Prevalence and Characterization of Apramycin-Resistant Salmonella Enterica Serotype Typhimurium Isolated from Healthy and Diseased Pigs in Korea During 1998 Through 2009

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Journal of Food Protection, Vol. 76, No. 8, 2013, Pages 1443–1446 doi:10.4315/0362-028X.JFP-13-069 Copyright G, International Association for Food Protection

Research Note Prevalence and Characterization of Apramycin-Resistant Salmonella enterica Serotype Typhimurium Isolated from Healthy and Diseased Pigs in Korea during 1998 through 2009

SUK-KYUNG LIM,1* HYANG-MI NAM,1 HEE-SOO LEE,1 AE-RAN KIM,1 GUM-CHAN JANG,1 SUK-CHAN JUNG,1 AND TAE-SUN KIM2 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/76/8/1443/1687379/0362-028x_jfp-13-069.pdf by guest on 30 September 2021

1Animal, Plant, and Fisheries Quarantine and Inspection Agency, 175 Anyang-ro, Manan-gu, Anyang 430-757, Republic of Korea; and 2Public Health and Environment Institute of Gwangju, Gwangju, Republic of Korea

MS 13-069: Received 22 February 2013/Accepted 7 April 2013

ABSTRACT Apramycin resistance was observed in 22.8% (81 of 355) of Salmonella Typhimurium isolates collected from pigs from 1998 through 2009 in Korea. All apramycin-resistant Salmonella Typhimurium isolates also were cross-resistant to gentamicin and tobramycin. Among the seven types of aminoglycoside resistance genes tested, only four types were detected in the apramycin-resistant Salmonella Typhimurium isolates: aac (3)-IV, aac (3)-II, aac (3)-III, and ant (20)-I. Although the aac (3)-IV gene was found in all apramycin-resistant Salmonella Typhimurium isolates, aac (3)-II, aac (3)-III, and ant (20)-I genes were detected in five (6.2%), two (2.5%), and three (3.7%) isolates, respectively. The apramycin-resistant isolates comprised six phage types, of which PT193 (16 of 81 isolates, 19.8%) was most commonly observed. To our knowledge, this is the first report describing characteristics of apramycin-resistant Salmonella Typhimurium isolates in Korea. Further study is warranted to determine whether apramycin use in animals results in cross-resistance to gentamicin, which may affect public health when gentamicin is required for disease treatment in humans.

Apramycin, a veterinary aminoglycoside antimicrobial, isolated from pigs in Korea and to characterize the has been used in livestock around the world (3, 16). apramycin-resistant isolates. Resistance to this antibiotic is conferred by the aminoglycoside-modifying enzyme (AME) 3-N-aminoglycoside ace- MATERIALS AND METHODS tyltransferase type-IV (aac (3)-IV) (2). This enzyme also Salmonella strains. A total of 355 Salmonella Typhimurium confers resistance to tobramycin and gentamicin, which are isolates collected between 1998 and 2009 were investigated in this used to treat serious infections in humans. Apramycin- study: 185 from feces or lymph nodes of healthy pigs and 170 from resistant Enterobacteriaceae strains, such as those of intestine or diarrheic feces of diseased pigs. Samples from healthy Escherichia coli and Salmonella, have been reported in pigs were obtained at slaughterhouses throughout Korea. Diarrheic animals (5, 10, 12, 15) and humans (14, 16), although samples were obtained on random occasions at slaughterhouses apramycin has not been used in human medicine. and from the diagnostic laboratory of Animal, Plant, and Fisheries Quarantine and Inspection Agency (Anyang, Korea). Salmonella In Korea, apramycin has been used for treatment or Typhimurium isolates also were received during 2008 and 2009 prevention of enteric diseases in animals since 1983. About from participating laboratories and centers of the Korean 90% (3.4 to 5.0 tons [3,084 to 4,536 kg]) of apramycin Veterinary Antimicrobial Resistance Monitoring System. One consumed in animals has been used for pigs, and resistance isolate per pig was selected for inclusion in this study. to this antimicrobial agent in E. coli also was much higher in pig isolates than in those from other animals (5). Antimicrobial susceptibility testing. MICs of apramycin, Apramycin-resistant Salmonella from pigs also has been gentamicin, and tobramycin were determined by an agar dilution reported in Korea (13); however, the majority of apramycin method, according to the Clinical and Laboratory Standards resistance studies have been conducted on E. coli in Korea Institute (CLSI) guidelines (6). Antimicrobials were obtained from (5, 14) and other countries (11, 12). Sigma Chemical Co. (St. Louis, MO). Apramycin-resistant transconjugants were examined for susceptibility using the agar The aims of this study were to determine the prevalence disk diffusion method according to the CLSI guidelines (6) for the of apramycin resistance in Salmonella Typhimurium following 14 antimicrobials: ampicillin (10 mg), amoxicillin plus clavulanic acid (20 plus 10 mg), cephalothin (30 mg), cefoxitin (30 mg), cefotaxime (30 mg), cefepime (30 mg), amikacin (30 mg), * Author for correspondence. Tel: 82-31-467-1770; Fax: 82-31-467-1778; neomycin (30 mg), streptomycin (10 mg), tetracycline (30 mg), E-mail: [email protected]. ciprofloxacin (5 mg), trimethoprim plus sulfamethoxazole (1.25 1444 LIM ET AL. J. Food Prot., Vol. 76, No. 8 plus 23.75 mg), nalidixic acid (30 mg), and chloramphenicol (30 mg). all serotypes, including serogroup B. In the present study, Cartridges of discs containing the antimicrobials were obtained apramycin resistance was much more prevalent in isolates from BD (Sensi-Disk, BBL, BD, Sparks, MD). E. coli ATCC from diseased pigs than those from healthy pigs (P , 25922 and E. coli ATCC 35218 were used as quality control 0.001). This finding could be related to the amount of strains. Inhibition zone diameters were interpreted in accordance antimicrobials used for the treatment of diseased pigs. with the CLSI guidelines (6). Breakpoints for gentamicin and Although Garcı´a-Feliz et al. (10) reported higher resistance tobramycin ($8 mg/ml), and apramycin (.16 mg/ml) were used as to apramycin in healthy pigs than in ill pigs, other authors described by CLSI (6) and the Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (7), respectively. have reported that Salmonella isolates from diseased pigs were more likely to have antimicrobial resistance than were Aminoglycoside resistance gene profile. The presence of those from healthy animals or from processed foods of aminoglycoside-modifying enzymes was determined for all animal origin (17). apramycin-resistant Salmonella Typhimurium by PCR assay as All apramycin-resistant Salmonella Typhimurium iso- previously described (11, 19) using primer sets for seven different lates were also resistant to gentamicin and tobramycin. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/76/8/1443/1687379/0362-028x_jfp-13-069.pdf by guest on 30 September 2021 genes: aac (3)-I, aac (3)-II, aac (3)-III, aac (3)-IV, ant (20)-I, Cross-resistance is common for the aminoglycoside group armA, and aac (6)-Ib. in E. coli (5, 12, 14) and Salmonella (21). Among nontyphoidal Salmonella isolates from humans, gentamicin Transferability. Transferability of apramycin resistance was resistance was observed in 5.5% (2 of 36 isolates) in Korea investigated with the filter mating method using E. coli J53 (sodium azide resistant) as the recipient. Overnight cultures of (4), 3% (258 of 7,370 isolates) in the United States (20), and 0.05 ml of donor and 0.45 ml of recipient were each added to 4.5 ml 0.5% (2 of 397 isolates) in the United Kingdom (18). of fresh tryptic soy broth (BD, Franklin Lakes, NJ) and then Although gentamicin resistance is still low in human trapped on a 13-mm nitrocellulose membrane filter (Millipore isolates, resistance might increase by transmission of Corp., Billerica, MA). Membranes were incubated at 37uC apramycin-resistant bacteria or acquisition of their genes overnight. Tenfold serial dilutions of 1021 to 1023 were plated from nonhuman sources. onto MacConkey agar (BD) containing apramycin (16 mg/ml) and Of the seven different types of aminoglycoside sodium azide (150 mg/ml) and incubated at 37uC for 16 h. Putative resistance genes tested, four were detected in apramycin- transconjugant strains were confirmed as E. coli with the indole resistant Salmonella Typhimurium: aac (3)-IV, aac (3)-II, test. Three transconjugant strains from each mating were aac (3)-III, and ant (20)-I. The aac (3)-II gene was detected subcultured on tryptic soy agar and characterized by antimicrobial in all apramycin-resistant Salmonella Typhimurium isolates, resistance phenotype with the disc diffusion method (14). and aac (3)-II, aac (3)-III, and ant (20)-I were detected in Phage typing. Phage types were determined by the extended five (6.2%), two (2.5%), and three (3.7%) isolates, phage typing scheme of Anderson et al. (1) by using 30 Salmonella respectively (Table 1). None of the isolates harbored aac Typhimurium phages obtained from the Health Protection Agency (3)-I, aac (6)-Ib, or armA genes. Frana et al. (9) reported (London, UK). All Salmonella Typhimurium isolates with unusual that a single gene is responsible for conferring the phage types or a pattern that did not match with any of the standard gentamicin and apramycin resistance. We found that most patterns were categorized as untypeable. of tested isolates (88.9%, 72 of 81) carried only the aac (3)- IV gene. Although we could not determine whether the Statistical analyses. The significance of differences in gentamicin and tobramycin resistance was attributed to this resistance between groups was determined using a chi-square test. gene, we assume that gentamicin and tobramycin resistance Differences were considered significant at P # 0.05. might be conferred by aac (3)-IV based on the fact that RESULTS AND DISCUSSION about 90% of strains that had no acetyltransferase genes other than the aac (3)-IV also were resistant to these two From 1998 through 2009 in Korea, apramycin antibiotics. resistance was observed in 14.1% (26 of 185) and 32.4% Apramycin resistance and ampicillin resistance were (55 of 170) of Salmonella Typhimurium isolates recovered transferred from 18 (22.2%) isolates by filter mating to the from healthy and diseased pigs, respectively. Although few E. coli J53 recipient strain. Apramycin-resistant E. coli and data are available for comparison, apramycin resistance Salmonella strains have been reported from humans in the observed in this study is much higher than that reported in European Union (3, 11, 16) and Korea (14), even though England (0.6% of Salmonella isolates from animals in 1982 humans have not been exposed to this antimicrobial agent. and 2.6% in 1984 (21)), the United States (0 to 15% of The reason for the emergence of apramycin resistance in Salmonella isolates from pigs from 1995 through 2004 humans might be horizontal gene transfer between human (15)), and Spain (6.2% of Salmonella isolates from and animal isolates (3, 16). Pohl et al. (16) reported that a clinically ill pigs and 9.3% from healthy pigs (10)). plasmid highly similar to that encoding apramycin resis- The reason for higher prevalence of apramycin tance was detected in E. coli from hospital patients and resistance in the present study might be the Salmonella cattle in Belgium. Although transferability of apramycin serotype examined. Overall, isolates belonging to serogroup resistance in the present study was relatively low compared B have exhibited significantly higher frequencies of with that in a previous report (21), genes encoding resistance to antimicrobials (10, 13). In this study, we apramycin resistance may be transferred by conjugation to investigated the apramycin resistance in only Salmonella other strains of Enterobacteriaceae found in the normal pig Typhimurium, whereas most other investigators evaluated intestinal flora. J. Food Prot., Vol. 76, No. 8 APRAMYCIN RESISTANCE OF SALMONELLA TYPHIMURIUM FROM PIGS 1445

The apramycin-resistant Salmonella Typhimurium iso-

1), lates comprised six definitive phage types. A large ~ a proportion of the 81 isolates (40.7%, 33 isolates) were n 1), 1),

1) either untypeable or reacted nonspecifically with the phages 2) ~ ~ CM ( ~ used for typing. The most common phage type among the n n ~ n 6), 2), z

n 81 isolates was PT193 (19.8%, 16 isolates) followed by 1) 2), 1) ~ ~ TC ( N( SXT PT203 (12.3%, 10 isolates), PT194 (11.1%, 9 isolates), n n ~ ~ ~ z z z

Transfer of other PT195 (6.2 , 5 isolates), U288 (4.9 , 4 isolates), and n n n % % AM AM ( AM AM ( antimicrobial resistance AM U302 (4.9%, 4 isolates) (Table 1). Emborg et al. (8) found that some definitive phage types (DT120 and DT170) were strongly associated with tetracycline resistance. In another study, apramycin resistance increased in a definitive phage

type (DT204C) following the introduction of apramycin for Downloaded from http://meridian.allenpress.com/jfp/article-pdf/76/8/1443/1687379/0362-028x_jfp-13-069.pdf by guest on 30 September 2021 veterinary usage (21). In the present study, the most 1) AM ( 3) AM ( 1) AM ( 10) No resistance ( 3) No resistance ( prevalent phage type was PT193, which was the fourth ~ ~ ~ ~ ~ most frequently observed phage type from pigs in Korea n n n n n (13). In the present study, apramycin resistance was not correlated with a specific phage type; however, more 30.3 ( attention on these common phage types is needed to control transferability of apramycin the apramycin and gentamicin resistance in Salmonella %

resistance (no. of transferred strains) isolates found in the pig production system.

1) We found a high prevalence of apramycin resistance in

~ Salmonella Typhimurium isolates from pigs in Korea. All n ( apramycin-resistant Salmonella Typhimurium isolates also )-I

0 were resistant to gentamicin and tobramycin. Our results 2) 1) 2) 1) 1) 1) suggest that further study is warranted to determine whether ~ ant (2 ~ ~ ~ ~ ~ apramycin use in animals results in cross-resistance to n n n n ( n n ( ( ( z ( ( gentamicin, which may affect public health when gentami- )-I )-I 0 0 cin is required for disease treatment in humans. 16) 6.3 ( 3) 0 Not tested 7) 30.0 ( 4) 0 Not tested 29) 8) 11.1 ( 5) 60.0 ( Resistant gene(s) aac (3)-II aac (3)-II ant (2 aac (3)-II aac (3)-III ant (2 Typhimurium isolates from pigs aac (3)-II ~ ~ ~ ~ ~ ~ ~ ACKNOWLEDGMENT n n n n n n n ( ( z ( ( z z ( z z z ( ( z This work was supported by a grant from the Animal, Plant and Fisheries Quarantine and Inspection Agency, Ministry of Food, Agriculture, Forestry and Fisheries, Republic of Korea. Salmonella aac (3)-IV aac (3)-IV aac (3)-IV aac (3)-IV aac (3)-IV aac (3)-IV aac (3)-IV aac (3)-IV aac (3)-IV aac (3)-IV aac (3)-IV aac (3)-IV aac (3)-IV aac (3)-IV REFERENCES 1. Anderson, E. S., L. R. Ward, M. J. Saxe, and J. D. de Sa. 1977. Bacteriophage-typing designations of Salmonella Typhimurium. 256 256 256 . . . J. Hyg. (Lond.) 78:297–330. 2. Bra¨u, B., U. Pilz, and E. Piepersberg. 1984. Genes for gentamicin- (3)-N-acetyltransferases III and IV. I. Nucleotide sequence of the AAC(3)-IV gene and possible involvement of an IS140 element in its expression. Mol. Gen Genet. 193:179–187. g/ml)

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