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Antimicrobial Resistance
in Bacterial Pathogens Isolated From Turkeys Minnesota
Yashpal S. Malik, BVSc, PhD*
Karen Olsen, BS*
Yogesh Chander, PhD†
Sagar M. Goyal, BVSc, PhD*
*Departments of Veterinary Diagnostic Medicine and †Soils, Water
and Climate,
KEY WORDS: antimicrobial resistance, Escherichia
coli, Salmonella spp, Mannheimia hemolytica, Bordetella avium,
surveillance, turkeys,
Abstract
Antimicrobials are commonly used as feed additives in turkey husbandry either as a growth enhancer or to minimize risks from bacterial infections. This practice is believed to contribute toward the development of antimicrobial resistance in bacterial pathogens. Perusal of the literature revealed the availability of exhaustive data on antimicrobial resistance in chicken pathogens but a limited amount of information on the prevalence of antimicrobial resistance in bacterial pathogens of turkeys. The purpose of this study was to determine the presence of antimicrobial resistance among bacterial pathogens isolated from turkeys in Minnesota during 1998 through 2002. The in vitro antimicrobial resistance of Escherichia coli, Salmonella spp., Bordetella avium, and Mannheimia hemolytica was evaluated using the disc diffusion antimicrobial susceptibility test. Antimicrobial agents tested were amikacin, enrofloxacin, gentamicin, ampicillin, penicillin, ceftiofur, trimethoprim sulfa, erythromycin, spectinomycin, tetracycline, clindamycin, sulfadimethoxine, and sulfachloropyridiazine. The frequency of bacterial isolation from 1998 to 2002 was E. coli (191 or 58.4%) > Salmonella spp. (87 or 26.6%) > B. avium (35 or 10.7%) > M. haemolytica (14 or 4.3%). A majority of the isolates was susceptible to amikacin, whereas variable resistance was observed against gentamicin, ampicillin, ceftiofur, and trimethoprim sulfa. The resistance to erythromycin, spectinomycin, tetracycline, sulfadimethoxine, and sulfachloropyridiazine was high. In general, antimicrobial resistance appeared to be increasing from year to year, indicating the need for continued surveillance to effectively monitor and control the emergence of resistance in turkey pathogens.
INTRODUCTION
The control of infectious diseases is of basic economic importance for animal farming to be successful. The use of antibiotics in feed during the past decades has led to an improvement in the health and safety of farm animals and poultry. Antibiotics are widely used for the treatment of infectious diseases or as growth promoters in the poultry industry,1–4 which is believed to contribute toward the development of antibiotic resistance in both the pathogens and normal microflora of poultry.5,6
Zoonotic pathogens could acquire antibiotic resistance while inhabiting the gastrointestinal tracts of food animals and could then transfer this resistance to humans via the food chain.6–8 Although transfer of resistant bacteria from turkeys to farmers, caretakers, slaughterers, and area residents has been reported,9,10 there is no consensus over the use and/or effects of antimicrobials in food animals.1 The development of antimicrobial resistance is of clinical importance and requires ongoing surveillance to effectively monitor its spread. As a part of global surveillance programs, it is important to determine local patterns of antimicrobial resistance on a regular basis to ascertain if antimicrobial resistance is increasing or decreasing. Perusal of literature reveals the existence of exhaustive data on antimicrobial resistance in chickens but limited information is available on the prevalence of antimicrobial resistance in turkeys.
This retrospective study was conducted to determine
the prevalence of 4 different pathogens (Escherichia coli, Salmonella spp., Bordetella avium,
and Mannheimia hemolytica) and their associated drug resistance in turkeys submitted to the Minnesota
Veterinary Diagnostic Laboratory during the last 5 years (1998–2002).
Pathogens were selected on the basis of their direct impact on poultry
and/or humans. It is well known that animals can serve as reservoirs
of E. coli for
humans and transfer of antibiotic resistance genes from poultry to humans
has been documented.10,11 Turkeys have also been reported to be a vehicle
for outbreaks of salmonellosis in humans.12 B. avium is an opportunistic pathogen of chickens and turkeys
and has been isolated from humans.13,14 M. haemolytica is
one of the most prominent pathogens of domestic animals, causing severe
disease and major economic losses in cattle, swine, sheep, and poultry.15
MATERIALS AND METHODS
Source of Samples
Tracheal and sinus swabs and lung tissues from affected turkeys are routinely submitted to the Minnesota Veterinary Diagnostic Laboratory for disease diagnosis. These samples were initially inoculated on sheep blood agar (SBA) followed by incubation at 37˚C for 18 to 24 hours. Suspect colonies of bacteria were then subjected to standard biochemical tests and were further tested with API-ZYM system (BioMeriuex-France, Lyon, France) for confirmation.
Antibiotic Susceptibility
Antimicrobial susceptibility of bacterial isolates was determined by methods described in the National Committee for Clinical Laboratory Standards (NCCLS).16–18 Briefly, an isolated colony of bacteria was inoculated in Mueller Hinton broth followed by overnight incubation at 37˚C. The overnight culture was than swabbed on the surface of blood-Mueller Hinton agar followed by the application of antibiotic discs. In 1998, ampicillin, gentamicin, spectinomycin, sulfadimethoxine, sulfachloropyridiazine, trimethoprim sulfa, and tetracycline were used whereas in 1999, amikacin, ceftiofur, clindamycin, erythromycin, penicillin, and enrofloxacin were also included. Antibiotic resistance was determined using the criteria for Gram-negative organisms as established by the NCCLS.18
RESULTS
A total of 327 isolates of E. coli, Salmonella spp., B. avium, and M. hemolytica were isolated from 1998 through 2002 (Table 1). The frequency of bacterial isolation was E. coli (58.4%) > Salmonella spp. (26.6%) > B. avium (10.7%) > M. haemolytica (4.3%) (Fig.1). No M. hemolytica was isolated in 1998. Yearly prevalence of all bacterial isolates is shown in Figure 1.
E. coli
As shown in Table 2, a majority of E. coli isolates was susceptible to amikacin except for one isolate each in 2000 and 2002. Resistance to ceftiofur (3.8–8.0%), enrofloxacin (4.0–9.7%), and trimethoprim sulfa (7.5–19.2%) was low (Table 2). Resistance to ampicillin (29.0–42.0%) and gentamicin (48.4–69.2%) was consistent from year to year (Table 2), whereas resistance to spectinomycin, sulfadimethoxine, and tetracycline was very high (88.7–100%).
Salmonella spp.
Amikacin and enrofloxacin were the most effective antibiotics in vitro against Salmonella (Table 3). Thus, none of the 87 isolates was resistant to amikacin, whereas only one isolate was resistant to enrofloxacin (Table 3). Resistance to ceftiofur (0.0–20.8%) and trimethoprim sulfa (0.0–14.3%) was also low. Resistance to gentamicin (21.4–61.1%), ampicillin (7.1–61.1 %), and tetracycline (20.0–77.8%) was highly variable from year to year and resistance to spectinomycin and sulfadimethoxine was almost absolute varying from 94.4% to 100%.
B. avium
As shown in Table 4, B. avium isolates were highly susceptible to amikacin. Although all isolates showed absolute resistance to gentamicin from 1998 to 2000, none was found resistant in 2001 and 2002. Similarly, all isolates were susceptible to tetracycline in 2002 while showing variable resistance (42.9–100%) from 1998 to 2001. An increasing trend of resistance was seen against erythromycin (from 37.5–100%) and trimethoprim sulfa (from 12.5–57.1%), whereas a decreasing trend was observed against ampicillin (from 75–40%). Resistance to sulfachloropyridiazine, clindamycin, and enrofloxacin was very high (71.4–100%).
M. hemolytica
All isolates were susceptible to amikacin, ampicillin, ceftiofur, trimethoprim sulfa, and gentamicin (Table 5). During 2001 and 2002, a sudden increase in resistance to enrofloxacin (42.9–100%) and complete resistance to tetracycline was observed, although these antibiotics were very effective during 1999 and 2000 (Table 5). Resistance to sulfadimethoxine, spectinomycin, erythromycin, and penicillin was very high (66.6–100%).
DISCUSSION
In general, isolation rate of E. coli, Salmonella, B. avium, and M. haemolytica continued to increase from 1998 through 2001 with a decreasing trend in 2002. It would be interesting to determine if this decreasing trend continues in the coming years. Limited information is available in the literature on antimicrobial resistance in bacterial pathogens of turkeys whereas exhaustive information has been published on antimicrobial resistance in chickens. Therefore, in this study, the results for some of the antimicrobials against bacterial isolates of turkeys are compared with resistance data available for chicken isolates.
High resistance to
spectinomycin and tetracycline seen in E. coli isolates is in accordance with earlier reports11,19
in which high resistance to these antibiotics (57.0–99.0%) was reported in chicken isolates. Low resistance
to trimethoprim sulfa and amikacin observed in the present study is
in contrast to the reports by Al-Ghamdi et al.19 and Over et al.,20
respectively, in which high resistance to these antibiotics was reported
in turkey isolates. Our results on resistance of E. coli isolates
to gentamicin (48.4–69.2%) are similar
to those observed in an earlier report21 in which 86% of the E. coli isolates from turkeys were resistant. High resistance
to sulfadimethoxine (93.5–100%) is in accordance
with the report of Takahashi et al.22 in which high resistance to this
antimicrobial was observed in chicken isolates. Results on resistance
to ampicillin (29–42.3%) and enrofloxacin (4.0–9.7%) are in accordance with a report by Amara et al.23
in which low to medium resistance (15–40%) was
reported in E. coli isolates of chickens. A search of the literature
revealed no data on ceftiofur resistance in E. coli isolates
from chickens and turkeys, although very low resistance (3.8–8.0%) was seen in this study.
Most of the Salmonella isolates showed high resistance to sulfadimethoxine and spectinomycin, which is in accordance with the findings of Rajashekera et al.24 in which high resistance to sulfonamides was reported. However, results on ampicillin resistance are in contrast to their findings because a decreasing trend of resistance was seen in the present study. The resistance to gentamicin and ampicillin decreased from year to year, which is in contrast to the findings of Hirsh et al.25 who reported an increasing trend of resistance to gentamicin and ampicillin in turkeys. Our results on tetracycline resistance are in contrast to the findings of Ekperigin et al.26 in which tetracycline was reported to be the most effective drug in vitro for Salmonella in chickens. In the present study, Salmonella isolates appeared to show higher resistance against tetracycline. These results are more close to those of Poppe et al.27 in which resistance to amikacin, gentamicin, and spectinomycin was low, medium, and high, respectively. Low resistance (0.0–14.3%) against trimethoprim sulfa is similar to that reported in a study by Nayak and Kenney28 in which 25% of the Salmonella isolates from turkeys were resistant. Low resistance to enrofloxacin (0–4.8%) and ceftiofur (0–20.8%) is also in accordance with a report by Pedersen et al.29 in which Salmonella isolates from Danish turkeys were reported to be highly susceptible to ceftiofur and enrofloxacin.
B. avium isolates showed high susceptibility to amikacin, which is in agreement with Mortensen et al.30 who found B. avium to be highly sensitive to amikacin. The organism was highly resistant to clindamycin (72.7–100%), enrofloxacin (71.4–100%), and erythromycin (37.5–100%). None of the isolates during 2001 and 2002 showed resistance to gentamicin, whereas complete resistance was seen during 1998 through 2000. Similarly high resistance (42.9–100%) against tetracycline was seen from 1998 through 2001 but not in 2002. These results are in contrast to those reported by Blackall et al.31 in which tetracycline was found to be effective. In the present study, B. avium isolates showed a decreasing trend of resistance to ampicillin from 1998 to 2002 (75.0–40.0%). These results are in agreement with those of Blackall et al.31 in which B. avium isolates were reported to be sensitive to these 2 antimicrobials. The results on erythromycin resistance (37.5–100%) are similar to those reported by Blackall et al.31 in which increasing resistance to erythromycin was reported. Data on amikacin and sulfachloropyridiazine resistance in turkey and chicken isolates have not been reported. In the present study, however, none of the isolates were resistant to amikacin, whereas 81.8% to 100% were resistant to sulfachloropyridiazine.
M. haemolytica does not appear to be an important pathogen of turkeys because only 14 isolates were obtained during the 5 years of the study. The presence of high resistance in M. haemolytica against erythromycin and tetracycline is in accordance with the findings of Watt et al.32 who reported high tetracycline and erythromycin resistance in M. haemolytica isolates. However, the absence of resistance to ampicillin is in contrast to the findings of Watt et al.32 in which high resistance to ampicillin was found. The results on ceftiofur resistance are in agreement with those of Watt et al.32 who reported M. haemolytica isolates to be highly sensitive to ceftiofur. Our results are in agreement with those of Hormandorfer and Bauer33 as far as resistance to sulfadimethoxine, tetracycline, and enrofloxacin is concerned. Our results are also in agreement with those of Diker et al.34 for sensitivity to ampicillin but not for erythromycin. No data are available on amikacin and penicillin resistance in turkey and chicken isolates, although none of the isolate was resistant to amikacin in his study and none was sensitive to penicillin.
These results clearly indicate the in vitro efficacy of amikacin, ceftiofur, and trimethoprim sulfa against turkey pathogens whereas spectinomycin and sulfadimethoxine appeared to be useless in vitro. It is rather easier to explain an increasing trend of resistance to tetracycline and erythromycin in B. avium, but why this organism became susceptible to gentamicin in 2001 and 2002 is not clear. These data on antimicrobial resistance patterns and distribution frequency of bacterial pathogens in turkeys could prove to be useful in developing strategies for the control of bacterial infections in the turkey industry. In summary, E. coli and Salmonella appeared to be the major pathogens of turkeys in Minnesota. A majority of the isolates showed resistance to erythromycin, spectinomycin, tetracycline, sulfadimethoxine, and sulfachloropyridiazine, whereas variable resistance was seen against gentamicin, ampicillin, ceftiofur, and trimethoprim sulfa. Amikacin appeared to be the most effective antibiotic in vitro. In general, antimicrobial resistance appeared to be increasing from year to year.
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Figure 1. Occurrence of Escherichia coli, Salmonella
spp., Bordetella avium, and Mannheimia hemolytica in turkeys from 1998
to 2002.
Table 1.
Isolation of Bacterial Pathogens From
Turkeys From 1998 to 2002
Number isolated in
Organism
1998 1999 2000 2001 2002 Total
Escherichia coli 31 26 50 53 31 191
Salmonella spp. 18 14 24 21 10 87
Bordetella avium 4 8 11 7 5 35
Mannheimia hemolytica 0 2 3 7 2 14
Total Total = 327
Table 2. Percent Antibiotic Resistance in Escherichia
coli Isolated From Turkeys
Percent resistant during the indicated year
(number of isolates tested)
1998 1999 2000
2001 2002
Antimicrobial agents (31) (26) (50) (53) (31)
Amikacin NT 0.0 2.0 0.0 3.2
Ceftiofur NT 3.8 8.0 7.5 6.4
Enrofloxacin NT 7.7 4.0 7.5 9.7
Trimethoprim sulfa 12.9 19.2 10.0 7.5 16.1
Ampicillin 29.0 42.3 34.0 34.0 38.7
Gentamicin 48.4 69.2 62.0 66.0 58.1
Spectinomycin 100.0 100.0 100.0 90.6 96.8
Sulfadimethoxine 93.5 100.0 98.0 100.0 100.0
Tetracycline 93.5 96.1 96.0 88.7 100.0
NT, not tested.
Table 3.
Percent Antibiotic Resistance in Salmonella
spp. Isolated From Turkeys
Percent
resistant during the indicated year
(number of isolates tested)
1998 1999 2000
2001 2002
Antimicrobial agents (18)
(14) (24) (21)
(10)
Amikacin NT 0.0 0.0 0.0 0.0
Enrofloxacin NT 0.0 0.0 4.8 0.0
Ceftiofur NT 7.1 20.8 4.8 0.0
Trimethoprim sulfa 11.1 14.4 0.0 4.8 0.0
Gentamicin 61.1 21.5 54.2 33.3 50.0
Ampicillin 61.1 7.1 37.5 19.0 30.0
Tetracycline 77.8 21.4 70.8 71.4 20.0
Spectinomycin 94.4 100.0 100.0 100.0 100.0
Sulfadimethoxine 100.0 100.0 95.8 100.0 100.0
NT, not
tested.
Table 4.
Percent Antibiotic Resistance in Bordetella
avium Isolated From Turkeys
Percent
resistant during the indicated year
(number of isolates tested)
1998 1999 2000
2001 2002
Antibiotics (4) (8) (11) (7) (5)
Amikacin NT 0.0 0.0 0.0 0.0
Gentamicin 100.0 100.0 100.0 0.0 0.0
Tetracycline 50.0 100.0 81.8 42.9 0.0
Erythromycin NT 37.5 45.5 100.0 100.0
Trimethoprim sulfa 25.0 12.5 36.4 57.1 20.0
Ampicillin 75.0 50.0 27.3 14.3 40.0
Sulfachloropyridiazine 100.0 100.0 81.8 NT NT
Clindamycin NT 100.0 72.7 100.0 100.0
Enrofloxacin NT 100.0 100.0 71.4 80.0
NT, not
tested.
Table 5. Percent Antibiotic Resistance in Mannheimia
hemolytica Isolated
From Turkeys*
Percent resistant
during the indicated year
(number
of isolates tested)
1999 2000
2001 2002
Antibiotics (2) (3) (7) (2)
Amikacin 0.0 0.0 0.0 0.0
Ampicillin 0.0 0.0 0.0 0.0
Ceftiofur 0.0 0.0 0.0 0.0
Trimethoprim sulfa 0.0 0.0 0.0 0.0
Gentamicin 0.0 0.0 0.0 0.0
Enrofloxacin 0.0 0.0 42.9 100.0
Tetracycline 0.0 0.0 100.0 100.0
Sulfadimethoxine 100.0 100.0 85.7 100.0
Spectinomycin 100.0 66.7 85.7 100.0
Erythromycin 100.0 100.0 85.7 100.0
Penicillin 100.0 100.0 100.0 100.0
*No M. hemolytica was isolated in 1998.
ISSN# 1542-2666