NOTE

Public Health

Antimicrobial Susceptibilities of Listeria monocytogenes Isolated in Japan

Yumiko OKADA1)*, Akiko OKUTANI1) **, Hodaka SUZUKI1), Hiroshi ASAKURA1), Shuko MONDEN1), Akiko NAKAMA2), Tsutomu MARUYAMA3) and Shizunobu IGIMI1)
1) 2)

Division of Biomedical Food Research, National Institute of Health Sciences, 1181 Kamiyoga, Setagaya-ku, Tokyo 1588501, Division of Food Microbiology, Department of Microbiology, Tokyo Metropolitan Institute of Public Health, 3241 Hyakuninncho, Shinjyuku-ku, Tokyo 1690073 and 3)Japan Food Hygiene Association, 261 Jingumae, Shibuya-ku, Tokyo 1500001, Japan

(Received 9 February 2011/Accepted 31 July 2011/Published online in J-STAGE 12 August 2011)

ABSTRACT. The antimicrobial susceptibility of 201 Listeria monocytogenes isolates from foods, environments, animals and human patients in Japan was determined. All isolates were susceptible to ampicillin, the first choice of drug for listeriosis treatment, chloramphenicol, dihydrostreptomycin, erythromycin, enrofloxacin, gentamicin, kanamycin, lincomycin, nosiheptide, salinomycin, vancomycin, and virginiamycin. A human strain was resistant to oxytetracycline. The Minimum Inhibitory Concentration (MIC) for 50% of the strains and the MIC for 90% of the strains were comparable in all the isolates. This is the first investigation to compare antibiotic resistances between isolates from foods and isolates from human patients in Japan. The result showed that most of the isolates were susceptible to antibiotics used in this study. KEY WORDS: antibiotics, Listeria, resistance. J. Vet. Med. Sci. 73(12): 16811684, 2011

Listeria monocytogenes causes listeriosis, a severe disease which can lead to abortion, stillbirth, and meningoencephalitis in human and animals. Contaminated foods are the main causative agents for humans. Many outbreaks of human listeriosis have been reported in the U.S.A. and European countries. In Japan, the case numbers of human listeriosis have been remained relatively low [11]; nevertheless, the contamination rates of retail foods are at similar levels to those in the U.S.A. and Europe [12]. To date, most cases of human listeriosis in Japan are sporadic, and only one recognized foodborne outbreak has been reported [9]. Antimicrobial drugs have been used routinely to treat listeriosis in humans and animals. The resistant isolates against ampicillin (ABPC), the drug of first choice for treatment in many cases, began to be observed from 1984 [13]. Previous researches suggested that veterinary use of antibiotics may cause increased resistance in human pathogens via food exposure (reviewed by [10]). However, there have been very few studies on antibiotic resistance of L. monocytogenes isolated from foods in Japan [7], and most studies were performed with small numbers of isolates from sporadic cases of human or animal listeriosis [16, 17, 19, 20]. In this study, we investigated the susceptibility of 201 isolates to 3 categories of antibiotics: (1) antibiotics used for the treatment of human bacterial infection, (2) antibiotics used for livestock, to determine the level of diffusion of resistance via dairy and fishery products, and other types of food, to humans, and (3) antibiotics used as feed additives. Antibiotic feed additives are supplemented to animal feed contin* CORRESPONDENCE TO: OKADA, Y., Division of Biomedical Food Research, National Institute of Health Sciences, 1181 Kamiyoga, Setagaya-ku, Tokyo 1588501, Japan. e-mail: yokada@nihs.go.jp **PRESENT ADDRESS: Department of Veterinary Science, National Institute of Infectious Diseases, 1231 Toyama, Shinjyuku-ku, Tokyo 1626840, Japan.

uously at low concentrations to increase animal growth. Use of feed additives is permitted in Japan and some Asian countries; however, the EU has banned the use of antibiotics as growth promoters. There are no reports of susceptibility of L. monocytogenes to these antibiotics. Therefore, we investigated the susceptibility to feed additives to assess the influence of continuous antibiotic use during livestock fattening phases on bacterial susceptibilities. L. monocytogenes isolates are listed in Table 1. One hundred and one isolates were from human patients of listeriosis, and 100 isolates were from foods, the environment, and animals (collectively called as FE isolates in this study). The sources of these latter isolates were as follows: 93 isolates from foods; 1 isolate from a cow with listeriosis; 1 isolate from feces of a cow, and 5 isolates from the environments for food-processing, such as a refrigerator and cutting boards. All isolates were obtained in Japan from
Table 1.
Origin Patient Pork Chicken Beef Non-cooked ham Fish Pickles Sausage Other types of food Cow of listeriosis Feces from cow Cutting board Refrigerator Floor Cockroach Total

L. monocytogenes strains used in this study

Number of isolate 101 25 24 11 12 8 5 3 5 1 1 2 1 1 1 201

1682

Y. OKADA ET AL.

1974 to 2003. The L. monocytogenes isolates were stored in brain heart infusion (BHI) broth (Becton, Dickinson and Company, Sparks, MD, U.S.A.) containing 20% glycerol at 80C until analysis. Susceptibilities of all isolates were tested against 13 different antibiotics. The antibiotics were selected from a wide range of those used as medication for humans and animals or as feed additives. ABPC (Wako Chemicals, Osaka, Japan), chloramphenicol (CP; Wako), erythromycin (EM; Sigma, St. Louis, MO, U.S.A.), gentamicin (GM; Sigma), kanamycin (KM; Wako), lincomycin (LCM), and vancomycin (VCM) are major antibiotics used for human bacterial infection. Dihydrostreptomycin (DSM), enrofloxacin (ERFX; Wako) and oxytetracycline (OTC) are used for animal diseases, and the susceptibility of L. monocytogenes have been investigated in the previous studies [1, 2, 6, 17]. Nosiheptide (NHT), salinomycin (SLM) and virginiamycin (VGM) are used as feed additives for livestock. The antibiotics without reference to companies were provided by the National Veterinary Assay Laboratory, Japan. All antibiotics were used in 2-fold serial dilutions. ABPC, CP, EM, GM, KM, and LCM were diluted to concentrations ranging from 0.125 to 16 g/ml; DSM, ERFX, OTC, SLM, VCM, and VGM were diluted to concentrations ranging from 0.125 to 128 g/ml; and NHT was diluted to concentrations ranging from 0.125 to 32 g/ml. The susceptibility tests were performed using the agar dilution method, modifying the method by Lyautey et al. [8]. Briefly, L. monocytogenes cells were grown in BHI broth at 37C for 24 hr and diluted 1:10 with saline for the antibiotic resistance tests. Bacterial cells were inoculated onto Muller-Hinton agar (Difco Laboratories, Detroit, MI, U.S.A.) plates, which were made with 20 ml of agar solution in 90 mm diameter Petri dishes, by using a MicroPlanter MIT-P multipoint inoculator (SAKUMA, Tokyo, Japan), according to the manufacturers instructions. The inoculation volume of each spot was 5 l; therefore, approximately 105 Colony Forming Unit per spot were inoculated. All plates were incubated at 37C. The formation of colonies was observed at 48 hr after inoculation, and the Minimum Inhibitory Concentration (MIC), the MIC for 50% of the strains (MIC50), and the MIC for 90% of the strains (MIC90) were determined for each antibiotic. The breakpoint for the susceptibility of L. monocytogenes to ABPC was obtained from the Clinical and Laboratory Standards Institute guideline, M31-A3 [5]. For the other antibiotics that did not have breakpoints specified in this guideline, and having bimodal MIC distributions, the microbiological breakpoints were applied as well as research results of the Japanese Veterinary Antimicrobial Resistance Monitoring System [21]. The microbiological breakpoint is defined as the intermediate MIC between the 2 peak distributions. When the MIC distribution was monomodal, the breakpoint was not determined. The MIC distributions of CP (2->16 g/ml), DSM (416 g/m l), EM (0.251 g/ml), ERFX (0.54 g/ml), GM (0.252 g/ml), KM (0.54 g/ml), LCM (2>16 g/ml), NHT (0.25 g/ml), SNM (0.251 g/ml), VCM (0.52 g/

ml), and VGM (0.54 g/ml) were monomodal, suggesting that all the isolates were susceptible to these antibiotics (Table 2). ABPC is the most frequently used antibiotic for the treatment of human listeriosis [14]. In our study, all of the isolates were susceptible, and the MIC50 and MIC90 values were 0.5 g/ml (Table 2), indicating that the MICs of isolates for ABPC were very low. In previous studies in Japan, there were only 2 reports on ABPC-resistant isolates from human patients. One was from a materno-infant case, and the isolate from the mother showed an MIC of >8 g/ ml. On the other hand, the MIC values of the isolates from blood, stool, ear, pharynx and cerebrospinal fluid of the infant were 0.5 g/ml [22], suggesting that the ABPC-resistance of the isolate from the mother was acquired after delivery. In another report, 1 of 5 isolates from human patients with cancer, from 1995 to 2007, was ABPC-resistant [16]. In all other papers including this study, concerning the Japanese situation, no resistant isolates were reported. It is considered that ABPC-resistant isolates may be rare in Japan, compared to Turkey [3, 4, 23] or the U.S. [15, 18], although the reasons are unclear. The MIC distribution of OTC was bimodal, and the microbiological breakpoint was determined as 16 g/ml (Table 2). One clinical strain isolated in 1988 was resistant to OTC (MIC=64 g/ml), which belongs to the tetracyclines class of antibiotics and is often used for livestock. In an additional confirmatory test, this isolate showed the same MIC to OTC and a very high MIC (128 g/ml) to tetracycline, belonging to the same group of antibiotics and used for human. In conclusion, our results indicate that most of these L. monocytogenes isolates from humans and other origins remain susceptible to antibiotics used as drugs for human, animal, or feed additives. Resistance in L. monocytogenes is not critical in Japan at present; however, because antimicrobial chemotherapy is the only available treatment for listeriosis in humans and animals, considered use of antibiotics in veterinary medicine and continuous surveillance of antibiotic susceptibility is essential. ACKNOWLEDGMENTS. This study was supported by Health and Labour Sciences Research Grants, Research on Food Safety, from the Ministry of Health, Labour and Welfare of Japan. We thank Dr. Michinori Terao for providing the clinical isolates of L. monocytogenes.
REFERENCES
1. Aarestrup, F. M., Knchel, S. and Hasman, H. 2007. Antimicrobial susceptibility of Listeria monocytogenes from food products. Foodborne Pathog. Dis. 4: 216221. Antunes, P., Ru, C., Sousa, J. C., Pestana, N. and Peixe, L. 2002. Incidence and susceptibility to antimicrobial agents of Listeria spp. and Listeria monocytogenes isolated from poultry carcasses in Porto, Portugal. J. Food Protect. 65: 18881893. Arslan, S. and Ozdemir, F. 2008. Prevalence and antimicrobial resistance of Listeria spp. in homemade white cheese. Food Control 19: 360363.

2.

3.

ANTIBIOTIC RESISTANCE OF L. MONOCYTOGENES

1683

Table 2.

MIC distributions, MIC50 and MIC90 of L. monocytogenes isolates

Origina) C FE C FE C FE C FE C FE C FE C FE C FE C FE C FE C FE C FE C FE Number of strains with MIC (g/ml) of 0.25 6 47 0.5 85 48 1 10 5 1 4 7 17 12 97 87 2 1 44 46 4 7 15 10 7 1 10 31 20 3 101 100 7 27 2 75 82 67 56 4 22 11 13 22 16 91 71 5 1b) 1 9 80 85 19 (>16) 3 (>16) 77 69 90 93 17 14 1 1 (>16) 2 4 8 16 32 64
c)

Antibiotic ABPC CP DSM EM ERFX GM KM LCM NHT OTC SNM VCM VGM

(g/ml) MIC50 0.5 0.5 8 8 8 8 0.5 0.5 2 1 0.5 0.5 2 2 16 16 0.25 0.25 4 4 0.5 0.5 1 1 1 1 MIC90 0.5 0.5 16 16 8 8 0.5 0.5 2 2 1 1 4 4 >16 16 0.25 0.25 4 4 1 0.5 2 2 2 4

6 42 54 39 42 9 11

56 35 1 57 69 1

1 1

73 97 15 5 12 15

13

a) C in origins indicates the isolates from patients. FE indicates the isolates from foods, animals and the environment. b) Numbers of resistant isolates are indicated in Bold. c) Test for the MIC ranges shown in gray were not performed.

4.

5.

6.

7.

8.

9.

10.

Bilir Ormanci, F. S., Erol, I., Ayaz, N. D., Iseri, O. and Sariguzel, D. 2008. Immunomagnetic separation and PCR detection of Listeria monocytogenes in turkey meat and antibiotic resistance of the isolates. British Poultry Science 49: 560565. Clinical and Laboratory Standards Institute. 2008. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals; Approved StandardThird Edition. M31-A3, vol. 28, No. 8, Clinical and Laboratory Standard Institutde, Wagne. Hara, F., Ueno, H., Shiraishi, T., Okada Y. and Ohfuku, S. 1983. Listeria monocytogenes associated with premature death of a bovine fetus. J. Jpn. Vet. Med. Assoc. 36: 205209. Kitazume, H., Suzuki, M., Suzuki, M., Matsumoto, Y., Yamada, M., Mutoh, T. and Fujii, K. 2002. Mukanetsusessyusyokuhinn kara kennsyutu sareta Listeria monocytogenes. Ann. Rep. Yokohama Inst. Health 41: 9193. Lyautey, E., Hartmann, A., Pagotto, F., Tyler, K., Lapen, D. R., Wilkes, G., Piveteau, P., Rieu, A., Robertson, W. J., Medeiros, D. T., Edge, T. A., Gannon, V. and Topp, E. 2007. Characteristics and frequency of detection of fecal Listeria monocytogenes shed by livestock, wildlife, and humans. Can. J. Microbiol. 53: 11581167. Makino, S. I., Kawamoto. K., Takeshi, K., Okada, Y., Yamasaki, M., Yamamoto, S. and Igimi, S. 2006. An outbreak of food-borne listeriosis due to cheese in Japan, during 2001. Int. J. Food Microbiol. 104: 189196. Mathew, A. G., Cissell, R. and Liamthong, S. 2007. Antibiotic resistance in bacteria associated with food animals: a United States perspective of livestock production. Foodborne Pathog.

11.

12.

13.

14.

15.

16.

17.

18.

Dis. 4: 115133. Okutani, A., Okada, Y., Yamamoto, S. and Igimi, S. 2004. Nationwide survey of human Listeria monocytogenes infection in Japan. Epidemiol. Infect. 132: 769772. Okutani, A., Okada, Y., Yamamoto, S. and Igimi, S. 2004. Overview of Listeria monocytogenes contamination in Japan. Int. J. Food Microbiol. 93: 131140. Rapp, M. F., Pershadsingh, H. A., Long, J. W. and Pickens, J. M. 1984 Ampicillin-resistant Listeria monocytogenes meningitis in a previously healthy 14-year-old athlete. Arch. Neurol. 41: 1304. Rivero, G. A., Torres, H. A. and Rolston, K. V. I. 2003. Listeria monocytogenes infection in patients with cancer. Diagn. Microbiol. Infect. Dis. 47: 393398. Safdar, A. and Armstrong, D. 2003. Antimicrobial activities against 84 Listeria monocytogenes isolates from patients with systemic listeriosis at a comprehensive cancer center. J. Clin. Microbiol. 41: 483485. Sakai, C., Kumagai, K., Tsujimura, H., Ise, M., Mimura, N. and Satomura, H. 2008. Akuseisyuyou ni gappeisita Listeria monocytogenes kinnketusyou, seijinn 5 syourei no rinnsyouteki saikingakuteki kentou. Clin. Microbiol. 35: 103107. Serikawa, S., Kusakari, N., Ohgi, T., Senna, K., Yonemichi, H., Kishi, K. and Nagai, T. 1989. An outbreak of listeriosis in ewe flock in Hokkaido. J. Jpn. Vet. Med. Assoc. 42: 781785. Srinivasan, V., Nam, H. M., Nguyen, L. T., Tamilselvam, B. and Murinda, S. E. 2005. Prevalence of antimicrobial resistance genes in Listeria monocytogenes isolated from dairy farms. Foodborne Pathog. Dis. 2: 201211.

1684
19.

Y. OKADA ET AL.
Sunaga, S., Nakauchi, K., Nakajima, H., Kataoka, Y. and Morozumi, T. 1991. Isolation and some properties of Listeria spp. from pig carcasses in an abattoir. J. Jpn. Vet. Med. Assoc. 44: 10511055. Tago, K., Tomono, N., Matsushima, T. and Shiro, H. 2008. Three cases of purulent meningitis caused by Listeria monocytogenes. J. Pediatr. Infect. Dis. Immun. 20: 814. Takahashi, T., Asai, T., Kojima, A., Harada, K., Ishihara, K., Morioka, A., Kijima, M. and Tamura, Y. 2006. Present situation of national surveillance of antimicrobial resistance in bacteria isolated from farm animals in Japan and correspondence to the issue. J. Jpn. Assoc. Infect. Dis. 80: 185195. Tsubakio, Y., Iwamura, C., Yoshimura, A., Asada, M. and Furukawa, K. 1998. Hahaoya oyobi kanji kara kinn ga kennsyutu sareta sinnseiji Listeria syou no 1 rei. Jpn. J. Pediatr. 51: 15971600. Ycel, N., itak, S. and nder, M. 2005. Prevalence and antibiotic resistance of Listeria species in meat products in Ankara, Turkey. Food Microbiol. 22: 241245.

22.

20.

23.

21.