Nel_239-246.qxd INTRODUCTION Antimicrobial drugs have been used in animals since shortly after their introduction to human medicine. They have been of great benefit to animals in terms of alleviation of suffering and to humans in helping to meet the growing demand for animal protein and in controlling the agents of potentially serious zoo- noses (Martel, Chaslus-Dancla, Coudert, Poumart & Lafont 1995). However, through the increasing use of antimicrobial drugs in humans, animals, fish and crops, an antimicrobial resistance problem has been created that is rapidly moving internationally to the forefront of public health concerns (Anon. 1999) with numerous governmental and non-gov- 239 Onderstepoort Journal of Veterinary Research, 71:239–246 (2004) Towards the establishment and standardization of a veterinary antimicrobial resistance surveillance and monitoring programme in South Africa H. NEL1, M. VAN VUUREN1 and G.E. SWAN2 ABSTRACT NEL, H., VAN VUUREN, M. & SWAN, G.E. 2004. Towards the establishment and standardization of a veterinary antimicrobial resistance surveillance and monitoring programme in South Africa. Onder- stepoort Journal of Veterinary Research, 71:239–246 The objective of this study was to establish a repeatable, standardized laboratory procedure for mon- itoring the development of antimicrobial resistance in bacteria isolated from animals and food of ani- mal origin in South Africa, with reagents prepared in-house. The emergence of resistance and the spread of resistant bacteria can be limited by implementing a veterinary antimicrobial drug policy, in which inter alia systematic monitoring and prudent use play essential roles. The bacteria included in this study represented three different categories, namely zoonotic bacteria (Salmonella), indicator bacteria (Escherichia coli, Enterococcus faecalis and Enterococcus faecium) and veterinary pathogens (Mannheimia haemolytica). Thirty isolates of each species were collected with the aim of standardizing the laboratory methodology for a future national veterinary surveillance and monitoring programme. Susceptibility to ten selected antimicrobial drugs was determined by means of minimum inhibitory concentrations (MICs) using the microdilution method. The method according to the National Committee for Clinical Laboratory Standards was used as the standard. Multi-well plates containing varying dilutions of antimicrobial drugs and prepared in-house for MIC determinations, yielded repeatable results. Storage of plates for 2 months at –70 °C did not influence results meaningfully. Within this limited sample of bacteria, MIC results did not indicate meaningful resistance against any of the ten selected antimicrobial drugs. The findings of the study will be used to establish a national veterinary antimicrobial resistance sur- veillance and monitoring programme in South Africa. To allow for international comparison of data, harmonisation of the surveillance and monitoring programme in accordance with global trends is encouraged. Ideally it should be combined with a programme monitoring the quantities of antimicro- bial drugs used. The aim is to contribute to slowing down the emergence of resistance and the prob- lems associated with this phenomenon by means of the rational use of antimicrobial drugs. Keywords: Antimicrobial resistance, harmonisation, minimum inhibitory concentration, monitoring 1 Department of Veterinary Tropical Diseases, Faculty of Vet- erinary Science, Private Bag X04 Onderstepoort, 0110 South Africa 2 Department of Paraclinical Sciences, Faculty of Veterinary Science, Private Bag X04, Onderstepoort, 0110 South Africa Accepted for publication 7 April 2004—Editor ernmental organizations being involved (Caprioli, Busani, Martel & Helmuth 2000). The role and impact of the use of antimicrobial drugs in animals on the development of bacterial resist- ance against these drugs have not clearly been delineated. While there is general concern about the emergence of antimicrobial resistance, the impor- tant aspect for the animal industries is the concern about the human health aspects of antimicrobial resistance that result from the use of antimicrobial drugs in animals, particularly their use for growth enhancement and disease prophylaxis and the rel- ative volumes of antimicrobial drugs used in ani- mals vs those used in humans. Two critical issues that linked the use of antimicrobial drugs in animals and public health concerns were the use of antimi- crobial drugs as digestive enhancing drugs (growth promoters) and work done in Denmark that demon- strated the emergence of vancomycin-resistant enterococci and its association with the use of a gly- copeptide antibiotic, avoparcin in animals (Woodford 1998). More recently, the potential impact of resist- ant Campylobacter infections in humans owing to fluoroquinolone use in chickens also contributed to concerns relating to the use of antimicrobial drugs in food producing animals. It is therefore necessary that the risk of development of resistance be con- sidered by the animal industries and that surveil- lance for resistance and guidelines for the prudent use of antimicrobial drugs be established. Antimicrobial drugs are considered essential for controlling bacterial infection and are among the most commonly used drugs in veterinary medicine. To safeguard the efficacy of their use in veterinary medicine and to minimize possible public health risks, the emergence of resistance and the spread of resistant bacteria must be limited by implement- ing a veterinary antibiotic policy in which, inter alia, systematic monitoring for the development of resist- ance and prudent use of such drugs play essential roles (Martel et al. 1995; Anon. 1999). The monitoring of antimicrobial resistance in bacte- ria from animal sources in South Africa is in its infancy. However, the veterinary profession in this country recognizes it as an emerging problem and seeks to address the problem by developing and implementing a national antimicrobial strategy for the use of antimicrobials in animals which inter alia will include the need for a standardized monitoring programme. The ultimate goal for a national antimi- crobial strategy is to prolong the efficacy of existing and new antimicrobial agents that are needed to control both human and animal infectious diseases and to minimize infections with zoonotic pathogens in humans (Tollefson, Angulo, Fedorka-Cray 1998). The objectives of the study were to establish a repeatable, standardized laboratory procedure that can be used for monitoring the development of anti- microbial resistance in bacteria isolated from ani- mals and food of animal origin in South Africa, and to make recommendations for the practical imple- mentation of a monitoring programme that can pro- vide information on a national scale. MATERIALS AND METHODS Specimens Specimens for the isolation of bacteria and stored isolates of bacteria were obtained from the bacteri- ology laboratory and poultry reference laboratory, Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, the Onderstepoort Veterinary Institute and Du Buisson, Bruinette & Kramer, Medical Pathologists, Pretoria. In addition, lung specimens were obtained from var- ious cattle feedlots in South Africa. Thirty isolates of each species of bacterium identified for possible inclusion in a future surveillance programme were collected and stored for testing. They included rep- resentatives of zoonotic pathogens (Salmonella), indicator bacteria of animal origin (Escherichia coli, Enterococcus faecium and Enterococcus faecalis) and a veterinary pathogen (Mannheimia haemolyti- ca). Some strains of E. faecalis of human origin were also included to make up the selected number of thirty isolates per bacterial species. The specimens were randomly collected but formal randomization was not carried out. When isolates of bacteria were received from participating laborato- ries, they were examined for purity and viability, and their identification confirmed by performing relevant tests. Pure strains of overnight growth of each organ- ism were inoculated into Brain Heart Infusion broth (CA Milsch), transferred to sterile vials and stored at –70 °C. Microdilution susceptibility test Direct phenotypic susceptibility testing in which the lowest concentration of an antimicrobial drug that can effectively inhibit bacterial cell division is deter- mined, was used. Sterile, plastic, microdilution plates with round wells (Sterilab), each containing 0.05 ml of cation-adjusted Mueller-Hinton broth (CA Milsch), was used. Enhanced growth and greater consisten- 240 Veterinary antimicrobial resistance surveillance and monitoring programme in South Africa cy was obtained when testing Mannheimia haemo- lytica by supplementing the Mueller-Hinton broth with 0.1 ml inactivated bovine serum prior to inoculation. The inoculum was prepared by emulsifying bacteri- al colonies in a tube containing 5 ml of Mueller- Hinton broth and standardizing the concentrations spectrophotometrically. Each well of a freshly pre- pared or stored plate was inoculated with 0.05 ml of inoculum within 15 min after it was standardized. Plates were incubated at 35 °C for 16–20 h in an aerobic incubator in stacks of not more than four plates to ensure that even incubation temperatures were kept. Antimicrobial drugs The plates containing the varying dilutions of the antimicrobial drugs for testing were prepared in- house. The document entitled Performance Stan- dards for Antimicrobial Disk and Dilution Susceptibil- 241 H. NEL, M. VAN VUUREN & G.E. SWAN TABLE 1 Minimum Inhibitory Concentrations (MIC) recorded for antimicrobial drugs tested against the different isolates Antimicrobial Organism MIC results in µg/ml Enrofloxacin Salmonella < 0.03 – 2 E. coli < 0.03 – > 4 M. haemolytica < 0.03 – 0.5 E. faecalis, E. faecium 0.125 – > 4 Oxytetracycline Salmonella 2 – > 64 E. coli 4 – > 64 M. haemolytica < 0.5 – > 64 E. faecalis, E. faecium < 0.5 – > 64 Gentamicin Salmonella 0.5 – > 32 E. coli 0.5 – 8 M. haemolytica 0.5 – 8 E. faecalis, E. faecium 1 – 32 Florfenicol Salmonella 4 – > 16 E. coli 4 – > 16 M. haemolytica 0.5 – 4 E. faecalis, E. faecium 1 – 4 Amoxycillin Salmonella < 0.5 – > 32 E. coli < 0.5 – > 32 M. haemolytica < 0.5 – 16 E. faecalis, E. faecium < 0.5 – 4 Neomycin Salmonella < 1 – 32 E. coli < 1 – 128 M. haemolytica 2 – 32 E. faecalis, E. faecium 8 – > 128 Tilmicosin Salmonella 64 – > 64 E. coli 64 – > 64 M. haemolytica < 0.5 – 32 E. faecalis, E. faecium < 0.5 – > 64 Trimethoprim/ Salmonella < 0.125 – > 16 Sulfamethoxazole E. coli < 0.125 – > 16 M. haemolytica < 0.125 E. faecalis, E. faecium < 0.125 – 1 Sulfadimethoxine Salmonella < 0.125 – > 512 E. coli 32 – > 512 M. haemolytica < 4 – 256 E. faecalis, E. faecium < 4 – > 512 Cephalexin Salmonella 4 – > 8 E. coli 4 – > 8 M. haemolytica < 0.125 – 4 E. faecalis, E. faecium 0.5 – > 8 ity Tests for Bacteria Isolated from Animals was used as the guidance document (National Committee for Clinical Laboratory Standards 1999). Briefly, antimicrobial stock solutions were prepared by weighing the powders and dissolving them to yield the required concentrations based on the potency of the respective antimicrobial drugs. Some of the drugs had to be dissolved in solvents other than water. In those cases only enough solvent was used to solubilize the antimicrobial powder to give a translucent solution. It was diluted further to the final stock concentration with water or the appropri- ate diluent. Aliquots of 1 ml of the stock solution were dispensed into sterile 2 ml Eppendorf tubes, sealed and stored at –70 °C. Vials were removed when needed and used the same day. Any unused drugs were discarded at the end of the day. Two-fold dilutions were used to dilute the antimicro- bial drugs and their concentration ranges were deter- mined by published breakpoints for the different antimicrobial drugs. The concentration ranges of the different doubling dilutions in µg/ml were as follows: enrofloxacin (0.03–4), oxytetracycline (0.5–64), gen- tamicin (0.25–32), florfenicol (0.125–16), amoxycillin (0.5–32), neomycin (1–128), tilmicosin (0.5–64), tri- methoprim/sulfamethoxazole (0.125/304–16/2.4), sulfadimethoxine (4–512) and cephalexin (0.125– 8). For each antimicrobial concentration in the dilution range, aliquots of 0.05 ml were dispensed to the corresponding wells in each plate. Quality control The following reference strains of bacteria obtained from the American Type Culture Collection were used: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, and Staphylococcus aureus ATCC 29213. The antimicrobial susceptibility of reference organisms was tested on each occasion that a new batch of microdilution plates was prepared. When the minimum inhibitory concentrations (MICs) of the reference strains did not fall between the ranges according to the requirements of the National Com- mittee for Clinical Laboratory Standards (NCCLS), the plates were discarded. The results of these tests were compared with the expected values given by the NCCLS in Table 4 of Document M31-A, for accuracy. Results were recorded on a quality-con- trol record sheet. Other control procedures included growth control, purity control and inoculum control (Sahm & Washington 1991; NCCLS 1999). Repeatability Susceptibility tests were done twice for each individ- ual bacterial organism to determine the inter-plate variation of microdilution plates. In addition, prepared plates were stored frozen at –70 °C until required for use. As the plates were filled, they were stacked in groups of five plates and covered with an adhesive seal. In this way, each tray fitted on top of the other tightly enough to provide a cover that minimizes evaporation and contamination. Each stack was then sealed in a plastic bag. These were thawed and tested 2 months after freezing to determine the influence of storage temperature on the repeatabil- ity of the test. Prior to inoculation, the plates were thawed at room temperature for approximately 1 h before use. RESULTS The MIC range of each organism tested against each antimicrobial drug is indicated in Table 1. 242 Veterinary antimicrobial resistance surveillance and monitoring programme in South Africa TABLE 2 Repeatability results of duplicate testing of selected antimicrobial drugs against four bacterial species Percentage of repeat analyses within one dilution Antimicrobial drug Salmonella E. coli Mannheimia E. faecalis, haemolytica E. faecium Enrofloxacin 93.33 100.0 90.0 100.0 Oxytetracycline 93.33 93.33 90.0 86.67 Gentamicin 96.67 93.33 100.0 96.67 Florfenicol 96.67 100.0 100.0 100.0 Amoxycillin 90.0 90.0 96.67 100.0 Neomycin 100.0 96.67 100.0 93.33 Tilmicosin 100.0 100.0 100.0 93.33 Trimethoprim/Sulfa 96.67 90.0 100.0 100.0 Sulfadimethoxine 100.0 90.0 96.67 96.67 Cephalexin 100.0 100.0 100.0 100.0 Repeatability of test Table 2 shows the repeatability in percentage of the duplicate test results, allowing a difference in MIC values corresponding to one log2 dilution step. All the antimicrobial drugs, except for oxytetracycline, when tested against E. faecalis and E. faecium gave a repeatability percentage of 90 % and higher. An overall agreement of 96.5 % was obtained between the 120 specimens and panel of antimicrobial drugs. Effect of storage The results given in Table 3 represent the data col- lected after testing the reference strains on plates that were stored at –70 °C for 2 months. DISCUSSION The microdilution minimal inhibitory concentration antimicrobial susceptibility test was chosen as the test of choice for future antimicrobial resistance sur- veillance and monitoring because it overcomes sev- eral limitations of the disk diffusion test. It is a quan- titative determination of the degree of susceptibility, not dependent on subjective interpretation, measure- ment of zones of growth inhibition or extrapolation of MIC values from zone sizes. The precise amount of antimicrobial drug required to inhibit bacterial growth can be determined (Fales, Morehouse, Mittal, Bean-Knudsen, Nelson, Kintner, Turk, Turk, Brown & Shaw 1989). The MIC is generally a repro- ducible quantitative characteristic of the bacterial isolate that can be measured readily (Prescott & Baggot 1985). In addition, quantitative susceptibility testing using the microdilution method is preferred when testing bacteria of frequently unpredictable susceptibility, such as Salmonella and E. coli. It is also the method of choice when testing bacteria that have developed multiple drug resistance (Baggot 1998). The guidelines of the NCCLS were used as the ref- erence method for preparing the varying dilutions of antimicrobial drugs and determining the minimum inhibitory concentrations. It is the quantitative meth- od used by most countries for the determination of susceptibility of bacteria. The NCCLS has also de- veloped protocols for susceptibility testing of bacte- ria of animal origin and the determination of the interpretive criteria. The World Organisation for Ani- mal Health (OIE) (Anon. 1999) has endorsed the use of NCCLS standards and guidelines (Franklin, Acar, Anthony, Gupta, Nicholls, Tamura, Thompson, Threlfall, Vose, Van Vuuren, White, Wegener & Costarrica 2001). Using the microdilution method standardized by the NCCLS will contribute to har- monisation of laboratory methodologies and allow more meaningful comparisons of data. Values obtained for each of the ten antimicrobial agents used against each isolate were generally 243 H. NEL, M. VAN VUUREN & G.E. SWAN TABLE 3 Minimum inhibitory concentrations of reference strains tested after plates were stored frozen for 2 months Staphylococcus aureus Enterococcus faecalis Escherichia coli Pseudomonas aeruginosa ATCC 29213 ATCC 29212 ATCC 25922 ATCC 27853 Drugs Control Resultsb Control Results Control Results Control Results Limitsa Limits Limits Limits 1 0.03–0.12 0.06 0.12–1 0.25 0.008–0.03 < 0.03 1–4 4 2 0.25–1 0.5 8–32 8 0.05–2 2 8–32 8 3 0.12–1 0.5 4–16 16 0.25–1 1 0.5–2 2 4 2–8 2 2–8 2 2–8 8 > 16 > 16 5 0.25–1 0.5 0.5–2 0.5 2–8 4 – – 6 1–4 2 16–64 16 1–4 2 – – 7 1–4 1 > 32 32 > 64 > 64 > 16 > 16 8 < 0.5/9.5 0.5 < 0.5/0.9 < 0.125 < 0.5/9.5 < 0.125 8/152– 32 32/608 9 32–128 32 32–128 32 8–32 8 – – 10 0.12–0.5 0.5 – – 4–16 4 – – a Accepted quality control ranges of MICs (µg/ml) for reference strains, derived from NCCLS, Table 4, Document M31-A, vol. 19, no. 11, June 1999 b Results (µg/ml) after storage for 2 months at –70 °C (1) enrofloxacin; (2) oxytetracycline; (3) gentamicin; (4) florfenicol; (5) amoxycillin; (6) neomycin; (7) tilmicosin; (8) trimethoprim/sulfa- methoxazole; (9) sulfadimethoxine; (10) cephalexin similar to those reported in related studies (Post, Cole & Raleigh 1991; Burrows, Morton & Fales 1993; Watts, Salmon, Yancey, Nersession & Kounev 1993; Hirsh & Jang 1994; Bengtsson, Franklin, Greko, Karlsson & Wallen 2000). However, different MIC values were obtained for neomycin against E. coli and Salmonella. Some of the results fell within com- parable ranges, but a high percentage of MICs were also found to be < 1 µg/ml. A particularly large devi- ation was found with sulfadimethoxine against M. haemolytica. The highest recorded MIC value in this study was > 256 µg/ml whilst the greatest distribu- tion of MICs were obtained at a dilution of < 64 µg/ml and 128 µg/ml. This could be owing to the use of samples from diagnostic submissions in this study, whereas comparative studies made use of specimens taken from healthy animals. The results of microdilution susceptibility testing are considered to be satisfactory if the results of indi- vidual tests vary no more than plus or minus one serial dilution. The results of the two replicate tests in this project revealed agreement within one dilution for most organisms tested. To determine whether repeatability is adequate, it is considered necessary to calculate the coefficients of variation (SD of repli- cates divided by mean of replicates) (Jacobson 1998). However, to do these calculations continu- ous data are required but not discrete data as was the case in this study. This is owing to the fact that the results are also considered to be concordant when the independent tests vary by one serial dilu- tion. Using the standardized method of the NCCLS, with all the quality control measures, the results showed 90 % comparability, except for oxytetracy- cline that showed 86.67 % comparability. The data thus indicated a high degree of correlation between the two sets of test results. Stability was shown for plates prepared in-house and frozen for a period of 2 months. Therefore, microtitre plates containing the antimicrobial drugs in suspen- sion can be safely stored at –70 °C for at least 2 months following preparation. The results point to the fact that custom-made microdilution plates are as good as commercially produced plates provided that universally accepted quality control strains are always included. In this regard, the ATCC control strains are satisfactory as far as susceptible strains are concerned. However, national reference labora- tories should also strive to measure results against resistant strains. Most clinical specimens from which the bacterial species used in this study were isolated were diag- nostic submissions and the results must therefore be interpreted with caution. Bacteria from these types of submissions tend to be species from severe and/or recurrent clinical cases and some may pos- sess some degree of antimicrobial resistance. Thus, the prevalence of resistant strains may be overesti- mated and may not reflect the resistance situation in the animal population as a whole (Bengtsson et al. 2000). The number of bacterial species used in this study was relatively small and cannot be con- sidered as representative of resistance patterns for the bacteria tested. Based on knowledge of antimi- crobial drug use in production animals in South Afri- ca, a future surveillance programme should ideally include specimens from adult cattle and calves, slaughter pigs, broiler chickens and layer hens. In addition, it is recommended that active surveillance as opposed to passive surveillance be conducted. The former represents a sampling scheme where the nature and number of specimens are defined and collection takes place at regular intervals to assure consistency. The structure of a veterinary antimicrobial surveil- lance and monitoring programme will of necessity have a different emphasis when compared with sim- ilar programmes for humans. The amount of data regularly generated in human hospitals is uncom- mon in the veterinary field, probably because a vari- ety of animal species must be included. Veterinary antimicrobial surveillance and monitoring pro- grammes should aim at detecting the level of resist- ance in bacteria isolated both from animals and animal-derived foods, at evaluating the risks of the use of antimicrobial drugs in animals and at quanti- fying the impact of these findings on human health (Moreno, Dominguez, Teshager, Herrero & Porrero 2000). Specimen collection can include diagnostic submis- sions that are readily available, but the emphasis should be on field specimens taken from healthy an- imals, food of animal origin and abattoirs. All spec- imens collected should preferably be from food pro- ducing animals, and sampling done randomly to avoid bias. Specimens can be collected weekly, monthly, or otherwise as required. The OIE guid- ance document (Franklin et al. 2001) is not pre- scriptive about the exact number of specimens to be collected. Thus, it is recommended that an opti- mal number of specimens as determined by avail- able resources be collected and included to give a true reflection and valid statistical results. The OIE has recommended certain bacterial spe- cies that could be included in surveillance pro- grammes. These are grouped under the following 244 Veterinary antimicrobial resistance surveillance and monitoring programme in South Africa three major categories of organisms (Franklin et al. 2001): • Veterinary pathogens: Pasteurella spp., Haemoph- ilus somnus, Actinobacillus pleuropneumoniae, E. coli, Salmonella serotypes, Staphylococcus aureus, Streptococcus spp. from udder speci- mens, Streptococcus suis, Brachyspira spp. and Aeromonas and Vibrio spp. from fish specimens. • Zoonotic bacteria: Salmonella Typhimurium, Sal- monella Enteritidis, Campylobacter jejuni, Cam- pylobacter coli and an enterohaemorrhagic E. coli such as O157. • Commensal/indicator bacteria: E. coli and entero- cocci especially, E. faecium. The latter bacteria are known to be indicators of antimicrobial resist- ance and should be obtained from healthy ani- mals. For a national surveillance and monitoring pro- gramme in South Africa, it is recommended that the bacterial species tested in this study are included. They represent the three major categories of organ- isms and, in the developmental stages of the pro- gramme, will not provide a formidable challenge in terms of isolation and identification. Additional organisms and types of specimens can be included when available resources and capacity make it pos- sible. For example, it may be worthy of considera- tion to restrict Salmonella monitoring to bovine and porcine specimens and include Campylobacter spp. isolations from poultry specimens. The antimicrobial drugs included in this study are similarly recommended for the initial phase of the programme. They represent most of the important antimicrobial classes used in animals and humans. The selection of two aminoglycosides within the panel of antimicrobials was based on the gastro- intestinal specific action of neomycin compared to systemic antibacterial indications of gentamicin (Prescott 2000). The difference in the aminoglyco- side-modifying enzymes in R plasmid-bearing bac- teria between neomycin and gentamicin was also considered (Lambert & O’ Grady 1992). Explosive outbreaks of nosocomial infections caused by gen- tamicin-resistant bacteria of many species have been reported in human hospitals (Prescott 2000). Neomycin plasmid-mediated resistance is relatively common in enteric commensals and pathogens but less common among other opportunist pathogens. Other drugs can be considered for inclusion at later stages, for example antimicrobial drugs that are used in humans and not in animals (e.g. glycopep- tides and streptogramins). It would be ideal to develop antimicrobial panels for each category of bacteria to be tested. Antimicrobial panels can also be designed specifically for Gram-negative and Gram-positive bacteria or for each bacterial species tested in the surveillance programme (Aarestrup, Bager, Jensen, Madsen, Meyling & Wegener 1998; Moreno et al. 2000; Aarestrup 2000; Franklin et al. 2001). Information on the consumption of antimicrobial drugs used in different animal species is also need- ed to assess the impact on the occurrence of resist- ance and to determine where and for which infec- tions most antimicrobial drugs are used. A key use of surveillance data is to relate this to antimicrobial use so that where resistance emerges, appropriate feedback exists and measures can be taken by reg- ulatory authorities to address use patterns that may be contributing to the emergence of resistance. 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