Sheryl P. Gow

Associations between Antimicrobial Resistance Genes in Fecal Generic Escherichia coli Isolates from Cow-Calf Herds in Western Canada

ABSTRACT The objective of this study was to examine associations among the genetic determinants of antimicrobial resistance (AMR) in 207 fecal generic Escherichia coli isolates obtained from 77 cow-calf herds in western Canada. Twenty-three resistance genes corresponding to six different antimicrobial families were assessed using DNA hybridization and PCR. The most common resistance genes in the study sample (207 isolates) were sul2 (48.3%), tet(B) (45.4%), and ant(3″)-Ia (aadA1) (19.3%). Several statistically significant associations between the examined resistance genes were detected. The strongest associations observed were those between genes for resistance to chloramphenicol (catI) and trimethoprim (dhfrI) (odds ratio [OR] = 214; P = 0.0001), sulfonamide (sul1) and chloramphenicol (catI) (OR = 96.9; P = 0.0001), streptomycin [ant(3″)-Ia (aadA1)] and trimethoprim (dhfrI) (OR = 96.2; P = 0.0001), sulfonamide (sul1) and streptomycin [ant(3″)-Ia (aadA1)] (OR = 79.3; P = 0.0001), and tetracycline [tet(B)] and sulfonamides (sul2) (OR = 25.7; P = 0.0001). At least one of the resistance genes corresponding to each nonaminoglycoside family of antimicrobials examined in this study was associated with the two aminoglycoside resistance genes ant(3″)-Ia (aadA1) and aph(3′)-Ia. The multiple, strong associations between genes and the diverse nature of the associations described in this study demonstrate the complexity of resistance gene selection in cow-calf herds and should be considered in the planning of AMR control practices for cow-calf operations.

Clostridium difficile and methicillin-resistant Staphylococcus aureus shedding by slaughter-age pigs.

BackgroundClostridium difficile and methicillin-resistant Staphylococcus aureus are critical human pathogens and of increasing concern in food animals. Because of the apparent impact of age on prevalence of these organisms, studies of slaughter age pigs are important when considering the potential for contamination of food. This study evaluated C. difficile and MRSA shedding by slaughter age pigs from farms across Canada.ResultsClostridium difficile was isolated from 30/436 (6.9%) samples from 15/45 (33%) farms. After adjusting for clustering at the herd level, the prevalence was 3.4%. Ribotype 078 (toxinotype V, North American Pulsotype 7) was the most common strain, accounting for 67% of isolates. MRSA was isolated from 21/460 (4.6%) pigs from 5/46 (11%) farms. The prevalence in pigs after adjusting for clustering at the herd level was 0.2%. Seven different spa types were identified, with 3 related spa types (t011, t034, new) accounting for 16 (76%) consistent with ST398 predominating.Both MRSA and C. difficile samples were collected from 45 farms. Both MRSA and C. difficile were detected on 2 (4.4%), with C. difficile only on 13 (29%), MRSA only on 3 (6.7%) and neither on 27 (60%).ConclusionsThe prevalence of C. difficile and MRSA in slaughter age pigs was relatively low, particularly in comparison with studies involving younger pigs. The predominance of C. difficile ribotype 078 and MRSA ST398 was not surprising, but there was diversity in strain types and the majority of isolates of both organisms were strains that can be found in humans. While the prevalence of C. difficile and MRSA in slaughter age pigs was relatively low, there is clearly potential for contamination of meat from healthy pigs carrying this pathogen into slaughterhouses.

Genetic characterization and antimicrobial susceptibility of Mannheimia haemolytica isolated from the nasopharynx of feedlot cattle.

A surveillance study was undertaken to examine the population dynamics and antimicrobial resistance of Mannheimia haemolytica isolated from feedlot cattle. A total of 416 isolates were collected from the nasopharynx either upon entry or exit from two feedlots in southern Alberta, Canada. Isolates were serotyped, characterized by pulsed-field gel electrophoresis and tested for susceptibility to ten antimicrobial agents via disk diffusion. Resistant isolates were screened by PCR for select antimicrobial-resistance gene determinants. Isolates were highly diverse, with 335 unique pulsed-field profiles identified among 147 strongly related clusters (similarity ≥ 85%). Clonal spread of isolates throughout the feedlots was limited and no clear association was found between genetic relatedness of M. haemolytica and sampling event (entry or exit). Pulsed-field profiles sharing a common serotype and resistance phenotype tended to cluster together. The majority of isolates were identified as serotype 2 (74.5%) although both serotype 1 (11.9%) and 6 (12.7%) were detected. Only 9.54% of isolates exhibited antimicrobial resistance. Resistance to oxytetracycline was most prevalent (n=16), followed by ampicillin (n=10), and amoxicillin/clavulanic acid (n=7). Multi-drug resistance was observed in five isolates. The tetH gene was detected in all but two oxytetracycline resistant isolates. Other detectable resistance determinates included ermX and bla(ROB-1). In the two feedlots examined, M. haemolytica exhibited considerable genetic diversity and limited resistance to common veterinary antibiotics. Garnering further information on the linkage between genotype and phenotype should contribute toward a better understanding of the pathogenesis and dissemination of M. haemolytica in feedlots.

Prevalence and molecular characterization of Clostridium difficile isolated from feedlot beef cattle upon arrival and mid-feeding period

BackgroundThe presence of indistinguishable strains of Clostridium difficile in humans, food animals and food, as well as the apparent emergence of the food-animal-associated ribotype 078/toxinotype V as a cause of community-associated C. difficile infection have created concerns about the potential for foodborne infection. While studies have reported C. difficile in calves, studies of cattle closer to the age of harvest are required. Four commercial feedlots in Alberta (Canada) were enrolled for this study. Fecal samples were collected at the time of arrival and after acclimation (< 62, 62-71 or > 71 days on feed). Selective culture for Clostridium difficile was performed, and isolates were characterized by ribotyping and pulsed-field gel electrophoresis. A logistic regression model was built to investigate the effect of exposure to antimicrobial drugs on the presence of C. difficile.ResultsClostridium difficile was isolated from 18 of 539 animals at the time of feedlot arrival (CI = 2.3-6.1) and from 18 of 335 cattle at mid-feeding period (CI = 2.9-13.1). Overall, there was no significant difference in the prevalence of C. difficile shedding on arrival versus mid-feeding period (P = 0.47). No association between shedding of the bacterium and antimicrobial administration was found (P = 0.33). All the isolates recovered were ribotype 078, a toxinotype V strain with genes encoding toxins A, B and CDT. In addition, all strains were classified as NAP7 by pulsed field gel electrophoresis (PFGE) and had the characteristic 39 base pairs deletion and upstream truncating mutation on the tcdC gene.ConclusionsIt is apparent that C. difficile is carried in the intestinal tracts of a small percentage of feedlot cattle arriving and later in the feeding period and that ribotype 078/NAP7 is the dominant strain in these animals. Herd management practices associated with C. difficile shedding were not identified, however further studies of the potential role of antimicrobials on C. difficile acquisition and shedding are required.

A multiplex polymerase chain reaction assay for the identification of Mannheimia haemolytica, Mannheimia glucosida and Mannheimia ruminalis.

The objective of this study was to design a multiplex PCR assay to identify Mannheimia haemolytica, Mannheimia glucosida and Mannheimia ruminalis. The multiplex PCR included primer sets HP, amplifying a DNA region from an unknown hypothetical protein, Lkt and Lkt2, amplifying different regions of the leukotoxinD gene, and 16S to amplify universal bacterial sequences of the 16S rRNA gene. Based on positive amplification, isolates were delineated as M. haemolytica (HP, Lkt, 16S), M. glucosida (HP, Lkt, Lkt2, 16S), or M. ruminalis (HP, 16S). The validity of the assay was examined against 22 reference strains within the family Pasteurellaceae and 17 field isolates (nasal) that had been collected previously from feedlot cattle and tentatively identified as M. haemolytica based on morphology and substrate utilization. Additionally, 200 feedlot cattle were screened for M. haemolytica using multiplex PCR. Forty-four isolates from 25 animals were identified as M. haemolytica. The PCR assay positively identified all M. haemolytica, as confirmed by phenotypic tests and clustering based upon cellular fatty acid methyl ester (FAME) profiles. Selected nasal isolates that exhibited evidence of haemolysis, but were M. haemolytica-negative based on PCR, were also confirmed negative by phenotypic and FAME analyses. The multiplex PCR assay required no additional phenotypic tests for confirmation of M. haemolytica, within the group of bacteria tested.

Response to experimentally induced infection with bovine respiratory syncytial virus following intranasal vaccination of seropositive and seronegative calves.

OBJECTIVE To determine whether a combination modified-live bovine respiratory syncytial virus (BRSV) vaccine can stimulate protective immunity in young BRSV-seropositive calves following intranasal (IN) administration. DESIGN Controlled challenge study. ANIMALS 66 Holstein bull calves, 3 to 8 days old. PROCEDURES In experiment 1, BRSV-seropositive and -seronegative calves were vaccinated IN with a commercially available combination modified-live virus vaccine formulated for SC administration; calves underwent BRSV challenge 4.5 months later. In experiment 2, BRSV-seronegative calves were vaccinated IN or SC (to examine the effect of route of administration) with the same combination vaccine that instead had a 1/100 dose of BRSV (to examine the effect of dose); calves underwent BRSV challenge 21 days later. RESULTS In experiment 1, BRSV challenge resulted in severe respiratory tract disease with low arterial partial pressures of oxygen and lung lesions in most calves from all groups. Maximum change in rectal temperature was significantly greater in seropositive IN vaccinated calves, compared with seronegative IN vaccinated and seropositive control calves. Number of days of BRSV shedding was significantly lower in seronegative IN vaccinated calves than in seropositive IN vaccinated and seropositive control calves. In experiment 2, maximum change in rectal temperature was significantly greater in seronegative control calves, compared with seronegative IN and SC vaccinated calves. Shedding of BRSV was significantly reduced in seronegative IN and SC vaccinated calves, compared with control calves; also, lung lesions were reduced in seronegative IN and SC vaccinated calves. CONCLUSIONS AND CLINICAL RELEVANCE Maternal antibodies may inhibit priming of protective responses by IN delivered BRSV vaccines.

Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) Farm Program: results from finisher pig surveillance.

In 2006, the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) Farm Program was implemented in sentinel grower‐finisher swine herds in Québec, Ontario, Manitoba, Saskatchewan and Alberta. Herds were visited 1–3 times annually. Faecal samples were collected from pens of close‐to‐market (CTM) weight (>80 kg) pigs and antimicrobial use (AMU) data were collected via questionnaires. Samples were cultured for generic Escherichia coli and Salmonella and tested for antimicrobial susceptibility. This paper describes the findings of this program between 2006 and 2008. Eighty‐nine, 115 and 96 herds participated in this program in 2006, 2007 and 2008 respectively. Over the 3 years, antimicrobial resistance (AMR) levels remained consistent. During this period, resistance to one or more antimicrobials was detected in 56–63% of the Salmonella spp. isolates and 84–86% of E. coli isolates. Resistance to five or more antimicrobials was detected in 13–23% of Salmonella and 12–13% of E. coli. Resistance to drugs classified as very important to human health (Category I) by the Veterinary Drug Directorate (VDD), Health Canada, was less than or equal to 1% in both organisms. AMU data were provided by 100 herds in 2007 and 95 herds in 2008. Nine herds in 2007 and five herds in 2008 reported no AMU. The most common route of antimicrobial administration (75–79% of herds) was via feed, predominantly macrolides/lincosamides (66–68% of herds). In both 2007 and 2008, the primary reasons given for macrolide/lincosamide use were disease prevention, growth promotion and treatment of enteric disease. The Category I antimicrobials, ceftiofur and virginiamycin were not used in feed or water in any herds in 2008, but virginiamycin was used in feed in two herds in 2007. Parenteral ceftiofur was used in 29 herds (29%) in 2007 and 20 herds (21%) in 2008. The reasons for ceftiofur use included treatment of lameness, respiratory disease and enteric disease.

Resistome diversity in cattle and the environment decreases during beef production

Antimicrobial resistant determinants (ARDs) can be transmitted from livestock systems through meat products or environmental effluents. The public health risk posed by these two routes is not well understood, particularly in non-pathogenic bacteria. We collected pooled samples from 8 groups of 1741 commercial cattle as they moved through the process of beef production from feedlot entry through slaughter. We recorded antimicrobial drug exposures and interrogated the resistome at points in production when management procedures could potentially influence ARD abundance and/or transmission. Over 300 unique ARDs were identified. Resistome diversity decreased while cattle were in the feedlot, indicating selective pressure. ARDs were not identified in beef products, suggesting that slaughter interventions may reduce the risk of transmission of ARDs to beef consumers. This report highlights the utility and limitations of metagenomics for assessing public health risks regarding antimicrobial resistance, and demonstrates that environmental pathways may represent a greater risk than the food supply. DOI: http://dx.doi.org/10.7554/eLife.13195.001

Characterization of the resistome in manure, soil and wastewater from dairy and beef production systems.

It has been proposed that livestock production effluents such as wastewater, airborne dust and manure increase the density of antimicrobial resistant bacteria and genes in the environment. The public health risk posed by this proposed outcome has been difficult to quantify using traditional microbiological approaches. We utilized shotgun metagenomics to provide a first description of the resistome of North American dairy and beef production effluents, and identify factors that significantly impact this resistome. We identified 34 mechanisms of antimicrobial drug resistance within 34 soil, manure and wastewater samples from feedlot, ranch and dairy operations. The majority of resistance-associated sequences found in all samples belonged to tetracycline resistance mechanisms. We found that the ranch samples contained significantly fewer resistance mechanisms than dairy and feedlot samples, and that the resistome of dairy operations differed significantly from that of feedlots. The resistome in soil, manure and wastewater differed, suggesting that management of these effluents should be tailored appropriately. By providing a baseline of the cattle production waste resistome, this study represents a solid foundation for future efforts to characterize and quantify the public health risk posed by livestock effluents.

Mannheimia haemolytica in Feedlot Cattle: Prevalence of Recovery and Associations with Antimicrobial Use, Resistance, and Health Outcomes

Background Mannheimia haemolytica is an important etiological agent in bovine respiratory disease. Objectives Explore risk factors for recovery of susceptible and resistant M. haemolytica in feedlot cattle and explore associations with health outcomes. Animals Cattle (n = 5,498) from 4 feedlots sampled at arrival and later in feeding period. Methods Susceptibility of M. haemolytica isolates tested for 21 antimicrobials. Records of antimicrobial use and health events analyzed using multivariable regression. Results M. haemolytica recovered from 29% of cattle (1,596/5,498), 13.1% at arrival (95% CI, 12.3–14.1%), and 19.8% at second sampling (95% CI, 18.7–20.9%). Nearly half of study cattle received antimicrobial drugs (AMDs) parenterally, mostly as metaphylactic treatment at arrival. Individual parenteral AMD exposures were associated with decreased recovery of M. haemolytica (OR, 0.2; 95% CI, 0.02–1.2), whereas exposure in penmates was associated with increased recovery (OR, 1.5; 95% CI, 1.05–2.2). Most isolates were pan‐susceptible (87.8%; 95% CI, 87.0–89.4%). AMD exposures were not associated with resistance to any single drug. Multiply‐resistant isolates were rare (5.9%; 95% CI, 5.1–6.9%), but AMD exposures in pen mates were associated with increased odds of recovering multiply‐resistant M. haemolytica (OR, 23.9; 95% CI, 8.4–68.3). Cattle positive for M. haemolytica on arrival were more likely to become ill within 10 days (OR, 1.7; 95% CI, 1.1–2.4). Conclusions and Clinical Importance Resistance generally was rare in M. haemolytica. Antimicrobial drug exposures in penmates increased the risk of isolating susceptible and multiply‐resistant M. haemolytica, a finding that could be explained by contagious spread.