Escolar Documentos
Profissional Documentos
Cultura Documentos
7 Kentaro Kawatsua
8
a
9 Osaka Institute of Public Health, Osaka, Japan; bOsaka Shijonawate public Health
11 Sanitation Laboratory, Osaka, Japan; dOsaka Tondabayashi public Health Center, Osaka
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19
1
20 Abstract
21 In September 2016, 140 patients with primary symptoms of sore throat and fever were
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
33
34 Introduction
35 The genus Streptococcus consists of Gram-positive bacteria that form chains or pairs and
37 respiratory infections
38 (https://web.archive.org/web/20071219224215/http://www.cdc.gov/ncidod/dbmd/diseas
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
2
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
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
55
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.
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
64
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.
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68
70 Epidemiological investigation
71 The Osaka Institute of Public Health received a report from the Osaka Prefectural
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).
80
82 Samples
83 Specimens of blood and pharyngeal swabs from patients were collected. Detection of
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,
89
90 Cultures
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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
94 enrichment and selection medium. The medium, comprising 2.5% heat infusion broth
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,
105
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
112
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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
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
124
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
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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
141
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
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.
153
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
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.
161
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162 Results
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
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).
179
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
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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
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
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
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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
215
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
221
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
231
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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
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
243
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
251
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
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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,
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
276
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
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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
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
291 during storage is strongly suspected to be the cause of the spread of GGS to rice.
292
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.
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305
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
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.
312
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
317 symptoms; rather, fever and sore throat were the main symptoms. From the symptoms,
319 enteropathogenic bacteria and respiratory pathogenic bacteria. This case indicates the
322
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
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329
330 References
331
332 1. Lancefield RC. 1933. A serological differentiation of human and other groups of
336 3. Bisno AL, Collins CM, Turner JC. 1996. M proteins of group C streptococci
337 isolated from patients with acute pharyngitis. J Clin Microbiol 34:2511–2515.
338 4. Campo RE, Schultz DR, Bisno AL. 1995. M proteins of group G streptococci:
340 5. Carapetis JR, Steer AC, Mulholland EK, Weber M. 2005. The global burden of
342 6. Stevens DL. 1992. Invasive group A streptococcus infections. Clin Infect Dis
343 14:2–11
350 9. Lother SA, Jassal DS, Lagacé-Wiens P, Keynan Y. 2017. Emerging group C and
352 65:128–132
15
353 10. Tsakris A, Pournaras S, Hathi D, Douboyas J, Efstratiou A. 1999. Outbreak of
355 353:1585–1586.
363 streptococcal infection in Fukuoka Prefecture, Japan. Jpn J Infect Dis 67:321–322.
365 outbreak of group G streptococcal sore throat in an Israeli military base. Epidemiol
367 14. Hill HR, Caldwell GG, Wilson E, Hager D, Zimmerman RA. 1969. Epidemic
369 15. Zaoutis T, Attia M, Gross R, Klein J. 2003. The role of group C and group G
371 16. Lindbaek M, Høiby EA, Lermark G, Steinsholt IM, Hjortdahl P. 2005.
372 Clinical symptoms and signs in sore throat patients with large colony variant
374 55:615–619.
375 17. Watsky KL, Kollisch N, Densen P. 1985. Group G Streptococcal bacteremia: The
376 clinical experience at Boston university medical center and a critical review of the
16
377 Literature. Arch Intern Med 145:58–61.
378 18. Lopardo HA, Vidal P, Sparo M, Jeric P, Centron D, Facklam RR, Paganini H,
379 Pagniez NG, Lovgren M, Beall B, the Argentinian Streptococcus Study Group.
383 19. Ekelund K, Skinhøj P, Madsen J, Konradsen HB. 2005. Invasive group A, B, C
389 21. Vartian C, Lerner PI, Shlaes DM, Gopalakrishna KV. 1985. Infections due to
391 22. Sato M. 1972. A new selective enrichment broth for detecting beta-hemolytic
392 streptococci in throat cultures: quinoline derivate and three percent salt as an
397 24. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH,
399 produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J
17
401 25. ISO 7128:1996 Microbiology of food and animal feeding stuffs- General
403 26. Facklam R. 2002. What happened to the streptococci: overview of taxonomic and
408 28. Kittang BR, Skrede S, Langeland N, Haanshuus CG, Mylvaganam H. 2010.
409 emm gene diversity, superantigen gene profiles and presence of SlaA among
410 clinical isolates of group A, C and G streptococci from western Norway. Eur J Clin
416 30. Gallo G, Berzero R, Cattai N, Recchia S, Orefici G. 1992. An outbreak of group
419 KEP, Mølbak K. 2007. Outbreak of group A streptococcal throat infection: don't
421 32. Levy M, Johnson CG, Kraa E. 2003. Tonsillopharyngitis caused by foodborne
423 33. Decker MD, Lavely GB, Hutcheson RH, Schaffner W. 1985. Food-borne
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425 34. Kaplan EL, Huew BB. 1980. The sensitivity and specificity of an agglutination
427 and comparison of the Streptozyme test with the anti-streptolysin O and
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430
No. patients %
Clinical symptom 140
Sore throat 125 89.3
Fever 118 84.3
Cooked rice
stG652.0 and stC36.0 Broccoli salad
1 patient
8 patients
stG652.0
1 cook
stC36.0 10 patients
434
435
436
437
20
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