed by the population isolated from the conchylicole station of Menzel Jmil (ARI?=?0.645). The results obtained by the MIC tests confirmed the resistance of the V. alginolyticus to ampicillin, erythromycin, kanamycin, cefotaxime, streptomycin and trimethoprim. Plasmids were found in 79.48?% of the strains analyzed and 30 different plasmid profiles were observed. The strains had a high difference in the size of plasmids varying between 0.5 and 45?kb. Our study reveals that the antibiotic-resistant bacteria are widespread in the aquaculture and conchylicole farm relatively to others strains isolated from seawater.