Y. S. Malik,* Y. Chander, S. C. Gupta, and S. M. Goyal*,1

2005 Poultry Science Association, Inc. A Retrospective Study on Antimicrobial Resistance in Mannheimia (Pasteurella) haemolytica, Escherichia coli, Salmonella Species, and Bordetella avium from Chickens in Minnesota Y. S. Malik,* Y. Chander, S. C. Gupta, and S. M. Goyal*,1 *Department of Veterinary Diagnostic Medicine, and Department of Soil, Water, and Climate, University of Minnesota, St. Paul, Minnesota 55108 Primary Audience: Researchers, Microbiologists, Poultry Farmers, Veterinarians, Public Health Workers, Nutritionists SUMMARY A 5-yr retrospective study (1998 to 2002) was conducted to determine the rate of isolation of 4 different bacterial pathogens and their antimicrobial resistance from chickens submitted to the Minnesota Veterinary Diagnostic Laboratory, St. Paul, Minnesota. The in vitro antimicrobial resistance was evaluated using the disc diffusion method. A total of 218 bacteria were isolated with the frequency of isolation being Mannheimia (Pasteurella) haemolytica 92 (42.2%) > Escherichia coli 80 (36.7%) > Salmonella spp., 42 (19.3%) > Bordetella avium 4 (1.8%). All isolates were resistant to macrolides and tetracycline antimicrobials but showed varied resistance to aminoglycoside and beta-lactam antibiotics. A majority of the isolates showed high resistance to spectinomycin, sarafloxacin, tetracycline, sulfadimethoxine, and sulfachloropyridiazine. These results emphasize the need for continued surveillance of chicken pathogens to monitor their resistance patterns with a view to control the infections caused by these pathogens. Key words: antimicrobial resistance, Bordetella avium, Escherichia coli, Mannheimia (Pasteurella) haemolytica, Minnesota, Salmonella spp., surveillance 2005 J. Appl. Poult. Res. 14:506 511 DESCRIPTION OF PROBLEM Antibiotics have been used in veterinary medicine since their discovery. In animal production systems, antibiotics are used for both therapeutic and nontherapeutic purposes. Nontherapeutically, antibiotics are used as growth promoters in livestock and poultry [1, 2, 3, 4, 5] and also to improve the general hygiene in barns. This nontherapeutic use of antibiotics in feed may lead to increased levels of antibiotic resistance in both the pathogens and fecal microflora of poultry, e.g., Salmonella, Shigella, Campylobacter, and Listeria [1, 6, 7, 8, 9, 10, 1 To whom correspondence should be addressed: goyal001@umn.edu.

MALIK ET AL.: ANTIMICROBIAL RESISTANCE IN CHICKEN PATHOGENS 507 11]. The transfer of antibiotic resistant Escherichia coli from chickens to humans has also been reported [12, 13, 14]. The development of resistance in poultry pathogens may undermine the efficacy and utility of antibiotics used to control these infections. Few countries have banned the use of some or all antibiotics as growth promoters [8, 10]. However, there have been mixed reports about decline in antimicrobial resistance in bacteria and the impact on the poultry industry as a result of this ban [15]. Since antibiotic usage and management practices differ geographically, data on local patterns of antimicrobial resistance over a period of time are needed to make any meaningful suggestions for their control. In this retrospective study, we have analyzed the prevalence of 4 different pathogens of chickens [Mannheimia (Pasteurella) haemolytica, E. coli, Salmonella spp., and Bordetella avium] and their associated resistance patterns in Minnesota from 1998 to 2002. MATERIALS AND METHODS Tracheal and sinus swabs and lung tissue from chickens are routinely submitted to the Minnesota Veterinary Diagnostic Laboratory, St. Paul, Minnesota, for disease diagnosis. These samples (approximately 600 cases or swab and tissue samples) were initially inoculated on sheep blood agar followed by incubation at 37 C for 18 to 24 h. Suspect colonies of bacteria were then subjected to standard biochemical tests and were further tested with API-ZYM system [16] for confirmation. Susceptibility testing of bacterial isolates was carried out by the disc diffusion method as proposed by the National Committee for Clinical Laboratory Standards [17, 18, 19]. Briefly, an isolated colony of bacteria was inoculated in Mueller Hinton broth followed by overnight incubation at 37 C. The overnight culture was then swabbed on to the surface of blood-mueller Hinton agar followed by the application of antibiotic discs. In 1998, ampicillin, gentamicin, spectinomycin, sulfadimethoxine, sulfachloropyridiazine, trimethoprim sulfa, erythromycin, penicillin, and tetracycline were used, while in 1999 amikacin, ceftiofur, enrofloxacin, and clindamycin were also used. Antibiotic resistance or susceptibility was determined using the criteria for gram-negative organisms as established by the National Committee for Clinical Laboratory Standards. RESULTS AND DISCUSSION A total of 218 isolates of M. haemolytica, E. coli, Salmonella spp., and B. avium were isolated from 1998 to 2002 (Table 1). The frequency of isolation was M. haemolytica 92 (42.2%) > E. coli 80 (36.7%) > Salmonella spp., 42 (19.3%) > B. avium 4 (1.8%). No B. avium was isolated from 2000 to 2002. The prevalence of M. haemolytica was consistent from year to year (n = 13 to 18), except in 2002, when the number of isolated increased to 29. For the remaining 3 organisms (E. coli, Salmonella, and B. avium), a decreasing trend was seen from 1998 to 2002. It would be interesting to determine if these trends continue in the coming years. Interestingly, B. avium (n = 4) was isolated only during 1998 and 1999, and all isolates were highly susceptible to a broad range of antimicrobials. This may have accounted for the absence of this organism in 2000, 2001, and 2002. M. haemolytica The number of M. haemolytica isolated during the 5-yr study was higher than all other bacteria. As shown in Table 2, M. haemolytica isolates showed no or little resistance to amikacin, ceftiofur, enrofloxacin, gentamicin, trimethoprim sulfa, and ampicillin. These results are in agreement with those of Watts et al. [20] and Diker et al. [21] in which M. haemolytica isolates were found to be highly susceptible to ceftiofur and ampicillin. The results on enrofloxacin, however, are in contrast to the findings of Hormandorfer and Bauer [22] in which M. haemolytica isolates were found to be resistant to enrofloxacin. Resistance to spectinomycin (47 to 100%), sulfadimethoxine (72.2 to 100%), penicillin (61.1 to 100%), and tetracycline (84.6 to 100%) was very high, while resistance to clindamycin and erythromycin was absolute. These results are in general agreement with those of Hormandorfer and Bauer [22] and Watts et al. [20]. In the latter study, the minimum inhibitory concentration of 90% of the isolates from cattle to ceftiofur was found to be 0.06 µg/ ml, 1 while resistance to spectinomycin varied

508 JAPR: Research Report TABLE 1. Distribution of bacterial pathogens in chickens from 1998 to 2002 1 Organism 1998 1999 2000 2001 2002 Number (%) Mannheimia hemolytica 18 15 13 17 29 92 (42.2) Escherichia coli 20 18 20 10 12 80 (36.7) Salmonella spp. 12 15 5 4 6 42(19.3) Bordetella avium 3 1 0 0 0 4(1.8) Year 1 Total = 218. from year to year. The results on erythromycin resistance are in contrast to those of Diker et al. [21] who found that only 3.0 and 19.2% of M. hemolytica Biotype A and Biotype T, respectively, were resistant to erythromycin in turkeys. E. coli All of the E. coli isolates were susceptible to amikacin (Table 3), which is in contrast to the report of Over et al. [23] in which 49.7% of E. coli isolates from chickens were reported to be resistant to amikacin. Resistance to enrofloxacin, ceftiofur, and ampicillin was low until 2001 but increased in 2002. Resistance to trimethoprim sulfa was also low. A decreasing trend of resistance was seen against gentamicin (from 45 to 25%) and tetracycline (80 to 50%). However, Over et al. [23] reported very high resistance to gentamicin (94.5%). Lambie et al. [24] also reported an increasing trend of gentamicin resistance in their 8-yr study on the prevalence of E. coli in broiler chickens. Of the 603 isolates, 50% TABLE 2. Antibiotic resistance profiles of Mannheimia hemolytica in chickens were resistant to gentamicin. High resistance to spectinomycin (70 to 100%) and sulfadimethoxine (80 to 100%) is in accordance with Al- Ghamdi et al. [25] and Ojeniyi [26]. In a Danish study, Ojeniyi [26] found high resistance against spectinomycin, sulphafurazole, and tetracycline in E. coli isolates. The results on ampicillin were inconsistent from year to year with resistance varying from 0 to 33.3%. Al-Ghamdi et al. [25] have reported high resistance to ampicillin. Resistance to trimethoprim sulfa was low or absent (from 0 to 20%), which is in contrast to earlier reports by Al-Ghamdi et al. [25], Blanco et al. [27], and Lambie et al. [24]. Blanco et al. [27], in their study on clinically affected broiler chickens in Spain, found 63% of the E. coli isolates from septicemic chickens and 76% of the isolates from healthy chickens to be resistant to trimethoprim sulfa. Low level of resistance against ceftiofur and enrofloxacin and high level of resistance to sulfadimethoxine could not be com- Percentage resistant during the indicated year Antibiotic 1998 1999 2000 2001 2002 Number tested 18 15 13 17 29 Amikacin NT 1 0.0 0.0 0.0 0.0 Ceftiofur NT 0.0 0.0 0.0 0.0 Enrofloxacin NT 0.0 0.0 0.0 3.4 Gentamicin 0.0 0.0 7.7 0.0 0.0 Trimethoprim sulfa 0.0 6.7 0.0 0.0 0.0 Ampicillin 5.6 13.3 7.7 0.0 3.4 Spectinomycin 66.7 100.0 76.9 47.0 79.3 Sulfadimethoxine 72.2 93.3 92.3 100.0 77.8 Penicillin 61.1 100.0 100.0 100.0 100.0 Tetracycline 100.0 86.7 84.6 100.0 93.1 Clindamycin NT 100.0 NT 100.0 100.0 Erythromycin 100.0 100.0 NT 100.0 100.0 1 Not tested.

MALIK ET AL.: ANTIMICROBIAL RESISTANCE IN CHICKEN PATHOGENS 509 TABLE 3. Antibiotic resistance profiles of Escherichia coli in chickens Percentage resistant during the indicated year Antibiotic 1998 1999 2000 2001 2002 Number tested 20 18 20 10 12 Amikacin NT 1 0.0 0.0 0.0 0.0 Enrofloxacin NT 6.0 0.0 0.0 16.3 Ceftiofur NT 6.0 0.0 0.0 33.3 Trimethoprim sulfa 10.0 0.0 20.0 0.0 0.0 Ampicillin 0.0 11.1 15.0 0.0 33.3 Gentamicin 45.0 38.9 40.0 0.0 25.0 Tetracycline 80.0 77.8 65.0 50.0 50.0 Spectinomycin 100.0 88.9 85.0 70.0 91.7 Sulfadimethoxine 100.0 94.4 90.0 80.0 91.7 1 Not tested. pared with earlier studies because of the lack of previous studies on these antibiotics. Salmonella spp. As shown in Table 4, none of the Salmonella isolates (n = 42) showed resistance to amikacin, ceftiofur, enrofloxacin, and trimethoprim sulfa during the study period. The results on amikacin susceptibility are in accordance with Poppe et al. [28] who tested 2,620 Salmonella isolates from 270 turkey flocks in Canada and found that none of them was resistant to amikacin. Results on enrofloxacin susceptibility are in accordance with Jacob-Reitsma et al. [29] who reported no resistance to this antibiotic in Salmonella isolates (n = 94) obtained from 40 flocks of chickens. Resistance to ampicillin and tetracycline varied from year to year; none of the isolates showed resistance to ampicillin in 1998, 1999, TABLE 4. Antibiotic resistance profiles of Salmonella spp., in chickens and 2002, while none was resistant to tetracycline in 2000 and 2002. Resistance to sulfadimethoxine (83.3 to 100%) and spectinomycin (100%) was very high. Although the isolates showed low resistance to gentamicin in 1998 and 1999, they were uniformly susceptible to gentamicin in 2000, 2001, and 2002. These results are similar to those reported by Lee et al. [30] but not to those of Ekperigin et al. [31]. In the latter study, Salmonella isolates were found to be completely resistant to gentamicin. The results on sulfonamide and tetracycline resistance are in accordance with those of Rajashekara et al. [32] but are in contrast to their observations of higher resistance against ampicillin. Ekperigin et al. [31] reported tetracycline to be the most effective in vitro drug, whereas in the present study tetracycline had inconsistent but higher resistance. Our results for resistance Percentage resistant during the indicated year Antibiotics 1998 1999 2000 2001 2002 Number tested 12 15 5 4 6 Amikacin NT 1 0.0 0.0 0.0 0.0 Ceftiofur NT 0.0 0.0 0.0 0.0 Enrofloxacin NT 0.0 0.0 0.0 0.0 Trimethoprim sulfa 0.0 0.0 0.0 0.0 0.0 Gentamicin 8.3 33.3 0.0 0.0 0.0 Ampicillin 0.0 0.0 20.0 25.0 0.0 Tetracycline 33.3 46.7 0.0 50.0 0.0 Sulfadimethoxine 100.0 100.0 100.0 100.0 83.3 Spectinomycin 100.0 100.0 100.0 100.0 100.0 1 Not tested.

510 to spectinomycin are very similar to those of Poppe et al. [28] who reported complete resistance to spectinomycin in Salmonella from chicken. JAPR: Research Report TABLE 5. Antibiotic resistance profile of Bordetella avium in chickens 1 Percentage resistant during the indicated year B. avium All of the B. avium (n = 4) isolates were uniformly susceptible to amikacin, ampicillin, enrofloxacin, gentamicin, neomycin, sulfachloropyridiazine, tetracycline, and trimethoprim sulfa (Table 5). Clindamycin and erythromycin were the only antibiotics against which resistance was seen. These results are in agreement with those of Mortensen et al. [33] in which B. avium isolates were found to be sensitive to gentamicin and amikacin. Blackall et al. [34] reported sensitivity of B. avium isolates to ampicillin and tetracycline and resistance to erythromycin, spectinomycin, and trimethoprim sulfa. In the present study, B. avium isolates showed sensitivity to ampicillin and tetracycline. Our results are in contrast with these finding for trimethoprim sulfa, as in the present study, B. CONCLUSIONS AND APPLICATIONS 1. Amikacin, ceftiofur, enrofloxacin, and trimethoprim sulfa were found to be effective in vitro antibiotics against most of the tested chicken pathogens. 2. Different bacterial pathogens showed different patterns of antimicrobial resistance. 3. More studies are needed to monitor the pattern of antimicrobial resistance of various poultry pathogens and to evaluate the use of antibiotics in the poultry industry. 4. It is equally important to continuously monitor antimicrobial profiles in pathogenic, zoonotic, and commensal bacteria of chickens with a view to eventually controlling the development of resistance in bacteria. 1. McEwen, S. A., and P. J. Fedorka-Cray. 2002. Antimicrobial use and resistance in animals. Clin. Infect. Dis. 34:S93 S106. 2. Johnston, A. M. 2001. Animals and antibiotics. Int. J. Antimicrob. Agents 18:291 294. 3. van Veen, L., E. Hartman, and T. Fabri. 2001. In vitro antibiotic sensitivity of strains of Ornithobacterium rhinotracheale isolated in the Netherlands between 1996 and 1999. Vet. Rec. 149:611 613. 4. Witte, W. 2000. Selective pressure by antibiotic use in livestock. Int. J. Antimicrob. Agents 16:S19 S24. 5. Swezey, J. L., B. B. Baldwin, and M. C. Bromel. 1981. Effects of oxytetracycline as a turkey feed additive. Poult. Sci. 60:738 743. 6. Manie, T., S. Khan, V. S. Brozel, W. J. Veith, and P. A. Gouws. 1998. Antimicrobial resistance of bacteria isolated from REFERENCES AND NOTES Antibiotic 1998 1999 Number tested 3 1 Amikacin NT 2 0.0 Ampicillin 0.0 0.0 Enrofloxacin NT 0.0 Gentamicin 0.0 0.0 Neomycin 0.0 0.0 Sulfachloropyridiazine 0.0 NT Tetracycline 0.0 0.0 Trimethoprim sulfa 0.0 0.0 Clindamycin NT 100.0 Erythromycin 100.0 100.0 1 Not isolated in 2000, 2001, and 2002. 2 Not tested. avium isolates were found highly sensitive to trimethoprim sulfa. Results for erythromycin are in accordance with the findings of Blackall et al. [34] for B. avium isolates. slaughtered and retail chickens in South Africa. Lett. Appl. Microbiol. 26:253 258. 7. van den Bogaard, A. E., and E. Stobberingh. 1999. Antibiotic usage in animals impact on bacterial resistance and public health. Drugs 58:589 607. 8. Evans, M. C., and H. C. Wegener. 2003. Antimicrobial growth promoters and Salmonella spp., Campylobacter spp. in poultry and swine, Denmark. Emerg. Infect. Dis. 9:489 492. 9. White, D. G., S. Zhao, S. Simjee, D. D. Wagner, and P. F. McDermott. 2002. Antimicrobial resistance of food borne pathogens. Microbes Infect. 4:405 412. 10. Butaye, P., L. A. Devriese, and F. Haesebrouck. 2003. Antimicrobial growth promoters used in animal feed: Effects of less wellknown antibiotics in gram positive bacteria. Clin. Microbiol. Rev. 16:175 188.

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