sign in sign up
<<
Home , Staphylococcus hyicus

NOTE Bacteriology

Antimicrobial Susceptibilities of Exfoliative Toxigenic and Non-Toxigenic hyicus Strains in Japan

Keiko FUTAGAWA-SAITO1)*, William BA-THEIN1,2) and Tsuguaki FUKUYASU1)

1)Department of Animal Health 2, School of Veterinary Medicine, Azabu University, 1–17–71 Fuchinobe, Sagamihara, Kanagawa 229– 8501, Japan and 2)Department of Microbiology and Immunology, Shantou University Medical College, Shantou, Guangdong, 515041, P. R. China

(Received 17 June 2008/Accepted 29 December 2008)

ABSTRACT. Staphylococcus hyicus isolates (n=207), including 150 exfolitative toxigenic and 57 non-toxigenic strains, were examined for their susceptibility to 13 agents by using the dehydrated 96-well MIC panel system. The frequency of their resistance to penicillin and was 76.8% (159/207), followed by erythromycin (56%, 116/207), trimethoprim-sulfamethoxazole (28.5%, 59/ 207), chloramphenicol (24.2%, 50/207), kanamycin (19.8%, 41/207), and doxycycline (1.4%, 3/207). Resistance to chloramphenicol and trimethoprim-sulfamethoxazole was significantly higher in toxigenic strains than non-toxigenic strains (p<0.01), whereas kanamycin and erythromycin resistance was significantly higher in non-toxigenic strains (p<0.01 and <0.05, respectively). Resistance to two or more was observed in 85.5% (177/207) of total strains, with a significantly higher occurrence in toxigenic strains (89.3%, 134/ 150 vs. 75.4%, 43/57; p<0.05). KEY WORDS: antimicrobial resistance, bacterial infection, staphylococcus. J. Vet. Med. Sci. 71(5): 681–684, 2009

Exfoliative toxins-producing Staphylococcus hyicus (tox- penicillin (0.06–8 g/ml), ampicillin (0.12–16 g/ml), igenic strains) have been implicated in causing porcine exu- oxacillin (0.25–4 g/ml), cefazolin (0.5–64 g/ml), dative epidermitis particularly in suckling and weaned cefuroxime (0.5–64 g/ml), (0.5–64 g/ml), [4, 9, 15]. The rate of toxigenic strains is report- kanamycin (1–128 g/ml), erythromycin (0.25–32 g/ml), edly four times higher in the pigs with exudative epidermitis doxycycline (0.25–32 g/ml), (0.5–16 g/ml), than the healthy pigs [8]. Exudative epidermitis is fre- chloramphenicol (1–128 g/ml), trimethoprim-sul- quently treated with antimicrobial agents, but successful famethoxazole (0.5 and 8–9.5 and 152 g/ml, respectively), treatment is complicated by the occurrence of antimicrobial and ofloxacin (0.12–16 g/ml). resistance among S. hyicus strains. JCM 2874 (ATCC29213) and Escherichia coli JCM 5491 of the isolates from various animals including diseased pigs (ATCC25922) were included as controls. MIC breakpoints in different countries have been reported [1–3, 14, 16, 17]. of susceptible (S), intermediate (I), and resistant (R), were However, antimicrobial resistance as to the toxin genes car- taken from the CLSI [5]. Significance of differences riage in S. hyicus is not known. between the variables was tested with the 2 test or the This study aimed to determine the antimicrobial suscepti- Fisher’s extract test. bility of toxigenic and non-toxigenic S. hyicus strains from Antimicrobial resistance profile of 207 S. hyicus strains is healthy pigs and pigs with exudative epidermitis in Japan, shown in Table 1. Intermediate resistance was observed and to investigate the association between antimicrobial only for doxycycline (1/207, 0.5%) and chloramphenicol (5/ resistance and the carriage of toxin genes. 207, 2.4%), and is therefore not shown in the table. For total A total of 207 S. hyicus strains, including 150 exfolitative S. hyicus strains, the highest frequency of resistance was toxigenic strains and 57 non-toxigenic strains from exuda- observed with penicillin and ampicillin (76.8%, 159/207), tive epidermitis and healthy pigs, isolated in 8 years followed by erythromycin (56%, 116/207), trimethoprim- between 1994 and 2002 from 17 prefectures in Japan and sulfamethoxazole (28.5%, 59/207), chloramphenicol previously studied for their exfoliative toxigenes [8] , were (24.2%, 50/207), kanamycin (19.8%, 41/207), and doxycy- included in this study. Minimal inhibitory concentration cline (1.4%, 3/207). Resistance to chloramphenicol and tri- (MIC) determination was done by broth dilution method methoprim-sulfamethoxazole was significantly higher in using the dehydrated 96-well MIC panel system (Eiken, toxigenic strains than non-toxigenic strains (p<0.01), Tokyo) following the Clinical and Laboratory Standards whereas kanamycin and erythromycin resistance was signif- Institute (CLSI) guidelines. The following antimicrobials, icantly higher in non-toxigenic strains (p<0.01 and <0.05, the representatives of commonly used drug classes in Japan, respectively). were tested (with tested ranges indicated in parentheses); Based on their antimicrobial resistance patterns, S. hyicus strains could be categorized into 14 distinctive groups (data *CORRESPONDENCE TO: FUTAGAWA-SAITO, K., Department of Animal Health 2, School of Veterinary Medicine, Azabu University, 1– not shown), with 5 predominant groups exhibiting resis- 17–71 Fuchinobe, Sagamihara, Kanagawa 229–8501, Japan. tance to at least 3 antimicrobials (Table 2). Out of five pre- e-mail: [email protected] dominant resistance patterns, penicillin/ampicillin/ 682 K. FUTAGAWA-SAITO, W. BA-THEIN AND T. FUKUYASU

Table 1. Antimicrobial resistance profile of 207 S. hyicus strains Antimicrobial Antimicrobial MIC Toxigenic strains Non-toxigenic strains Total class agent resistant n=150 n=57 n=207 P (tested range in g/ml) breakpoint* MIC Range n (%) MIC Range n (%) MIC Range n (%) value** Penicillins Penicillin (0.06–8) 0.25 0.06–>8 118(78.7) 0.06–>8 41(71.9) 0.06–>8 159(76.8) 0.357 Ampicillin (0.12–16) 0.5 0.12–>16 118(78.7) 0.12–>16 41(71.9) 0.12–>16 159(76.8) 0.357 Oxacillin (0.25–4) 0.5 <0.25 0 <0.25 0 <0.25 0 – Cephalosporins Cefazolin (0.5–64) 32 0.5 0 0.5 0 0.5 0 – Cefuroxime (0.5–64) 32 0.5–2 0 0.5–1 0 0.5–2 0 – Aminoglycosides Gentamicin (0.5–64) 16 0.5 0 0.5 0 0.5 0 – Kanamycin (1–128) 64 1–>128 14(9.3) 1–>128 27(47.4) 1–>128 41(19.8) <0.01 Macrolides Erythromycin (0.25–32) 8 0.25–>32 71(47.3) 0.25–>32 45(78.9) 0.25–>32 116(56.0) <0.05 Tetracyclines Doxycycline (0.25–32) 16 0.25–16 2(1.3) 0.25–16 1(1.8) 0.25–16 3(1.4) 1 Glycopeptides Vancomycin (0.5–16) 16 0.5–1 0 0.5–1 0 0.5–1 0 – Phenicols Chloramphenicol (1–128) 32 4–64 50(33.3) 4–8 0 4–64 50 (24.2) <0.01 Folate pathway inhibitors Trimethoprim (0.5–8)– 0.5/9.5– 0.5/9.5– 0.5/9.5– 56(37.3) 3(5.3) 59(28.5) sulfamethoxazole (9.5–152) 4/76 >8/152 >8/152 >8/152 <0.01 Fluoroquinolones Ofloxacin (0.12–16) 4 0.25–1 0 0.25–1 0 0.25–1 0 – Note: MIC resistant breakpoints and ranges are in g/ml. * MIC resistant breakpoints taken from the CLSI. ** p values account for the difference in the number of resistant isolates observed between toxigenic and non-toxigenic strains.

Table 2. Antimicrobial resistance patterns among 207 S. hyicus strains Number of isolates (%) Antimicrobial resistance patterns Toxigenic Non-toxigenic Total n=150 n=57 n=207 Penicillin/ampicillin/trimethoprim-sulfamethoxazole 29 (19.3) 0 29 (14.0) Penicillin/ampicillin/erythromycin/trimethoprim-sulfamethoxazole 27 (18.0) 0 27 (13.0) Penicillin/ampicillin/chloramphenicol 25 (16.7) 0 25 (12.1) Penicillin/ampicillin/kanamycin/erythromycin 9 (6.0) 24 (42.1) 33 (15.9) Penicillin/ampicillin/erythromycin 4 (2.7) 12 (21.1) 16 (7.7) Other patterns 56 (37.3) 21 (36.8) 77 (37.2) Resistance to 2 antimicrobials 134 (89.3)* 43 (75.4)* 177 (85.5) Resistance to <2 antimicrobials 16 (10.7) 14 (24.6) 30 (14.5) * p<0.05. trimethoprim-sulfamethoxazole, penicillin/ampicillin/eryth- (177/207) of total strains, with a significantly higher occur- romycin/trimethoprim-sulfamethoxazole, and penicillin/ rence in toxigenic strains (89.3%, 134/150 vs. 75.4%, 43/57; ampicillin/chloramphenicol were restricted to 16.7–19.3% p<0.05). All toxigenic and non-toxigenic strains were sus- of toxigenic strains only, whereas penicillin/ampicillin/kan- ceptible to oxacillin, cefazolin, cefuroxine, gentamicin, van- amycin/erythromycin and penicillin/ampicillin/erythromy- comycin, and ofloxacin, and all non-toxingenic strains were cin patterns were common to both toxigenic and non- susceptible to chloramphenicol. Actually, most non-toxi- toxigenic strains, with a slightly higher occurrence in non- genic strains (95%) are also susceptible to trimethoprim-sul- toxigenic strains. Resistance to two or more antimicrobials famethoxazole. Notably, all resistant strains displayed a (multi-antimicrobial resistance) was observed in 85.5% double resistance to penicillin and ampicillin. ANTIMICROBIAL SUSCEPTIBILITIES OF S. HYICUS 683

There were only a few reports about antimicrobial sus- 745–770. ceptibility of S. hyicus in the past. In a previous study from 2. Aarestrup, F. M., Bager, F., Jensen, N. E., Madsen, M., Mey- Japan in 1987 [14] , the resistance rates for penicillin, eryth- ling, A. and Wegener, H. C. 1998. Surveillance of antimicro- romycin, and doxycycline were 38.7%, 41.1%, and 55.6%, bial resistance in isolated from food animals to respectively, by using the MIC breakpoints of 1.56 g/ml antimicrobial growth promoters and related therapeutic agents in Denmark. APMIS 106: 606–622. (penicillin), 12.5 g/ml (erythromycin), and 0.39 g/ml 3. Aarestrup, F. M. and Jensen, L. B. 2002. Trends in antimicro- (doxycycline), whereas in our study, using the correspond- bial susceptibility in relation to antimicrobial usage and pres- ing MIC breakpoints of 0.25 g/ml, 8.0 g/ml, and 16.0 g/ ence of resistance genes in Staphylococcus hyicus isolated ml, the resistance rates were 76.8%, 56%, 1.4%, respec- from exudative epidermitis in pigs. Vet. Microbiol. 89: 83–94. tively. Finding very low doxycycline resistance was unex- 4. Ahrens, P. and Andresen, L. O. 2004. Cloning and sequence pected because tetracycline compounds share 43% of analysis of genes encoding Staphylococcus hyicus exfoliative animal antimicrobial sales in Japan [13], and oxytetracy- toxin types A, B, C, and D. J. Bacteriol. 186: 1833–1837. cline has been one of the feed additives. 5. Clinical and Laboratory Standard Institute (CLSI). 2008. Per- Chloramphenicol resistance was 24.2% (50/207) over the formance standards for antimicirobial susceptibility testing; study period. In fact, most chloramphenicol resistant strains eighteenth informational supplement, vol. 28, Wayne. 6. Dale, G. E., Broger, C., Hartman, P. G., Langen, H., Page, M. (82%, 41/50) were isolated from 1994 to 1998; only nine G., Then, R. L. and Stuber, D. 1995. Characterization of the strains (18%) were of 2002. Among the tested antimicrobi- gene for the chromosomal dihydrofolate reductase (DHFR) of als chloramphenicol, oxacillin, and vancomycin are not Staphylococcus epidermidis ATCC 14990: the origin of the tri- commonly used in food-producing animals. Chlorampheni- methoprim-resistant S1 DHFR from Staphylococcus aureus? J. col has been prohibited for use in food-producing animals Bacteriol. 177: 2965–2970. since 1998. Increasing resistance up to 82% of chloram- 7. Dale, G. E., Langen, H., Page, M. G., Then, R. L. and Stuber, phenicol from indiscriminate use until 1998 was effectively D. 1995. Cloning and characterization of a novel, plasmid- averted by ban of its use, which is evident from only 18% encoded trimethoprim-resistant dihydrofolate reductase from resistance rate in 2002. Staphylococcus haemolyticus MUR313. Antimicrob. Agents Our finding of high resistance to penicillin (76.8%) and Chemother. 39: 1920–1924. 8. Futagawa-Saito, K., Ba-Thein, W., Higuchi, T., Sakurai, N. erythromycin (56%) during 1994 to 2002 is comparable to and Fukuyasu, T. 2007. Nationwide molecular surveillance of the corresponding resistance (54–75% and 15–62%) in a exfoliative toxigenic Staphylococcus hyicus on farms Danish study over the period 1996–2001 [3]. across Japan. Vet. Microbiol. 124: 370–374. Staphylococcal bacterial toxins and antimicrobial resis- 9. Jones, L. D. 1956. Exudative epidermitis of pigs. Am. J. Vet. tance determinants are carried on mobile genetic elements Res. 17: 179–193. such as plasmids, transposons, and prophages [10]. 10. Novick, R. P., Schlievert, P. and Ruzin, A. 2001. Pathogenicity Chloramphenicol acetyltransferase genes (cat genes) and a and resistance islands of staphylococci. Microbes Infect. 3: trimethoprim-resistant dihydrofolate reductase have been 585–594. reported in chromosomes and plasmids in a wide variety of 11. Schwarz, S. and Noble, W. C. 1994. Structure and putative ori- Staphylococci [6, 7, 11, 12]. Despite there is a possibility of gin of a plasmid from Staphylococcus hyicus that mediates chloramphenicol and streptomycin resistance. Lett. Appl. antimicrobial resistance genes residing together with exfoli- Microbiol. 18: 281–284. ative toxin genes in S. hyicus, we did not find any such 12. Schwarz, S., Werckenthin, C., Pinter, L., Kent, L. E. and occurrence in this study. Noble, W. C. 1995. Chloramphenicol resistance in Staphylo- In conclusion, we did not find any significant correlation intermedius from a single veterinary centre: evidence between the antimicrobial resistance and the toxin genes for plasmid and chromosomal location of the resistance genes. carriage or the isolation origin (i.e., from healthy or diseased Vet. Microbiol. 43: 151–159. pigs) among S. hyicus strains. Although methicillin-resis- 13. Takahashi, T., Asai, T., Kojima, A., Harada, K., Ishihara, K., tant Staphylococcus hyicus (MRSH) or vancomycin-resis- Morioka, A., Kijima, M. and Tamura, Y. 2006. Present situa- tant Staphylococcus hyicus (VRSH) were not detected in tion of national surveillance of antimicrobial resistance in bac- this study, like other staphylococci, S. hyicus strains showed teria isolated from farm animals in Japan and correspondence to the issue. Kansenshogaku Zasshi 80: 185–195. a tendency toward increasing resistance to many antimicro- 14. Teranishi, H., Shimizu, A., Kawano, J. and Kimura, S. 1987. bials. Therefore, sustained monitoring of resistance devel- resistance of Staphylococcus hyicus subsp. hyicus opment in organisms of animal origin would be a prudent strains isolated from pigs, and chickens. Jpn. J. Vet. Sci. preventive measure against zoonotic infections in human. 49: 427–432. 15. Watanabe, T., Sato, H., Hatakeyama, Y., Matsuzawa, T., REFERENCES Kawai, M., Aizawa, C., Danbara, H. and Maehara, N. 2000. Cloning of the gene coding for Staphylococcus hyicus exfolia- 1. Aarestrup, F. M., Bager, F., Jensen, N. E., Madsen, M., Mey- tive toxin B and its expression in Escherichia coli. J. Bacteriol. ling, A. and Wegener, H. C. 1998. Resistance to antimicrobial 182: 4101–4103. agents used for animal therapy in pathogenic-, zoonotic- and 16. Wegener, H. C., Watts, J. L., Salmon, S. A. and Yancey, R. J. indicator bacteria isolated from different food animals in Den- Jr. 1994. Antimicrobial susceptibility of Staphylococcus hyicus mark: a baseline study for the Danish Integrated Antimicrobial isolated from exudative epidermitis in pigs. J. Clin. Microbiol. Resistance Monitoring Programme (DANMAP). APMIS 106: 32: 793–795. 684 K. FUTAGAWA-SAITO, W. BA-THEIN AND T. FUKUYASU

17. Werckenthin, C., Cardoso, M., Martel, J. L. and Schwarz, S. porcine Staphylococcus hyicus, and canine Staphylococcus 2001. Antimicrobial resistance in staphylococci from animals intermedius. Vet. Res. 32: 341–362. with particular reference to bovine Staphylococcus aureus,