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Article(id=1151881501227496002, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1151881493552394994, articleNumber=null, orderNo=null, doi=10.19812/j.cnki.jfsq11-5956/ts.20241219006, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1734537600000, receivedDateStr=2024-12-19, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1752559550816, onlineDateStr=2025-07-15, pubDate=1748102400000, pubDateStr=2025-05-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1752559550816, onlineIssueDateStr=2025-07-15, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1752559550816, creator=13701087609, updateTime=1752559550816, updator=13701087609, issue=Issue{id=1151881493552394994, tenantId=1146029695717560320, journalId=1149652044408987649, year='2025', volume='16', issue='10', pageStart='1', pageEnd='324', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1752559548986, creator=13701087609, updateTime=1756202008453, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1167159075906265916, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1151881493552394994, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1167159075906265917, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1151881493552394994, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=112, endPage=118, ext={EN=ArticleExt(id=1151923892731343483, articleId=1151881501227496002, tenantId=1146029695717560320, journalId=1149652044408987649, language=EN, title=Analysis of drug resistance and virulence genes of Campylobacter jejuni in Xuzhou City from 2023 to 2024, columnId=1151923892655846010, journalTitle=Journal of Food Safety & Quality, columnName=Special Topic: Food Safety Risk Assessment and Risk Monitoring, runingTitle=null, highlight=null, articleAbstract=

Objective To understand the drug resistance phenotypes,, carriage of drug resistance genes and virulence genes, serotypes and sequence type (ST) classification of Campylobacter jejuni in Xuzhou from 2023 to 2024. Methods Whole genome sequencing was conducted on Campylobacter jejuni isolated from Xuzhou City in 2023—2024 to understand the molecular characteristics such as virulence genes, drug resistance genes, and multilocus sequence typing. The serotypes related to Guillain-Barré syndrome were identified by real-time fluorescent quantitative polymerase chain reaction (PCR), and the drug resistance phenotypes were determined by agar dilution method. Results Forty-eight strains of Campylobacter jejuni were classified into 24 ST types. ST137 was the most numerous, accounted for 18.75% (9/48). Fifteen strains were found to have serotypes related to Guillain-Barré syndrome. A total of 24 drug resistance genes and 121 virulence genes were detected. The top 3 antibiotics with the highest resistance rates were nalidixic acid (100.00%), ciprofloxacin (100.00%), and tetracycline (97.92%). They were 100.0% sensitive to erythromycin, and the multi-drug resistance rate reached 62.50% (30/48). Conclusion Campylobacter jejuni in Xuzhou area is characterized by a high rate of multi-drug resistance, a relatively high carrying rate of some drug resistance genes, a high carrying rate of virulence genes, a large number of sequence types (ST types), and shows relatively high genetic diversity.

, correspAuthors=Feng-Ling YU, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Yan-Ping XU, Shu-Qing QU, Xiao WANG, Huan-Huan FENG, Wen-Wen CUI, Xin-Chi ZHANG, Jing TONG, Feng-Ling YU), CN=ArticleExt(id=1151923905918234687, articleId=1151881501227496002, tenantId=1146029695717560320, journalId=1149652044408987649, language=CN, title=2023—2024年徐州市空肠弯曲菌耐药及毒力基因分析, columnId=1151923892995109553, journalTitle=食品安全质量检测学报, columnName=专题：食品安全风险评估与风险监测, runingTitle=null, highlight=null, articleAbstract=

目的 探究2023—2024年徐州市空肠弯曲菌的耐药表型、耐药基因及毒力基因携带情况、血清型、序列类型(sequence type, ST)分型。方法 对2023—2024年徐州市分离的空肠弯曲菌进行全基因组测序分析了解毒力基因、耐药基因、多位点序列分型等分子特征, 通过实时荧光定量聚合酶链式反应(polymerase chain reaction, PCR)法了解格林-巴利综合征相关的血清型, 通过琼脂稀释法了解其耐药表型。结果 48株空肠弯曲菌共有24个ST型, ST137最多, 占比18.75% (9/48), 有15株菌检出格林-巴利综合征相关的血清型, 共检出24种耐药基因和121种毒力基因。耐药率排前3位的抗生素分别是萘啶酸(100.00%)、环丙沙星(100.00%)、四环素(97.92%), 对红霉素100.0%敏感, 多重耐药率达到62.50% (30/48)。结论 徐州地区空肠弯曲菌具有多重耐药率高、部分耐药基因携带率高等特征, 并且毒力基因携带率高, ST型较多, 表现出较高的遗传多样性。

, correspAuthors=余峰玲, authorNote=null, correspAuthorsNote=
* 余峰玲(1986—), 女, 硕士, 副主任技师, 主要研究方向为食品风险监测和食源性致病菌检测。E-mail:
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许艳平(1990—), 女, 主管技师, 主要研究方向为食源性致病菌。E-mail:

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许艳平(1990—), 女, 主管技师, 主要研究方向为食源性致病菌。E-mail:

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许艳平(1990—), 女, 主管技师, 主要研究方向为食源性致病菌。E-mail:

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Jiangsu Journal of Preventive Medicine, 2024, 35(3): 324-327, 387., articleTitle=Epidemiological status of human-derived Campylobacter and analysis of virulence genes and antibiotic resistance in Fengtai District of Beijing from 2018 to 2022, refAbstract=null)], funds=[Fund(id=1167158619456942320, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1151881501227496002, awardId=KC22225, language=CN, fundingSource=徐州市重点研发计划(社会发展)项目(KC22225), fundOrder=null, country=null), Fund(id=1167158619511468273, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1151881501227496002, awardId=Y12023007, language=CN, fundingSource=江苏省卫生健康委科研项目(Y12023007), fundOrder=null, country=null), Fund(id=1167158619561799922, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1151881501227496002, awardId=Ym2023072, language=CN, fundingSource=江苏省卫生健康委科研项目(Ym2023072), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1167158616659341490, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1151881501227496002, xref=null, ext=[AuthorCompanyExt(id=1167158616663535795, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1151881501227496002, companyId=1167158616659341490, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=Xuzhou Center for Disease Control and Prevention, Xuzhou 221006, China), AuthorCompanyExt(id=1167158616671924404, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1151881501227496002, companyId=1167158616659341490, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=徐州市疾病预防控制中心, 徐州 221006)])], figs=[ArticleFig(id=1167158618823602406, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1151881501227496002, language=EN, label=Fig.1, caption=Distribution map of drugresistance genes in 48 Campylobacter jejuni strains, figureFileSmall=XqxDl1rMuNCIm8ktZQi79g==, figureFileBig=ovqKRxqKL2rUfLOOpLkTjA==, tableContent=null), ArticleFig(id=1167158618869739751, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1151881501227496002, language=CN, label=图1, caption=48株空肠弯曲菌耐药基因分布热图, figureFileSmall=XqxDl1rMuNCIm8ktZQi79g==, figureFileBig=ovqKRxqKL2rUfLOOpLkTjA==, tableContent=null), ArticleFig(id=1167158618915877096, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1151881501227496002, language=EN, label=Fig.2, caption=Distribution map of virulence genes in 48 Campylobacter jejuni strains, figureFileSmall=TOKv6uzXagxxs7qOpsdd3g==, figureFileBig=/pGI4SHQV2OHLij2fIGitA==, tableContent=null), ArticleFig(id=1167158618991374569, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1151881501227496002, language=CN, label=图2, caption=48株空肠弯曲菌部分毒力基因分布热图

注: 白色方块为未检测, 彩色方块为检出。动力模块中的pseH、flgL、flaC、flhB、fliQ、Cj0371、flgG、flgD、fliK、motB、flhF、flgJ、flgR、flgS、pseA、fliI、flgB、flgC、fliF、cheA、cheW、fliM、fliN、flhA、pseC、fliY、fliG、fliH、rpoN、fliE、flgI、flgP、flgQ、fliW、cheY、flgH、flgG2、fliD、pseG、flgK、flgM、Cj0883c、flaG、eptC、fliL、pflA、motA、flhG、pseB等56种基因携带率均为100.00%, 未在上图列出。

, figureFileSmall=TOKv6uzXagxxs7qOpsdd3g==, figureFileBig=/pGI4SHQV2OHLij2fIGitA==, tableContent=null), ArticleFig(id=1167158619041706218, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1151881501227496002, language=EN, label=Fig.3, caption=Phylogenetic analysis of 48 Campylobacter jejuni strains, figureFileSmall=xxvnMmT/wSWMyVaf54Ir5A==, figureFileBig=oEPmU92G/QGmESzIv//88g==, tableContent=null), ArticleFig(id=1167158619092037867, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1151881501227496002, language=CN, label=图3, caption=48株空肠弯曲菌系统进化分析

注: -表示未知。

, figureFileSmall=xxvnMmT/wSWMyVaf54Ir5A==, figureFileBig=oEPmU92G/QGmESzIv//88g==, tableContent=null), ArticleFig(id=1167158619146563820, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1151881501227496002, language=EN, label=Table 1, caption=

Resistance results of 48 Campylobacter jejuni strains to 11 kinds of antibiotics of 7 categories

, figureFileSmall=null, figureFileBig=null, tableContent=
类别 药物名称 耐药判定
/(μg/mL)		 耐药率/%
大环内酯类 ERY* ≥32 0.00 (0/48)
AZM ≥1 8.33 (4/48)
喹诺酮及氟喹诺酮类 NAL ≥32 100.00 (48/48)
CIP* ≥4 100.00 (48/48)
氨基糖苷类 GEN ≥4 6.25 (3/48)
STR ≥16 6.25 (3/48)
氯霉素类 CHL ≥32 8.33 (4/48)
FLO ≥8 54.17 (26/48)
四环素类 TET ≥16 97.92 (47/48)
酮内酯类 TEL* ≥8 27.08 (13/48)
林可酰胺类 CLI ≥1 16.67 (8/48)
), ArticleFig(id=1167158619209478381, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1151881501227496002, language=CN, label=表1, caption=

48株空肠弯曲菌对7类11种抗生素的耐药结果

, figureFileSmall=null, figureFileBig=null, tableContent=
类别 药物名称 耐药判定
/(μg/mL)		 耐药率/%
大环内酯类 ERY* ≥32 0.00 (0/48)
AZM ≥1 8.33 (4/48)
喹诺酮及氟喹诺酮类 NAL ≥32 100.00 (48/48)
CIP* ≥4 100.00 (48/48)
氨基糖苷类 GEN ≥4 6.25 (3/48)
STR ≥16 6.25 (3/48)
氯霉素类 CHL ≥32 8.33 (4/48)
FLO ≥8 54.17 (26/48)
四环素类 TET ≥16 97.92 (47/48)
酮内酯类 TEL* ≥8 27.08 (13/48)
林可酰胺类 CLI ≥1 16.67 (8/48)
), ArticleFig(id=1167158619272392942, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1151881501227496002, language=EN, label=Table 2, caption=

Detection results of 9 kinds of Campylobacter jejuni serotypes associated with GBS

, figureFileSmall=null, figureFileBig=null, tableContent=
血清型 菌株编号 检出菌株数
HS:19 — 0
HS:41 — 0
HS:2 2023030 1
HS:1 2023001、2023059、2023156、2024042 4
HS:5-31 2023028、2023029、2024031、2024065、2024068 5
HS:23-36 — 0
HS:44 — 0
HS:4 2024052 1
HS:37 2023007、2023015、2023119、2023120 4
), ArticleFig(id=1167158619343696111, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1151881501227496002, language=CN, label=表2, caption=

9种GBS综合征相关的空肠弯曲菌血清型检出结果

, figureFileSmall=null, figureFileBig=null, tableContent=
血清型 菌株编号 检出菌株数
HS:19 — 0
HS:41 — 0
HS:2 2023030 1
HS:1 2023001、2023059、2023156、2024042 4
HS:5-31 2023028、2023029、2024031、2024065、2024068 5
HS:23-36 — 0
HS:44 — 0
HS:4 2024052 1
HS:37 2023007、2023015、2023119、2023120 4
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2023—2024年徐州市空肠弯曲菌耐药及毒力基因分析
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许艳平 , 渠淑晴 , 王潇 , 冯欢欢 , 崔雯雯 , 张心驰 , 童晶 , 余峰玲 *
食品安全质量检测学报 | 专题：食品安全风险评估与风险监测 2025,16(10): 112-118
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食品安全质量检测学报 | 专题：食品安全风险评估与风险监测 2025, 16(10): 112-118
2023—2024年徐州市空肠弯曲菌耐药及毒力基因分析
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许艳平 , 渠淑晴, 王潇, 冯欢欢, 崔雯雯, 张心驰, 童晶, 余峰玲*
作者信息
  • 徐州市疾病预防控制中心, 徐州 221006

    • 许艳平(1990—), 女, 主管技师, 主要研究方向为食源性致病菌。E-mail:

通讯作者:

* 余峰玲(1986—), 女, 硕士, 副主任技师, 主要研究方向为食品风险监测和食源性致病菌检测。E-mail:
Analysis of drug resistance and virulence genes of Campylobacter jejuni in Xuzhou City from 2023 to 2024
Yan-Ping XU , Shu-Qing QU, Xiao WANG, Huan-Huan FENG, Wen-Wen CUI, Xin-Chi ZHANG, Jing TONG, Feng-Ling YU*
Affiliations
  • Xuzhou Center for Disease Control and Prevention, Xuzhou 221006, China
出版时间: 2025-05-25 doi: 10.19812/j.cnki.jfsq11-5956/ts.20241219006
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摘要
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目的 探究2023—2024年徐州市空肠弯曲菌的耐药表型、耐药基因及毒力基因携带情况、血清型、序列类型(sequence type, ST)分型。方法 对2023—2024年徐州市分离的空肠弯曲菌进行全基因组测序分析了解毒力基因、耐药基因、多位点序列分型等分子特征, 通过实时荧光定量聚合酶链式反应(polymerase chain reaction, PCR)法了解格林-巴利综合征相关的血清型, 通过琼脂稀释法了解其耐药表型。结果 48株空肠弯曲菌共有24个ST型, ST137最多, 占比18.75% (9/48), 有15株菌检出格林-巴利综合征相关的血清型, 共检出24种耐药基因和121种毒力基因。耐药率排前3位的抗生素分别是萘啶酸(100.00%)、环丙沙星(100.00%)、四环素(97.92%), 对红霉素100.0%敏感, 多重耐药率达到62.50% (30/48)。结论 徐州地区空肠弯曲菌具有多重耐药率高、部分耐药基因携带率高等特征, 并且毒力基因携带率高, ST型较多, 表现出较高的遗传多样性。

空肠弯曲菌  /  全基因组测序  /  血清型  /  多重耐药

Objective To understand the drug resistance phenotypes,, carriage of drug resistance genes and virulence genes, serotypes and sequence type (ST) classification of Campylobacter jejuni in Xuzhou from 2023 to 2024. Methods Whole genome sequencing was conducted on Campylobacter jejuni isolated from Xuzhou City in 2023—2024 to understand the molecular characteristics such as virulence genes, drug resistance genes, and multilocus sequence typing. The serotypes related to Guillain-Barré syndrome were identified by real-time fluorescent quantitative polymerase chain reaction (PCR), and the drug resistance phenotypes were determined by agar dilution method. Results Forty-eight strains of Campylobacter jejuni were classified into 24 ST types. ST137 was the most numerous, accounted for 18.75% (9/48). Fifteen strains were found to have serotypes related to Guillain-Barré syndrome. A total of 24 drug resistance genes and 121 virulence genes were detected. The top 3 antibiotics with the highest resistance rates were nalidixic acid (100.00%), ciprofloxacin (100.00%), and tetracycline (97.92%). They were 100.0% sensitive to erythromycin, and the multi-drug resistance rate reached 62.50% (30/48). Conclusion Campylobacter jejuni in Xuzhou area is characterized by a high rate of multi-drug resistance, a relatively high carrying rate of some drug resistance genes, a high carrying rate of virulence genes, a large number of sequence types (ST types), and shows relatively high genetic diversity.

Campylobacter jejuni  /  whole genome sequencing  /  serotype  /  multi-drug resistance
许艳平, 渠淑晴, 王潇, 冯欢欢, 崔雯雯, 张心驰, 童晶, 余峰玲. 2023—2024年徐州市空肠弯曲菌耐药及毒力基因分析. 食品安全质量检测学报, 2025 , 16 (10) : 112 -118 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20241219006
Yan-Ping XU, Shu-Qing QU, Xiao WANG, Huan-Huan FENG, Wen-Wen CUI, Xin-Chi ZHANG, Jing TONG, Feng-Ling YU. Analysis of drug resistance and virulence genes of Campylobacter jejuni in Xuzhou City from 2023 to 2024[J]. Journal of Food Safety & Quality, 2025 , 16 (10) : 112 -118 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20241219006
正文
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0 引言
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空肠弯曲菌(Campylobacter jejuni)为革兰阴性菌, 属于弯曲菌属, 生长营养要求高, 在微需氧环境中生长, 最适生长温度为42 ℃, 是一种重要的人畜共患病病原菌[1], 该菌一般寄居于禽畜的肠道中[2], 在禽畜之间相互感染, 人主要通过摄入被污染的食物(如未煮熟的鸡肉、牛奶等)和水而感染[3], 也可通过接触感染动物及其排泄物传播, 人感染空肠弯曲菌后, 可引起腹泻、腹痛等胃肠道疾病, 严重时可引发周围神经系统的严重疾病, 如格林-巴利综合征(Guillain-Barresyndrome, GBS)和米勒综合征[4-7]。空肠弯曲菌是导致人类弯曲菌病的主要病原菌之一, 全球发病率近十年来逐渐增加[8], 有研究显示空肠弯曲菌引发的腹泻病例数量超过了食源性沙门氏菌所致的病例数[9-10]。据世界卫生组织报道, 空肠弯曲菌是腹泻病的全球四大主要病因之一, 也是最常见的食源性致病菌之一[11]。
空肠弯曲菌的耐药性和多重耐药性的快速增加已成为全球关注焦点[12]。空肠弯曲菌对多种常用的抗生素的耐药性呈上升趋势, 尤其是对氟喹诺酮类抗生素(如环丙沙星)的耐药率增长明显。全基因组测序(whole genome sequencing, WGS)属于基因层面的检测技术, 能应用于菌种鉴定、分型、基因突变、耐药基因、毒力基因分析以及聚类分析等方面[13]。了解徐州市空肠弯曲菌耐药性及基因组特征, 对于监测及防控其引发的感染性疾病具有重要意义, 但目前关于徐州市空肠弯曲菌的研究较少, 因此, 本研究借助耐药表型、血清型及全基因组测序, 对徐州市2023和2024年分离出的空肠弯曲菌开展检测分析, 包括其耐药表型、耐药基因、毒力基因、多位点序列分型(multi-locussequence typing, MLST)以及GBS相关血清型等分子特征, 揭示徐州地区空肠弯曲菌的流行特点、潜在致病风险与耐药状况, 构建徐州市空肠弯曲菌分子分型数据库, 为防控空肠弯曲菌及追溯暴发源头给予数据支撑。
1 材料与方法
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1.1 材料与试剂
药敏质控菌株空肠弯曲菌ATCC33560(美国MicroBioLogics公司); 哥伦比亚血琼脂平板(青岛高科技工业园海博生物技术有限公司); 质谱基质液(郑州安图生物工程股份有限公司); 空肠弯曲菌核酸扩增荧光检测试剂盒(F20240514, 北京卓诚惠生生物科技股份有限公司); GBS高风险空肠弯曲菌血清型筛查试剂盒(240527)、弯曲菌药敏板、菌种保存管(青岛中创汇科生物科技有限公司)。
1.2 仪器与设备
Bugbox M型厌氧培养箱(英国Ruskinn公司); VITEK DENSIMAT浊度仪(法国梅里埃公司); Thermo Fisher 1300生物安全柜(美国赛默飞世尔科技有限公司); Autof ms1000全自动微生物质谱检测系统(郑州安图生物工程股份有限公司); KingFisher Flex全自动核酸提取系统(美国Thermo公司); CFX96 Deepwell实时荧光聚合酶链式反应(polymerase chain reaction, PCR)仪(美国伯乐生命医学产品有限公司)。
1.3 实验方法
1.3.1 菌株来源
48株空肠弯曲菌, 其中41株分离自2023—2024年生禽样本, 7株分离自2023—2024年徐州市儿童医院及徐州市肿瘤医院腹泻病人粪便样本。所有菌株按照年度分离顺序进行本实验室编号。
1.3.2 菌株复苏及鉴定
从-80 ℃冰箱取出菌株保存管, 待室温下保存液溶解后, 用无菌接种环取一环菌液接种于哥伦比亚血平板, 在微需氧(5% O2、10% CO2、85% N2的混合气体)条件下42 ℃培养36~48 h后, 将生长良好的单个菌落再次转种血平板进行微需氧培养。纯培养的空肠弯曲菌利用全自动核酸提取系统提取核酸, 具体方法按仪器操作规程操作。采用空肠弯曲菌核酸扩增荧光检测试剂盒和全自动微生物质谱检测系统鉴定相结合的方法进行菌株鉴定[14-15]。
1.3.3 药敏实验
空肠弯曲菌(ATCC33560)为本次药敏实验的质控菌株, 使用96孔板琼脂稀释法来检测, 选用7类共11种抗生素。分别为大环内酯类: 红霉素(erythromycin, ERY)、阿奇霉素(azithromyein, AZM); 喹诺酮及氟喹诺酮类: 萘啶酸(nalidixicacid, NAL)、环丙沙星(ciprofloxacin, CIP); 氨基糖苷类: 庆大霉素(gentamicin, GEN)、链霉素(streptomycin, STR); 氯霉素类: 氯霉素(chloramphenicol, CHL)、氟苯尼考(florfenicol, FLO); 四环素类: 四环素(tetracyeline, TET); 酮内酯类: 泰利霉素(telithromycin, TEL); 林可酰胺类: 克林霉素(clindamycin, CLI); 检测过程: 用无菌棉签挑取生长良好的待测菌与质控菌菌落3~4个, 悬浮于3 mL无菌稀释液中, 充分混匀, 将悬液调整到0.5麦氏单位后再用无菌稀释液进行10倍稀释, 此时弯曲菌的浓度大约为104 CFU/mL; 将稀释好的待检菌液点加到药敏培养板检测列以及质控孔中进行检测; 将药敏板置于微需氧环境下42 ℃培养 24 h, 肉眼判断检测结果, 读取最低抑菌浓度(minimum inhibitory concentration, MIC)值, 药敏结果判定依据美国临床与实验室标准化协会(Clinicaland Laboratory Standards Institute, CLSI)[16]以及美国国家抗生素耐药监测系统(National Antimicrobial Resistance Monitoring, NARMS)(https://www.cdc.gov/narms/antibiotics-tested.html)所推荐的耐药标准来执行。菌株对3类及3类以上的抗生素产生耐药性, 则被判定为多重耐药(multi-drug resistance, MDR)。
1.3.4 血清型实验
根据空肠弯曲菌的DNA序列鉴定9类GBS相关的空肠弯曲菌的血清型(包括HS:2, HS:19, HS:41, HS:1, HS:23-36, HS:5-31, HS:44, HS:4和HS:37共11种血清型), 将提取好的核酸采用GBS高风险空肠弯曲菌血清型筛查试剂盒通过荧光PCR鉴定; PCR反应操作如下: 分别吸取2 μL的待测菌液核酸标本、阴性对照、阳参试剂(试剂阳性对照品)以及提取好的标准菌株的核酸, 将其分别加入已配制好反应液的PCR反应管内, 使每管的终体积达到20 μL。扩增条件: 94 ℃预变性30 s; 94 ℃变性5 s, 60 ℃退火、延伸及检测荧光30 s, 40个循环; Ct值<35为阳性。
1.3.5 全基因组测序
把提取好的核酸运送至北京诺禾致源有限公司开展二代测序分析。先是在CLC Genomics Workbench 23.0.5软件上运用Trimming模块, 将测序所得的原始序列(Raw reads)里的adapter及数据质量低的去除, 接着进行从头拼接(De Novo Assembly), 且用本研究的菌株编号为组装后的contigs命名。
1.4 数据处理
测序数据由北京诺禾智源生物信息科技有限公司提供。毒力基因分析以毒力因子数据库(virulence factors database, VFDB)数据库为基础, 耐药基因分析则依据ResFinder数据库, 同时把基因结果比对到PubMLST数据库(https://pubmlst.org/), 用以完成空肠弯曲菌的MLST分型注释。利用CLC Genomics Workbench 23.0.5软件构建单核苷酸多态性(single nucleotide polymorphisms, SNP)系统发育树, 参考菌株为ATCC33560 (GenBank ID: CP019838.1), 采用最大似然法。使用ChiPlot工具进行图形的注释与美化[17]。
2 结果与分析
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2.1 耐药表型及耐药基因携带情况
48株空肠弯曲菌对11种抗生素耐药性检测结果显示, 对红霉素最敏感, 没有耐药菌株, 对其余抗生素均有不同程度耐药, 耐药率排前3位分别是萘啶酸(100.00%)、环丙沙星(100.00%)、四环素(97.92%)。多重耐药率高达62.50% (30/48), 多重耐药模式较多, 其中检出最多的为喹诺酮及氟喹诺酮类, 四环素类, 氯霉素类同时存在耐药, 占多重耐药的80.00% (24/30), 见表1。
耐药基因分析结果发现, 48株菌一共检测出了属于5类的24种耐药基因。分别为氨基糖苷类、β-内酰胺类、大环内酯类、氯霉素类、四环素类, 其中blaOXA-193、blaOXA-448、blaOXA-450、blaOXA-451、blaOXA-452、blaOXA-453、blaOXA-489携带率最高为87.50% (42/48), 其次为blaOXA-489携带率为85.42% (41/48), 第三为tet (O)携带率为81.25% (39/48), 携带最低的为cat (pC194)、tet (39)、tet (L)、blaOXA-185、blaOXA-460、blaOXA-461、blaOXA-465、blaOXA-466, 携带率为2.08% (1/48), 详见图1。
2.2 血清型结果
48株空肠弯曲菌共有15株检出GBS综合征相关的空肠弯曲菌血清型, 其中1株检出HS:2, 4株检出HS:1, 5株检出HS:5-31, 1株检出HS:4, 4株检出HS:37, 其余均未检出, 见表2。
2.3 毒力基因携带情况
在对48株空肠弯曲菌的检测中, 共发现了121种毒力基因, 包括黏附、侵袭、动力、免疫调节、外毒素5大功能模块。每一株菌株所携带的毒力基因数量均不少于71种, 部分菌株多达103种。在这5大功能模块当中, 动力模块所检出的毒力基因数量尤为突出, 位列各模块之首, 占54.55% (66/121); 其次为免疫调节模块, 占37.19% (45/121); 外毒素基因中cdtB、cdtC、cdtA的携带率分别为95.83% (46/48)、97.92% (47/48)、89.58% (43/48), 侵袭相关基因中ciaB的携带率为95.83% (46/48); 免疫调节相关基因wlaN携带率为29.17% (14/48)。48株菌中全部携带的毒力基因有63种, 包括waaC、waaV、ciaC、gmhA、kpsD、hldD、hldE以及运动力相关的56种毒力基因。见图2。
2.4 MLST分型结果
空肠弯曲菌MLST[18]是按照7个管家基因进行分类的, 结果发现48株空肠弯曲菌共检出24种序列类型(sequence type, ST)型, 有1株菌未能通过PubMLST数据库鉴定出ST型。ST型呈现出多样化的特征。其中, ST137所占比例最高, 达到18.75% (9/48); 其次为ST2274和ST6500, 它们的占比均为8.33% (4/48)。30株多重耐药菌, 共有21种ST型, 7株人源型菌株共5种ST型, 多重耐药占42.86% (3/7), 41株食源型菌株共21种ST型, 多重耐药占65.85% (27/41)。两种不同来源的菌株型别较为分散, 无明显的聚类特征。详见图3。
3 讨论与结论
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空肠弯曲菌是导致人类患胃肠炎的主要病原菌, 此问题已演变为全球瞩目的食品安全焦点[19], 给世界范围内的疾病防控与公共卫生体系带来了极为沉重的负担[20-21]。相关文献报道近年来弯曲菌耐药率一直在增长[22-23], 治疗弯曲菌病的抗生素为大环内酯类和喹诺酮类及氨基糖苷类药物, 本研究分离的48株空肠弯曲菌对萘啶酸和环丙沙星耐药率为100.00%, 四环素耐药率为97.92%, 呈现出高度的耐药性, 与相关研究一致[24-25], 大环内酯类抗生素是当下临床方面用于治疗弯曲菌感染而被推荐的一线用药类别[24]。据其他不同地区的研究发现[26-27], 从不同地区、不同来源所分离出的空肠弯曲菌研究发现, 其对红霉素的耐药率具有明显差异, 耐药率在8.20%到100.00%的范围内, 本研究发现, 全部菌株均呈现出对红霉素的高度敏感性, 未发现有耐药情况, 这与王小龙等[28]研究一致, 提示在本地区针对空肠弯曲菌感染开展治疗时, 红霉素可作为优先选用的药物。本研究发现多重耐药率达到62.50% (30/48), 多种多重耐药模式并存, 其中喹诺酮类、四环素类与氯霉素类的组合模式最为常见, 说明徐州地区空肠弯曲菌耐药严重, 本研究中, 耐药基因与耐药表型存在着特定的相关性, 不过二者并非全然匹配。一方面, 存在表型耐药率低于耐药基因携带率的情形, 例如氨基糖苷类的两种抗生素, 其耐药率均为6.25%, 而耐药基因aph (2'')-If的携带率则达10.42%, 这可能是由于部分菌株的相关耐药基因并未表达[29]。另一方面, 还存在耐药基因携带率低于耐药表型的情况, 比如四环素耐药率为97.92%, 耐药基因tet (O)携带率为81.25%, 氯霉素类中氟苯尼考和氯霉素耐药率分别为54.17%、8.33%, 耐药基因cat携带率为4.20%, 大环内酯类中阿奇霉素耐药率为8.33%, 耐药基因lnu (C)携带率为2.08%。可能由于耐药基因数据库的局限性, 没有覆盖全部的耐药基因, 还可能与细菌存在复杂的耐药机制有关。
48株空肠弯曲菌共有15株检出GBS相关的空肠弯曲菌的血清型, 提示在这些空肠弯曲菌中, 大约31.25% (15/48)的菌株属于可能引发GBS的特定血清型, 存在引起GBS的风险, 它们可能具有独特的表面抗原或其他特性, 这些特性可以激发人体免疫系统的交叉反应, 进而攻击自身的周围神经系统, 最终引发GBS。有研究证实弯曲菌的黏附、动力、侵袭能力以及产毒能力等均与细菌的毒力及致病能力密切相关[30]。本研究发现48株菌共检出121种毒力基因, 48株菌共含有66种动力相关基因, 细菌活动能力是致病能力的关键, 细菌通过运动达到肠道的黏液层[31]。本研究中, 空肠弯曲菌未检测出格林巴利综合征相关基因cst-Ⅱ, 部分菌株携带了诸如cstIII、neuA1、neuB1、neuC1、waaF、waaV以及wlaN等格林巴利综合征相关基因, 或许存在因空肠弯曲菌感染而诱发格林巴利综合征的潜在风险, 提示本地区空肠弯曲菌分离株有较强的致病潜力。
48株空肠弯曲菌共检出24种ST型, 总体呈现多样性, 排前3的ST型为ST137、ST6500、ST2274, 这与张志敏等[32]报道不一致, 检出最多的为ST137的9株菌均来源于食品, 两年检测均有此型别, 提示菌株可能有延续性, 有2株空肠弯曲菌的型别为ST50, 它们均来自同一家医院的2例患者, 且这2例患者的就诊时间较为相近。从遗传进化的角度来看, 这2株菌具有较高的相似度。然而, 由于流行病学调查数据存在缺失, 难以获取更具参考价值的信息, 在后期研究中建议加强信息收集, 进行完整的动态监测, 以便对空肠弯曲菌遗传特性做更深入的研究。本研究发现有一株为ST未分型菌株, 新ST型的出现提示空肠弯曲菌发生新的变异, 需要加强监测。本研究表明, 相同ST型的菌株在遗传进化层面未必比不同ST型的菌株更具亲缘性。原因在于ST型仅仅是依据7个管家基因的序列来确定的, 无法全面体现整个基因组的差异状况。
本研究显示徐州市空肠弯曲菌的ST型较多, 携带较多的毒力基因, 部分菌株携带GBS相关血清型, 总体耐药较严重, 相关部门应加强监督, 防止抗生素在人和禽畜中的过度使用, 所有菌株对红霉素敏感, 可继续加强监测。由于本研究中检测的菌株数量有限, 在后续的研究进程中, 宜进一步扩充检测样本的规模, 从而提升数据的可信度与可靠性。
基金
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  • 徐州市重点研发计划(社会发展)项目(KC22225)
  • 江苏省卫生健康委科研项目(Y12023007)
  • 江苏省卫生健康委科研项目(Ym2023072)
文献
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doi: 10.19812/j.cnki.jfsq11-5956/ts.20241219006
  • 接收时间：2024-12-19
  • 首发时间：2025-07-15
  • 出版时间：2025-05-25
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  • 收稿日期：2024-12-19
基金
徐州市重点研发计划(社会发展)项目(KC22225)
江苏省卫生健康委科研项目(Y12023007)
江苏省卫生健康委科研项目(Ym2023072)
作者信息
    徐州市疾病预防控制中心, 徐州 221006

通讯作者:

* 余峰玲(1986—), 女, 硕士, 副主任技师, 主要研究方向为食品风险监测和食源性致病菌检测。E-mail:
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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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