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JCM Accepted Manuscript Posted Online 28 February 2018

J. Clin. Microbiol. doi:10.1128/JCM.01884-17

Copyright © 2018 American Society for Microbiology. All Rights Reserved.

1 Food-Borne Outbreak of Group G Streptococcal Pharyngitis in a School

2 Dormitory in Osaka, Japan

4 Takahiro Yamaguchia#, Ryuji Kawaharaa, Chihiro Katsukawaa, Masashi Kankia,

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5 Tetsuya Haradaa, Shinya Yonogia, Satomi Iwasakib, Hirokazu Ueharac, Saori Okajimab,

6 Hiroshi Nishimuraa, Kazushi Motomuraa, Masaya Miyazonod, Yuko Kumedae and

7 Kentaro Kawatsua

9 Osaka Institute of Public Health, Osaka, Japan; bOsaka Shijonawate public Health

10 Center, Osaka Prefectural Government, Osaka, Japan; cOsaka Prefectural Market

11 Sanitation Laboratory, Osaka, Japan; dOsaka Tondabayashi public Health Center, Osaka

12 Prefectural Government, Osaka, Japan; eResearch Center of Microorganism Control,

13 Osaka Prefecture University, Osaka, Japan


15 Running Title: Food-Borne Outbreak of Group G Streptococcus


17 #Address correspondence to Takahiro Yamaguchi, yamaguchi@iph.osaka.jp

18 Takahiro Yamaguchi and Ryuji Kawahara contributed equally to this work.


20 Abstract

21 In September 2016, 140 patients with primary symptoms of sore throat and fever were

22 identified in a school dormitory in Osaka, Japan. Epidemiological and laboratory

23 investigations determined that this symptomatic condition was a food-borne outbreak of

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24 group G Streptococcus (GGS), with GGS being isolated from samples from patients,

25 cooks and foods. The strain of GGS was identified as Streptococcus dysgalactiae subsp.

26 equisimilis (SDSE) of two emm types (stG652.0 and stC36.0). The causative food, a

27 broccoli salad, was contaminated with the two types of SDSE, totaling 1.3×104 CFU/g.

28 Pulsed-field gel electrophoresis (PFGE) of samples from patients, cooks and foods

29 produced similar band patterns among samples with the same emm type. This result

30 suggested the possibility of exposure from the contaminated food. The average onset

31 time was 44.9 hours and the prevalence rate was 62%. This is the first report to identify

32 the causative food of a food-borne outbreak by SDSE.


34 Introduction

35 The genus Streptococcus consists of Gram-positive bacteria that form chains or pairs and

36 are catalase negative. β-hemolytic streptococci are known causative bacteria of

37 respiratory infections

38 (https://web.archive.org/web/20071219224215/http://www.cdc.gov/ncidod/dbmd/diseas

39 einfo/groupastreptococcal_g.htm). β-hemolytic streptococci are classified according to

40 the polysaccharide antigen in the cell wall (1). The main β-hemolytic streptococci that

41 cause pharyngitis are group A (GAS), C (GCS) and G (GGS). Numerous superficial

42 antigenic factors have been described for GAS, GCS and GGS. Among these, the M

43 protein is type-specific and is widely used as an epidemiological marker (2-4).

44 Streptococcus pyogenes (GAS) is a widely prevalent bacterial pathogen whose global

45 distribution reflects the fact that humans constitute its primary biological host (5). In

46 most cases GAS infects shallow tissue sites, including the upper respiratory tract

47 mucosal epithelium or the epidermal layer of the skin, and causes pharyngitis or

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48 impetigo. GAS are monitored at approximately 3,000 cooperating hospitals, and over

49 250,000 cases of GAS pharyngitis are reported annually in Japan

50 (https://www.niid.go.jp/niid/en/aboutniid-2/865-iasr/5866-tpc426.html). S. pyogenes

51 can also cause skin and soft tissue infection, septic arthritis, bacteremia, and

52 endocarditis (6). Although GAS causes most streptococcal infections, GCS- and

53 GGS-mediated infections have also been reported(7-9). On the other hand, the

54 surveillance of β hemolytic streptococci excluding GAS is not sufficient.


56 Streptococci, including GAS and GGS, cause food-borne outbreaks (10-14). Although

57 GAS food-borne outbreaks are rare, large-scale outbreaks exceeding 100 patients have

58 been reported, requiring risk management. Similar to GAS, GGS also causes

59 pharyngitis and STSS (15-19). GGS, which include Streptococcus dysgalactiae subsp.

60 equisimilis (SDSE) and Streptococcus dysgalactiae subsp. dysgalactiae, are common

61 components of the normal flora of human skin, pharynx, and gastrointestinal tract (20,

62 21). Food-borne outbreaks caused by GGS are also rare (13, 14) and have not been

63 reported by isolation from causative foods.


65 Here, we provide the first report of a case of food-borne outbreak caused by GGS in

66 Osaka, Japan, in September 2016, that was identified by isolating SDSE from the

67 causative food.


69 Materials and Methods

70 Epidemiological investigation

71 The Osaka Institute of Public Health received a report from the Osaka Prefectural

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72 Affairs Division on September 12, 2016 that students of a vocational training school

73 had developed symptoms of fever and pharyngitis. To determine whether these

74 symptoms were due to an infectious disease or food-borne outbreak, we conducted an

75 epidemiological investigation comprising interviews regarding patients’ activities and a

76 health survey to identify foods eaten, symptoms, and onset date for all students, teachers

77 and cooks. This school had a dormitory, and the investigation also included a survey

78 about meals eaten by students in the dormitory (see Table S1 in the supplemental

79 material).


81 Environmental and laboratory investigation

82 Samples

83 Specimens of blood and pharyngeal swabs from patients were collected. Detection of

84 Streptococcus was conducted by evaluating pharyngeal swabs from 25 patients and 11

85 cooks, samples from 69 meals (supplied for five days, three meals a day) and drinking

86 water, and 11 samples collected by swabbing various parts of the kitchen. All food

87 samples that were stored frozen after cooking were collected and examined. In addition,

88 blood specimens and pharyngeal swabs were examined for viruses.


90 Cultures

91 Pharyngeal swabs were inoculated onto blood agar and incubated at 35°C under

92 microaerophilic conditions overnight. Each of the food samples (2 g) and swabs were

93 incubated in 10 mL of Q medium at 35°C overnight (22). Q medium was used as the

94 enrichment and selection medium. The medium, comprising 2.5% heat infusion broth

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95 (BD), 0.5% tryptose (DB), 0.5% yeast extract (BD), 2.8% sodium chloride, 5.0 mL of

96 0.1% aqueous sodium azide solution (per 100 mL) and 0.22 mL of aqueous crystal

97 violet solution (per 100 mL), was autoclaved at 121°C for 15 min. The pH was adjusted

98 to 7.0 using a 10% sodium hydroxide solution. Defibrinated horse blood was added to

99 make a final concentration of 5%. Cultures of hemolytic colonies that were confirmed to

100 be catalase-negative were used for confirmation of the bacteria’s Lancefield group using

101 a Seroiden® Strepto Kit (Eiken Chemical Co., Ltd., Japan). Bacterial isolates were

102 identified using Rapid ID32 Strep (bioMerieux, Marcy l’Etoile, France). Trypticase soy

103 agar with 5% sheep blood (Becton, Dickinson and Company (BD), Franklin Lakes, NJ,

104 USA) was used as blood agar for isolation of SDSE.


106 M protein gene (emm) typing

107 For DNA extraction, colonies were picked from the blood agar, suspended in 100 μL of

108 Tris-EDTA buffer in a 1.5-mL tube, and heated at 95°C for 10 minutes on a heat block.

109 The suspension was centrifuged at 8,500 g for 5 minutes and the supernatant was used as

110 the DNA template. emm typing was carried out according to the Centers for Disease

111 Control and Prevention (CDC) method (https://www.cdc.gov/streplab/index.html).


113 Pulsed-field gel electrophoresis

114 All isolates were characterized by pulsed-field gel electrophoresis (PFGE) after DNA

115 digestion with SmaI (Roche Diagnostics GmbH, Mannheim, Germany) using a

116 CHEF-DR system (Bio-Rad, CA) (23). The DNA fragments were separated on 1%

117 Seakem® Gold agarose gel (Lonza, Basel, Switzerland) in 0.5× Tris-Borate-EDTA

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118 (TBE). The electrophoresis conditions were as follows: block 1 was run at 12°C and 6

119 V/cm for 11 h with time intervals between 5 and 15 s, and block 2 was run at 12°C and

120 6 V/cm for 8.5 h with time intervals between 5 and 45 s. The DNA of Salmonella

121 enterica serovar Braenderup strain H9812 was digested with XbaI and used as the

122 molecular marker for PFGE analysis. PFGE patterns were interpreted according to the

123 criteria described by Tenover et al (24).


125 Determination of the amount of SDSE in causative food

126 The number of SDSE in food was calculated by direct plate colony counting and the most

127 probable number (MPN) method. Broccoli salad and rice were examined using the

128 three-tube and five-tube methods, respectively. In direct plate colony counting, 5–10 g of

129 SDSE-positive samples was added to a 10-fold volume of Heart Infusion (HI) broth and

130 stirred. A portion of this sample (1 mL) was used for direct plate colony counting.

131 Mixtures were diluted 10–1000-fold and applied to Columbia CNA with 5% sheep blood

132 agar (CNA agar) (BD). After incubation at 35°C for 22±2 hours, colonies were counted to

133 calculate the number of colony-forming units per gram (CFU/g). For the MPN method,

134 the remaining SDSE sample solution in HI broth was diluted in a series of decimal

135 dilutions with HI broth and incubated at 35°C for 6 hours for recovery of SDSE. We

136 previously prepared modified Q medium containing reduced HI broth so that the required

137 Q medium composition would be obtained upon addition of the sample solution. The

138 modified Q medium was added to each dilution solution, and incubated at 35°C for 22±2

139 hours. MPN was calculated with reference to the International Organization for

140 Standardization (25)


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142 Growth of SDSE in food

143 To confirm growth of SDSE in food, a pseudo-positive sample of broccoli salad was

144 cooked using the same recipe as that of the causative food, and the isolates were

145 inoculated at estimated concentrations of 100–1000 CFU/g. Pseudo-positive samples

146 were frozen immediately after cooking, thawed and used for analysis. The average

147 temperature in Osaka at the time of the outbreak (September 2016) was 25.6°C. Given

148 that the temperature was likely even higher in the kitchen during food preparation, we

149 conducted our experiment at 30°C. Samples taken from these foods were incubated at

150 30°C, and cultured samples were picked after 2, 4, 8, 12 and 24 hours in triplicate. The

151 picked samples were diluted with sterile saline, applied to CNA blood agar, incubated at

152 35°C for 22±2 hours, and used to calculate the colony count.


154 Measure of ASO titers

155 Anti-streptolysin O (ASO) antibody titer was measured using sera from 10 patients at

156 onset and 1 month after the outbreak. ASO titers were measured using a Latex

157 agglutination turbidimetric immunoassay by LSI Medience Corporation (Tokyo, Japan).

158 Collection of sera was approved by the ethics review committee of the Osaka Institute of

159 Public Health. ASO titers at the time of the outbreak were compared with that collected 1

160 month after the outbreak in each of the 10 patients using a paired t-test.


162 Results

163 Epidemiological investigation

164 The affected school was a vocational training school where students were in attendance

165 for six months out of the year. Students were aged 18 to 31 years old. All students

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166 stayed in the dormitory from Monday to Friday and almost all students ate the same

167 meals. Our interviews revealed that many patients had symptoms of sore throat and

168 fever. The doctor who examined the patients also received reports of suspected

169 streptococcal infection. The dormitory consisted of three floors, and four to six students

170 lived in one room. Among 227 students and two teachers, 140 people developed mainly

171 pharyngitis, fever and headache from September 9-13. The epidemiological

172 investigation revealed no bias among dormitory rooms, floors or groups. One hundred

173 and forty patients had clinical symptoms such as sore throat (89.3%), fever (84.3%),

174 headache (46.4%), malaise (38.6%), arthralgia (36.4%), chills (27.9%), diarrhea

175 (11.4%), nausea (6.4%), and stomachache (3.6%) (Table 1). The causative food was a

176 broccoli salad with soy sauce (broccoli salad), which was served on September 9 during

177 morning meals and eaten by most of the patients. The occurrence was unimodal (Fig. 1).

178 The mean progression time was 44.9 hours.


180 Our environmental and epidemiological investigations indicated that the broccoli salad

181 had been contaminated. The broccoli salad was prepared using 16.45 kg of frozen

182 broccoli, 235 g of dried bonito shavings, and 0.7 L of soy sauce and cooked as follows:

183 frozen broccoli was placed in the refrigerator at noon on the previous day to thaw; the

184 broccoli was boiled for 10 min from 5 am, cooled under tap water and the water was

185 drained; the dried bonito shavings were toasted in a frying pan for 20 min; and the

186 broccoli, dried bonito shavings and soy sauce were then mixed in the kitchen at room

187 temperature from 6 am. The broccoli salad was served for breakfast at 8 am. The

188 examined Broccoli salad was that had left at room temperature for 2 hours or more

189 before storing in a freezer.

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191 Environmental and laboratory investigation

192 Culture

193 We inspected the patient’s specimens for Legionella, Bordetella pertussis, hemolytic

194 streptococcus and viruses of respiratory disease. Because measles was prevalent in

195 Osaka at the time of this case, we also conducted an examination for the measles virus.

196 Real-time PCR was used to detect viruses of respiratory disease (parainfluenza viruses

197 1–4, respiratory syncytial virus, human metapneumovirus, enterovirus/rhinovirus,

198 human bocavirus, human parechovirus, adenovirus, and human coronavirues OC43,

199 NL63, 229E and HKU-1) and the measles virus (18, 19). DNA of Legionella and B.

200 pertussis were extracted using a QIAamp DNA Mini kit (Qiagen, Hilden, Germany),

201 and loop-mediated isothermal amplification (LAMP) was conducted using Loopamp

202 Legionella Detection Kit C and Loopamp Bordetella pertussis Detection Kit D (Eiken

203 Chemical Co., Ltd., Tokyo, Japan). Viral genes of respiratory infection viruses were

204 detected in some specimens but were not common pathogens in patients; bacterium of

205 the genus Legionella and B. pertussis and the measles virus were not detected in

206 patients. We therefore concluded that these pathogens were not the cause of the

207 outbreak. In addition, because the doctor’s diagnosis strongly suggested streptococcal

208 infection, patient’s specimens were cultured on blood agar medium. Cultures of

209 pharyngeal swab samples on blood agar medium revealed β-hemolytic colonies. Further

210 examination of the β-hemolytic colonies led to the isolation of GGS from 19 out of 25

211 patients, 1 of 11 cooks, and 2 of 69 foods. Drinking water and 11 swab samples from

212 various parts of the kitchen were negative. GGS was isolated from the patients, a cook,

213 and from broccoli salad and cooked rice served for breakfast on September 9. All

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214 isolated GGS were identified as SDSE.


216 Molecular typing

217 Two kinds of SDSE with different emm types were isolated from foods, patients and a

218 cook (stC36.0, stG652.0). SDSE of both emm types were isolated from the foods and

219 one patient, while SDSE of one or the other emm type was found in 18 patients and the

220 cook (Table 2).


222 PFGE analysis requires digesting genomic DNA with a restriction enzyme,

223 electrophoresing the fragmented DNA molecules, and comparing the band patterns.

224 This analysis was used to estimate the difference in strains according to the DNA band

225 patterns. PFGE produced unique band patterns among samples isolated from patients,

226 the cook and foods for each of the two emm types (stG652.0 and stC36.0). All DNA

227 band patterns of SDSE stG652.0 isolated from foods, patients and a cook were the same.

228 Although we observed two DNA band patterns for stC36.0 isolated from foods and

229 patients, these patterns differed by only one band. We therefore regarded these two

230 patterns as belonging to the same strain (24).


232 Estimation of the amount of SDSE in the causative foods

233 Direct plate counting showed that cooked rice contained <10 CFU/g while the broccoli

234 salad contained 1.3×104 CFU/g. The MPN method showed that cooked rice had 3.3

235 MPN/g (95% confidence limits: 1.1–8.9 MPN/g), while the broccoli salad contained

236 4600 MPN/g (95% confidence limits: 900–19800 MPN/g).

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238 Growth of SDSE in food

239 Additional experiments were conducted to confirm the growth of SDSE in broccoli

240 salad. The two SDSE emm types were added to broccoli salad and the resulting growth

241 of both SDSEs was observed. The results confirmed that SDSE proliferates even in

242 vegetables (Fig. 3).


244 Measure of ASO titers

245 ASO is an antibody against streptolysin-o, a representative extracellular product

246 produced by β-hemolytic streptococci of groups A, C, and G, and is used as an infection

247 index. ASO titer was measured in sera collected from SDSE-positive patients at the

248 time of the outbreak and after 1 month. Analysis using the paired t-test showed that the

249 ASO titer was significantly higher one month after the outbreak than at the time of the

250 outbreak (p-value < 0.01; data not shown).


252 Discussion

253 We describe a food-borne outbreak of GGS from a school dormitory meal. Similar

254 cases have been reported from an Israeli military base and an American college (13, 14),

255 and were caused by meals served in dormitories or school cafeterias, as in the present

256 case. The incidence rate of these previous reports was 46% and 31%, respectively. In

257 contrast, the incidence rate in the present study was 62%, which is much higher than

258 that in previous reports. We speculate that this may be due to the large amount of

259 bacteria in the contaminated food. The incubation period in the current case was 2 days,

260 which is the same as that in the previous cases.

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262 emm typing revealed that two types of SDSE (stG652.0, stC36.0) were present in the

263 food samples and one patient, while one or the other type was found in 18 patients and

264 one cook (Table 2). Eight patients had stG652.0, while 10 patients had stC36.0. Patient

265 symptoms were similar for the two SDSE types, suggesting that both types of SDSE

266 were the causative bacteria. The PFGE band patterns of the emm types isolated in the

267 present study differed from those from other clinical isolates (Fig. 2a, b). Isolates No.

268 24, 25, 26 differed from all other patterns by only one band. Tenover et al. described

269 that the PFGE pattern had a difference of one to three bands at the time of outbreak (24).

270 Therefore, we concluded that all isolates of stC36.0 were of the same strain. All isolates

271 containing stG652.0 produced the same band pattern. SDSE is known to be part of the

272 normal flora of the skin and oral cavity (26). Both stG652.0 and stC36.0 have been

273 isolated from invasive or noninvasive GGS infections globally (18, 27-29), and are

274 therefore not unique. Reports have indicated that multiple emm types cause

275 GAS-mediated food-borne outbreaks (30, 31).


277 Broccoli salad was contaminated with 1.3×104 CFU/g SDSE. GAS food-borne

278 outbreak cases have mainly been reported in military bases and schools, in which the

279 infected foods were salads containing eggs (13, 14, 32). This is similar to the present

280 case, where the outbreak occurred at a vocational training school. In GGS- and

281 GAS-mediated food-borne outbreaks, the cook or a patient is often reported as a carrier.

282 Cases of causative “dressing” have also been reported (33), in which the cook was also

283 a carrier of GAS. The one SDSE-positive cook in the present study ate but did not cook

284 the contaminated foods, indicating that the cook was not the source of the

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285 contamination. Moreover, cooked rice was contaminated with low concentrations of

286 GGS in the present study. From our investigation of the cooking facility,

287 cross-contamination between the cooked rice and broccoli salad was unlikely to have

288 occurred during the cooking process. However, the possibility of cross-contamination

289 during storage remains because the two foods were frozen and stored in the same box

290 wrapped in cellophane for post inspection of meals. Therefore, cross-contamination

291 during storage is strongly suspected to be the cause of the spread of GGS to rice.


293 We considered the broccoli salad, which contained a small amount of dried bonito

294 shavings, to be a uniquely Japanese dish. Moreover, there are limited reports on the

295 proliferation of SDSE in food, and the amount of SDSE required to cause a food-borne

296 outbreak is unknown. Therefore, our growth examination confirmed that SDSE

297 proliferates even in vegetables (Fig. 3). We estimated that the amount of SDSE in the

298 contaminated food was 1,000 CFU/g, and confirmed that it had reached more than

299 10,000 CFU/g in about 4 hours. This high level of SDSE in broccoli salad suggests that

300 the food had been stored at room temperature after cooking. It also indicates that

301 contaminated food requires only a short period of time to accumulate the amount of

302 bacteria required to cause a food-borne outbreak. This result indicates that it is

303 necessary to store cooked food at low temperatures to prevent a food-borne outbreak of

304 SDSE.


306 ASO titer is an indicator of streptococcal infection (34). ASO has been reported to

307 continue to rise 3 to 5 weeks after infection. ASO titer was measured in sera collected

308 from 10 patients at onset and 1 month after the outbreak. Values exceeding the

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309 reference titer would suggest that the patient was infected with hemolytic streptococci.

310 All patient ASO titers were significantly elevated 1 month after the outbreak (Fig. 4),

311 suggesting that the infections occurred from the same exposure.


313 This study is the first to report a case of food poisoning caused by GGS in Japan, and

314 revealed that SDSE is able to grow in broccoli salad. Our findings indicate that cooked

315 foods need to be protected from contamination by SDSE. A food-borne outbreak caused

316 by Streptococcus was considered unlikely because there were no gastrointestinal

317 symptoms; rather, fever and sore throat were the main symptoms. From the symptoms,

318 we considered that we could respond quickly by examining both general

319 enteropathogenic bacteria and respiratory pathogenic bacteria. This case indicates the

320 importance of investigating the possibility of a food-borne outbreak in cases of

321 epidemic respiratory symptoms.


323 Acknowledgments

324 We wish to thank the members of the Shijonawate Health Center and Division of Food

325 Safety Promotion of Osaka Prefecture for help with the epidemiological investigation

326 and sampling. We also thank the members of the Division of Microbiology, Osaka

327 Institute of Public Health for help with the laboratory investigation, and Dr. K. Tomono

328 for clinical advice.


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Table 1. Clinical symptoms of patients falling ill.

No. patients %
Clinical symptom 140
Sore throat 125 89.3
Fever 118 84.3

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Headache 65 46.4
Malaise 54 38.6
Arthralgia 51 36.4
Chills 39 27.9
Diarrhea 16 11.4
Nausea 9 6.4
Stomachache 5 3.6

Table 2. SDSE emm type from specimens.

Isolated SDSE emm type Source

Cooked rice
stG652.0 and stC36.0 Broccoli salad
1 patient
8 patients
1 cook
stC36.0 10 patients

431 Figure legends

432 Fig. 1 Number of patients between September 9-15, 2016.

433 Fig. 2 Growth of SDSE in broccoli salad.





Downloaded from http://jcm.asm.org/ on March 6, 2018 by FUDAN UNIVERSITY
Downloaded from http://jcm.asm.org/ on March 6, 2018 by FUDAN UNIVERSITY