多重耐药铜绿假单胞菌群体感应相关基因表达及其对生物膜形成和耐药性的影响

doi:10.13481/j.1671-587X.20250408

Abstract

Abstract:

Objective To discuss the effect of quorum sensing-related gene expression on biofilm formation and drug resistance in clinically multidrug-resistant Pseudomonas aeruginosa， and to clarify the mechanism of enhacing drug resistance. Methods A total of 77 strains of Pseudomonas aeruginosa were collected. Based on drug resistance， the strains were divided into multidrug-resistant group and sensitive group. The optimal biofilm formation conditions were determined using the microtiter plate method； biofilm formations of the stains in both groups was observed under an optical microscope； crystal violet staining was used to semiquantitatively detect biofilm formation ability of P. aeruginosa in both groups； microbroth dilution method was used to determine the minimal inhibitory concentration （MIC） values of the quorum sensing inhibitor （C-30） against Pseudomonas aeruginosa in both groups； RNA was extracted from two groups using a commercial kit， while RNA from planktonic state and biofilm state of multidrug-resistant strains was extracted using modified TRIzol method； real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the mRNA expression levels of quorum sensing-related genes （lasR/I， RhlR/I， PqsR/A） of the stains in multidrug-resistant group and sensitive group， as well as before and after adding the quorum sensing inhibitor C-30. Results Among 77 strains of Pseudomonas aeruginosa， 56 were multidrug-resistant （multidrug-resistant group） and 21 were fully sensitive（sensitive group）. Optimal biofilm formation occurred at a bacterial concentration of 1.5×10⁸ CFU·mL^-1 with 48 h incubation. The biofilm positivity rate was 91%， with strongly positive， moderately positive， weakly positive， and negative biofilms accounting for 16%， 34%， 41%， and 9%， respectively. The biofilm positivity rate in multidrug-resistant strains was 96%， and biofilm formation ability in multidrug-resistant group was higher than that in sensitive group （P<0.05）. When the concentration of C-30 was 8 mg·L^-1 the biofilm formation in most Pseudomonas aeruginosa was inhibited， with enhanced suppression at higher concentrations. The absorbtion （A） value of both planktonic-state and biofilm-state RNA ranged from 1.8 to 2.0. The RT-qPCR results showed that compared with planktonic state， the expression levels of lasR/I， RhlR/I， and PqsR/A mRNA of the stains in biofilm state were significantly increased （P<0.01）. Compared with non-inhibitor group， the expression levels of lasR/I， RhlR/I， and PqsR/A mRNA in biofilm state of inhibitor-treated group were significantly decreased （P<0.01）. Conclusion High expression of quorum sensing-related genes in multidrug-resistant P. aeruginosa promotes biofilm formation， thereby enhancing drug resistance.

Key words: Pseudmonas aeruginosa, Biofilms, Quorum sensing system, Real-time fluorescence quantitative PCR, Drug resistance

CLC Number:

R378.99

Yan LIU,Beizhen PAN,Jifei YANG,Xianyu ZHANG,Wenbo DING,Lingli SONG,Yundong ZHAO. Expression of quorum sensing related genes in multidrug-resistant Pseudomonas aeruginosa and its effects on biofilm formation and drug resistance[J].Journal of Jilin University(Medicine Edition), 2025, 51(4): 921-928.

Figures/Tables 7

Tab.1

Tab.2

Fig. 1

Fig. 2

Tab.3

Tab.4

Fig. 3

References 27

[1]	CRONE S， VIVES-FLÓREZ M， KVICH L， et al. The environmental occurrence of pseudomonas aeruginosa［J］. APMIS， 2020， 128（3）： 220-231.
[2]	ALMEIDA V F， DANTAS R C， FERREIRA M L， et al. Relationship between antimicrobial use and the highest number of multidrug-resistant-Pseudomonas aeruginosa： a 10-year study［J］. J Infect Dev Ctries， 2024， 18（8）： 1227-1232.
[3]	吴贤丽，祝司霞. 2017-2021年临床分离多重耐药铜绿假单胞菌的分布、耐药性变迁及OprD2基因突变分析［J］. 国外医药（抗生素分册）， 2022， 43（6）： 415-420.
[4]	尹佳锋，郭智，耿燕，等. 西安某院近8年铜绿假单胞菌的临床分布特征及耐药性分析［J］. 中国抗生素杂志， 2022， 47（9）： 957-961.
[5]	AVAKH A， GRANT G D， CHEESMAN M J， et al. The art of war with Pseudomonas aeruginosa： targeting mex efflux pumps directly to strategically enhance antipseudomonal drug efficacy［J］. Antibiotics （Basel）， 2023， 12（8）： 1304.
[6]	NASSARAWA S S， NAYIK G A， GUPTA S D， et al. Chemical aspects of polyphenol-protein interactions and their antibacterial activity［J］. Crit Rev Food Sci Nutr， 2023， 63（28）： 9482-9505.
[7]	SHASTRY R P， ABHINAND C S. Targeting the Pseudomonas aeruginosa quorum sensing system to inhibit virulence factors and eradicate biofilm formation using AHL-analogue phytochemicals［J］. J Biomol Struct Dyn， 2024， 42（4）： 1956-1965.
[8]	HUMPHRIES R， BOBENCHIK A M， HINDLER J A， et al. Overview of changes to the clinical and laboratory standards institute performance standards for antimicrobial susceptibility testing， M100［J］. J Clin Microbiol， 2021， 59（12）： 10.1128/jcm. 00213-21.
[9]	李小凤. 碳青霉烯类耐药铜绿假单胞菌生物膜与外排泵相关性研究［D］. 昆明：昆明医科大学， 2022.
[10]	YU H， LIU Y， YANG F W， et al. The combination of hexanal and geraniol in sublethal concentrations synergistically inhibits quorum sensing in Pseudomonas fluorescens-In vitro and in silico approaches［J］. J Appl Microbiol， 2022， 133（4）： 2122-2136.
[11]	段薇，刘思琪，黄云，等. 浮游状态和生物膜状态白念珠菌总RNA提取方法的比较研究［J］. 口腔医学， 2022， 42（5）： 416-419， 430.
[12]	CAO P B， FLEMING D， MOUSTAFA D A， et al. A Pseudomonas aeruginosa small RNA regulates chronic and acute infection［J］. Nature， 2023， 618（7964）： 358-364.
[13]	王艳，曹洪兵，丁妍. 2437株临床病原菌分布及其耐药性分析［J］. 中国病原生物学杂志， 2024， 19（2）： 209-212.
[14]	MENDES PEDRO D， PAULO S E， SANTOS C M， et al. Extensively drug-resistant Pseudomonas aeruginosa： clinical features and treatment with ceftazidime/avibactam and ceftolozane/tazobactam in a tertiary care university hospital center in Portugal-A cross-sectional and retrospective observational study［J］. Front Microbiol， 2024， 15： 1347521.
[15]	MA L Z， WANG D， LIU Y W， et al. Regulation of biofilm exopolysaccharide biosynthesis and degradation in Pseudomonas aeruginosa ［J］. Annu Rev Microbiol， 2022， 76： 413-433.
[16]	ELSHAFIEE E A， KHALEFA H S， AL-ATFEEHY N M， et al. Biofilms and efflux pump regulatory gene （mexR） in multidrug-resistant Pseudomonas aeruginosa isolated from migratory birds in Egypt［J］. Vet World， 2022， 15（10）： 2425-2431.
[17]	ANDERSEN J B， KRAGH K N， HULTQVIST L D， et al. Induction of native c-di-GMP phosphodiesterases leads to dispersal of Pseudomonas aeruginosa biofilms［J］. Antimicrob Agents Chemother， 2021， 65（4）： e02431-20.
[18]	WANG W Q， FENG X C， SHI H T， et al. Biofilm inhibition based on controlling the transmembrane transport and extracellular accumulation of quorum sensing signals［J］. Environ Res， 2023， 221： 115218.
[19]	MÉSINÈLE J， RUFFIN M， KEMGANG A， et al. Risk factors for Pseudomonas aeruginosa airway infection and lung function decline in children with cystic fibrosis［J］. J Cyst Fibros， 2022， 21（1）： 45-51.
[20]	UGWUANYI F C， AJAYI A， OJO D A， et al. Evaluation of efflux pump activity and biofilm formation in multidrug resistant clinical isolates of Pseudomonas aeruginosa isolated from a Federal Medical Center in Nigeria［J］. Ann Clin Microbiol Antimicrob， 2021， 20（1）： 11.
[21]	ALI A S B E， OZLER B， BADDAL B. Characterization of virulence genes associated with type Ⅲ secretion system and biofilm formation in pseudomonas aeruginosa clinical isolates［J］. Curr Microbiol， 2023， 80（12）： 389.
[22]	CHIMI L Y， NOUBOM M， BISSO B N， et al. Biofilm formation， pyocyanin production， and antibiotic resistance profile of Pseudomonas aeruginosa isolates from wounds［J］. Int J Microbiol， 2024， 2024： 1207536.
[23]	KUMAR R， SINGH N， CHAUHAN A， et al. Mycobacterium tuberculosis survival and biofilm formation studies： effect of D-amino acids， D-cycloserine and its components［J］. J Antibiot （Tokyo）， 2022， 75（8）： 472-479.
[24]	MIRANDA S W， ASFAHL K L， DANDEKAR A A， et al. Pseudomonas aeruginosa quorum sensing［J］. Adv Exp Med Biol， 2022， 1386： 95-115.
[25]	李镛，刘嘉琳，周莲. 泛耐药铜绿假单胞菌群体行为与群体密度感应系统基因的相关性分析［J］. 蚌埠医学院学报， 2018， 43（3）： 288-291， 295.
[26]	谭芳，佘鹏飞，李诗佳，等. 铜绿假单胞菌生物膜和浮游菌的毒力表达差异［J］. 中国微生态学杂志， 2020， 32（6）： 646-650.
[27]	VETRIVEL A， VETRIVEL P， DHANDAPANI K， et al. Inhibition of biofilm formation， quorum sensing and virulence factor production in Pseudomonas aeruginosa PAO1 by selected LasR inhibitors［J］. Int Microbiol， 2023， 26（4）： 851-868.

Specific primer	Specific primer sequence （5'-3'）	Fragment size（bp）
lasR	F:TCGAACATCCGGTCAGCAAA R:GTTCACATTGGCTTCCGAGC	128
lasI	F:CGTGCTCAAGTGTTCAAGGA R:AAACCTGGGCTTCAGGAGTA	130
RhlR	F:GAATGACGGAGGCTTTTTGC R:CGTAGTAATCGAAGCCCAGG	125
RhlI	F:CAGTTCGACCATCCGCAAAC R:TAGCGCGAAAGCTCCCATAC	170
pqsR	F:TCGTTCTGCGATACGGTGAG R:CACTGGTTGAAGCGGGAGAT	167
pqsA	F:TTTCCTGCAATACACCTCGG R:GCTGAACCAGGGAAAGAACA	193
16S RNA	F:CGTAGGTGGTTCAGCAAGTT R:TCTACGCATTTCACCGCTAC	125

Antibacterial drug	Drug resistancerate	Intermediaterate	Sensitivity rate
TZP	24（31.17）	13（16.88）	40（51.95）
TIM	48（62.34）	8（10.39）	21（27.27）
CAZ	33（42.86）	4（5.19）	40（51.95）
FEP	24（31.17）	11（14.29）	42（54.54）
IPM	31（40.26）	1（1.30）	45（58.44）
MEM	29（37.66）	1（1.30）	47（61.04）
TOB	3（3.90）	0（0）	74（96.10）
AK	3（3.90）	0（0）	74（96.10）
CIP	27（35.06）	8（10.39）	42（54.55）
LEV	30（38.96）	8（10.39）	39（50.65）
SCF	19（24.68）	12（15.58）	46（59.74）

Strain number	A(570)	Biofilm formation ability
7	1.772±0.027
8	1.687±0.063
50	1.623±0.156
55	1.409±0.185
69	1.331±0.080	+
70	1.520±1.136	+

Group	Biofilm formation ability				χ2	P
Group	Feminine character	Weakly positive	Moderately positive	Strongly positive	χ2	P
Drug resistant	2	24	21	9	7.843	<0.05
Sensitive	5	8	5	3	7.843	<0.05

Expression of quorum sensing related genes in multidrug-resistant Pseudomonas aeruginosa and its effects on biofilm formation and drug resistance

RichHTML

PDF (PC)

Like

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 7

References 27

Related Articles 15

Metrics

Comments

Recommended 10

[1]	Yao ZHENG,Mingxia FU,Weichen WANG,Weiwei CHEN,Yuchen HAN,Yu BAI,Jiajia AN. Effect of sodium selenite on biological behaviors of breast cancer doxorubicin-resistant MCF-7/ADR cells [J]. Journal of Jilin University(Medicine Edition), 2026, 52(2): 410-417.
[2]	Yunxiu XIA,Shuo ZHANG,Huanhai ZHANG,Fei WANG,Hongliang DONG,Jing DU. Long non-coding RNA LINC00973 promotes migration, invasion and distal metastasis of epithelial ovarian cancer and its molecular mechanism [J]. Journal of Jilin University(Medicine Edition), 2025, 51(4): 866-878.
[3]	Shuo ZHANG,Yunxiu XIA,Weiwei CHEN,Hongliang DONG,Bingjie CUI,Cuilan LIU,Zhiqiang LIU,Fei WANG,Jing DU. Effect of over-expression of NR2F2 on biological behaviors of human ovarian cancer SKOV3 cells [J]. Journal of Jilin University(Medicine Edition), 2025, 51(1): 58-67.
[4]	Bingbing HU,Kangning LUO,Su PENG,Yuzhong ZHOU,Maoliang CHEN,Changhua LIU. Improvement effect of down-regulation of miR-208a on 5-FU resistance in colorectal cancer cells through targeting SFRP1 for mediating Wnt signaling pathway [J]. Journal of Jilin University(Medicine Edition), 2024, 50(4): 947-955.
[5]	Zhibo XU,Xinmiao JIANG,Yuqi ZHEN,MA Quzhen BAI,Mengyao SUN,Xiuping MENG. Effect of chlorhexidine on drug resistance and pathogenicity of Enterococcus faecalis and its mechanism [J]. Journal of Jilin University(Medicine Edition), 2024, 50(3): 676-681.
[6]	Ying ZHANG,Zhaohui WAN,Xianxun JIANG. Effect of over-expression of NDRG1 on resistance of castration-resistant prostate cancer resistant cell line C4-2/ENZA and its mechanism [J]. Journal of Jilin University(Medicine Edition), 2024, 50(3): 708-717.
[7]	ERDEMUTU,Yanyan WANG,Yutong LI,Guilin CHEN,Junrui WANG. Comparison of drug resistance of MRSA clinical strains from respiratory tract and non-respiratory tract specimens and their molecular epidemiological characteristics [J]. Journal of Jilin University(Medicine Edition), 2024, 50(2): 422-430.
[8]	Chunyan KANG,Xiuzhi ZHANG,Huicong ZHOU,Jie CHEN. Effect of downregulating proline-rich protein 11 expression on drug resistance of esophageal cancer drug resistant cell EC9706/DDP and its mechanism [J]. Journal of Jilin University(Medicine Edition), 2024, 50(1): 113-119.
[9]	Shaoting HUANG,You LI,Zhaochun WU,Jiawen HE,Keqi LIAO,Shengnan LI. Construction of SOX17 over-expression lentiviral vector and stably transfected cell line [J]. Journal of Jilin University(Medicine Edition), 2023, 49(6): 1424-1430.
[10]	Jiaojiao XUE,Lei HAO,Yuxiu ZHANG,Heyang DAI,Lixia ZHANG,Shaowei GUO,Jingjing ZHANG,Yang LI,Qingxia LI. Effect of Huaier aqueous extract on breast cancer tamoxifen-resistant LCC2 cells and its mechanism [J]. Journal of Jilin University(Medicine Edition), 2023, 49(3): 714-721.
[11]	Yanan MA,Bing TAN,Lei XU,Jiandong ZHANG. Detection of drug resistance genes of Acinetobacter baumannii in sputum samples of ICU patients by real-time fluorescence quantitative PCR method and its evaluation [J]. Journal of Jilin University(Medicine Edition), 2022, 48(6): 1623-1628.
[12]	Shunjia SONG,Xinying WANG,Feifei JIANG,Huijian JIA,Yuan ZHAO,Zhiping YANG,Liyuan SUN,Yundong ZHAO. Resistance rates of Helicobacter pylori to clarithromycin, metronidazole, and levofloxacin and mutation characteristics of their related drug resistance genes [J]. Journal of Jilin University(Medicine Edition), 2022, 48(3): 630-637.
[13]	Qinghui WANG,Bo LI,Chuancui HU,Mingchao NIE,Xiaomei ZHENG. Effects of miR-107 on regulation of immune escape and taxol resistance of ovarian cancer cells [J]. Journal of Jilin University(Medicine Edition), 2022, 48(3): 734-743.
[14]	Shuqing FENG,Yanhong YAO,Yue SHI,Zhibin LIU,Feng GAO. Application of real-time fluorescence quantitative PCR for detection of BCR/ABL gene expression in peripheral blood in hematopoietic stem cell transplantation therapy in patients with Ph⁺ acute lymphoblastic leukemia [J]. Journal of Jilin University(Medicine Edition), 2022, 48(1): 180-187.
[15]	Lisong CHEN,Hui FANG,Hefei WANG,Chengyuan HE,Xiaodong GAI,Chun LI. Effects of Notch1 signaling pathway on invasion and cisplatin resistance of lung adenocarcinoma cells and their mechanisms [J]. Journal of Jilin University(Medicine Edition), 2021, 47(5): 1171-1177.