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J Zhejiang Univ (Med Sci)  2021, Vol. 50 Issue (3): 345-351    DOI: 10.3724/zdxbyxb-2021-0187
    
Relationship of biofilm-forming ability of Pseudomonas aeruginosa with swimming motility, twitching motility and virulence gene distribution
SHUI Jian1,2(),WANG Haichen1,TAO Xiaoyan1,MIN Changhang1,LI Jun1,ZOU Mingxiang1,*()
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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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 wordsPseudomonas aeruginosa      Bacterial biofilm      Swimming motility      Twitching motility      Virulence gene     
Received: 21 January 2021      Published: 16 August 2021
CLC:  Q939.112  
Corresponding Authors: ZOU Mingxiang     E-mail: shuijian2005_2005@163.com;zoumingxiang@126.com
Cite this article:

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.

URL:

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


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

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,exoSexoUexoYexoT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exoSexoUexoT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 

基因

引物序列(5′→3′)

产物大小(bp)

退火温度(℃)

exoS

正向:ATCCTCAGGCGTACATCC反向:ACGACGGCTATCTCTCCAC

328

55

exoU

正向:AGTGCTTACATTCCCCAGATTAAG反向:TGTCAAAATTCTTGTCGATCATCT

271

53

exoY

正向:CTTTCATCGTTCTGTCTCCTTCTT反向:GTTAGAAACCACCTGACGATGAC

272

55

exoT

正向:AATCGCCGTCCAACTGCATGCG反向:TGTTCGCCGAGGTACTGCTC

152

58

Table 1 PCR primers of virulence genes
Figure 1 Crystal violet quantification of strains

成膜能力

株数(%)

吸光度值(xˉ±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

Table 2 Biofilm-forming ability of 192
Figure 2 Swimming motility and twitching motility of strains
Figure 3 Virulence genes electrophoregram of strains

成膜能力

n

exoS

exoU

exoY

exoT

不成膜

6

5(83.3)

0(0.0)

5(83.3)

5(83.3)

弱成膜

36

35(97.2)

2(5.6)

35(97.2)

35(97.2)

中等成膜

84

76(90.5)

15(17.9)

79(94.0)

84(100.0)

强成膜

66

47(71.2)*#

23(34.8)*

64(97.0)

65(98.5)

χ2

15.349

14.221

3.140

7.072

P

<0.01

<0.01

>0.05

<0.05

Table 3 Analysis of difference in virulence genes for strains with different biofilm-forming abilities
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