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Article(id=1322954340364853961, tenantId=1146029695717560320, journalId=1319373162502258839, issueId=1322954308836270447, articleNumber=null, orderNo=null, doi=10.16016/j.2097-0927.202507058, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1752768000000, receivedDateStr=2025-07-18, revisedDate=1761062400000, revisedDateStr=2025-10-22, acceptedDate=null, acceptedDateStr=null, onlineDate=1793346494046, onlineDateStr=2026-10-30, pubDate=1763136000000, pubDateStr=2025-11-15, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1793346494046, onlineIssueDateStr=2026-10-30, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1793346494046, creator=13701087609, updateTime=1793346494046, updator=13701087609, issue=Issue{id=1322954308836270447, tenantId=1146029695717560320, journalId=1319373162502258839, year='2025', volume='47', issue='21', pageStart='2581', pageEnd='2724', issueExtLink='null', onlineDate='null', pubDate='1763136000000', pubDateStr='2025-11-15', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1793346486529, creator='13701087609', updateTime=1793346486529, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext=null, issueFiles=null, downloadFileDto=null}, startPage=2706, endPage=2716, ext={EN=ArticleExt(id=1322954342428451530, articleId=1322954340364853961, tenantId=1146029695717560320, journalId=1319373162502258839, language=EN, title=Antimicrobial resistance profiles of Escherichia coli based on molecular typing and public health prevention and control strategy, columnId=1320434453051199700, journalTitle=Journal of Army Medical University, columnName=Think Tank for Public Health, runingTitle=null, highlight=null, articleAbstract=
Objective

The pathogenic characteristics of Escherichia coli (E.coli) in bacterial infections were analyzed using a combination of multiple molecular typing techniques in order to provide evidence for the management of clinical medication safety.

Methods

Samples from some bacterial infection-related cases in a district of Chongqing in 2021 were collected. A total of 30 E.coli strains were selected by a completely random method, and phoA gene PCR assay was performed for identification. Molecular typing of the strains was analyzed using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Antimicrobial susceptibility testing was conducted to determine the drug resistance of the strains, and four β-lactamase-encoding genes (blaCTX-M, blaTEM, blaSHV, blaZ) were selected to detect the carriage of resistance genes.

Results

All 30 E.coli strains displayed the phoA gene target band. Their PFGE banding patterns, with a similarity of 50%~98%, could be classified into 8 clusters. Cluster C was the dominant group, accounting for 53.3% (16/30). C1 and C2 exhibited high genetic correlation, indicating a close phylogenetic relationship. One E.coli strain could not be assigned a sequence type (ST) by MLST, while the remaining 29 E.coli isolates were classified into 16 different STs, demonstrating a polymorphic distribution. Among them, 10 isolates belonged to ST131 (10/30, 33.33%). Evolutionary analysis of the 10 ST131 E.coli strains revealed their distribution across different branches, indicating varying degrees of genetic relatedness. Antimicrobial susceptibility testing revealed that all 30 E.coli strains exhibited varying degrees of resistance, with the highest resistance rate observed against the β-lactam antibiotic ampicillin (25/30,83.33%). Among them, 60.0% were multidrug-resistant bacteria (MDRB). These MDRB strains exhibited 16 distinct resistance profiles, displaying a scattered distribution without a dominant resistance pattern. 50.0% (9/18) of the MDRB strains exhibited six-drug resistance, while the most drug-resistant strain showed eight-drug resistance. Furthermore, the blaCTX-M gene carriage rate among the 30 E.coli strains was 86.67% (26/30), while no blaZ gene was detected.

Conclusion

E.coli related to bacterial infections from a Chongqing district exhibited diverse PFGE/MLST patterns and significant drug resistance. The application of multiple molecular typing techniques can reveal the genetic diversity, evolutionary relationships, and antimicrobial resistance characteristics of pathogenic bacteria. Countermeasures　It is recommended to enhance the molecular typing and drug resistance surveillance network for pathogenic bacteria, establish an early warning mechanism, and implement hierarchical management of antibiotics, thereby improving targeted prevention and epidemic traceability capabilities for key drug-resistant bacteria such as ST131.

, authors=Hua ZONG1, 2, Caiyun LI1, 2, Lingyu GONG2, Yi LUO2, Hong XIAO1, 3, authorsList=Hua ZONG, Caiyun LI, Lingyu GONG, Yi LUO, Hong XIAO, authorCompany=null, correspAuthors=Hong XIAO, authorNote=null, correspAuthorsNote=null, copyrightStatement=© 2025 Journal of Army Medical University, copyrightOwner=Editorial Office of Journal of Army Medical University, 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, fund=null), CN=ArticleExt(id=1322954343950983895, articleId=1322954340364853961, tenantId=1146029695717560320, journalId=1319373162502258839, language=CN, title=基于分子分型监测的大肠埃希菌耐药性分析与公共卫生防控策略, columnId=1320434453520961751, journalTitle=陆军军医大学学报, columnName=论著·公共卫生智库, runingTitle=null, highlight=null, articleAbstract=
目的

采用多种分子分型技术联合分析细菌性感染相关大肠埃希菌（Escherichia coli， E.coli）的特征，为临床用药安全性管理提供参考。

方法

收集2021年重庆市某区部分细菌性感染相关病例样本，采用完全随机方法选择30株E.coli进行phoA基因PCR鉴定，采用脉冲场凝胶电泳（pulsed-field gel electrophoresis，PFGE）、多位点序列分析（multilocus sequence typing，MLST）分析菌株的分子分型，采用药敏试验分析菌株的耐药性，选择4种编码β-内酰胺酶的基因（blaCTX-M、blaTEM、blaSHV、blaZ）分析菌株的耐药基因携带情况。

结果

30株E.coli均出现phoA基因目的条带。PFGE带型图谱显示条带相似度为50%~98%，按照相似度分为8聚类，其中C聚类为优势菌群，占比达53.3%（16/30）；C1/C2属于高度相关的菌株，存在亲缘关系。1株E.coli的MLST未分出型别，其余29株可分为16种ST型，呈多态性分布；其中10株（33.33%）为ST131，但进化分析显示同为ST131也存在不同分支，亲缘关系远近不一。药敏试验结果显示30株E.coli表现出不同程度耐药，对β-内酰胺类的氨苄西林耐药率（83.33%）最高；其中多重耐药菌（multidrug-resistant bacteria，MDRB）为18株（60.0%），表现出16种耐药谱型，其分布呈分散态势，无明显优势耐药谱型；MDRB中50.0%（9/18）为六重耐药，最严重的一株为八重耐药。30株E.coli中blaCTX-M基因携带率为86.67%（26/30），但无blaZ基因。

结论

重庆市某区细菌性感染相关E.coli的PFGE和MLST分子分型呈多态性分布，存在较严重的耐药情况。联合应用多种分型技术，可以揭示致病菌的遗传多样性、进化关系和耐药性等特征。对策 建议通过强化致病菌分子分型与耐药监测网络、建立预警机制并分级管控抗菌药物，以提升对ST131等重点耐药菌的靶向防控与疫情溯源能力。

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肖 虹，E-mail：
XIAO Hong，E-mail:
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M：marker

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MLST typing results of 30 E.coli strains

, figureFileSmall=null, figureFileBig=null, tableContent=
Sample IDadkfumCgyrBicdmdhpurArecAST
20220105-569531811714ST1148
20220125-153404713362829ST131
20220125-33624913171125ST73
20220125-414141020017710ST1193
20220125-553404713362829ST131
20220125-66412120127ST88
20220125-72135276554ST69
20220125-837381937171126ST95
20220125-96412120127ST88
20220105-10924879670582ST648
20220125-1053404713362829ST131
20220125-118481148882ST8774
20220215-16412120127ST88
20220215-253404713362829ST131
20220215-353404713362829ST131
20220215-41342691316109ST4702
20220215-553404713362829ST131
20220215-6353729254573ST405
20220105-153404713362829ST131
20220105-95340--13362829--
20220125-237381937171126ST95
20220105-8362491317825ST5242
20220215-78718226846ST2172
20220105-72135276554ST69
20220105-653404713362829ST131
20220215-92135276554ST69
20220105-1197409313233466ST4456
20220105-4924879670582ST648
20220105-353404713362839ST131
20220105-253404713362829ST131
), ArticleFig(id=1322954349382607636, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1322954340364853961, language=CN, label=表1, caption=

30株E.coli的MLST分型结果

, figureFileSmall=null, figureFileBig=null, tableContent=
Sample IDadkfumCgyrBicdmdhpurArecAST
20220105-569531811714ST1148
20220125-153404713362829ST131
20220125-33624913171125ST73
20220125-414141020017710ST1193
20220125-553404713362829ST131
20220125-66412120127ST88
20220125-72135276554ST69
20220125-837381937171126ST95
20220125-96412120127ST88
20220105-10924879670582ST648
20220125-1053404713362829ST131
20220125-118481148882ST8774
20220215-16412120127ST88
20220215-253404713362829ST131
20220215-353404713362829ST131
20220215-41342691316109ST4702
20220215-553404713362829ST131
20220215-6353729254573ST405
20220105-153404713362829ST131
20220105-95340--13362829--
20220125-237381937171126ST95
20220105-8362491317825ST5242
20220215-78718226846ST2172
20220105-72135276554ST69
20220105-653404713362829ST131
20220215-92135276554ST69
20220105-1197409313233466ST4456
20220105-4924879670582ST648
20220105-353404713362839ST131
20220105-253404713362829ST131
), ArticleFig(id=1322954349437133589, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1322954340364853961, language=EN, label=Table 2, caption=

Antimicrobial susceptibility test results of E.coli

, figureFileSmall=null, figureFileBig=null, tableContent=
Category of antibioticsAntibioticResistance rate/%Intermediate rate/%Susceptibility rate/%
β-Lactams
PenicillinsAMP83.33016.67
CarbapenemsMEM03.3396.67
ETP3.33096.67
CephalosporinsCTZ16.6713.3370.00
CTX53.33046.67
Combination drugsAMS56.6720.0023.33
CZA3.33096.67
PolupeptidesCT3.3396.670
SulfonamidesSXT50.00050.00
PhenicolsCHL13.33086.67
TetracyclinesTET60.00040.00
TIG00100.00
QuinolonesCIP46.673.3350.00
NAL76.67023.33
MacrolidesAZM36.67063.33
AminoglycosidesAMK00100.00
STR53.339.9936.68
AMP：ampicillin；MEM：meropenem；ETP：ertapenem；CTZ：ceftazidime；CTX：cefotaxime；AMS：ampicillin/sulbactam；CZA：ceftazidime/avibactam；CT：colistin；SXT：sulfamehoxazole tablets；CHL：chloramphenicol；TET：tetracyline；TIG：tigecycline； CIP：ciprofloxacin； NAL：nalidixic acid；AZM：azithromycin；AMK：amikacin；STR：streptomycin
), ArticleFig(id=1322954351081300759, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1322954340364853961, language=CN, label=表2, caption=

E.coli的药敏试验结果

, figureFileSmall=null, figureFileBig=null, tableContent=
Category of antibioticsAntibioticResistance rate/%Intermediate rate/%Susceptibility rate/%
β-Lactams
PenicillinsAMP83.33016.67
CarbapenemsMEM03.3396.67
ETP3.33096.67
CephalosporinsCTZ16.6713.3370.00
CTX53.33046.67
Combination drugsAMS56.6720.0023.33
CZA3.33096.67
PolupeptidesCT3.3396.670
SulfonamidesSXT50.00050.00
PhenicolsCHL13.33086.67
TetracyclinesTET60.00040.00
TIG00100.00
QuinolonesCIP46.673.3350.00
NAL76.67023.33
MacrolidesAZM36.67063.33
AminoglycosidesAMK00100.00
STR53.339.9936.68
AMP：ampicillin；MEM：meropenem；ETP：ertapenem；CTZ：ceftazidime；CTX：cefotaxime；AMS：ampicillin/sulbactam；CZA：ceftazidime/avibactam；CT：colistin；SXT：sulfamehoxazole tablets；CHL：chloramphenicol；TET：tetracyline；TIG：tigecycline； CIP：ciprofloxacin； NAL：nalidixic acid；AZM：azithromycin；AMK：amikacin；STR：streptomycin
), ArticleFig(id=1322954351160992536, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1322954340364853961, language=EN, label=Table 3, caption=

Multidrug resistance profiles of 18 E.coli strains

, figureFileSmall=null, figureFileBig=null, tableContent=
Antibiotic resistance profileNumber of
resistant drugs		Number of resistant drug categoriesNumber of
resistant strains
AMP/TET/NAL331
AMP/CTX/TET/NAL431
AMP/AMS/TET/NAL/STR541
AMP/AMS/SXT/TET/STR541
SXT/CHL/TET/NAL/STR551
AMP/AMS/SXT/TET/AZM/STR651
AMP/CTX/SXT/TET/CIP/NAL/STR751
AMP/AMS/SXT/TET/NAL/AZM/STR761
AMP/AMS/SXT/TET/CIP/NAL/AZM/STR861
AMP/CTX/AMS/SXT/TET/NAL/AZM/STR862
AMP/CTX/SXT/TET/CIP/NAL/AZM/STR861
AMP/AMS/SXT/TET/CIP/NAL/AZM/STR861
AMP/CTZ/CTX/AMS/SXT/CHL/TET/CIP/NAL/STR1061
AMP/CTZ/CTX/AMS/SXT/TET/CIP/NAL/AZM/STR1062
AMP/CTZ/CTX/AMS/SXT/CHL/TET/CIP/NAL/AZM/STR1171
AMP/ETP/CTZ/CTX/AMS/CZA/CT/SXT/CHL/TET/CIP/NAL/AZM/STR1481
), ArticleFig(id=1322954351265850137, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1322954340364853961, language=CN, label=表3, caption=

18株E.coli的多重耐药谱型

, figureFileSmall=null, figureFileBig=null, tableContent=
Antibiotic resistance profileNumber of
resistant drugs		Number of resistant drug categoriesNumber of
resistant strains
AMP/TET/NAL331
AMP/CTX/TET/NAL431
AMP/AMS/TET/NAL/STR541
AMP/AMS/SXT/TET/STR541
SXT/CHL/TET/NAL/STR551
AMP/AMS/SXT/TET/AZM/STR651
AMP/CTX/SXT/TET/CIP/NAL/STR751
AMP/AMS/SXT/TET/NAL/AZM/STR761
AMP/AMS/SXT/TET/CIP/NAL/AZM/STR861
AMP/CTX/AMS/SXT/TET/NAL/AZM/STR862
AMP/CTX/SXT/TET/CIP/NAL/AZM/STR861
AMP/AMS/SXT/TET/CIP/NAL/AZM/STR861
AMP/CTZ/CTX/AMS/SXT/CHL/TET/CIP/NAL/STR1061
AMP/CTZ/CTX/AMS/SXT/TET/CIP/NAL/AZM/STR1062
AMP/CTZ/CTX/AMS/SXT/CHL/TET/CIP/NAL/AZM/STR1171
AMP/ETP/CTZ/CTX/AMS/CZA/CT/SXT/CHL/TET/CIP/NAL/AZM/STR1481
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基于分子分型监测的大肠埃希菌耐药性分析与公共卫生防控策略
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宗华 1, 2 , 李彩云 1, 2 , 龚玲玉 2 , 罗益 2 , 肖虹 1, 3
陆军军医大学学报 | 论著·公共卫生智库 2025,47(21): 2706-2716
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陆军军医大学学报 |论著·公共卫生智库 2025 , 47 (21) : 2706 -2716
基于分子分型监测的大肠埃希菌耐药性分析与公共卫生防控策略
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宗华1, 2, 李彩云1, 2, 龚玲玉2, 罗益2, 肖虹1, 3
作者信息
  • 1重庆医科大学公共卫生学院，重庆
  • 2重庆市南岸区疾病预防控制中心，重庆
  • 3高致病性病原微生物重庆市重点实验室，重庆
通讯作者:
肖 虹，E-mail：
XIAO Hong，E-mail:
Antimicrobial resistance profiles of Escherichia coli based on molecular typing and public health prevention and control strategy
Hua ZONG1, 2, Caiyun LI1, 2, Lingyu GONG2, Yi LUO2, Hong XIAO1, 3
Affiliations
  • 1School of Public Health，Chongqing Medical University，Chongqing
  • 2Nan’an Center for Disease Control and Prevention，Chongqing
  • 3Chongqing Key Laboratory of Highly Pathogenic Microbes，Chongqing，China
出版时间: 2025-11-15 doi: 10.16016/j.2097-0927.202507058
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摘要
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目的

采用多种分子分型技术联合分析细菌性感染相关大肠埃希菌（Escherichia coli， E.coli）的特征，为临床用药安全性管理提供参考。

方法

收集2021年重庆市某区部分细菌性感染相关病例样本，采用完全随机方法选择30株E.coli进行phoA基因PCR鉴定，采用脉冲场凝胶电泳（pulsed-field gel electrophoresis，PFGE）、多位点序列分析（multilocus sequence typing，MLST）分析菌株的分子分型，采用药敏试验分析菌株的耐药性，选择4种编码β-内酰胺酶的基因（blaCTX-M、blaTEM、blaSHV、blaZ）分析菌株的耐药基因携带情况。

结果

30株E.coli均出现phoA基因目的条带。PFGE带型图谱显示条带相似度为50%~98%，按照相似度分为8聚类，其中C聚类为优势菌群，占比达53.3%（16/30）；C1/C2属于高度相关的菌株，存在亲缘关系。1株E.coli的MLST未分出型别，其余29株可分为16种ST型，呈多态性分布；其中10株（33.33%）为ST131，但进化分析显示同为ST131也存在不同分支，亲缘关系远近不一。药敏试验结果显示30株E.coli表现出不同程度耐药，对β-内酰胺类的氨苄西林耐药率（83.33%）最高；其中多重耐药菌（multidrug-resistant bacteria，MDRB）为18株（60.0%），表现出16种耐药谱型，其分布呈分散态势，无明显优势耐药谱型；MDRB中50.0%（9/18）为六重耐药，最严重的一株为八重耐药。30株E.coli中blaCTX-M基因携带率为86.67%（26/30），但无blaZ基因。

结论

重庆市某区细菌性感染相关E.coli的PFGE和MLST分子分型呈多态性分布，存在较严重的耐药情况。联合应用多种分型技术，可以揭示致病菌的遗传多样性、进化关系和耐药性等特征。对策 建议通过强化致病菌分子分型与耐药监测网络、建立预警机制并分级管控抗菌药物，以提升对ST131等重点耐药菌的靶向防控与疫情溯源能力。

大肠埃希菌  /  细菌性感染  /  脉冲场凝胶电泳  /  多位点序列分型  /  耐药性
Objective

The pathogenic characteristics of Escherichia coli (E.coli) in bacterial infections were analyzed using a combination of multiple molecular typing techniques in order to provide evidence for the management of clinical medication safety.

Methods

Samples from some bacterial infection-related cases in a district of Chongqing in 2021 were collected. A total of 30 E.coli strains were selected by a completely random method, and phoA gene PCR assay was performed for identification. Molecular typing of the strains was analyzed using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Antimicrobial susceptibility testing was conducted to determine the drug resistance of the strains, and four β-lactamase-encoding genes (blaCTX-M, blaTEM, blaSHV, blaZ) were selected to detect the carriage of resistance genes.

Results

All 30 E.coli strains displayed the phoA gene target band. Their PFGE banding patterns, with a similarity of 50%~98%, could be classified into 8 clusters. Cluster C was the dominant group, accounting for 53.3% (16/30). C1 and C2 exhibited high genetic correlation, indicating a close phylogenetic relationship. One E.coli strain could not be assigned a sequence type (ST) by MLST, while the remaining 29 E.coli isolates were classified into 16 different STs, demonstrating a polymorphic distribution. Among them, 10 isolates belonged to ST131 (10/30, 33.33%). Evolutionary analysis of the 10 ST131 E.coli strains revealed their distribution across different branches, indicating varying degrees of genetic relatedness. Antimicrobial susceptibility testing revealed that all 30 E.coli strains exhibited varying degrees of resistance, with the highest resistance rate observed against the β-lactam antibiotic ampicillin (25/30,83.33%). Among them, 60.0% were multidrug-resistant bacteria (MDRB). These MDRB strains exhibited 16 distinct resistance profiles, displaying a scattered distribution without a dominant resistance pattern. 50.0% (9/18) of the MDRB strains exhibited six-drug resistance, while the most drug-resistant strain showed eight-drug resistance. Furthermore, the blaCTX-M gene carriage rate among the 30 E.coli strains was 86.67% (26/30), while no blaZ gene was detected.

Conclusion

E.coli related to bacterial infections from a Chongqing district exhibited diverse PFGE/MLST patterns and significant drug resistance. The application of multiple molecular typing techniques can reveal the genetic diversity, evolutionary relationships, and antimicrobial resistance characteristics of pathogenic bacteria. Countermeasures　It is recommended to enhance the molecular typing and drug resistance surveillance network for pathogenic bacteria, establish an early warning mechanism, and implement hierarchical management of antibiotics, thereby improving targeted prevention and epidemic traceability capabilities for key drug-resistant bacteria such as ST131.

Escherichia coli  /  bacterial infections  /  pulsed-field gel electrophoresis  /  multilocus sequence typing  /  antibiotic resistance
宗华, 李彩云, 龚玲玉, 罗益, 肖虹. 基于分子分型监测的大肠埃希菌耐药性分析与公共卫生防控策略. 陆军军医大学学报, 2025 , 47 (21) : 2706 -2716 . DOI: 10.16016/j.2097-0927.202507058
Hua ZONG, Caiyun LI, Lingyu GONG, Yi LUO, Hong XIAO. Antimicrobial resistance profiles of Escherichia coli based on molecular typing and public health prevention and control strategy[J]. Journal of Army Medical University, 2025 , 47 (21) : 2706 -2716 . DOI: 10.16016/j.2097-0927.202507058
正文
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2017年我国启动建设传染病检测的创新模式——国家致病菌识别网（Chinese Pathogen Identification Net，China PIN）［1］，它由覆盖全国疾控系统的县级疾控实验室为网络构成单元，依托网络化信息平台，以病原识别、分子分型和基因组溯源技术为核心，开展细菌性传染病以及感染相关的监测与研判，提高疫情发现和防控能力。China PIN在我国细菌性疾病的早期发现、精准溯源和有效防控中起到重要作用［2］。
细菌性感染性疾病是由病原菌通过消化道、呼吸道、接触等途径引发的疾病，涵盖呼吸道感染、感染性腹泻、急性肠胃炎、尿路感染、菌血症等多种疾病［3-4］。此类疾病因病原体种类繁多，影响因素混杂，精准确定病原存在一定难度。细菌性感染常见的病原菌有大肠埃希菌（Escherichia coli，E.coli）、葡萄球菌、铜绿假单胞菌等［5-6］，其中E.coli是人类肠道的正常寄居性菌群，当机体抵抗力下降或侵入肠外组织或器官时，可作为条件致病菌引发感染，能够引起从胃肠炎到泌尿道、血液和中枢神经系统等范围广泛的疾病［7］。有研究表明我国武汉市食源性致泻大肠埃希菌（Foodborne Diarrheagenic Escherichia coli ，FDEC）型别多样，产肠毒素大肠埃希菌（Enterotoxigenic Escherichia coli，ETEC）的优势克隆复合体CC-10被广泛检出［8］。ST131耐药克隆被广泛认为是引起全球范围内社区与医院获得性泌尿道感染和菌血症的首要原因，ST131也是我国E.coli中最优势的克隆型别［9］。这种分子型别的变迁与抗菌药物使用、人口流动等因素密切相关，也为E.coli感染的防控带来新挑战。因此，各地区开展本地E.coli分子分型研究具有重要现实意义。
传统的病原微生物可通过其生化反应、血清学、耐药性等特征进行分型，近年来，随着分子分型技术快速发展，可基于核酸、蛋白质、核糖体等进行致病菌分型，其中核酸分型技术主要包括PCR、脉冲场凝胶电泳（pulsed-field gel electrophoresis，PFGE）、多位点序列分析（multilocus sequence typing，MLST）、宏基因组测序（metagenomics sequencing，MGS）等［10-11］。多种分型技术联合使用，可更准确判定致病菌的来源和传播途径，更有效识别新的、潜在的流行菌株，有助于及时采取措施控制感染的传播。目前，国内对细菌性感染病原特征研究常见病原谱和耐药性研究，病原菌分子分型联合研究在逐渐开展。本文以2021年重庆市某区China PIN哨点医院上报的细菌性感染相关病例为研究对象，对经实验室确诊的感染相关的部分E.coli菌株，进行PCR、PFGE、MLST、耐药性和耐药基因联合分析，有助于确定该地区的病原特征及变异趋势，为该地区人群中E.coli感染的分析、研判和防控提供科学依据。
1　资料与方法
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1.1　样本来源
2021年重庆市某区4个China PIN哨点医院将部分临床诊断为细菌性感染相关的病例上报网络实验室（该区疾病预防控制中心），收集病例的血液、粪便样本418份，经分离培养、微生物质谱技术及PCR技术等，鉴定出样本中含E.coli共90株，采用完全随机方法选择30株进行后续的分型分析。操作步骤参照《国家致病菌识别网实验室监测技术手册（2022试用版）》。
1.2　定性PCR分析
菌株过夜培养，使用细菌基因组DNA提取试剂盒（北京天根生化科技有限公司）提取DNA，选择E.coli通用引物 phoA进行PCR扩增。扩增体系为ddH2O 9.5 μL、上下游引物各1 μL、模板1 μL、Taq/Pfu酶12.5 μL。PCR扩增条件为：94 ℃×5 min预变性；94 ℃×45 s变性，55 ℃×45 s退火，72 ℃×45 s延伸，共循环32次；72 ℃×10 min再延伸；4 ℃。PCR产物经1%琼脂糖凝胶电泳后，凝胶成像系统（基因有限公司）分析。
1.3　PFGE分析
参照《细菌分子分型实验操作规范（China PIN-2022-TYJS003）》。经菌株培养准备、DNA包埋、酶切、脉冲场电泳、染色成像等步骤，以H9812作为DNA标记物，XbaⅠ酶切，使用BioNumerics软件分析电泳成像结果。
1.4　MLST分析
选择7对管家基因（adk、fumC、gyrB、icd、mdh、purA、recA）设计引物，以菌株DNA为模板进行PCR扩增。对PCR产物用1%琼脂糖凝胶电泳后进行双向测序，测序结果用SeqMan软件拼接剪切。所有测序序列在MLST数据库（https://pubmlst.org/） 中进行比对分析，获取序列型（sequence-typing，ST）。使用mlst（https://github.com/tseemann/mlst）和PubMLST （https://pubmlst.org/）进行基因组序列分型及注释，从NCBI数据库（https://www.ncbi.nlm.nih.gov/）中下载E.coli 数据，使用进化树可视化在线工具（http://etetoolkit.org/treeview/）进行树结构和SNP队列的展示。
1.5　药敏试验和耐药基因分析
药敏试验：以革兰阴性需氧菌药物敏感性96孔检测板［复星诊断科技（上海）有限公司］为材料，采用微量肉汤稀释法对30株E.coli进行8大类17种抗生素的耐药谱筛查，选择E.coli ATCC25922作为质控菌株；药物耐药折点参照2017年临床与实验室标准化研究所药敏试验操作标准（CLSI M100-S27）。
耐药基因分析：根据药敏试验结果，选择4种编码β-内酰胺酶（β-lactamase）的基因（blaCTX-M、blaTEM、blaSHV、blaZ）设计引物，进行PCR扩增。PCR产物经1%琼脂糖凝胶电泳成像鉴定目的基因成像。
1.6　统计学分析
采用 SPSS 26.0 统计软件进行数据处理。计数资料以例（%）表示，耐药基因携带率与耐药表型的关联性分析采用χ²检验；不同ST型菌株耐药率的比较采用Fisher确切概率法。以P<0.05为差异具有统计学意义。
2　结果
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2.1　定性PCR结果
30株E.coli的phoA基因PCR产物均出现目的条带，其大小在622 bp，说明此30菌确为E.coli（图1）。
2.2　PFGE分析结果
30株E.coli通过使用XbaⅠ酶切后进行PFGE，除7号菌条带模糊，其余29株DNA片段获得较好的分离，可见从凝胶上端到下端清晰分离开的片段，分子量20.5~668.9 kb，各菌产生14~20条DNA指纹图谱。E.coli的PFGE酶切图谱见图2，30株E.coli的DNA分子产生30个酶切带型。
使用BioNumerics软件分析E.coli的PFGE带型图谱，其聚类分析结果见图3。30株E.coli显示条带相似度为50%~98%，按照相似度不同分为8聚类（A、B、C、D、E、F、G、H），呈多态性分布。其中C聚类最多，占比达53.3%（16/30），为优势菌群。聚类C分为C1和C2两种亚聚类，C1/C2（条带相似度分别为92.3%、98.0%）的条带只有1~3条差异，属于高度相关的菌株，存在亲缘关系。
2.3　MLST分析结果
30株E.coli的7对管家基因adk、fumC、gyrB、icd、mdh、purA、recA电泳结果如图4所示。
30株E.coli有1株（20220105-9）未分出型别，是由于其gyrB管家基因测序为重叠峰，无分型结果；其余29株E.coli可分为16种ST型，呈多态性分布，多样性占比达53.33%（16/30），其中10株为ST131（33.33%）；其次为3株ST69（10.00%）；3株ST88（10.00%）。见表1。
10株同为ST131型的E.coli菌株进化分析显示存在不同分支，亲缘关系远近不一，详见图5。
2.4　药敏试验和耐药基因分析
2.4.1　药敏试验结果
本研究中药敏试验结果显示，30株E.coli中28株表现出不同程度耐药，耐药率达93.33%（28/30）。30株E.coli表现出不同程度耐药（表2），其中β-内酰胺类的氨苄西林耐药率最高，为25株（83.33%），其次是耐喹诺酮类的萘啶酸23株（76.67%）、耐四环素类的四环素18株（60.00%）；30株E.coli全部对美罗培南、替加环素和阿米卡星敏感；1株E.coli对多粘菌素E耐药。
2.4.2　多重耐药情况
多重耐药表现为耐3类或3类以上抗菌药物。30株E.coli对17种常见抗菌药物的多重耐药结果见表3，可见30株E.coli中多重耐药菌（multidrug-resistant bacteria，MDRB）为18株（60.00%），这些多重耐药菌全部表现出对四环素耐药（18/18，100%），其次为对氨苄西林耐药（17/18，94.44%）、对萘啶酸和链霉素耐药（16/18，88.89%）。18株多重耐药菌表现出16种耐药谱型，其分布呈现为分散态势，无明显优势耐药谱型。其中，六重耐药最为常见，占比50.0%（9/18），最严重的一株为八重耐药。
2.4.3　耐药基因情况
30株E.coli中3种编码β-内酰胺酶的（blaCTX-M、blaTEM、blaSHV）的携带率分别为86.67%（26/30）、20.00%（6/30）、6.67%（2/30），无blaZ基因（0/30）。部分菌株的blaCTX-M、blaTEM、blaSHV基因PCR扩增结果见图6。
3　讨论
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3.1　分子分型技术在感染性疾病监测和病原溯源中的应用价值
病原微生物核酸分子分型技术（如PCR、PFGE、MLST、MGS等）是通过分析微生物的核酸特征，对其进行分类和溯源的技术［12-13］。这些技术在流行病学调查、感染控制、疫苗开发和致病菌耐药性监测中具有重要应用。在我国基层疾病预防机构的实际工作中，病原微生物分子分型技术的选择需结合具体病原体和研究目标，权衡分辨率、速度和成本。WGS因其具有极高的分辨率且能精准溯源，正逐渐成为新一代分子分型“金标准”，但其高昂的成本、专业的生物信息学分析等缺点［13］限制了其在基层单位的应用。在资源有限地区，进行病原微生物分子分型选择成本相对低廉的PFGE、MLST技术较WGS更加可行。
3.2　PFGE和MLST联合应用对感染相关E.coli分子分型的优势
PFGE和MLST作为两种常用的分子分型技术，在感染相关致病菌的监测、溯源和流行病学研究中发挥了重要作用。PFGE技术具有高分辨率和可重复性，适用于致病菌的快速溯源和监测，广泛应用于细菌的分子分型及溯源分析［14-16］；而MLST技术则以其高度的可比性和可移植性在全球范围内得到了广泛应用，适用于长期监测和流行病学研究［17-18］。两种分型技术各具优势。本研究根据China PIN的PFGE分析溯源要求，联合应用国际公认的MLST分型技术，从不同维度对致病菌的来源和分型进行综合分析，显著提升了分型结果的准确性和可靠性。本研究中PFGE分析显示，30株E.coli显示条带相似度在50%~98%，MLST分型成功的29株E.coli可分为16种ST型，表明该区的E.coli菌株基因型呈现高度多态性，与我国东南部［19］和加纳、西班牙等国家［20-21］的情况类似，且该区E.coli以ST131为优势流行型别，是全球肠外致病性E.coli分离株中的主要谱系，也与王晓驰等国内外研究结果相似［17-19，22］。根据PFGE和MLST联合分型结果，基层传染病防控机构可对相似度高的菌株开展追溯性的流行病学调查，明确感染来源与传播链，对于遏制感染相关致病菌引起的疾病扩大及隐匿传播具有积极作用。
3.3　 E.coli存在较严重的耐药情况
E.coli是最常见的革兰氏阴性细菌病原体，在临床和流行病学领域均构成重大挑战。近年来，由于抗菌药物使用带来的选择性压力持续增强，以ST131为代表的高耐药高风险E.coli不断进化并广泛传播［23］，进一步加剧了感染控制难度。本研究中药敏试验结果表明E.coli存在较严重的耐药现象，93.33%的感染相关菌株表现出不同程度耐药，其对β-内酰胺类的氨苄西林耐药率最高（83.33%），与国内江苏（71.85%）［24］、辽宁（85.9%）［25］等地和国外赞比亚卢萨卡（83.4%）［26］的研究结果接近。此外，本研究中的E.coli有60.0%（18/30）为MDRB，这一比例与近年来国内外感染相关E.coli的多重耐药流行趋势一致［24-27］，提示该地区感染相关E.coli的耐药问题已较为严峻。对于这种现象，应结合实际情况采取预防措施，规范临床使用抗生素，有效遏制MDRB甚至超级细菌的产生［28-29］。而不同研究中的药敏试验使用的抗生素种类不同，使研究结果之间相互比较存在一定的困难。
本研究对多粘菌素的药敏谱分析发现，所有30株大肠杆菌未表现出敏感特性。值得注意的是其中绝大多数（29/30，96.7%）为中介，仅1株（3.3%）E.coli对多粘菌素E耐药。而多粘菌素有着悠久的抗菌历史，曾长期被用作治疗由高耐药性革兰氏阴性细菌引起的严重感染的最后一道防线［30］。然而，E.coli对多粘菌素耐药性的出现［31］，对多粘菌素的应用提出了挑战。mcr基因是一类质粒介导的多粘菌素耐药基因，最早于2015年在中国动物源E.coli中发现（mcr-1），随后在全球传播，目前已有多个变种（mcr-1至mcr-10）［32］。由于本研究未开展mcr基因相关检测，无法明确该地区感染相关E.coli对多粘菌素耐药的分子机制，这将成为本课题组后续研究的重点方向，以期为多粘菌素耐药性的精准防控提供更全面的科学依据。
E.coli耐药性的产生既与抗生素频繁使用有关，也与耐药基因携带情况相关［33-34］，编码β-内酰胺酶的基因通常是bla基因，其表达的酶能够水解β-内酰胺类抗生素（如青霉素、氨苄西林、头孢菌素等），导致细菌耐药。本研究的30株E.coli中编码超广谱β-内酰胺酶（extended-spectrum β-lactamases，ESBL）的blaCTX-M基因携带率为86.67%，明显高于2017年河南孙文萱等［35］研究中的52.17%，研究者推测可能是地域或时间不同造成的差异。而blaZ基因［36］是编码 β-内酰胺酶（青霉素酶）的基因，主要存在于革兰氏阳性菌（如金黄色葡萄球菌），赋予细菌对青霉素类抗生素（如青霉素G、氨苄西林）的耐药性。本研究的30株E.coli中没有检出blaZ基因，验证了耐药基因试验检测结果正确。
3.4　公共卫生相关问题的政策建议
针对本研究揭示的重庆市某区细菌性感染相关E.coli多态性分布及严重耐药问题，为切实提升区域感染性疾病防控能力，特向卫生政策制定部门提出以下建议：①国家卫生健康行政部门继续强化国家致病菌监测网络建设，将分子分型与耐药监测纳入常规公共卫生监测体系，建立长期动态监测机制，以系统掌握菌株遗传演化与耐药变迁规律。②省/市级卫生健康行政部门制定抗菌药物分级管控专项政策，严格限制氨苄西林等高耐药率药物的不合理使用，完善处方审核制度，推动药敏结果与临床用药有效衔接，降低耐药选择压力。③省/市级卫生健康行政部门建立区域耐药基因数据库与疫情预警机制，整合分子分型、耐药数据与临床信息，构建信息共享平台，针对ST131等优势克隆实施靶向防控，实现早期预警与快速响应。④市/区县级卫生健康行政部门加强基层实验室能力建设，重点支持分子分型技术的推广与人员培训，提升菌株鉴定与溯源能力，强化医防协同与信息共享，构建“监测-分析-预警-防控”闭环管理体系。
综上所述，重庆市某区细菌性感染相关E.coli的PFGE和MLST分子分型呈多态性分布，存在较严重的耐药情况。联合应用多种分型技术，可以揭示致病菌的遗传多样性、进化关系和耐药性等病原特征，为细菌性感染相关疾病的监测、临床治疗方案优化、疫情暴发追踪溯源及防控策略制定提供有力技术支持。

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2025年第47卷第21期
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doi: 10.16016/j.2097-0927.202507058
  • 接收时间：2025-07-18
  • 首发时间：2026-10-30
  • 出版时间：2025-11-15
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  • 收稿日期：2025-07-18
  • 修回日期：2025-10-22
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    1重庆医科大学公共卫生学院，重庆
    2重庆市南岸区疾病预防控制中心，重庆
    3高致病性病原微生物重庆市重点实验室，重庆

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肖 虹，E-mail：
XIAO Hong，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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