Jpn. J. Infect. Dis., 59, 397-399, 2006

Short Communication Characteristics of suis Isolated from Patients in Japan Bin Chang, Akihito Wada, Tadayoshi Ikebe, Makoto Ohnishi, Kazuhito Mita1, Miyoko Endo2, Hirosuke Matsuo3, Yoshinori Asatuma4, Sanae Kuramoto5, Hiroshi Sekiguchi6, Motoyosi Yamazaki7, Hiroko Yoshikawa8, Nobuei Watabe9, Hideko Yamada9, Shohachi Kurita9, Yumiko Imai9 and Haruo Watanabe* Department of Bacteriology, National Institute of Infectious Diseases, Tokyo 162-8640; 1Department of Traumatology and Critical Care Medicine, Matsudo City Hospital, Matsudo 271-8511; 2Department of Bacteriology, Tokyo Metropolitan Institute of Public Health, Tokyo 169-0073; 3Central Laboratory, Sasebo Chuo Hospital, Sasebo 857-1195; 4Department of Bacterial Laboratory, Nagaoka Red Cross Hospital, Nagaoka 940-2085; 5Department of Health and Food Safety, Ishikawa Prefectural Institute of Public Health and Environmental Science, Kanazawa 920-1154; and 6Critical Care Medical Center, 7Department of Neurology, 8Department of Infectious Diseases, and 9Clinical Laboratory, Niigata Municipal Hospital, Niigata 950-8739, Japan (Received June 20, 2006. October 23, 2006)

SUMMARY: Seven cases of Streptococcus suis infection in Japan during 1994 and 2006 were summarized. All cases had porcine exposure and five of them had hand skin injury during the exposure. Five cases presented symptoms of , three presented symptoms of , and one resulted in sudden death. All of the isolated S. suis belonged to Lancefield’s group D and to serotype 2. They were susceptible to G, ampicillin, cefotaxime, and ciprofloxacin. However, six of them were resistant to both erythromycin and clindamycin, and four were also resistant to minocycline. Multilocus sequence typing of six isolates showed that they belonged to sequence type (ST) 1, and their pulsed-field gel electrophoresis (PFGE) patterns were similar. The remaining isolate was ST28 and its PFGE pattern was distinct from those of the others.

Streptococcus suis is an important swine that has Gram staining was performed by the Bartholomew & been associated with cases of meningitis, endocarditis, Mittwer method. The Lancefield’s group was determined by arthritis, and other infections in (1). Thirty-five capsular using Streptex (Remel Inc., Lenexa, Kans., USA). API 20 serotypes based on capsular polysaccharides have been Strep Kit (bioMérieux, Marcy l’Etoile, France) was used for described for the bacterium, and the serotype 2 isolates are biochemical tests. The capsular types were determined most frequently associated with these diseases (2). The bac- by the Quellung method with Suis Type Antisera (Statens terium is also recognized as a zoonotic agent, with the first Serum Institut, Copenhagen, Denmark). Determination and human case of S. suis infection having been reported in 1968 homology searches of the 16S rDNA sequence were per- (3). Most patients have a contact history with infected pigs formed according to the instructions in MicroSeq (Applied or swine products. Clinical presentations in humans include Biosystems). acute bacterial arthritis, sepsis, meningitis, and so on (4,5). The antimicrobial susceptibility of the isolates to eight Vestibular dysfunction and/or permanent hearing loss are antibiotics was analyzed by the broth microdilution method commonly noted sequelae (4,6). S. suis infections in humans according to the Clinical and Laboratory Standards Institute have sporadically occurred without obvious seasonal change, (CLSI) (16,17) by using dry plates (Eiken Chemistry, Tokyo, and almost all of them are caused by serotype 2 isolates (7). Japan). The eight antibiotics were penicillin G, ampicillin, In China, two outbreaks caused by S. suis serotype 2 occurred cefotaxime, ciprofloxacin, minocycline, erythromycin, clindamy- in summer (8,9). The reasons why S. suis causes such out- cin, and telithromycin. Antimicrobial susceptibility breakpoints breaks are still unknown. In Japan, several sporadic cases were defined according to CLSI criteria, while those of in humans have been reported (10-14). In this study, we ciprofloxacin were the same as levofloxacin, and those of summarize the clinical features of these infection cases in telithromycin were from Streptococcus pneumoniae (17). To Japan, and perform a bacteriological analysis of the isolates. clarify the molecular mechanism of the drug resistance, the All nucleic acid manipulations were accomplished accord- presence of the ermB and mefA/E genes, which responded ing to the standard molecular biology techniques (15). Poly- to macrolide- and lincosamide-antibiotics resistance, was merase chain reaction (PCR) amplifications were carried out investigated by PCR as described previously (18). In addi- with Ex Taq polymerase (Takara, Otsu, Japan) by the tion to these two genes, we also investigated the presence of GeneAmp PCR System 9700 (Applied Biosystems, Foster point mutation(s) in rrl, rplD, and rplV, encoding 23S rRNA, City, Calif., USA). The DNA sequencing was performed with L4, and L22 ribosomal proteins, respectively. Primers were Genetic Analyzer 310 and Genetic Analyzer 3130 (Applied designed according to the sequences of S. pneumoniae (19). Biosystems). Molecular typing of the isolates was done by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing *Corresponding author: Mailing address: Department of Bacte- (MLST). PFGE was performed according to a method de- riology, National Institute of Infectious Diseases, Toyama 1-23- scribed previously (20). Restriction enzymes, SmaI (Takara) 1, Shinjuku-ku, Tokyo, 162-8640, Japan. Tel & Fax: +81-3-5285- and ApaI (Takara), were used for digestion of the genomic 1171, E-mail: [email protected] DNAs. Electrophoresis was carried out at 6 V/cm for 17 h,

397 with the pulse time ranging from 1.2 to 30 s. MLST was Sequences of the 16S rDNA of these isolates shared 99.4- performed and primers were designed as described by King 100% homology with that of the S. suis type strain ATCC et al. (21). We sequenced both strands of the amplicons and 43765, which also belongs to serotype 2. trimmed the sequences of the seven genes (aroA, cpn60, dpr, All of the seven S. suis isolates were susceptible to peni- gki, mutS, recA, and thrA). The allelic numbers and sequence cillin G (minimum inhibitory concentration [MIC], 0.015 - types of the isolates were determined by comparing their 0.06 μg/ml), ampicillin (MIC, 0.015 μg/ml), cefotaxime sequences with those in the database provided by King and (MIC, 0.03-0.06 μg/ml), and ciprofloxacin (MIC, 0.5-1 Whatmore (http://ssuis.mlst.net/). μg/ml). The six isolates other than SUIS2 were resistant to From 1994 to January 2006, seven cases of S. suis infec- erythromycin (MIC, ≥2 μg/ml) and clindamycin (MIC, ≥4 tions emerged in Japan. Six of them have been published as μg/ml); four (SUIS1, SUIS4, SUIS5, and SUIS6) of them case reports (10-14). The remaining case occurred in January were also resistant to minocycline (MIC, ≥8 μg/ml). All the 2006. The patient was a 56-year-old previously healthy six resistant isolates were positive for ermB, but negative for woman. She had come into contact with raw viscera from mefA/E in the PCR. SUIS2, which was isolated in 1994 (11), swine, and suffered from sepsis, disseminated intravascular carried neither the ermB nor the mefA/E genes. Among the coagulation (DIC), arthritis, and endophthalmitis. Blood six resistant isolates, four (SUIS4, SUIS5, SUIS6, and SUIS7) culture on admission yielded S. suis. After treatment with showed reduced susceptibility to telithromycin (MIC, 1-2 effective antibiotics, she recovered and had no aftereffects. μg/ml). It had been reported that telithromycin-resistant Clinical characteristics of these cases are summarized in S. pneumoniae possessed ermB, and showed a combination Table 1. The ages of the seven patients (4 males and 3 of mutations in the rrl, rplD, and/or rplV genes (18,22). females) ranged from 47 to 58 and the average age was 53.7. Therefore, the sequences of rrl (position 31 - 2891), rplD All of them had history of exposure to swine materials and (position 1-625), and rplV (position 1-345) of the S. suis five of them had skin injury on their hands during the isolates were determined. No difference was observed be- exposure. Five patients showed meningitis, which is the most tween the telithromycin-susceptible and the telithromycin- prevalent (75.2 -84.6%) symptom in the reported S. suis- reduced-susceptible isolates (data not shown). The molecular infected cases (7). Four patients suffered from DIC, three mechanism(s) for the reduced susceptibility to telithromycin from sepsis, and three from arthritis. Endophthalmitis and of these isolates is still unclear. epidural abscess, two unusual symptoms in S. suis infections PFGE analysis was performed and the SmaI-digested (7), were also present in these cases. Five patients who suf- profiles of S. suis are shown in Figure 1. The PFGE patterns fered from meningitis had deafness as a sequela. One patient of six isolates (SUIS1, SUIS2, SUIS3, SUIS4, SUIS5, and died suddenly; the symptoms of this patient were not recorded, SUIS6) were similar to each other; in particular, those of but a forensic autopsy revealed sepsis and DIC (11). The SUIS1, SUIS4, SUIS5, and SUIS6 were indistinguishable, mortality rate of the seven cases was 14.3%. Enteritis, pneu- although they were isolated in different years and/or regions monia, or endocarditis, which are frequently present in cases (10,13,14). However, the pattern of SUIS7 was clearly distinct of S. suis infection (7), were not observed in these patients. from those of the others (Fig. 1). SUIS7 was isolated from Patients with asplenia, diabetes mellitus, alcoholism, or the case occurring in January 2006, as described above. The malignancy are at high risk for S. suis infection (7), but none same result was obtained when restriction enzyme ApaI was of these underlying diseases was noted in the seven patients. used for digestion of the genomic DNAs (data not shown). Streptococcal etiological agents (SUIS1, SUIS2, SUIS3, The MLST revealed that SUIS1, SUIS2, SUIS3, SUIS4, SUIS4, SUIS5, SUIS6, and SUIS7) were isolated from indi- SUIS5, and SUIS6 belonged to sequence type (ST) 1, whereas vidual patients: four (SUIS1, SUIS4, SUIS5, and SUIS6) were SUIS7 belonged to ST28. isolated from cerebrospinal fluid (CSF) and the other three STs of six of the seven isolates in Japan were the same as from blood. All seven isolates were Gram-positive spindle- shaped cocci, belonged to Lancefield’s group D, and were identified as S. suis by using API 20 Strep Kit. All of the isolates reacted specifically with the anti-serotype 2 serum.

Table 1. Summary of clinical characteristics of patients with Streptococcus suis infection Cases reported (n = 7)1) Characteristics no. (%) Exposure history Porcine exposure 7 (100.0) Skin injury during exposure 5 ( 71.4) Type of infection Meningitis 5 ( 71.4) Sepsis 3 ( 42.9) Arthritis 3 ( 42.9) Endophthalmitis 2 ( 28.6) Epidural abscess 1 ( 14.3) Complication Deafness 5 ( 71.4) Fig. 1. PFGE profiles of the Streptococcus suis isolates from patients Death 1 ( 14.3) in Japan. Genomic DNAs of the isolates were digested with SmaI. 1): Data of six cases are adapted from references (10-14). Molecular size markers are indicated on the left.

398 ST1, which has been reported to be the most common ST of Med., 3, e151. S. suis, and were isolated in European nations, the United 10. Asatsuma, Y., Yanagida, Y., Hoshi, S., Matsunaga, K., States, and Hong Kong (21). Based on these observations, Yagi, K., Shibata, N. and Arakawa, Y. (2003): Two the ST1 isolates are recognized as a highly successful clone bacterial meningitis cases caused by Streptococcus suis. that arose relatively recently and has rapidly spread world- J. Jpn. Soc. Clin. Microbiol., 13, 105 (in Japanese). wide (21). 11. Endo, M., Okuno, R. and Yanagawa, Y. (2005): Identifition In this study, we summarized the clinical characteristics of of two Streptococcus suis strains isolated from blood of seven cases of S. suis infection in Japan, determined the patients. J. Jpn. Soc. Clin. Microbiol., 15, 110 (in Japa- serotypes and antimicrobial susceptibilities, and compared nese). PFGE patterns and STs of the isolates. Since all of the patients 12. Ibaraki, M., Fujida, N., Tada, M., Ohtaki, O. and Nagai, had come into contact with swine materials, enhanced pro- H. (2003): A Japanese case of Streptococcus suis men- tection during work will be the best defense against S. suis ingitis associated with lumbar epidural abscess. Clin. infection. The observed mortality rate of these cases was high Neurol., 43, 176-179 (in Japanese). (14.3%), and was comparable to those reported previously 13. Matsuo, H. and Sakamoto, S. (2003): Purulent menin- (13.2-20.2%) (7). Therefore, early diagnosis and adminis- gitis caused by Streptococcus suis in a breeder. J. Jpn. tration of appropriate antibiotics will be indispensable for Assoc. Infect. Dis., 77, 340-342 (in Japanese). effective treatment and reduction in mortality of S. suis 14. Sekiguchi, H., Honda, H., Miyajima, M., Tanaka, T., infections. 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