产志贺毒素大肠埃希菌生物被膜形成能力与毒力基因表达的关系

doi:10.3969/j.issn.1673-8640.2015.06.018

摘要/Abstract

摘要： 目的

探讨临床腹泻患者产志贺毒素大肠埃希菌(STEC)生物被膜形成能力与致病毒力基因表达的关系。

方法

收集从520例临床腹泻患者标本中检测分离到的STEC,进行体外药敏试验和生物被膜形成能力定量分析;应用多重聚合酶链反应(PCR)检测产志贺毒素相关基因stx1、stx2,实时定量逆转录PCR(RT-PCR)检测eae、ehxA基因表达。以健康体检人群粪便标本中分离的98株大肠埃希菌作为对照菌株。

结果

从520例腹泻患者标本中分离出328株大肠埃希菌,其中stx1、stx2基因阳性菌(即STEC)78株(23.8%)。78株STEC中产生物被膜菌57株(73.1%),eae和ehxA基因的相对表达量分别为6.57(2.82~27.8)、1.81(1.07~5.28);不产生物被膜菌21株(26.9%),eae和ehxA基因的相对表达量分别为3.41(0.96~12.1)、1.76(0.89~6.06);另分离出副溶血弧菌18株[其中产生物被膜菌10株(55.6%)]、奇异变形杆菌3株。98株对照菌中产生物被膜菌39株(39.7%),eae和ehxA基因的相对表达量分别为1.02(0.86~3.47)、1.03(0.88~3.03)。产生物被膜STEC对氨苄西林/舒巴坦、复方磺胺甲噁唑、环丙沙星的耐药率明显高于不产生物被膜STEC和对照菌株。

结论

STEC生物被膜形成率明显高于健康人群定植大肠埃希菌,产生物被膜STEC的eae基因表达明显提高;ehxA基因表达与不产生物被膜的STEC比较无明显变化。

关键词: 大肠埃希菌, 志贺毒素, 生物被膜, 基因表达

Abstract: Objective

To investigate the relationship between the biofilm formation ability of the patients with diarrhea of Shiga toxin-producing Escherichia coli (STEC) and virulence gene expression.

Methods

STEC were detected and isolated from 520 patients with diarrhea. The antibiotic drug sensitivity in vitro and ability to form biofilm were quantitatively analyzed. Shiga toxin related gene stx1 and stx2 were amplified and detected by multiplex polymerase chain reaction (PCR). The expressions of eae and ehxA genes were detected by real-time quantitative reverse transcriptase (RT)-PCR. A total of 98 isolates of Escherichia coli from fecal specimens in healthy subjects were included as control isolates.

Results

A total of 328 isolates of Escherichia coli were isolated from patients with diarrhea. The stx1 and stx2 gene positive bacteria (STEC) had 78 isolates, accounting for 23.8%. There were 57 isolates of STEC formed biofilm (73.1%). The relative expression of eae gene was 6.57(2.82-27.8), and the expression of ehxA gene was 1.81(1.07-5.28). There were 21 isolates of STEC unformed biofilm (26.9%).The relative expression of eae gene was 3.41(0.96-12.1), and the expression of ehxA gene was 1.76(0.89-6.06). A total of 18 isolates of Vibrio parahaemolyticus were isolated, and 10 isolates were formed biofilm (55.6%). There were 3 isolates of Proteus mirabilis.Among 98 control isolates, 39 isolates were formed biofilm (39.7%). The relative expression of eae gene was 1.02(0.86-3.47), and the expression of ehxA gene was 1.03(0.88-3.03). Biofilm-formed STEC resistance to ampicillin / sulbactam, trimethoprim and ciprofloxacin was significantly higher than that of biofilm-unformed STEC and control isolates.

Conclusions

The STEC biofilm formation rate is significantly higher than that in healthy subjects' colonization Escherichia coli. The expression of eae gene in biofilm-formed STEC is significantly improved. The expression of ehxA gene has no significant difference with biofilm-unformed STEC.

Key words: Escherichia coli, Shiga toxin, Biofilm, Gene expression

中图分类号:

R378.2

解范迪, 牛术孟, 施锦杰, 张雯雁. 产志贺毒素大肠埃希菌生物被膜形成能力与毒力基因表达的关系[J]. 检验医学, 2015, 30(6): 617-621.

XIE Fandi, NIU Shumeng, SHI Jinjie, ZHANG Wenyan. Relationship between STEC biofilm formation ability and the expression of virulence genes[J]. Laboratory Medicine, 2015, 30(6): 617-621.

图/表 5

参考文献 9

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引物	序列 (5'→3')	产物(bp)
stx1 F	TGG ATT TAA TCT CGC ATA GTG GAA	81
stx1 R	CAG CTG TCA CAG TAA CAA ACC GTA A
stx2 F	CAA CGG ACA GCA GTT ATA CCA CTC T	102
stx2 R	TTA ACG CCA GAT ATG ATG AAA CCA
eaeA F	AAG CGA CTG AGG TCA CT	159
eaeA R	ACG CTG CTC ACT ACT AGA TGT
ehxA F	CAC ACC GAC CTT ATA ATA TTC TGT CA	204
ehxA R	AAT GTT ATC CCA TTG AGA TCA TTT GAC T
16S rRNAF	GGA GGA AGG TGG GGA TGA CG	61
16S rRNAR	ATG GTG TGA CGG GCG GTG TG

引物	序列 (5'→3')	产物(bp)
stx1 F	TGG ATT TAA TCT CGC ATA GTG GAA	81
stx1 R	CAG CTG TCA CAG TAA CAA ACC GTA A
stx2 F	CAA CGG ACA GCA GTT ATA CCA CTC T	102
stx2 R	TTA ACG CCA GAT ATG ATG AAA CCA
eaeA F	AAG CGA CTG AGG TCA CT	159
eaeA R	ACG CTG CTC ACT ACT AGA TGT
ehxA F	CAC ACC GAC CTT ATA ATA TTC TGT CA	204
ehxA R	AAT GTT ATC CCA TTG AGA TCA TTT GAC T
16S rRNAF	GGA GGA AGG TGG GGA TGA CG	61
16S rRNAR	ATG GTG TGA CGG GCG GTG TG

致病菌	菌株数(%)
STEC	78(78.8)
副溶血弧菌	18(18.2)
奇异变形杆菌	3(3.0)
合计	99(100.0)

致病菌	菌株数(%)
STEC	78(78.8)
副溶血弧菌	18(18.2)
奇异变形杆菌	3(3.0)
合计	99(100.0)

被膜形成能力	菌株数	阴性(-)	弱阳性(+)	中等阳性(++)	强阳性(+++)
STEC	78	21(26.9)^*	12(15.4)	26(33.3)	19(24.4)
副溶血弧菌	18	8(44.4)	3(16.7)	5(27.8)	2(11.1)
对照菌株	98	59(60.3)^*	6(6.1)	11(11.2)	22(22.4)