Relationships Between Multidrug-Resistant Salmonella

Jpn. J. Infect. Dis., 62, 198-200, 2009

Short Communication Relationships between Multidrug-Resistant Salmonella enterica Serovar Schwarzengrund and Both Broiler Chickens and Retail Chicken Meats in Japan Tetsuo Asai*, Koichi Murakami1, Manao Ozawa, Ryoji Koike, and Hitoshi Ishikawa National Veterinary Assay Laboratory, Ministry of Agriculture, Forestry and Fisheries, Tokyo 185-8511, and 1Division of Pathology and Bacteriology, Fukuoka Institute of Health and Environmental Sciences, Fukuoka 818-0135, Japan (Received September 22, 2008. Accepted February 10, 2009) SUMMARY: We examined 29 isolates of Salmonella enterica subspecies enterica serovar Schwarzengrund from broiler chickens (n = 19) and retail chicken meats (n = 10) in Japan for antimicrobial susceptibility and pulsed-field gel electrophoresis (PFGE) profiling. All isolates exhibited resistance to both bicozamycin and sulfadimethoxine (minimum inhibitory concentration of both antimicrobial agents: ≥512 μg/ml). Nalidixic acid resistance was found in only one broiler chicken isolate. PFGE analysis showed that there were two geno- types among S. Schwarzengrund isolates. Isolates from 11 of 19 broiler chickens and from 6 of 10 retail chicken meats exhibited resistance to dihydrostreptomycin, kanamycin, oxytetracycline, bicozamycin, trimethoprim, and sulfadimethoxine, and had an identical PFGE pattern classified into a predominant genotype. Thus, our results indicate that genetically identical multidrug-resistant S. Schwarzengrund appeared to be disseminated among broiler chickens and retail chicken meats in Japan.

Salmonella enterica, a common cause of human gastro- electrophoresis (PFGE) to study the features of Japanese iso- enteritis and bacteremia, is prevalent in a wide variety of ani- lates of both origins. mals. Out of many S. enterica serovars, serovars Typhimurium A total of 29 isolates of S. Schwarzengrund were included, and Enteritidis are the most common causes of human sal- 19 from fecal samples of broiler chickens obtained from 1999 monellosis worldwide. In the United States, Denmark, and to 2007 and 10 from retail chicken meats obtained from 2005 Southeast Asia, S. Schwarzengrund has been reported as a to 2007 (Table 1). Ten broiler chicken isolates were derived causative agent of the disease (1-4). In addition, a large-scale from 4 prefectures in Kyushu, 6 from 2 prefectures in Kinki, outbreak of this serovar caused by contaminated dry dog food and 3 from 2 prefectures in Chugoku. All of the chicken meat has been reported in humans (5). isolates were from retail meat samples of domestic chicken In the United States, S. Schwarzengrund was the 10th origin collected at different meat shops in Fukuoka Prefec- most frequently isolated serovar that caused invasive sal- ture (Kyushu). They were stored at –80°C in 10% skim milk. monellosis in humans between 1996 and 1999 (6). In Japan, Minimum inhibitory concentrations (MICs) of the 11 anti- S. Schwarzengrund was the 10th most frequent cause of microbial agents (ampicillin, dihydrostreptomycin, kanamy- food-borne illness in 2007 but was not in the top 10 from cin, gentamicin, oxytetracycline, chloramphenicol, bicozamycin, 2005 to 2006 (https://hasseidoko.mhlw.go.jp/Byogentai/Pdf/ colistin, nalidixic acid, trimethoprim, and sulfadimethoxine) data81e.pdf). Broiler chickens had a low incidence of S. were determined by using the agar dilution method of the Schwarzengrund prior to 2000: 0.4% of 452 poultry isolates Clinical and Laboratory Standards Institute (CLSI, for- in 1980-1995 (7) and 2.0% of 197 retail chicken meat iso- merly NCCLS) (11). All the antimicrobial agents except for lates in 1992-1999 (8). S. Infantis is the predominant serovar bicozamycin and nalidixic acid were purchased from Sigma- isolated from broiler chickens and retail meats in Japan (9). Aldrich (St. Louis, Mo., USA). Bicozamycin and nalidixic The proportion of S. Schwarzengrund with broiler chicken acid were kindly supplied by Fujisawa Co. (Osaka, Japan) origin, however, increased from 0% in 2000-2003 (10) to and Daiich Pharmaceutical Co. (Tokyo, Japan), respectively. 28.1% in 2005-2007 (unpublished data). The increase in the The MIC of each antimicrobial agent was interpreted by us- incidence of S. Schwarzengrund among broiler chickens ing the recommendations of the CLSI (12). The breakpoints is considered a threat to chicken meat hygiene. Recently, not seen in the CLSI were defined in a previous study (13). Aarestrup et al. (1) showed the international spread of Escherichia coli ATCC 25922 and Pseudomonas aeruginosa multidrug-resistant S. Schwarzengrund in humans caused by ATCC 27853 were used as quality control strains. According contaminated food products imported into Denmark and the to the CDC PulseNet protocol (14), isolates were analyzed United States. In the present study, S. Schwarzengrund for genetic relatedness by PFGE with XbaI and BlnI restric- isolates from broiler chickens and retail chicken meats were tion enzymes. Electrophoresis was performed by the CHEF- subjected to antimicrobial susceptibility and pulsed-field gel DR III System (Bio-Rad Laboratories, Hercules, Calif., USA) with a running condition of 1 phase from 2.2 to 63.8 s at 180 *Corresponding author: Mailing address: National Veterinary As- V for 19 h. Molecular Analyst Fingerprinting Plus software, say Laboratory, Ministry of Agriculture, Forestry and Fisheries, version 1.6 (Bio-Rad Laboratories), was used to compare the 1-15-1 Tokura, Kokubunji, Tokyo 185-8511, Japan. Tel: +81-42- PFGE profiles. Similarity and diversity were assessed using 321-1841, Fax: +81-42-321-1769, E-mail: [email protected] the Dice coefficient.

198 Table 1. Pulsed-field gel electrophoresis (PFGE) type and antimicrobial susceptibility of Salmonella serovar Schwarzengrund isolates from broiler chickens and retail chicken meats in Japan Broiler chicken Broiler chicken meat PFGE type1) Resistance pattern2) Total 1999 2005 2006 2007 Subtotal 2005 2006 2007 Subtotal Ia DSM-OTC-TMP 2 (KS, 2)3) 2 2 DSM-OTC 2 (CG, 2) 2 2 Ib – 1 (CG, 1) 1 1 IIa DSM-KM-OTC-TMP 5 (KS, 5) 6 (KK, 6) 11 1 (KS, 1) 2 (UK, 2) 3 (KS, 2; UK, 1) 6 17 DSM-OTC-TMP 1 (KS, 1) 1 1 IIb DSM-KM-OTC-TMP 1 (KS, 1) 1 1 DSM-OTC-NA-TMP 1 (KS, 1) 1 1 IIc DSM-KM-OTC 3 (KS, 1; UK, 2) 3 3 DSM-OTC-TMP 1 (KS, 1) 1 1 Total 2 5 8 4 19 1 2 7 10 29 1): Refer to Figure 1. 2): Bicozamycin and sulfadimethoxine were excludes from resistance patterns of this table because all isolates tested exhibited resistance to both anti- microbials. DSM, dihydrostreptomycin; KM, kanamycin; OTC, oxytetracycline; NA, nalidixic acid; TMP, trimethoprim. 3): District and number of isolates are in parenthesis. KS, Kyushu; CG, Chugoku; KK, Kinki; UK, unknown.

All S. Shwarzengrund isolates used in this study exhibited resistance to both bicozamycin and sulfadimethoxine (≥512 μg/ml). In addition, most of the isolates showed resistance to dihydrostreptomycin and oxytetracycline with or without kanamycin and/or trimethoprim (Table 1). Such resistance patterns, except for bicozamycin resistance, were frequently found in S. Infantis isolates from broiler chickens in Japan (9,10). On the other hand, bicozamycin resistance was rarely found in S. Infantis isolates from broiler chickens between 2000 and 2003 (10). Bicozamycin has been approved as a veterinary medicine in cattle and pigs but not in poultry; also between 1998 and 2007, it was not used to promote the growth of food animals (http://www.famic.go.jp/ffis/feed/sub2_ kentei.html [in Japanese]) (15,16). Thus, the prevalence of bicozamycin resistance in S. Schwarzengrund is not likely to be result from bicozamycin use on broiler chicken farms. Epidemic infection of Salmonella serovars, such as S. Enteritidis PT4 and S. Typhimurium DT104, is a worldwide concern in human and animal health. These serovars have been prevalent in Japan (17,18). Recently, Aarestrup et al. (1) showed that multidrug-resistant S. Schwarzengrund, exhibit- ing resistance to ampicillin, chloramphenicol, gentamicin, kanamycin, nalidixic acid, sulfamethoxazole, tetracycline, and Fig. 1. Pulsed-field gel electrophoresis (PFGE) types among Salmonella enterica serovar Schwarzengrund isolates from broiler chickens and trimethoprim/sulfamethoxazole, was found worldwide and retail chicken meats in Japan. PFGE types by XbaI and BlnI digestion was believed to be disseminated internationally by chicken were shown in (A) and (B), respectively. A DNA molecular weight products from Thailand. In the present study, nalidixic acid marker (MW) derived from S. Braenderup strain H9812 was used as a resistance was found in only one isolate, from broiler chick- control. (A) Lane 1, MW; lane 2, Ia and Ib; lane 3, IIa, IIb, and IIc. (B) ens in 2007. Even though only a small number of isolates Lane 1, MW; lane 2, Ia; lane 3, Ib; lane 4, IIa; lane 5, IIb; lane 6, IIc. were tested in this study, resistance to ampicillin, gentamicin, chloramphenicol, and colistin was not found in any of them, Japan after 2005 exhibited a common resistance to dihydro- implying that S. Schwarzengrund isolates in Japan were dif- streptomycin, kanamycin, oxytetracycline, bicozamycin, and ferent from the worldwide endemic strains. trimethoprim (Table 1). Thus, these results indicate that a Among the 29 isolates, two unique PFGE patterns were genotype of multidrug-resistant S. Schwarzengrund appeared observed by digestion with XbaI restriction enzyme. In addi- to be disseminated in common among broiler chickens and tion, PFGE analysis revealed that isolates of one PFGE pat- retail chicken meats in Japan. Until now, S. Schwarzengrund tern, classified by XbaI digestion, were divided into three BlnI- is a less common cause of human salmonellosis in Japan, but digested PFGE patterns; and that those of another pattern, the increased incidence of this serovar in broiler chickens classified by XbaI digestion, were divided into two PFGE may become a threat to human health through the contamina- BlnI-digested PFGE patterns (Figure 1). PFGE typing revealed tion of chicken meats. a single genotype in 11 of 19 broiler chickens and 7 of 10 retail chicken meats after 2005 (Table 1). The predominant ACKNOWLEDGMENTS PFGE pattern IIa could be clearly distinguished from that We would like to acknowledge the staff members of Livestock Hygiene (Ia) of broiler chicken isolates in 1999. In addition, the 11 Service Centers for providing S. enterica serovar Schwarzengrund isolates. broiler chicken isolates derived from two different regions of We also thank J. Terajima, H. Izumiya, and H. Watanabe for providing the S.

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