Scott A. McEwen

Amplification of an invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella

Amplification of nucleotide sequences within the invA gene of Salmonella typhimurium was evaluated as a means of detecting Salmonella. A collection of 630 strains of Salmonella comprising over 100 serovars, including the 20 most prevalent serovars isolated from animals and humans in Canada, was examined. Controls consisted of 142 non-Salmonella strains comprising 21 genera of bacteria. Cultures were screened by inoculating a single colony of bacteria directly into a polymerase chain reaction (PCR) mixture which contained a pair of primers specific for the invA gene. The specific PCR product was a 284 bp DNA fragment which was visualized in 2% agarose gels. With the exception of two S. litchfield and two S. senftenberg strains, all Salmonella strains were detected. In contrast, none of the non-Salmonella strains yielded the specific amplification product. Non-specific amplification of a few non-Salmonella strains resulted in a product that was distinctly different in size from the specific 284 bp product. Specificity of amplification was further confirmed by demonstration of hybridization of a 32P-labelled invA gene fragment only to the specific 284 bp product. The detection of 99.4% of Salmonella strains tested and the failure to specifically amplify DNA from non-Salmonella strains confirm that the invA gene contains sequences unique to Salmonella and demonstrate that this gene is a suitable PCR target, with potential diagnostic applications.

Antimicrobial Use and Resistance in Animals

Food animals in the United States are often exposed to antimicrobials to treat and prevent infectious disease or to promote growth. Many of these antimicrobials are identical to or closely resemble drugs used in humans. Precise figures for the quantity of antimicrobials used in animals are not publicly available in the United States, and estimates vary widely. Antimicrobial resistance has emerged in zoonotic enteropathogens (e.g., Salmonella spp., Campylobacter spp.), commensal bacteria (e.g., Escherichia coli, enterococci), and bacterial pathogens of animals (e.g., Pasteurella, Actinobacillus spp.), but the prevalence of resistance varies. Antimicrobial resistance emerges from the use of antimicrobials in animals and the subsequent transfer of resistance genes and bacteria among animals and animal products and the environment. To slow the development of resistance, some countries have restricted antimicrobial use in feed, and some groups advocate similar measures in the United States. Alternatives to growth-promoting and prophylactic uses of antimicrobials in agriculture include improved management practices, wider use of vaccines, and introduction of probiotics. Monitoring programs, prudent use guidelines, and educational campaigns provide approaches to minimize the further development of antimicrobial resistance.

Growing concerns and recent outbreaks involving non-O157:H7 serotypes of verotoxigenic Escherichia coli

Verocytotoxin-producing E. coli (VTEC) of serotype O157:H7 have been shown to be important agents of foodborne disease in humans worldwide. While the majority of research effort has been targeted on this serotype it is becoming more evident that other serotypes of VTEC can also be associated with human disease. An increasing number of these non-O157:H7 VTEC have been isolated from humans suffering from HUS and diarrhea. Recently a number of foodborne outbreaks in the USA, Australia, and other countries have been attributed to non-O157:H7 VTEC serotypes. Surveys of animal populations in a variety of countries have shown that the cattle reservoir contains more than 100 serotypes of VTEC, many of which are similar to those isolated from humans. The diversity and complexity of the VTEC family requires that laboratories and public health surveillance systems have the ability to detect and monitor all serotypes of VTEC.

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

ABSTRACT 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.

Human Health Risk Assessment (HHRA) for Environmental Development and Transfer of Antibiotic Resistance

Background: Only recently has the environment been clearly implicated in the risk of antibiotic resistance to clinical outcome, but to date there have been few documented approaches to formally assess these risks. Objective: We examined possible approaches and sought to identify research needs to enable human health risk assessments (HHRA) that focus on the role of the environment in the failure of antibiotic treatment caused by antibiotic-resistant pathogens. Methods: The authors participated in a workshop held 4–8 March 2012 in Québec, Canada, to define the scope and objectives of an environmental assessment of antibiotic-resistance risks to human health. We focused on key elements of environmental-resistance-development “hot spots,” exposure assessment (unrelated to food), and dose response to characterize risks that may improve antibiotic-resistance management options. Discussion: Various novel aspects to traditional risk assessments were identified to enable an assessment of environmental antibiotic resistance. These include a) accounting for an added selective pressure on the environmental resistome that, over time, allows for development of antibiotic-resistant bacteria (ARB); b) identifying and describing rates of horizontal gene transfer (HGT) in the relevant environmental “hot spot” compartments; and c) modifying traditional dose–response approaches to address doses of ARB for various health outcomes and pathways. Conclusions: We propose that environmental aspects of antibiotic-resistance development be included in the processes of any HHRA addressing ARB. Because of limited available data, a multicriteria decision analysis approach would be a useful way to undertake an HHRA of environmental antibiotic resistance that informs risk managers. Citation: Ashbolt NJ, Amézquita A, Backhaus T, Borriello P, Brandt KK, Collignon P, Coors A, Finley R, Gaze WH, Heberer T, Lawrence JR, Larsson DG, McEwen SA, Ryan JJ, Schönfeld J, Silley P, Snape JR, Van den Eede C, Topp E. 2013. Human health risk assessment (HHRA) for environmental development and transfer of antibiotic resistance. Environ Health Perspect 121:993–1001; http://dx.doi.org/10.1289/ehp.1206316

Antimicrobial drug residues in milk and meat: causes, concerns, prevalence, regulations, tests, and test performance.

This paper presents a historical review of antimicrobial use in food animals, the causes of residues in meat and milk, the types of residues found, their regulation in Canada, tests used for their detection, and test performance parameters, with an emphasis on immunoassay techniques. The development of residue detection methods began shortly after the introduction of antimicrobials to food animal production in the late 1940s. From initial technical concerns expressed by the dairy industry to the present public health and international trade implications, there has been an ongoing need for reliable, sensitive, and economical methods for the detection of antimicrobial residues in food animal products such as milk and meat. Initially there were microbial growth inhibition tests, followed by more sensitive and specific methods based on receptor binding, immunochemical, and chromatographic principle. An understanding of basic test performance parameters and their implications is essential when choosing an analytical strategy for residue testing. While each test format has its own attributes, none test will meet all the required analytical needs. Therefore the use of a tiered or integrated system employing assays designated for screening and confirmation is necessary to ensure that foods containing violative residues are not introduced into the food chain.

Persistence of Escherichia coli O157:H7 in dairy cattle and the dairy farm environment.

The persistence of Escherichia coli O157:H7 in cattle and the farm environment was investigated on eight Ontario dairy farms positive for E. coli O157:H7 in a longitudinal study commenced one year previously. Faecal samples from cows, calves, humans, cats, rodents, wild birds, a composite fly sample and numerous composite and individual environmental samples were cultured and tested for verotoxin-producing E. coli (VTEC). VTEC isolates were serotyped and E. coli O157:H7 isolates were phage typed. E. coli O157:H7 phage type 34 was isolated from one calf on each of two farms. The same phage type had been isolated on one of these farms 12 months earlier. Most E. coli O157:H7-positive animals and farms became culture-negative within 2 and 3 months, respectively. E. coli O157:H7 was not isolated from any environmental samples, although evidence of VTEC was found in composite samples from calf feeders (19.1%), calf barn surfaces (18%), cow feeders (14.9%), flies (12.5%), cow barn surfaces (11.3%), and individual milk filters (12.5%). VTEC belonging to 21 non-O157 serotypes were isolated from 24 cows (8.2%), 21 calves (18.3%), 2 cow feeder samples (3.0%), and 1 calf feeder sample (4.8%). Shedding of E. coli O157:H7 by infected dairy cattle appears to be transient and persistence of E. coli O157:H7 was not demonstrated from the farm environment sites tested.

Temporal and geographical distributions of reported cases of Escherichia coli O157:H7 infection in Ontario.

The distribution of 3001 cases of verocytotoxigenic Escherichia coli (VTEC) reported in the Province of Ontario, Canada, were examined to describe the magnitude of this condition geographically and to evaluate the spatial relationship between livestock density and human VTEC incidence using a Geographical Information System. Incidence of VTEC cases had a marked seasonal pattern with peaks in July. Areas with a relatively high incidence of VTEC cases were situated predominantly in areas of mixed agriculture. Spatial models indicated that cattle density had a positive and significant association with VTEC incidence of reported cases (P = 0.000). An elevated risk of VTEC infection in a rural population could be associated with living in areas with high cattle density. Results of this study suggested that the importance of contact with cattle and the consumption of contaminated well water or locally produced food products may have been previously underestimated as risk factors for this condition.

Associations between Indicators of Livestock Farming Intensity and Incidence of Human Shiga Toxin-Producing Escherichia Coli Infection

The impact of livestock farming on the incidence of human Shiga toxin-producing Escherichia coli (STEC) infection was assessed by using several livestock density indicators (LDI) that were generated in a systematic approach. A total of 80 LDI were considered suitable proxy measures for livestock density. Multivariate Poisson regression identified several LDI as having a significant spatial association with the incidence of human STEC infection. The strongest associations with human STEC infection were the ratio of beef cattle number to human population and the application of manure to the surface of agricultural land by a solid spreader and by a liquid spreader showed. This study demonstrates the value of using a systematic approach in identifying LDI and other spatial predictors of disease.

Magnitude and distribution of acute, self-reported gastrointestinal illness in a Canadian community

To estimate the magnitude and distribution of self-reported, acute gastrointestinal illness in a Canadian-based population, we conducted a retrospective, cross-sectional telephone survey of approximately 3500 randomly selected residents of the city of Hamilton (Ontario, Canada) from February 2001 to February 2002. The observed monthly prevalence was 10% (95 % CI 9.94-10.14) and the incidence rate was 1.3 (95 % CI 1.1-1.4) episodes per person-year; this is within the range of estimates from other developed countries. The prevalence was higher in females and in those aged < 10 years and 20-24 years. Overall, prevalence peaked in April and October, but a different temporal distribution was observed for those aged < 10 years. Although these data were derived from one community, they demonstrate that the epidemiology of acute gastrointestinal illness in a Canadian-based population is similar to that reported for other developed countries.