Appendix A: 2008 Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) annual report – Methods

Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS)

Appendix A - Methods

Categorization of Antimicrobials Based on Importance in Human Medicine

Categories of antimicrobials used in this report were taken from the document Categorization of Antimicrobial Drugs Based on Importance in Human MedicineFootnote 36 by Health Canada's Veterinary Drugs Directorate (Table A.1).

Antimicrobials are considered to be of Very High Importance in Human Medicine (Category I) when they are essential for the treatment of serious bacterial infections and there is no or limited availability of alternative antimicrobials for effective treatment. Antimicrobials of High Importance in Human Medicine (Category II) consist of those that can be used to treat a variety of infections, including serious infections, and for which alternatives are generally available. Bacteria resistant to antimicrobials of this category are generally susceptible to Category I antimicrobials, which could be used as alternatives. Antimicrobials of Medium Importance in Human Medicine (Category III) are used in the treatment of bacterial infections for which alternatives are generally available. Infections caused by bacteria resistant to these antimicrobials can, in general, be treated with Category II or I antimicrobials. Antimicrobials of Low Importance in Human Medicine (Category IV) are currently not used in human medicine.

Table A.1. Categorization of antimicrobial drugs based on importance in human medicine.
Category of importance in human medicine Antimicrobial class
I Very High Importance Carbapenems
Cephalosporins - the 3rd and 4th generations
Fluoroquinolones
Glycopeptides
Glycylcyclines
Ketolides
Lipopeptides
Monobactams
Nitroimidazoles (metronidazole)
Oxazolidinones
Penicillin-β-lactamase inhibitor combinations
Polymyxins (colistin)
Therapeutic agents for tuberculosis (e.g. ethambutol, isoniazid, pyrazinamide, and rifampin)
II High Importance Aminoglycosides (except topical agents)
Cephalosporins - the first and second generations (including cephamycins)
Fusidic acid
Lincosamides
Macrolides
Penicillins
Quinolones (except fluoroquinolones)
Streptogramins
Trimethoprim-sulfamethoxazole
III Medium Importance Aminocyclitols
Aminoglycosides (topical agents)
Bacitracins
Fosfomycin
Nitrofurans
Phenicols
Sulfonamides
Tetracyclines
Trimethoprim
IV Low Importance Flavophospholipols
Ionophores

Antimicrobial Resistance

Sampling Design and Data Collection

Surveillance of Human Clinical Isolates

The objectives of the Surveillance of Human Clinical Isolates component of CIPARS are to provide a representative and methodologically unified approach to monitor temporal trends in the development of antimicrobial resistance in Salmonella isolated from humans.

Hospital-based or private clinical laboratories usually culture human Salmonella isolates in Canada. Although reporting is mandatory through laboratory notification of reportable diseases to the National Notifiable Disease Reporting System, forwarding of Salmonella cultures to provincial reference laboratories is voluntary and passive. A high proportion (84% in 2001)Footnote 37 of Salmonella isolates is forwarded to Provincial Public Health Laboratories (PPHLs), but this proportion may vary among laboratories. The Yukon, Northwest Territories, and Nunavut, which do not have a PPHL counterpart, also forward isolates to one of the PPHLs.

Prior to 2002, PPHLs forwarded a certain number of Salmonella isolates to the Enteric Diseases Program, National Microbiology Laboratory (NML), Public Health Agency of Canada (PHAC), Winnipeg, Manitoba for confirmation and subtype characterization. A letter of agreement by which provinces agreed to forward all or a subset of their Salmonella isolates to CIPARS was signed in 2002 by the PPHLs, the NML, the Laboratory for Foodborne Zoonoses (LFZ), and the Centre for Food-borne, Environmental and Zoonotic Infectious Diseases of the PHAC. This agreement officially launched the Surveillance of Human Clinical Isolates component of CIPARS.

To ensure a statistically valid sampling plan, all human Salmonella isolates (outbreak-associated and non-outbreak-associated) received passively by PPHLs in Saskatchewan, Manitoba, New Brunswick, Nova Scotia, Prince Edward Island, and Newfoundland and Labrador were forwarded to the NML. The PPHLs in more heavily populated provinces (British Columbia, Alberta, Ontario, and Québec) forwarded only the isolates received from the 1st to the 15th of each month. However, all human S. Newport and S. Typhi isolates were forwarded to the NML because of concerns of multidrug resistance and clinical importance, respectively.

The PPHLs were also asked to provide a defined set of data for each forwarded isolate, including serovar name, date collected, outbreak identification (if applicable), and patient age, sex, and province of residence. Provision of patient information on travel history, antimicrobial use, hospitalization status at the time of sample collection, and date of disease onset was optional. These optional data were not usually available to the NML in 2008. Although many outbreaks are identified by PPHLs prior to isolate submission, some outbreaks are identified after the isolates are forwarded to the NML. For 2008, there was no outbreak identification information available to accompany any isolates submitted to the NML.

Farm Surveillance

The objectives of the CIPARS Farm Surveillance component are to provide data on antimicrobial use (Antimicrobial Use, Appendix A) and resistance, monitor temporal trends in the development of antimicrobial resistance, investigate associations between antimicrobial use and resistance on grower-finisher pigs, and provide data for human-health risk assessments.

Farm Surveillance is the most recent component of CIPARS and complements existing abattoir and retail sample collection activities. This initiative focuses on a sentinel farm framework that provides data on antimicrobial use and fecal samples obtained from farms for bacterial isolation and antimicrobial susceptibility testing. It is administered and coordinated by the LFZ.

In 2006, the CIPARS Farm Surveillance component was implemented in swine herds across the 5 major pork-producing provinces in Canada (Alberta, Saskatchewan, Manitoba, Ontario, and Québec). The swine industry was selected as the pilot commodity for development of the farm surveillance infrastructure because the Canadian Quality Assurance (CQA®) program had been extensively implemented by the industry and because there has not been a recent outbreak of foreign animal disease in pigs. The Farm Surveillance component concentrates on grower-finisher hogs. Pigs in this stage of production were chosen because of their proximity to the consumer.

Nationally, 23 veterinarians and 96 sentinel grower-finisher sites were enrolled. In each of the 5 participating provinces, the number of CIPARS sentinel sites was proportional to the national total of grower-finisher units, except in Alberta, where 10 additional sentinel herds were included. Alberta Agriculture and Rural Development (AARD) provided laboratory testing for all samples collected from the CIPARS sentinel herds in Alberta.

To preserve the anonymity of participating producers, herd veterinarians collected the samples and data and submitted depersonalized information to PHAC. In the case of corporate herds, 2 private supervisory veterinarians ensured confidentiality by holding the key to corporate herd codes. This step was taken because knowing a corporate veterinarian's name could have identified the corporation associated with the herd, thereby breaking anonymity.

Veterinarians were purposively selected from the list of veterinarians practicing swine medicine in each province. Each veterinarian selected a predetermined number of sentinel farm sites by use of specific inclusion and exclusion criteria. To be included, herds were required to be CQA® validated, produce more than 2,000 market pigs per year, and be representative of the characteristics (i.e. similar production volumes and types of production systems) and geographic distribution of herds in the contractor's swine practice. Herds were excluded when they were regarded as organic with respect to animal husbandry, were fed edible residual material, or were raised on pasture. These criteria helped ensure that the herds enrolled were representative of most grower-finisher swine herds in Canada.

Pooled fecal samples were collected 3 times per year from pens of pigs that were close to market (CTM) weight (i.e. more than 175 lb; Figure A.1). In a subset of herds, specific cohorts of pigs were sampled twice: within 6 hours after pigs entered the grow-finisher unit and again when the same pigs reached CTM weight.

Antimicrobial resistance data for bacterial isolates recovered from pooled fecal samples of CTM pigs are presented in this report. Data are not presented for pooled fecal samples collected when pigs arrived in grower-finisher units; however, these data are available upon request. Overall prevalence estimates, which were calculated from data for arrival and CTM market samples, are also not presented here.

Figure A.1.
Figure A.1. - text equivalent

Pooled fecal samples were collected 3 times per year from pens of pigs that were close to market (CTM) weight (i.e. more than 175 lb; Figure A.1). In a subset of herds, specific cohorts of pigs were sampled twice: within 6 hours after pigs entered the grow-finisher unit and again when the same pigs reached CTM weight.

Abattoir Surveillance

The objectives of the CIPARS Abattoir Surveillance component are to provide nationally representative, annual antimicrobial resistance data for bacteria isolated from animals entering the food chain, and to monitor temporal trends in the development of antimicrobial resistance in these bacteria. Initially, this component targeted generic Escherichia coli and Salmonella from beef cattle, pigs, and broiler chickens. In 2003, the component was refined to discontinue Salmonella isolation from beef cattle because of the low prevalence of Salmonella in that population. An additional change was the addition of Campylobacter surveillance in beef cattle in late 2005.

In the Abattoir Surveillance component, the unit of concern (i.e. the subject of interest) was the bacterial isolate. The bacteria of interest were sampled from the caecal contents (not carcasses) of slaughtered food animals to avoid misinterpretation related to cross-contamination and to better reflect antimicrobial resistance in bacteria that originated on the farm.

The sampling method used was designed with the expectation that, across Canada, 150 isolates of each targeted bacterial species would be recovered from each of the 3 animal species over a 12-month period to avoid any potential seasonal bias in bacterial prevalence and antimicrobial susceptibility. The exception to this expectation was Campylobacter in beef cattle, for which it was estimated that 100 isolates would be recovered over the same period. These numbers represented a balance between acceptable statistical precision and affordability (Ravel, 2001). The actual number of samples collected was determined for each food animal species on the basis of the expected caecal prevalence of the bacteria in that animal species. For example, if the expected bacterial prevalence was 10%, then 1,500 samples would need to be collected and submitted for bacterial isolation.

The sampling design was based on a 2-stage sampling plan, with each commodity handled separately. The first stage consisted of random selection of federally inspected slaughterhouses. The probability of an abattoir being selected was proportional to its annual slaughter volume. Federally inspected abattoirs slaughter over 90% of all food animals in Canada.Footnote 38 The second stage involved systematic selection of animals on the slaughter line. The annual number of caecal samples collected at each abattoir was proportional to its slaughter volume.

To minimize shipping costs and allow each abattoir to maintain efficiency, the annual total number of samples to be collected in each abattoir was divided by 5, resulting in the number of collection periods. For each collection period, 5 caecal samples were collected within 5 days, at the convenience of the slaughterhouse staff, provided the 5 animals and associated samples originated from different groups. Sampling from different groups of animals was important to maximize diversity and avoid bias attributable to overrepresentation of particular producers. Collection periods were uniformly distributed throughout the year, leading to an abattoir-specific schedule for collection of caecal contents. The uniform distribution of the collection periods helped to avoid any bias that may have resulted from seasonal variation in bacterial prevalence and antimicrobial susceptibility test results.

Forty-two federally inspected slaughter plants (24 poultry plants, 12 swine plants, and 6 beef cattle plants) from across Canada participated in the 2008 CIPARS Abattoir Surveillance component. For pigs and chickens, numbers of samples collected were based on the aforementioned expectation of 150 Salmonella and 150 E. coli isolates and the expected prevalence of Salmonella and E. coli in each animal species. For beef cattle, the number of samples collected was based the expectation of 100 Campylobacter and 150 E. coli isolates and the expected prevalence of Campylobacter and E. coli in the cattle. Samples were obtained according to a predetermined protocol, with modifications to accommodate various production-line configurations in the different plants. Protocols were designed to avoid conflict with carcass inspection methods, plant-specific Food Safety Enhancement Programs, and Health and Safety requirements. They were also designed to avoid situations of potential cross-contamination. All samples were collected by industry personnel under the oversight of the Veterinarian-in-Charge of the Canadian Food Inspection Agency (CFIA).

Retail Meat Surveillance

The objectives of CIPARS Retail Meat Surveillance are to provide data on antimicrobial resistance and to monitor temporal variations in selected bacteria found in raw meat at the provincial/region level. Retail surveillance also provides a measure of human exposure to antimicrobial-resistant bacteria via undercooked meat consumption. Retail food represents a logical sampling point for surveillance of antimicrobial resistance because it is the endpoint of food animal production. The focus of the surveillance framework can be modified (e.g. food commodities, bacteria, or regions) as necessary and functions as a research platform for investigation of specific questions regarding antimicrobial resistance in the agri-food sector.

As with Abattoir Surveillance, the unit of concern in Retail Meat Surveillance was the bacterial isolate cultured from one of the commodities of interest. In this situation, the commodities were raw meat products commonly consumed by Canadians, which originated from the 3 animal species sampled in the Abattoir Surveillance component. These raw meat products consisted of poultry (chicken legs or wings [skin on]),Footnote 39 pork (chops), and beef (ground beef).

For ground beef, only samples of lean ground beef were collected in the first year of surveillance (2003); however, in 2004, the scope was widened to include systematic selection of extra-lean, lean, medium, and regular ground beef. This change was made to ensure representation of the heterogeneity of ground beef with respect to its origins (e.g. domestic vs. imported beef or raised beef cattle vs. culled dairy cattle). The meat cuts "legs or wings with skin on," "chops," and "ground beef" were chosen on the basis of suspected high prevalences of the targeted bacterial species within and the low purchase prices of these commodities (Ravel, 2002).

Bacteria of interest in chicken were Campylobacter, Salmonella, Enterococcus, and generic E. coli. In pork both Salmonella and E. coli were cultured, but only isolates of E. coli underwent antimicrobial susceptibility testing. Salmonella was isolated from pork mainly to provide recovery estimates from this commodity for other PHAC programs. Because the prevalence of Salmonella in pork is low, antimicrobial susceptibility results are not presented separately for each year but, rather, have been combined. Recovery of Campylobacter from pork was not attempted because of the low prevalence observed in the initial stages of Retail Meat Surveillance. In beef, only E. coli was cultured and then tested for antimicrobial susceptibility given the low prevalence of Campylobacter and Salmonella in these commodities at the retail level, as determined during the early phase of the program. Lastly, the presence of Enterococcus in beef and pork was not determined because of resource and budgetary constraints.

The sampling protocol was designed to evaluate antimicrobial resistance in certain bacterial species that contaminate retail meat and to which Canadian consumers may subsequently be exposed. It primarily involved continuous weekly submission of samples of retail meat from randomly selected geographic areas (i.e. census divisions defined by Statistics Canada), weighted by population, in each participating province. In 2008, retail meat samples were collected in British Columbia, Saskatchewan, Ontario, and Québec, and the Maritimes region (Nova Scotia, New Brunswick, and Prince Edward Island). Data from Statistics Canada were used to define strata. This was done by using cumulative population quartiles (or thirdtiles) from a list of census divisions in a province, sorted by population in ascending order. Between 15 and 18 census divisions per province were then chosen by means of stratified random selection and weighted by population within each stratum. The number of sampling days allocated to each stratum was also weighted by population and is summarized as follows:

Ontario and Québec
  • Stratum One - 10 divisions selected, with 2 sampling days per division per year
  • Stratum Two - 4 divisions selected, with 5 sampling days per division per year
  • Stratum Three - 2 divisions selected, with 10 sampling days per division per year
  • Stratum Four - 1 division selected, with 20 sampling days per year
Saskatchewan
  • Stratum One - 9 divisions selected, with 2 sampling days per division per year
  • Stratum Two - 5 divisions selected, with 3 sampling days per division per year
  • Stratum Three - 2 divisions selected, with 5 sampling days per division per year
  • Stratum Four - 1 division selected, with 7 sampling days per year
British Columbia
  • Stratum One - 10 divisions selected, with 1 sampling day per division per year
  • Stratum Two - 4 divisions selected, with 3 sampling days per division per year
  • Stratum Three - 1 division selected, with 20 sampling days per year.
Maritime Provinces

For the 3 Maritimes provinces, results are aggregated and presented at the Maritimes region level; however, sampling activities for this region were proportional to the population within each province as indicated below. Furthermore, as with the other provinces sampled in the retail component, sampling within each province was proportional to the census division subpopulations and is summarized as follows:

Nova Scotia
  • Stratum One - 5 divisions selected, with 1 sampling day per division per year (on average)
  • Stratum Two - 4 divisions selected, with 2 sampling days per division per year
  • Stratum Three - 1 division selected, with 10 sampling days per division per year
New Brunswick
  • Stratum One - 5 divisions selected, with 1 sampling day per division per year (on average)
  • Stratum Two - 4 divisions selected, with 2 sampling days per division per year
  • Stratum Three - 2 divisions selected, with 4 sampling days per division per year (on average)
Prince Edward Island
  • Stratum One - 1 division selected, with 1 sampling day per division per year
  • Stratum Two - 1 division selected, with 2 sampling days per division per year.

Field workers in Ontario and Québec conducted sampling on a weekly basis, and those in British Columbia, Saskatchewan, and Maritimes region conducted sampling every other week. Sampling was less frequent in British Columbia, Saskatchewan, and the Maritimes region because of funding constraints, limited laboratory capacity, and a desire to avoid over-sampling at particular stores. Samples were collected on Mondays or Tuesdays for submission to the LFZ, Saint-Hyacinthe, Québec (LFZ-Saint-Hyacinthe) by Wednesday. Samples submitted from outside Québec (with the exception of samples from the Maritimes region) were sent to the same laboratory via 24-hour courier. Samples from the whole Maritimes region were collected on Mondays or Tuesdays and submitted to a laboratory in Prince Edward Island within 24 hours.

In each province, 2 census divisions were sampled each sampling week. In each census division, 4 stores were selected prior to the sampling day, based on store type. Generally, 3 chain stores and 1 independent market or butcher shop were selected. An exception to this protocol was made in densely populated urban census divisions (e.g. Toronto or Montréal), where 2 chain stores and 2 independent markets or butcher shops were sampled to reflect the presumed shopping behaviour of that subpopulation. From each store type, 1 sample of each commodity of interest was collected, for a total of 11 meat samples (4 chicken, 4 pork, and 3 beef samples) per division per sampling day.Footnote 40 When possible, specific stores were sampled only once per sampling year.

Prevalence estimates were used to determine the numbers of samples to be collected, which were based on an expected yield of 100 isolates per commodity per province per year, plus 20% to account for lost or damaged samples. Because sampling was less frequent in British Columbia, Saskatchewan, and the Maritimes region than in Ontario and Québec, the target of 100 isolates per year may not have always be met in those provinces.

In 2008, personal digital assistants (PDAs) were used to capture the following store and sample data:

  • Type of store
  • Number of cash registers (surrogate measure of store volume)
  • "Sell-by" or packaging date
  • "May contain previously frozen meat" label - yes or no
  • Final processing in store - yes, no, or unknown
  • Air chilled - yes, no, or unknown (applied to chicken samples only)
  • Organic - yes, no, or unknown
  • Antimicrobial free - yes, no, or unknown
  • Price per kilogram.

Individual samples were packaged in sealed zipper-type bags and placed in 16-L thermal coolers for transport. The ambient environmental temperature was used to determine the number of ice packs placed in each cooler (i.e. 1 ice pack for temperatures below 20ºC and 2 ice packs for temperatures 20ºC or higher). In 1 or 2 coolers per sampling day, instruments for recording temperature data (Ertco Data Logger™, West Patterson, NJ, USA) were used to monitor temperatures to which samples were exposed.

Surveillance of Animal Clinical Isolates

The objective of Surveillance of Animal Clinical Isolates is to detect new and/or emerging antimicrobial resistance patterns or new serovar/antimicrobial resistance pattern combinations in Salmonella. This component of CIPARS is primarily based on veterinary diagnostic submissions collected by veterinarians and/or producers. Consequently, methods of sample collection and submission varied among laboratories. Salmonella isolates were sent by provincial and private animal health laboratories from across the country to the Salmonella Typing Laboratory (STL) at the LFZ, Guelph, Ontario (LFZ-Guelph). Salmonella isolates from the Direction des laboratoires d'expertises du Ministère de l'Agriculture, des Pêcheries et de l'Alimentation du Québec were sent to the Laboratoire d'épidémiosurveillance animale du Québec, Saint-Hyacinthe, Québec. However, unlike the Surveillance of Human Clinical Isolates component, not all isolates received by provincial animal health laboratories were necessarily forwarded to the LFZ, with the exception of the provinces of British Columbia, Ontario, and Québec. Therefore, coverage may have varied considerably among provinces.

Feed and Feed Ingredients

Data from the Feed and Feed Ingredients component of CIPARS were obtained from various sources, including monitoring programs of the CFIA and a few isolates from provincial authorities. Information on specimen collection methods was only available for the CFIA monitoring programs.

The CFIA collects samples of animal feed under 2 different programs: Program 15A (Monitoring Inspection - Salmonella ) and Program 15E (Directed Inspection - Salmonella ). Under Program 15A, feeds produced at feed mills, rendering facilities, ingredient manufacturers, and on-farm facilities are sampled and tested for Salmonella. Although this program makes use of a random sampling process, extra attention is paid to feeds that are more likely to have a higher degree of Salmonella contamination, such as those that contain rendered animal products, oilseed meals, fishmeals, grains, and mashes. Program 15E targets feeds or ingredients from establishments that (i) produce rendered animal products, other feeds containing ingredients in which Salmonella could be a concern (e.g. oilseed meal or fishmeal), or a significant volume of poultry feed; (ii) are known to have repeated problems with Salmonella contamination; or (iii) have identified a Salmonella serovar that is highly pathogenic (e.g. Typhimurium, Enteritidis, or Newport). Program 15E is a targeted program; samples are not randomly selected.

Bacterial Isolation

All samples were cultured by use of standard protocols as described below. All primary isolation of human Salmonella isolates was conducted by hospital-based or private clinical laboratories from across the provinces. Most primary isolation of Escherichia coli, Salmonella, Enterococcus, and Campylobacter from agri-food samples was conducted at the LFZ-Saint-Hyacinthe. Part of the primary isolation for Farm Surveillance was conducted at the Agri-Food Laboratory, AARD. Samples from the CIPARS Animal Clinical Isolates component were cultured by various participating laboratories. Most primary bacterial isolation from Feed and Feed Ingredients sample was conducted by the CFIA - Laboratory Services Division (Calgary or Ottawa).

Salmonella

Surveillance of Human Clinical Isolates: Hospital-based and private clinical laboratories isolated and identified Salmonella from human samples according to approved methods (Kauffman, 1966; Ewing, 1986; Le Minor, 2001; Murray et al., 2005).

Farm Surveillance and Abattoir Surveillance: The method used to isolate Salmonella was a modification of the MFLP-75 method of the Compendium of Analytical Methods, Health Protection Branch, Methods of Microbiological Analysis of Food, Government of Canada. This method allowed isolation of motile and viable Salmonella from fecal samples from pigs and caecal contents from broiler chickens and pigs. It was based on the ability of Salmonella to multiply and be motile in modified semi-solid Rappaport Vassiliadis (MSRV) medium at 42oC. A 10-g portion of each pig sample was mixed with 90 mL of buffered peptone water (BPW), which served as a non-selective pre-enrichment broth. For chickens, caecal contents were weighed and BPW was added at a ratio of 1:10. The pig and chicken samples were incubated at 35 ± 1°C for 24 hours. Afterward, an MSRV plate was inoculated with 0.1 mL of the pre-enrichment broth and incubated at 42 ± 1°C for 24 to 72 hours. Suspect colonies were screened for purity and used to inoculate triple-sugar-iron and urea agar slants. Presumptive Salmonella isolates were then assessed with the indole test, and their identities were verified by means of slide agglutination with Poly A-I and Vi Salmonella antiserum.

Retail Meat Surveillance: One chicken legFootnote 41 was added to 225 mL of BPW. One hundred and fifty millilitres of the peptone rinse was kept for isolation of Campylobacter, E. coli, and Enterococcus. Chicken samples were left in the remaining 75-mL BPW rinse and were incubated at 35 ± 1°C for 24 hours. Afterward, an MSRV plate was streaked with 0.1 mL of the incubated rinse, and the plate was incubated at 42 ± 1°C for 24 to 72 hours. Suspect colonies were screened for purity and used to inoculate triple-sugar-iron and urea agar slants. Presumptive Salmonella isolates were assessed with the indole test, and their identities were verified by means of slide agglutination with Poly A-I and Vi Salmonella antiserum.

Surveillance of Animal Clinical Isolates: Salmonella was isolated according to standard procedures, which varied among laboratories. Most methods for detecting Salmonella in animal clinical isolates were similar in principle and involved pre-enrichment, selective enrichment, differential and selective plating, isolation, and biochemical and serological confirmation of the selected isolates.

Feed and Feed Ingredients: Under both CFIA programs (15A and 15E), all samples were collected aseptically and submitted for bacterial culture and isolation. For Salmonella isolation, MSRV medium was used.

Escherichia coli

Farm Surveillance: One drop of the BPW mixture prepared for Salmonella isolation was streaked onto MacConkey agar and incubated at 35 ± 1oC for 18 to 24 hours. Suspect lactose-fermenting colonies were screened for purity and transferred onto Luria-Bertani agar. Presumptive E. coli colonies were assessed with Simmons citrate and indole tests. Isolates with negative indole results were identified with a test kit for identification of enteric bacteria (API®20E system, bioMérieux Clinical Diagnostics, Marcy l'Étoile, France).

Abattoir Surveillance: Generic E. coli was isolated from the caecal contents of broiler chickens, pigs, and beef cattle. Ten grams of each caecal sample was mixed with 90 mL of BPW. One drop of this mixture was streaked onto MacConkey agar and incubated at 35oC for 18 to 24 hours. Suspect lactose-fermenting colonies were screened for purity and transferred onto Luria-Bertani agar. Presumptive E. coli colonies were assessed with Simmons citrate and indole tests. Isolates with negative indole results were identified with a test kit for identification of enteric bacteria (API® 20E system).

Retail Meat Surveillance: One chicken leg,Footnote 41 1 pork chop, or 25 g of ground beef was added to 225 mL of BPW. Fifty millilitres of the peptone rinse was mixed with 50 mL of a double-strength broth for selective identification of coliform bacteria and E. coli (EC broth) and incubated at 45 ± 1°C for 24 hours. One loopful of the incubated mixture was streaked onto eosin methylene blue agar and incubated at 35 ± 1°C for 24 hours. Suspect colonies were screened for purity and transferred onto trypticase soy agar with 5% sheep blood. Presumptive E. coli colonies were assessed with Simmons citrate and indole tests. Isolates with negative indole results were identified with a bacterial identification test kit (API® 20E system).

Campylobacter

Abattoir Surveillance: For isolation of Campylobacter from beef cattle caecal samples, 1 mL of the BPW mixture prepared for isolation of E. coli was used. This volume was mixed with 9 mL of Hunt's enrichment broth (HEB) and incubated in a microaerophilic atmosphere at 35 ± 1°C for 4 hours. After this first incubation, 36 μL of sterile cefoperazone was added to the HEB. Tubes were then incubated in microaerophilic conditions at 42 ± 1°C for 20 to 24 hours. A loop of the incubated HEB was then used to inoculate a modified cefoperazone charcoal deoxylate agar (mCCDA) plate. Plates were incubated at 42 ± 1°C in microaerophilic conditions for 72 hours. Suspect colonies were streaked onto another mCCDA plate to obtain pure colonies and on Mueller Hinton agar supplemented with 5% sheep blood. Plates were incubated in a microaerophilic atmosphere at 42 ± 1°C for 48 to 72 hours. Presumptive Campylobacter colonies were identified by genus and species ( C. coli, C. jejuni, or other Campylobacter spp.) via the following tests: Gram stain, oxidase, catalase, growth at 25 ± 1°C, cephalothin resistance, and hippurate and indoxyl acetate hydrolysis.

Retail Meat Surveillance: One chicken legFootnote 41 or 2 wings were mixed with 225 mL of BPW. Fifty millilitres of the peptone rinse was mixed with 50 mL of double-strength Bolton broth and incubated in a microaerophilic atmosphere at 42 ± 1°C for 48 hours. The incubated broth was then streaked onto an mCCDA plate and incubated in a microaerophilic atmosphere at 42 ± 1°C for 24 hours. Suspect colonies were streaked onto another mCCDA plate and a Mueller Hinton plate. Plates were incubated in a microaerophilic atmosphere at 42 ± 1°C for 48 to 72 hours. Presumptive Campylobacter colonies were identified by genus and species ( C. coli, C. jejuni, or other Campylobacter spp.) via the following tests: Gram stain, oxidase, catalase, growth at 25 ± 1°C, cephalothin resistance, and hippurate and indoxyl acetate hydrolysis.

Enterococcus

Farm Surveillance: One drop of the BPW mixture prepared for Salmonella isolation was streaked onto enterococcal isolation agar (Enterococcosel™ agar, BD, Mississauga, ON) and incubated at 35 ± 1°C for 24 hours. Suspect colonies were screened for purity on Columbia agar with 5% sheep blood. Presumptive Enterococcus colonies were transferred onto Slaneth and Bartley agar and used to inoculate 3 tubes of phenol-red base broth containing 0.25% L-arabinose, 1% mannitol, or 1% α-methyl-D-glucoside. The plate and tubes were incubated at 35°C ± 1°C for 24 hours.

Retail Meat Surveillance: One chicken legFootnote 41 or 2 wings were added to 225 mL of BPW. Fifty millilitres of the peptone rinse was mixed with 50 mL of double-strength selective broth (Enterococcosel™ broth, BD) and incubated at 35 ± 1°C for 24 hours. One loopful of incubated broth was then streaked onto selective agar (Enterococcosel™ agar) and incubated at 35 ± 1°C for 24 hours. Suspect colonies were screened for purity on Columbia agar with 5% sheep blood. Presumptive Enterococcus colonies were transferred onto Slaneth and Bartley agar and used to inoculate 3 tubes of phenol-red base broth containing 0.25% L-arabinose, 1% mannitol, or 1% α-methyl-D-glucoside. The plate and tubes were incubated at 35 ± 1°C for 24 hours.

Serotyping and Phage Typing of Salmonella

Surveillance of Human Clinical Isolates : In general, clinical laboratories forwarded their Salmonella isolates to their PPHL for identification and serotyping. The PPHL further forwarded Salmonella isolates to NML according to the predefined testing scheme. Isolate identities were confirmed by the NML when isolates received did not have a serovar name (Le Minor and Popoff, 2001) or when inconclusive results arose during phage typing. The O or somatic antigens of the Salmonella isolates were serotyped by use of a slide agglutination method (Ewing, 1986). At the NML, Salmonella H or flagellar antigens were detected via slide and confirmatory tube agglutination methods. Salmonella isolates were maintained at room temperature (25° to 35°C) until typed.

All Salmonella Heidelberg, S. Typhimurium, S. Enteritidis, S. Hadar, S. Newport, S. Typhi, S. Paratyphi B, S. Paratyphi B variant. L(+) tartrate+, S. Infantis, S. Thompson, S. Oranienburg, S. Panama, S. I 4,[5],12:b:-, and S. I 4,[5],12:i:- isolates were phage following the standard technique described by Anderson and Williams (1956) was followed. Isolates were streaked onto nutrient agar plates and incubated at 37°C for 18 hours. One smooth colony was selected and used to inoculate 4.5 mL of phage broth (Difco phage broth, Difco Laboratories, Baltimore, MD; pH, 6.8), which was then incubated for 1.5 to 2 hours in a shaking water bath at 37°C to attain bacterial growth with a turbidity equivalent to 0.5 McFarland standard. Phage agar plates (Difco phage agar, Difco Laboratories) were flooded with approximately 2 mL of culture medium, and the excess liquid was removed with a Pasteur pipette. Flooded plates were allowed to dry for 15 minutes at room temperature. Afterward, approximately 20 L of each serovar-specific typing phage was used to inoculate the bacterial lawn by means of a multiple inoculating syringe method (Farmer et al., 1975). The plates were incubated at 37°C overnight, and lytic patterns were subsequently interpreted (Anderson and Williams, 1956).

Salmonella Enteritidis isolates were phage typed with typing phages obtained from the International Centre for Enteric Phage Typing (ICEPT), Central Public Health Laboratories, Colindale, UK (Ward et al., 1987). The phage typing scheme and phages for Salmonella Typhimurium developed by Callow (1959) and further extended by Anderson (1964) and Anderson et al. (1977) were obtained from the ICEPT. The Salmonella Heidelberg phage typing scheme and phages were supplied by the NML (Demczuk et al., 2003). Isolates that reacted with the phages but did not conform to any recognized phage type were designated as atypical. Strains that did not react with any of the typing phages were designated as untypable.

The Identification and Serotyping and the Phage Typing units at the NML have attained International Standards Organization (ISO) 17025 accreditation by the Standards Council of Canada. The Identification and Serotyping, Phage Typing, and Antimicrobial Resistance units at the NML participate in the annual Global Salmonella Surveillance (GSS), External Quality Assurance System of the World Health Organization, the Enter-net (a European network for the surveillance of human gastrointestinal infections) proficiency program for Salmonella , and a strain exchange with the LFZ (Salmonella and Escherichia coli). The NML has been a strategic planning member of the GSS program since 2002.

Surveillance of Agri-Food, Animal Clinical, and Feed Isolates: Animal clinical Salmonella isolates from Québec were serotyped by the Laboratoire d'épidémiosurveillance animale du Québec, Saint-Hyacinthe, Québec and were sent to the STLFootnote 43 for phage typing. All Salmonella isolates from other provinces were submitted to the STL for serotyping and phage typing. The serotyping method detects O or somatic antigens of the Salmonella isolates via slide agglutination (Ewing, 1986). The H or flagellar antigens were identified with a microtitre plate well precipitation method (Shipp and Rowe, 1980). The Antigenic Formulae of the Salmonella serovars by Grimont and Weill (2007) were used to identify and name the serovars. For phage typing, the standard technique by Anderson and Williams (1956) and described above was followed. The sources of the typing phages for Salmonella Enteritidis, Typhimurium and Heidelberg were the same as described above for Surveillance of Human Clinical Isolates.

Since 1995, the STL has participated in annual inter-laboratory exchange serotyping panels with up to 3 other laboratories. The STL began external proficiency testing for phage typing in 2003. Every year, the STL participates successfully in phage typing proficiency panels provided by the NML, which originate from the Central Public Health Laboratory, Colindale, England.

Antimicrobial Susceptibility Testing

All Salmonella isolates of human origin were tested for antimicrobial susceptibility at the NML, and all isolates of agri-food or feed origin were tested for antimicrobial susceptibility at the LFZ-Guelph. The majority of Enterococcus, Campylobacter, and Escherichia coli isolates from all agri-food components were tested by the LFZ-Saint-Hyacinthe. Escherichia coli isolates from Retail Meat Surveillance in Prince Edward Island were processed at the Atlantic Veterinary College, University of Prince Edward Island. In most instances, only 1 isolate per positive sample was tested for antimicrobial susceptibility. For Farm Surveillance, antimicrobial susceptibility testing was performed on 3 E. coli isolates, 3 Enterococcus isolates, and 1 Salmonella isolate per sample. A portion of the Enterococcus and E. coli isolates from Farm Surveillance in Alberta and Saskatchewan were processed by the Agri-Food Laboratory Branch, AARD. The LFZ-Guelph, LFZ-Saint-Hyacinthe, AARD, and Atlantic Veterinary College participate in external proficiency antimicrobial susceptibility testing for Salmonella, E. coli, and Enterococcus. Like the STL, the LFZ-Guelph laboratory for antimicrobial sensitivity testing is ISO/IEC 17025-accredited.

Salmonella, Escherichia coli, and Enterococcus

All Salmonella and E. coli isolates were tested for antimicrobial susceptibility with a panel of 15 antimicrobials (Table A.2) and for Enterococcus with a panel of 17 antimicrobials (Table A.3). The minimal inhibitory concentration (MIC) values for Salmonella, E. coli, and Enterococcus were determined by means of the broth microdilution method (Clinical and Laboratory Standards Institute [CLSI] M7-A7). This method was performed with an automated system (Sensititre™ Automated Microbiology System, Trek™ Diagnostic Systems Ltd, West Sussex, England). This system involves a commercially available broth dilution technique that makes use of dehydrated antimicrobials in the wells of microtitre plates. The CMV1AGNF susceptibility plates (Sensititre™, Trek™ Diagnostic Systems) of the National Antimicrobial Resistance Monitoring System were used for E. coli and Salmonella isolates, whereas CMV2AGPF plates were used for Enterococcus isolates.

Isolates were streaked onto a plate of Mueller Hinton agar (or Columbia blood agar or Mueller Hinton blood agar) and incubated in an inverted position at 36 ± 1°C for 18 to 24 hours to obtain isolated colonies. One colony was chosen from the plate and re-streaked onto agar plates for growth. The agar plates were subsequently incubated at 36 ± 1°C for 18 to 24 hours. A 0.5-McFarland suspension was prepared by transferring bacterial growth from the agar plates into 5.0 mL of sterile, demineralized water and suspending the organisms in the liquid by use of a vortex mixer. Ten microlitres of the water-bacteria suspension was transferred to a tube containing 10 mL of Mueller Hinton broth (MHB) and mixed with a vortex device. The MHB suspension was dispensed into plates at 50 µL per well. The plates were sealed with adhesive plastic sheets and incubated for 18 hours at 36 ± 1°C. Detection of possible vancomycin-resistant enterococci required 6 more hours of incubation for a total of 24 hours.

After incubation, the CMV1AGNF plates were read and interpreted with an automated reading and incubation system (ARIS®, Trek™ Diagnostic Systems Ltd), whereas the CMV2AGPF plates were read with the manual reader (Sensititre Sensitouch™, Trek™ Diagnostic Systems). In accordance with standards set by the CLSI (CLSI M100-S18), Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Enterococcus faecalis ATCC 29212 were used for quality assurance purposes to ensure validity and integrity of the MIC values of the CMV1AGNF susceptibility panels. Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, Enterococcus faecalis ATCC 29212, and Enterococcus faecalis ATCC 51299 were used as quality control organisms for Enterococcus antimicrobial susceptibility testing.

Campylobacter

All Campylobacter isolates were tested for antimicrobial susceptibility with a panel of 9 antimicrobials (Table A.4).The MIC values for Campylobacter isolates were determined by means of the broth microdilution method (CLSI M7-A7). Antimicrobial susceptibility testing was performed with CAMPY susceptibility panels (Sensititre™) from the National Antimicrobial Monitoring System. The colonies were streaked onto Mueller Hinton agar plates with 5% sheep blood and incubated in a microaerophilic atmosphere at 42 ± 1°C for 24 hours. A 0.5-McFarland suspension of bacterial growth was prepared by transferring selected bacterial colonies into a tube containing 5 mL of MHB and mixing the tube contents with a vortex device for at least 10 seconds. Afterward, 10 µL of the MHB mixture was transferred into a tube containing 11 mL of MHB with laked horse blood and mixed for 10 seconds. The MHB mixture was dispensed into plates at 100 µL per well. The plates were sealed with adhesive plastic sheets and incubated in a microaerophilic atmosphere at 42 ± 1°C for 24 hours. Campylobacter jejuni ATCC 33560 was used as quality control organism. The MIC values obtained were compared with those of CLSI standards (CLSI M45-A).

Antimicrobial Susceptibility Breakpoints

Table A.2. Breakpoints in antimicrobial susceptibility of Salmonella and Escherichia coli isolates; CMV1AGNF plate, 2008.
Antimicrobial Range tested (μg/mL) Breakpointsa (μg/mL)
S I R

Roman numerals I to IV indicate the ranking of antimicrobials based on importance in human medicine as outlined by the Veterinary Drugs Directorate.

S = Susceptible. I = Intermediate susceptibility. R = Resistant. N/A = Not applicable.

a CLSI M100-S20.

b No Clinical and Laboratory Standards Institute interpretive criteria for Enterobacteriaceae were available for this antimicrobial.

Breakpoints were based on the distribution of minimal inhibitory concentrations and were harmonized with those of the National Antimicrobial Resistance Monitoring System.

I Amoxicillin-clavulanic acid 1.0/0.5 - 32/16 ≤ 8/4 16/8 ≥ 32/16
Ceftiofur 0.12 - 8 ≤ 2 4 ≥ 8
Ceftriaxone 0.25 - 64 ≤ 1 2 ≥ 4
Ciprofloxacin 0.015 - 4 ≤ 1 2 ≥ 4
II Amikacin 0.5 - 32 ≤ 16 32 ≥ 64
Ampicillin 1 - 32 ≤ 8 16 ≥ 32
Cefoxitin 0.5 - 32 ≤ 8 16 ≥ 32
Gentamicin 0.25 - 16 ≤ 4 8 ≥ 16
Kanamycin 8 - 64 ≤ 16 32 ≥ 64
Nalidixic acid 0.5 - 32 ≤ 16 N/A ≥ 32
Streptomycinb 32 - 64 ≤ 32 N/A ≥ 64
Trimethoprim-sulfamethoxazole 0.12/2.38 - 4/76 ≤ 2/38 N/A ≥ 4/76
III Chloramphenicol 2 - 32 ≤ 8 16 ≥ 32
Sulfisoxazole 16 - 512 ≤ 256 N/A ≥ 512
Tetracycline 4 - 32 ≤ 4 8 ≥ 16
IV
Table A.3. Breakpoints in antimicrobial susceptibility of Enterococcus isolates; CMV2AGPF plate, 2008.
Antimicrobial Range tested (μg/mL) Breakpointsa (μg/mL)
S I R

Roman numerals I to IV indicate the ranking of antimicrobials based on importance in human medicine as outlined by the Veterinary Drugs Directorate.

S = Susceptible. I = Intermediate resistance. R = Resistant. N/A = Not applicable.

a CLSI M100-S18 Table 2D. M7-A7-MIC Testing section.

b No Clinical and Laboratory Standards Institute (CLSI) interpretive criteria for Enterococcus were available for this antimicrobial.

Breakpoints were based on the distribution of minimal inhibitory concentrations and were harmonized with those of the National Antimicrobial Resistance Monitoring System.

c Based on the resistance breakpoint from the European Committee on Antimicrobial Susceptibility Testing because no interpretative criteria were available from the CLSI for tigecycline.

I Ciprofloxacin 0.12 - 4 ≤ 1 2 ≥ 4
Daptomycinb 0.5 - 16 ≤ 4 N/A N/A
Linezolid 0.5 - 8 ≤ 2 4 ≥ 8
Tigecyclinec 0.015 - 0.5 ≤ 0.25 0.5 ≥ 1
Vancomycin 0.5 - 32 ≤ 4 8-16 ≥ 32
II Erythromycin 0.5 - 8 ≤ 0.5 1-4 ≥ 8
Gentamicin (high-level) 128 - 1,024 ≤ 500 N/A > 500
Kanamycin (high-level)b 128 - 1,024 ≤ 512 N/A ≥ 1,024
Lincomycinb 1 - 32 ≤ 2 4 ≥ 8
Penicillin 0.5 - 16 ≤ 8 N/A ≥ 16
Quinupristin-dalfopristin 1 - 32 ≤ 1 2 ≥ 4
Streptomycin (high-level)b 512 - 2,048 ≤ 1,000 N/A > 1,000
Tylosinb 0.25 - 32 ≤ 8 16 ≥ 32
III Chloramphenicol 2 - 32 ≤ 8 16 ≥ 32
Nitrofurantoin 2 - 64 ≤ 32 64 ≥ 128
Tetracycline 4 - 32 ≤ 4 8 ≥ 16
IV Flavomycinb 1 - 16 ≤ 8 16 ≥ 32
Table A.4. Breakpoints in antimicrobial susceptibility of Campylobacter isolates; CAMPY plate, 2008.
Antimicrobial Range tested (μg/mL) Breakpointsa (μg/mL)
S I R

Roman numerals I to IV indicate the ranking of antimicrobials based on importance in human medicine as outlined by the Veterinary Drugs Directorate.

S = Susceptible. I = Intermediate susceptibility. R = Resistant. N/A = Not applicable.

a CLSI M45-A.

b No Clinical and Laboratory Standards Institute interpretive criteria for Campylobacter were available for this antimicrobial.

Breakpoints were based on the distribution of minimal inhibitory concentrations and were harmonized with those of the National Antimicrobial Resistance Monitoring System.

c No resistance breakpoint was defined at the time this report was prepared.

I Ciprofloxacin 0.015 - 64 ≤ 1 2 ≥ 4
Telithromycinb 0.015 - 8 ≤ 4 8 ≥ 16
II Azithromycinb 0.015 - 64 ≤ 2 4 ≥ 8
Clindamycinb 0.03 - 16 ≤ 2 4 ≥ 8
Erythromycin 0.03 - 64 ≤ 8 16 ≥ 32
Gentamicinb 0.12 - 32 ≤ 2 4 ≥ 8
Nalidixic acidb 4 - 64 ≤ 16 32 ≥ 64
III Florfenicolb,c 0.03 - 64 ≤ 4 N/A N/A
Tetracycline 0.06 - 64 ≤ 4 8 ≥ 16
IV

Antimicrobial Resistance Data Analysis for Human and Agri-Food Isolates

Data from human and agri-food surveillance were integrated and maintained in 2 computer repositories (Oracle ®, Oracle Corp., Redwood Shores, CA, USA) and then transferred to a harmonized database (SAS® 9.1, SAS Institute Inc., Cary, NC, USA). For the Farm Surveillance component of CIPARS, the bacterial species, serovar, and MIC data were maintained in a relational database (Microsoft® Access, Microsoft Corp., Redmond, WA, USA).

Data were analyzed with statistical software programs (SAS® 9.1; and Stata® 8, Stata Corp., College Station, TX, USA), and outputs were exported into a spreadsheet application (Microsoft® Excel 2000, Microsoft Corp.). All tables and figures were generated with the spreadsheet application (Microsoft® Excel 2000). For Farm Surveillance, statistical analyses were performed to account for clustering of antimicrobial resistance within swine herds through generalized estimating equations (PROC GENMOD, SAS® 9.1). All statistical models for pig farms had a binary outcome, logit-link function, and an exchangeable correlation structure. Exact confidence intervals were computed by use of the BINOMIAL statement in PROC FREQ (SAS® 9.1) and an alpha level of 0.05. When the prevalence was 0%, an alpha level of 0.1 was used instead.

For the Farm Surveillance, Abattoir Surveillance, and Retail Meat Surveillance components, recovery rate was defined as the number of positive culture results divided by the total number of samples submitted for culture.

The percentage of isolates with resistance to antimicrobials was defined as the number of isolates resistant divided by the total number of isolates tested for each antimicrobial. The breakpoints used for the interpretation of antimicrobial susceptibility results are listed in Table A.2, Table A.3, and Table A.4. Intermediate MIC values were categorized as susceptible for all analyses. A new ceftriaxone breakpoint was officially adopted by the CLSI in January 2010. This new breakpoint was applied to all data, including historical data, and was used to perform the analysis for the 2008 Annual Report. The total number of antimicrobials in each resistance pattern was calculated by summing the number of antimicrobials to which each isolate was resistant.

For the provincial human incidence data, the number of Salmonella clinical cases in which a particular serovar was detected per 100,000 inhabitant-years was calculated by dividing the total number of isolates of each serovar received by CIPARS from that province by the provincial population (Statistics Canada post-census population estimates, Jan. 1, 2005) and then multiplying by 100,000. The national estimates for all serovars except S. Typhi and S. Newport were calculated as follows. In more heavily populated provinces, the number of isolates resistant and the total number of submitted isolates were multiplied by 2 each month. The numbers of isolates resistant (estimated value in larger provinces or actual value in smaller provinces) for all provinces were summed to obtain the total estimated number of isolates resistant. Total numbers of isolates submitted (estimated value in larger provinces or actual value in smaller provinces) for all provinces were summed to obtain the total estimated number of submissions. Finally, the total estimated number of isolates resistant was divided by the total estimated number of submissions for each antimicrobial tested to obtain a national estimate of resistance for each antimicrobial and each serovar.

Temporal analyses were performed for selected antimicrobials. Only 1 antimicrobial per antimicrobial class was selected among those antimicrobials commonly used in the agri-food and/or human sectors. Some antimicrobials were excluded from the temporal analyses for the following reasons:

  • Resistance to the antimicrobial was absent or at a very low prevalence, or the breakpoint was debatable, and other antimicrobials could be used to provide a surrogate measure of resistance or intermediate susceptibility (e.g. nalidixic acid for ciprofloxacin).
  • The isolate had cross-resistance to another selected antimicrobial (e.g. amoxicillin-clavulanic acid and ceftiofur).
  • The antimicrobial is banned for use in the agri-food sector, and resistance to this drug is maintained because of the use of another drug (e.g. chloramphenicol).

A logistic regression model was developed with year as an independent categorical variable. Data were analyzed with commercial software (Stata 9.1®; or R version 2.2.1, R Foundation for Statistical Computing, Vienna, Austria). Firth's penalized maximum likelihood estimation was performed (R version 2.2.1) when data separation (1 or more zero cells in the contingency table) was encountered. In most situations, the year 2003 was selected as the baseline period; therefore, comparisons between 2003 and 2008 were performed. Comparisons between 2004 and 2008 were also performed for resistance to ampicillin and ceftiofur in E. coli and Salmonella isolated from chicken samples to assess changes in antimicrobial resistance after the early 2005 voluntary withdrawal of ceftiofur by Québec chicken hatcheries. The year 2004 was also used as a reference for temporal comparisons of ceftiofur and ampicillin resistance in human S. Heidelberg isolates because S. Heidelberg in humans was suspected to be mainly of chicken origin. For analyses of temporal variations in retail data from Saskatchewan, 2005 was used as the comparison year because this was the first year of CIPARS retail surveillance in that province. At the request of data users, comparisons between 2007 (past year of surveillance) and current year 2008 are also presented in this report. For temporal analysis of ceftiofur and ampicillin resistance in Salmonella and E. coli from retail chicken, the year 2006 was compared with 2008 because of changes in use of those drugs in 2007. Values of P ≤ 0.05 were considered significant for all analyses.

Null binomial response models were used to estimate the prevalence of resistance to each antimicrobial. From each model, the intercept (β0) and 95% confidence intervals were used to calculate population-averaged prevalence estimates with the formula [1 + exp(-β0)]-1.

Antimicrobial Use

Data Collection and Analysis

Humans

Canadian CompuScript (CCS) is a database that records the number of prescriptions and number of units of product dispensed by pharmacists to consumers in Canada. Data fields include product name (including manufacturer), form, and strength as well as province, number of prescriptions, units of product, and dollars spent by month for each year.

The sampling frame (or "universe") for this dataset in 2008 consisted of approximately 7,980 pharmacies, covering nearly all retail pharmacies in Canada and excluding those in the Yukon, Northwest Territories, and Nunavut. The company Intercontinental Medical Statistics (IMS) Health uses a method of geospatial projection that creates projection factors for application to all non-participating stores on the basis of the number of stores in the area, distance between stores, and store size. In 2008, an average of 5,092 stores was included. The projection factor was used to extrapolate the number of prescriptions dispensed in the stores actually sampled to that of the "universe" (7,980 pharmacies).

Drugs were classified and defined daily doses (DDDs) were determined according to the Anatomical Therapeutic Chemical (ATC) classification system (Table A.5). Temporary DDDs (not yet approved but posted on the World Health Organization website) were used when available. For pediazole, the DDD for erythromycin ethyl succinate (2 g) was used. For oral administration of penicillin G, the DDD for benzylpenicillin by parenteral route (3.6 g) was used. Drugs with no DDDs were excluded, including trisulfaminic (drug discontinued in 2001; a total of 832,384 extended units were dispensed in 2000).

Although no hospital pharmacies participated in the CCS program, CCS data included a small volume of antimicrobials administered in non-oral forms such as injectable drugs or products administered by inhalation. Inconsistencies related to non-oral drugs, which represent a very small volume of the CCS data, were judged too common to include these drugs in the CIPARS analysis. Consequently, the 2008 report only describes orally administered drugs dispensed only by retail pharmacies. Only information regarding drugs of ATC group J01 (antimicrobials for systemic use) were retained in the analysis. Information regarding orally administered vancomycin (ATC group A07AA) was included in the analysis under class J01XA.

The total amount of active ingredient was obtained by multiplying the number of extended units (real or corrected) by the strength of the product in grams. For combination drugs, the active ingredients of all antimicrobial components were summed to obtain the total number of active ingredients. However, the amount of active ingredient used in the calculation of the total number of DDDs for combination drugs included only the compounds from which the DDDs were derived. For example, for drugs composed of trimethoprim-sulfamethoxazole, only the total number of grams of sulfamethoxazole was used to compute the number of DDDs.

The total number of DDDs per 1,000 inhabitant-days for a given year was obtained by summing all DDDs for each ATC class and each year. This number was further divided by the size of the population in thousands during that year, divided by the number of days in that year (365 or 366). The total number of prescriptions and total cost per 1,000 inhabitants was obtained by dividing the total number of prescriptions or the total cost by the population size in thousands for each year. Population data were obtained from updated and preliminary post-census estimates based on the results of the 2001 Census. Census counts were adjusted for net under-coverage (Statistics Canada).

In the 2002 and 2003 CIPARS reports, methenamine and linezolid were classified under "other antimicrobials." As of 2004, they have been reported separately to harmonize with reports from other surveillance programs such as the Danish Integrated Antimicrobial Resistance Monitoring and Research Program. The use of metronidazole (under J01XD imidazole) was added in 2005. Data from metronidazole could not be extracted at the time of analysis for year 2000. That information is therefore missing from the tables and is not included in any totals for year 2000.

Data were analyzed with statistical software programs (SAS® 9.1, SAS Institute Inc., Cary, NC, USA; Stata® 8, Stata Corp., College Station, TX, USA), and outputs were exported into a spreadsheet application (Microsoft® Excel 2000, Microsoft Corp., Redmond, WA, USA).

Table A.5. List of antimicrobials from the CompuScript database for each ATCFootnote 44 class.
ATC code ATC class Antimicrobial

Roman numerals I to III indicate the ranking of antimicrobials based on importance in human medicine as outlined by the Veterinary Drugs Directorate.

ATC = Anatomical Therapeutic Chemical. NC = Not classified.

I J01CR Combinations of penicillins, including β-lactamase inhibitors Amoxicillin-clavulanic acid
J01DD Third generation cephalosporins Cefixime
J01MA Fluoroquinolones Ciprofloxacin, gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin
J01XA Glycopeptides Vancomycin
J01XD Imidazoles Metronidazole
J01XX Linezolid Linezolid
II J01CA Penicillins with extended spectrum Amoxicillin, ampicillin, bacampicillin, pivampicillin, pivmecillinam
J01CE β-lactamase sensitive penicillins Penicillin G, penicillin V
J01CF β-lactamase resistant penicillins Cloxacillin, dicloxacillin, flucloxacillin
J01DB First generation cephalosporins Cefadroxil, cephalexin, cephradine
J01DC Second generation cephalosporins Cefaclor, cefprozil, cefuroxime axetil
J01EE Combinations of sulfonamides and trimethoprim, including derivatives Sulfadiazine-trimethoprim, sulfamethoxazole-trimethoprim
J01FA Macrolides Azithromycin, clarithromycin, erythromycin, spiramycin, telithromycine
J01FF Lincosamides Clindamycin, lincomycin
J01GB Aminoglycosides Neomycin
J01MB Other quinolones Nalidixic acid
J01RA Sulfonamide combinations, excluding
trimethoprim
Erythromycin-sulfisoxazole
J01XC Steroid antibacterials Fusidic acid
III J01AA Tetracyclines Demeclocycline, doxycycline, minocycline, tetracycline
J01BA Amphenicols Chloramphenicol
J01EA Trimethoprim and derivatives Trimethoprim
J01EB Short-acting sulfonamides Sulfamethizole, sulfapyridine, sulfisoxazole
J01EC Intermediate-acting sulfonamides Phenazopyridine-sulfamethoxazole, sulfadiazine, sulfamethoxazole
J01XE Nitrofuran derivatives Nitrofurantoin
J01XX Fosfomycin Fosfomycin
NC J01XX Methenamine Methenamine, methenamine-sodium-tartaric acid
Farm Surveillance in Pigs

The selection of swine herds is described in the subsection Antimicrobial Resistance in the Agri-Food Sector under Farm Surveillance (Appendix A). Data regarding these participating herds were collected through questionnaires completed by veterinarians, owners, or managers of the herds. The questionnaires included questions on antimicrobial use (AMU) within each herd, health of pigs, and farm characteristics.

The questionnaire for AMU was designed to collect data for herds of pigs in the grower-finisher production phase. No data on individual pigs were collected. Two pens representative of this population were selected for the collection of fecal specimens for bacterial culture and antimicrobial susceptibility testing. Thus, in herds with all-in-all-out (or batch) management, the population of interest included all pigs that entered and exited the barn in the same group as the sampled pigs. For herds with continuous-flow management, the population of interest for the first sampling period was defined as the grower-finisher pigs that were in the barn 4 months before the first fecal specimens were collected. In subsequent sampling periods, the population of interest was those pigs that had moved into the grower-finisher barn since the previous set of specimens was collected. The interval between sampling points was approximately 4 months (mean, 4.3 months; standard deviation, 2.1 months). The weight of pigs entering the grower-finisher production phase varied among herds.

Questions pertaining to the population of interest slightly varied in questionnaires, depending on whether continuous-flow management or all-in-all-out management was used, in order to accurately describe these different systems. All-in-all-out pig flow is a production system whereby animals are moved into and out of facilities in distinct groups. By preventing the commingling of groups, the hope is to reduce the spread of disease. Facilities are normally cleaned and disinfected thoroughly between groups of animals. This type of management is generally by room or by barn. In continuous-flow operations, animals are continually being removed and added and there is no distinct group of animals that stays together within each phase of production.

Herd owners/managers were asked about antimicrobial use (AMU) via feed, water, and injections. Data were collected on each diet fed to each population of interest, including diets that contained no antimicrobials. Because all pigs in each population of interest were exposed to the same diets, data on the number of pigs exposed to antimicrobials through feed were not collected. Diet-specific data included weight of the pigs at the start and end of the diet and duration of exposure and tonnes consumed for each diet. The following additional information was collected for diets containing antimicrobials: active ingredient(s), antimicrobial concentration(s), and reason(s) for AMU (categories included enteric disease, lameness, respiratory disease, disease prevention, growth promotion, and other). Exposure to antimicrobials though water was described by the active ingredient(s) of the drug(s), weight of the pigs at the start and end of exposure, duration of exposure, number of pigs exposed, and reason(s) for AMU. Data collected on AMU through injection included active ingredient(s) of the drug(s), the number of pigs exposed, and the reason(s) for AMU. No AMU data were collected for any production phase prior to the grower-finisher phase. Any data describing AMU in pigs weighing less than 15 kg were excluded because this weight is considered below the industry standard for grower-finisher pigs.

Antimicrobial exposures were summarized for each herd. An exposure was defined as any reported use of an active ingredient by a given administration route in 2008. Data were described by exposure to an active ingredient by a given administration route, as well as by exposure to an active ingredient by any administration route. These exposures were summarized by antimicrobial class.Footnote 45 It is important to note that typically, treatment through feed tends to be administered to a larger group of pigs and for longer periods than water treatment, whereas injectable drugs are generally administered on an individual basis to a limited number of pigs.

Data were entered into a database, and all descriptive statistics were obtained with commercially available software (Microsoft Excel® 2003 and Microsoft Access® 2003 [Microsoft Corp., Redmond, WA, USA] and Intercooled Stata® version 9.2 [R Foundation for Statistical Computing, Vienna, Austria]).

Data on AMU were provided for every herd for every route of antimicrobial administration. In Canada, pigs are typically maintained in the grower-finisher production phase for 16 to 20 weeks, and therefore the replacement rate of pigs in a grower-finisher barn is approximately 3 times per year. The surveillance program was designed for administration of the AMU questionnaire to each herd 3 times annually, at approximately 4-month intervals, so AMU during the calendar year could be described.

Data from the AMU questionnaires were compiled so that any reported exposure mentioned in a single questionnaire was classified as an exposure in that herd in 2008. The questionnaires were designed to collect quantitative AMU data for antimicrobial exposures through feed and water, but not through injection. However, the results reported in the CIPARS annual report are solely qualitative and do not include exposure rate, duration, or dose of antimicrobial.

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