铜绿假单胞菌泳动、蹭行能力及Ⅲ型分泌系统与成膜能力的关系

doi:10.3724/zdxbyxb-2021-0187

浙江大学学报（医学版）

2021, Vol. 50

Issue (3): 345-351 DOI: 10.3724/zdxbyxb-2021-0187

原著

铜绿假单胞菌泳动、蹭行能力及Ⅲ型分泌系统与成膜能力的关系

税剑^1,²(

),王海晨¹,陶晓燕¹,闵昌航¹,李军¹,邹明祥^1,*(

)

1.中南大学湘雅医院检验科，湖南长沙 410008
2.南华大学附属长沙中心医院检验科，湖南长沙 410004

Relationship of biofilm-forming ability of Pseudomonas aeruginosa with swimming motility, twitching motility and virulence gene distribution

SHUI Jian^1,²(

),WANG Haichen¹,TAO Xiaoyan¹,MIN Changhang¹,LI Jun¹,ZOU Mingxiang^1,*(

)

1. Clinical Laboratory, Xiangya Hospital, Central South University, Changsha 410008, China;
2. Clinical Laboratory, Affiliated Changsha Central Hospital, University of South China, Changsha 410004, China

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摘要：

目的：探讨铜绿假单胞菌（PA）泳动及蹭行能力对生物被膜形成的影响，并分析不同成膜能力PA菌株Ⅲ型分泌系统毒力基因差异。方法：收集中南大学湘雅医院临床分离的非重复PA 192株，96孔板法检测菌株成膜能力，平板法检测菌株的泳动及蹭行能力，聚合酶链反应检测Ⅲ型分泌系统毒力基因。结果：192株PA临床分离株中186株（96.9%）具有成膜能力，其中弱成膜36株，中等成膜84株，强成膜66株。不成膜、弱成膜、中等成膜、强成膜组泳动环直径分别为（9.12±6.76）、（18.42±7.51）、（19.10±4.77）、（17.80±5.27）mm；各组蹭行环直径分别为（8.38±1.50）、（17.21±7.42）、（18.49±5.62）、（20.44±6.43）mm，不成膜组泳动和蹭行能力均弱于各成膜组，差异具有统计学意义（均P<0.05）。192株PA中，exoS、exoU、exoY和exoT阳性分别为163株（84.9%）、40株（20.8%）、183株（95.3%）和189株（98.4%）。不同成膜能力PA毒力基因exoS、exoU、exoT阳性率不同，差异具有统计学意义（P<0.05）。其中强成膜组exoS阳性率低于中等成膜组（χ²=9.293，P<0.01）和弱成膜组（χ²=9.997，P<0.01）；强成膜组exoU阳性率高于弱成膜组（χ²=10.803，P<0.01）。结论：中南大学湘雅医院临床分离的铜绿假单胞菌多具有生物被膜形成能力；泳动、蹭行能力与是否形成生物被膜相关，而与成膜能力强弱相关不明显；Ⅲ型分泌系统毒力基因与生物被膜形成能力可能相关。

关键词： 铜绿假单胞菌; 细菌生物被膜; 泳动能力; 蹭行能力; 毒力基因

Abstract:

Objective:To investigate the relationship of biofilm-forming ability of Pseudomonas aeruginosa(PA) with swimming motility, twitching motility and virulence gene distribution. Methods:A total of 192 clinical isolates of PA were collected consecutively. Microtiter plate method was used to evaluate the ability to form biofilm. The swimming and twitching motilities were detected by plate method. Polymerase chain reaction (PCR) was used to detect virulence genes. Results:Of the 192 PA clinical isolates, 186 (96.9%) showed biofilm-forming ability. Among them, 36 isolates showed weak biofilm-forming ability, 84 exhibited moderate biofilm-forming ability and 66 showed strong biofilm-forming ability. The diameters of the swimming ring for PA with none biofilm-forming ability, weak biofilm-forming ability, moderate biofilm-forming ability, strong biofilm-forming ability were (9.12±6.76), (18.42±7.51), (19.10±4.77) and (17.80±5.27)?mm, respectively. The diameters of the twitching ring for PA in above groups were (8.38±1.50), (17.21±7.42), (18.49±5.62) and (20.44±6.43)?mm, respectively. The swimming motility and twitching motility of none biofilm-forming ability group were weaker than biofilm-forming ability groups (all P<0.05). Among 192 PA strains, 163 wereexoS positive (84.9%), 40 were exoUpositive (20.8%), 183 were exoY positive (95.3%), and 189 were exoT positive (98.4%). The positive rate of PA virulence gene exoS, exoU and exoT were different in strains with different biofilm-forming abilities (P<0.05). The rate ofexoS in the strong biofilm-forming ability group was lower than that in the moderate biofilm-forming ability group (χ²=9.293, P<0.01) and the weak biofilm-forming ability group (χ²=9.997, P<0.01). The rate ofexoU in the strong biofilm-forming ability group was higher than that in the weak biofilm-forming ability group (χ²=10.803, P<0.01).Conclusions:Most clinical isolates of PA can form biofilm. Swimming and twitching motilities are related to the formation of biofilm, but not significantly related to strength of biofilm-forming ability. The virulence genes of type Ⅲ secretion system for PA may be related to the biofilm-forming ability.

Key words: Pseudomonas aeruginosa Bacterial biofilm Swimming motility Twitching motility Virulence gene

收稿日期: 2021-01-21 出版日期: 2021-08-16

CLC:

Q939.112

基金资助: 湖南省自然科学基金（2018JJ6058，2020JJ5900）；湖南省科技创新项目（湘发改投资[2014]658号）；湖南省卫生健康委科研计划（202111000032）

通讯作者: 邹明祥 E-mail: shuijian2005_2005@163.com;zoumingxiang@126.com

作者简介: 税　剑，主管技师，主要从事临床微生物学研究；E-mail：shuijian2005_2005@163.com；https：//orcid.org/0000-0001-8809-0175

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引用本文:

税剑,王海晨,陶晓燕,闵昌航,李军,邹明祥. 铜绿假单胞菌泳动、蹭行能力及Ⅲ型分泌系统与成膜能力的关系[J]. 浙江大学学报（医学版）, 2021, 50(3): 345-351.

SHUI Jian,WANG Haichen,TAO Xiaoyan,MIN Changhang,LI Jun,ZOU Mingxiang. Relationship of biofilm-forming ability of Pseudomonas aeruginosa with swimming motility, twitching motility and virulence gene distribution. J Zhejiang Univ (Med Sci), 2021, 50(3): 345-351.

链接本文:

http://www.zjujournals.com/med/CN/10.3724/zdxbyxb-2021-0187 或 http://www.zjujournals.com/med/CN/Y2021/V50/I3/345

表 1 毒力基因引物序列

图 1 部分铜绿假单胞菌菌株结晶紫染色结果3、7、9为不成膜菌株；4、8为弱成膜菌株；5、6、10、12为中等成膜菌株；1、2、11为强成膜菌株；C为阴性对照.

成膜能力	株数（%）	吸光度值（ $x ˉ ± s$ ）
不成膜	6（3.1）	0.0956±0.0068
弱成膜	36（18.8）	0.1658±0.0354
中等成膜	84（43.7）	0.3265±0.0647
强成膜	66（34.4）	0.8346±0.5131

表 2 192株不同成膜能力铜绿假单胞菌半定量检测结果

图 2 部分铜绿假单胞菌菌株泳动、蹭行实验结果1：不成膜菌株；2：弱成膜菌株；3：中等成膜菌株；4：强成膜菌株.

图 3 部分铜绿假单胞菌毒力基因电泳图M：Marker 2000；1~16：PA菌株.

表 3 不同成膜能力铜绿假单胞菌毒力基因携带率比较

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