中国学校卫生

一起学校F型产气荚膜梭菌食物中毒事件分析

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郁旷明 , 彭健 , 杨素青 , 方叶珍 , 黄世旺 ^* , 蒋思清 , 王书菁 , 金轶卉

中国学校卫生 | 疾病控制 2025,46(10): 1493-1497

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中国学校卫生 |疾病控制 2025 , 46 (10) : 1493 -1497

一起学校F型产气荚膜梭菌食物中毒事件分析

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郁旷明, 彭健, 杨素青, 方叶珍, 黄世旺^*, 蒋思清, 王书菁, 金轶卉

作者信息

杭州市上城区疾病预防控制中心(杭州市上城区卫生监督所)，浙江 310004

通讯作者:

黄世旺，E-mail：hshw2003@163.com

作者简介:

郁旷明(1978-)，男，浙江杭州人，大学本科，副主任医师，主要从事学校食源性疾病防控工作

Analysis of food poisoning event by type F Clostridium perfringens in a school

Kuangming YU, Jian PENG, Suqing YANG, Yezhen FANG, Shiwang HUANG^*, Siqing JIANG, Shujing WANG, Yihui JIN

Affiliations

Hangzhou Shangcheng District Center for Disease Control and Prevention (Hangzhou Shangcheng District Health Supervision Institution), Hangzhou 310004, Zhejiang Province, China

出版时间: 2025-10-25 doi: 10.16835/j.cnki.1000-9817.2025296

文章导航

摘要

收起

目的

了解浙江省杭州市某校发生的一起食物中毒事件的流行病学及病原学特征，为相关突发性事件的科学处置提供参考依据。

方法

通过流行病学调查确定2024年5月浙江省杭州市某校一起食物中毒事件，开展病例对照研究以查找可疑食物，采用卫生学调查发现风险因素，采集患者和从业人员肛拭、食堂留样、环节涂抹等标本进行病原体快速初筛，根据筛查结果进行可疑病原体产气荚膜梭菌(CP)分离鉴定，对分离菌株进行毒素基因检测和全基因组测序分析。

结果

此次食物中毒事件共造成45人出现腹泻、腹痛等胃肠道症状，可疑食物为5月29日中餐学生食堂番茄炒蛋和玉米排骨。卫生学调查发现，该食堂在食物加工、制备、贮存环节存在污染风险。46份肛拭和10份食堂留样CP 16 S阳性，27份样本中分离出59株CP，10例病例和1名从业人员分离株的CPE(cpe)阳性(F型)、全基因组进化分析为同一来源。

结论

该起食物中毒事件由携带CPE(cpe)的F型CP感染引起，从业人员带菌为可能的暴发源头。建议食堂从业人员应加强培训并进行常见食源性病原体的定期监测。

关键词

梭菌，产气荚膜 / 食物中毒 / 基因 / 学生

Abstract

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Objective

To understand the epidemiological and etiological characteristics of food poisoning event occurred in a school in Hangzhou, Zhejiang, so as to provide reference for the scientific management of related emergencies.

Methods

By determining the nature of the event through epidemiological investigation, a case-control study was carried out to spot suspicious food in May 2024. The hygienic investigation was conducted to find out possible pollution links and factors, patients and canteen practitioners' anal swab, canteen retention samples, catering link daub and other specimens were collected, for rapid pathogen screening. And the suspected pathogen Clostridium perfringens (CP) were isolated and identified according to the screening results, and toxin gene detection and whole genome sequencing and cluster analysis of CP isolated strains were carried out.

Results

The incident resulted in 45 people experiencing gastrointestinal symptoms, such as diarrhea and abdominal pain. The suspicious food was tomato scrambled eggs and corn ribs provided by the student canteen for lunch on May 29. A hygiene investigation found that there was a risk of contamination in the food processing, preparation and storage. A total of 46 anal swabs and 10 canteen retention samples were positive for CP 16 S, 59 strains of CP were isolated from 27 samples, 10 cases and 1 practitioner isolate were positive for CPE (cpe) (F mode), and their whole genome evolution analysis was conducted based on the same source.

Conclusions

The food poisoning event is caused by CP infection carrying CPE (cpe) (F mode), and the possible sources of outbreak are the carriers of the CP by employees. It is recommended that cafeteria staff strengthen training on common foodborne diseases and conduct regular monitoring of pathogens.

Key words

Clostridium perfringens / Food poisoning / Genes / Students

引用本文

郁旷明, 彭健, 杨素青, 方叶珍, 黄世旺, 蒋思清, 王书菁, 金轶卉. 一起学校F型产气荚膜梭菌食物中毒事件分析. 中国学校卫生, 2025 , 46 (10) : 1493 -1497 . DOI: 10.16835/j.cnki.1000-9817.2025296

Kuangming YU, Jian PENG, Suqing YANG, Yezhen FANG, Shiwang HUANG, Siqing JIANG, Shujing WANG, Yihui JIN. Analysis of food poisoning event by type F Clostridium perfringens in a school[J]. Chinese Journal of School Health, 2025 , 46 (10) : 1493 -1497 . DOI: 10.16835/j.cnki.1000-9817.2025296

正文

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产气荚膜梭菌(Clostridium perfringens，CP)是导致食源性疾病暴发的重要病原菌之一^[1-4]，美国CP食物中毒事件每年影响约100万人并造成3.1亿美元的经济损失^[2]，英国8%~12%的胃肠道疾病与CP感染密切相关^[3]。我国也时有由CP引起的学校食源性疾病暴发事件^[5]。CP是一种培养条件苛刻的专性厌氧芽孢梭菌，常以共生菌形式存在于健康人体肠道中^[6]，在处理暴发事件时常被忽略导致漏检。随着各级实验室专性厌氧仪器的配备和荧光定量聚合酶链式反应(polymerase chain reaction, PCR)、高通量测序(next generation sequencing，NGS)等分子生物学技术的不断进步，近几年我国由CP感染引起的食源性疾病暴发事件的报道明显增多^[7-9]。

2024年5月，浙江省杭州市某高级中学发生一起聚集性腹泻事件。在流行病学调查、统计学分析、病例对照研究等的基础上，采用巢式多重PCR、荧光PCR、靶向高通量测序技术(target next generation sequencing，tNGS)确定CP致病因子，使用专性厌氧设备分离阳性菌株，对CP分离株全基因组测序、构建系统发育树进行溯源分析，证实为一起由F型CP感染引起的学校食物中毒事件，同时该事件也是浙江省报道的首起由CP感染引起的食源性疾病暴发事件，现报道如下。

1　对象与方法

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1.1　对象

2024年5月30日15：00，杭州市某区疾病预防控制中心(卫生监督所)接到属地某三甲医院报告，该院当日13：00—15：00陆续接诊3例腹泻、腹痛患者，均为某校学生，有共同就餐史。区疾病预防控制中心(卫生监督所)接报后，立即派出专业技术人员联合区市场监管局赶赴涉事学校开展联合调查。本次事件的搜索病例定义为：该校所有在校生、教职工、后勤人员。2024年5月28—30日，在学校食堂就餐后一天内出现腹泻≥3次，同时伴腹痛、腹胀、恶心、呕吐、发热(≥37.5℃)等症状之一者为临床诊断病例; 同时在肛拭/粪便中培养或PCR方法检测到相关病原体为实验室确诊病例; 另将一天腹泻 < 3次、流行病学调查信息不全或已确诊其他病原体的个体排除于本次病例统计范围; 共搜索发现45例病例。

1.2　方法

1.2.1　病例搜索及调查

通过询问、查阅学生因病缺课登记及教职工、后勤人员请假记录等方式在全校展开病例搜索，对搜索到的病例开展个案调查。使用《食物中毒事故个案调查登记表》^[10]，调查内容包括病例基本信息、发病症状、就诊情况和进餐史等。由现场流调人员进行电话询问或面对面调查。

1.2.2　现场卫生学调查

2024年5月30—31日对该校食堂进行现场卫生学调查，向食堂从业人员询问食材原料来源以及运输情况，对厨房卫生状况进行初步评估。同时调取5月29—30日食堂监控录像，查看食物加工、制作、贮存等环节是否存在风险因素。

1.2.3　样本采集

共采集各类样本252件，包含25名发病学生、27名食堂从业人员和4名咖啡吧服务员的肛拭标本，168份食堂留样、2份咖啡吧在售食品、8份教室直饮水、18份餐饮环节涂抹样本。

1.2.4　实验室检测

1.2.4.1　可疑病原体初筛

(1) Filmarray检测：挑选5例症状典型发病学生的肛拭子振荡混匀，各吸取200 μL放入同一离心管中混合，采用Filmarray GI Panel(生物梅里埃公司，RFIT-ASY-0116)22种胃肠道感染检测芯片进行巢式多重PCR检测。(2)荧光PCR筛查：将上述5份肛拭标本分别提取核酸进行其他相关胃肠道病原体荧光PCR筛查。

1.2.4.2　样本CP

对剩余247份样本进行CP 16 S种属基因荧光PCR检测。对CP 16 S阳性样本进行6种毒素基因检测，包括α毒素(plc/cpa基因)、β毒素(cpb基因)、ε毒素(etx基因)、ι毒素(iap和ibp基因)、肠毒素(clostridium perfringens enterotoxin，CPE)(cpe基因)和坏死性肠炎B毒素(Necrotic enteritis B-like toxin，NetB)(netB基因)。

1.2.4.3　CP菌株分离、毒素基因鉴定

按食品微生物学检验产气荚膜梭菌检验(GB 4789.13—2012)^[11]对CP 16 S阳性的肛拭标本和食堂留样进行厌氧培养分离。对CP分离株进行6种毒素基因检测。

1.2.4.4　菌株全基因组测序及组装

将菌株纯化培养后使用TIANamp Bacteria DNA Kit(T IAN GEN，DP302)试剂盒提取细菌全基因组DNA，使用Enzymic Universal DNAseq Library Prep Kit(KAITAI-BIO，AT4107)建库试剂盒进行文库构建，在Illumina NovaSeq 6000平台进行全基因组测序，对测序得到的原始数据进行质控(删除接头及碱基质量较低的测序读长后得到clean data，使用FastQC v0.12.1软件检测clean data的质量，使用Unicycler v0.4.8软件进行de novo组装^[12]，使用checkM v1.2.2软件检测组装后的基因组质量^[13]。

1.2.4.5　基因组溯源分析

以CPS 2a(NZ_CP075903.1)作为参考菌株，使用snippy(https://github.com/tseemann/snippy)将全基因组测序原始数据进行比对获得单核苷酸突变位点(single nucleotide variants, SNVs); 将SNVs位点连锁后，使用RAxML软件构建基因组系统发育进化树(模型为GTRGAMM，Bootstrap=1 000)^[14]，并采用GGTREE软件对系统发育树进行可视化^[15]。

1.3　统计学分析

采用Excel 2003软件建立数据库，使用SPSS 17.0软件对数据资料进行统计分析。潜伏期计量资料采用中位数和四分位数间距表示，分类资料采用频数和率/构成比表示。采用χ²检验比较不同组间暴露率，采用比值比(odds ratio, OR)及其95%CI评估病例组与对照组中某种食物食用的患病风险。以P < 0.05为有统计学意义。

2　结果

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2.1　学校基本情况

事发学校为杭州市某高级中学分校区，设高一至高三3个年级，共有33个班1 487名学生、128名教职工和32名后勤人员(从事食堂工作27名)。高一12个班544名学生，高二11个班496名学生，高三10个班447名学生。该校食堂委托第三方公司运营，人员和食材原料由其供应。设置学生和教师食堂各1个，工作人员27名，其中22名服务学生，5名服务老师，二者不混用。师生分开用餐，均为堂食。中餐、晚餐统一在校内食用，早餐不强制。食堂另设西点房、咖啡吧各1间，供应面包、甜点、三明治、饮品，工作人员4名。饮用水源为带有滤芯且具备沸腾功能的直饮水，师生共用，每层楼2台饮水设备。学校共有2幢教学楼(以下称A楼和B楼)和1幢行政楼，均为6层，2幢教学楼在行政楼的两侧，呈U字型结构。每层楼中间由走廊相连，各设男、女厕所1间。高一、高二各有10个班在A楼上课，剩余班级和高三10个班均在B楼; A楼学生总数为905名，B楼学生总数582名。该校为半走读学校，有宿舍楼3幢(校内1幢，校外2幢)。

2.2　流行病学特征

2.2.1　时间分布

首例病例的发病时间为5月29日16:00左右，发病高峰为5月30日1:00—4:00，末次病例为5月30日8:00，31日后无新发病例。首末病例间隔16 h，中位数为30日1：00。截止31日0：00时，共搜索发现45例腹泻病例，其中实验室确诊25例，临床诊断20例。根据病例共同就餐时间推测，最短潜伏期为4 h，最长20 h，中位数13 h。

2.2.2　人群分布

45例病例均为学生，男生17例，女生28例，男女比例为1∶1.65，学生罹患率为3.03%(45/1 487)。病例临床表现以腹泻(45例)、腹痛(43例)为主，部分病例伴有恶心症状(3例)，所有病例均无发热和呕吐。无重症和死亡病例。

2.2.3　空间分布

45例病例分布在高一至高三的18个班级，其中高一6个班，高二9个班，高三3个班; 发病人数最多的7人，最少的1人，18个班级罹患率为2.22%~15.56%。高一至高三学生罹患率分别为2.64%(14/530)，5.53%(26/470)，1.13%(5/442)，差异有统计学意义(χ²=14.22，P < 0.01)。

2.2.4　可疑暴露因素

该校中餐、晚餐供应时间分别为11:30—12:30，17:00—18:00，按高三、高一、高二的顺序错峰用餐，中间间隔20 min，各年级所用食物相同。从罹患率来看，高二>高一>高三，说明CP在食物中暴露时间越长，感染风险越大。首例病例于29日16：00发病，当时尚未进食当日晚餐，距28日晚餐供应时间23 h; 末次病例发病时间为30日8：00，与28日晚餐的间隔时长更是达到39 h，与产气荚膜梭菌常见的潜伏期为8~24 h^[16]相去甚远，因此本起腹泻事件可疑餐次集中在5月29日学生食堂的早餐和中餐。调查发现，45名发病学生5月29日均在学生食堂食用了当日中餐，而早餐仅2名食用，故推测可疑餐次为5月29日学生食堂提供的中餐。选择5月29日中午在学生食堂就餐的35名无任何症状学生作为对照组，以45名腹泻学生为病例组，对该餐次学生食用情况开展病例对照调查。结果显示食用番茄炒蛋、玉米排骨的学生患病风险分别是未食用者的6.70和9.71倍[OR值(95%CI)分别为6.70(1.40~32.09)，9.71(1.18~80.05)，P值均 < 0.05]。

2.3　卫生学调查

调查显示，该校食堂经营场地布局合理，食材原料来源正规，运输流程安全可靠; 食堂内设有留样柜、熟食间、洗消室等常用设施设备，均可正常运行，环境卫生整体较为整洁。但部分食物在加工、制作、贮存环节存在较大的污染风险。

2.4　实验室检测

2.4.1　病原体筛检结果

(1) Filmarray多重PCR初筛：5名症状典型学生肛拭子样本混合物采用Filmarray GI Panel胃肠道感染检测芯片对22种病原微生物进行初步筛查，结果均为阴性。(2)荧光PCR检测：将上述5份肛拭标本分别提取核酸，进行其他8种相关胃肠道病原体荧光PCR检测，结果显示5份标本均检出CP 16 S，其他病原体均阴性。

2.4.2　样本中CP 16 S与毒素基因检测结果

25例病例、20名食堂从业人员、1名咖啡吧服务员肛拭和10份食堂留样中均检出CP 16 S和α毒素(plc/cpa)，其中25例病例、14名食堂从业人员肛拭中同时检出CPE(cpe); 所有CP 16 S阳性样本的β毒素(cpb)、ε毒素(etx)、ι毒素(iap和ibp)和NetB(netB)均阴性。且2份可疑食物并未检出CP 16 S。

2.4.3　CP菌株分离及毒素基因检测情况

对CP 16S阳性样本进行厌氧培养，共从27份样本中分离出59株CP。其中CPE(cpe)阳性22株，来自10名病例和1名学生食堂从业人员; CPE(cpe)阴性37株，来自8名病例[2名同时CPE(cpe)阳性]、7名食堂从业人员、1名咖啡吧服务员和2份食堂留样。所有CP分离株的α毒素(plc/cpa)均阳性，β毒素(cpb)、ε毒素(etx)、ι毒素(iap和ibp)和NetB(netB)均阴性。

2.4.4　全基因组溯源分析

为分析CPE(cpe)阳性株、CPE(cpe)阴性株与暴发事件关联性，挑选本次分离的11株CPE(cpe)阳性株(来自10例学生病例和1名学生食堂从业人员)和12株CPE(cpe)阴性株(来自4例学生病例、5名学生食堂从业人员、1名咖啡吧工作人员、2份食堂留样)进行全基因组测序，以CPS 2a(NZ_CP075903.1)为参考菌株，构建基于全基因组的系统发育进化树，如图1。结果显示，11株CPE(cpe)阳性株基因组高度相似，在进化树中聚在了一簇，提示为共同来源，分析本次暴发事件的致病因子为携带CPE(cpe)的CP，从业人员带菌可能为该事件的感染源头; 其次，12株CPE(cpe)阴性株基因组差异明显，分散在进化树不同分支上，符合人体正常带菌多样性; 此外，2号和8号病例同时检出CPE(cpe)阳性株和CPE(cpe)阴性株，推测阴性株为患者发病前正常携带、阳性株为此次腹泻暴发事件新近感染。

3　讨论

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F型CP产生的主要毒素为α毒素和CPE，其中α毒素在所有CP中均携带^[17]，而CPE则是CP引起食源性疾病的唯一毒素^[18]。CPE毒素是一种分子量大小为35 kDa的孔形成毒素，可与claudin受体结合，引起肠上皮脱落、肠坏疽、肠液吸收障碍等胃肠道疾病^[19]。一旦摄入受污染的食物，F型CP会在肠道内迅速生长，形成孢子，这种体内孢子化在F型CP致病机制中起着关键作用，即仅在肠道内形成孢子时才会产生CPE^[20]。此外，由于缺乏分泌信号肽，只有在CP裂解释放成熟孢子时CPE才会进入肠腔^[19]。提示学校、餐厅等公共场所食品从业人员在腹泻症状发生时需立即停止工作，同时在执业上岗时除取得健康证外，还应进行常见食源性病原菌的检测以及岗前培训。

CP导致的食物中毒大多发生在集体用餐中，潜伏期为8~24 h，临床表现以腹泻、腹痛为主，常在较短的同一时间集中发病，中毒食物多为同批大量加热烹煮后在较高温度下长时间地缓慢冷却且不经再加热而直接供餐的肉、鸡、鸭、鱼或其他菜肴及其汤汁^[16]。本事件中患者均为学生，临床症状为腹泻、腹痛，首末病例间隔16 h，潜伏期介于4~20 h，可疑食物经烹制后较高温度存放数小时缓慢冷却、且食用前未重新加热，临床症状与流行病学均符合CP食物中毒判定原则。同时，在采集的25名病例肛拭标本中均检测到CPE(cpe)，与《产气荚膜梭菌食物中毒诊断标准及处理原则》(WS/T 7—1996)^[16]中实验室诊断部分相对应。在后续的分离鉴定中，共从27份样本中分离出59株CP，其中22株携带CPE(cpe)。11株来自10名病例和1名食堂工作人员的CPE(cpe)阳性株在进化树上聚为一簇，为同一来源; 12株CPE(cpe)阴性株分散在进化树不同分支上，为不同来源，符合人体正常带菌多样性。最终认为该事件为一起由携带CPE(cpe)的F型产气荚膜梭菌感染引起的学校食物中毒事件，从业人员带菌为可能的暴发源头。提示对学校食堂从业人员加强培训和进行常见食源性疾病病原体定期监测的必要性。

调查发现，学生食堂按高三、高一、高二的顺序错峰20 min用餐，最早用餐的高三就餐时CP在食物中暴露时间最短、发病人数也最少; 随着暴露时间的增加，越晚就餐感染风险越大，可能是罹患率高三 < 高一 < 高二的主要原因。病例对照研究显示，5月29日中餐学生食堂提供的番茄炒蛋和玉米排骨为可疑食品，后续的荧光PCR检测中两份食品CP 16 S均为阴性，未进一步检测CPE(cpe)和进行CP分离培养。

本研究存在一些不足之处：由于疾病预防控制中心工作人员介入时间晚，未在食品、餐饮环节中检出携带CPE(cpe)的CP，不能明确致病菌最初污染来源，提示校园食品安全事件早期报告的重要性。另外，国内大多数基层疾病预防控制中心并不具备开展动物实验的条件，此次事件中并没有从患者和从业人员体内检测到CP肠毒素的直接证据，无法判断CPE(cpe)阳性的食堂从业人员为事件施害方还是受害方。

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浙江省疾病预防控制科技计划项目(2025JK052)

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2025年第46卷第10期

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doi: 10.16835/j.cnki.1000-9817.2025296

接收时间：2025-03-28
首发时间：2026-11-06
出版时间：2025-10-25

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收稿日期：2025-03-28
修回日期：2025-07-14

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浙江省疾病预防控制科技计划项目(2025JK052)

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杭州市上城区疾病预防控制中心(杭州市上城区卫生监督所)，浙江 310004

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黄世旺，E-mail：hshw2003@163.com

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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