Detection of quorum sensing virulence factor genes and its consanguinity to antibiotic sensitivity profile in the clinical isolates of Pseudomonas aeruginosa

Document Type : Original Article

Authors

1 Department of Microbiology, School of Allied Health Sciences, Mallareddy University, Hyderabad, Telangana, India

2 Department of Microbiology, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences (SIMATS), Chennai, India

3 Department of Microbiology, Mallareddy Institute of Medical Sciences, Hyderabad, Telangana, India

Abstract

Objective(s): Virulent strains of Pseudomonas aeruginosa exhibit multidrug resistance by intrinsic and extrinsic mechanisms which are regulated by quorum sensing signalling systems. This includes the production of auto-inducers and their transcriptional activators to activate various virulence factors resulting in host infections. The present study is thus aimed to detect the virulence factor production, quorum sensing activity, and susceptibility pattern of P. aeruginosa to antibiotics from clinical specimens.
Materials and Methods: A total of 122 isolates of P. aeruginosa were phenotypically characterized by standard protocols and were categorized into MDR and non-MDR based on the antibiotic susceptibility profiles. Pyocyanin, alkaline protease and elastase production were assessed by qualitative and quantitative methods. Crystal violet assay was carried out for the quantification of biofilms. The genetic determinants of virulence were detected by PCR.
Results: Out of the 122 isolates, 80.3% of isolates were MDR and the production of virulence factors was in positive correlation with the presence of genetic determinants and 19.6% were non-MDR, but still showed the production of virulence factors, as confirmed by both phenotypic and genotypic methods. Few carbapenem-resistant strains were detected which did not show the production of virulence factors by both methods.
Conclusion: The study concludes, though the strains were non-MDR, they were still capable of producing the virulence factors which may be responsible for the dissemination and chronicity of the infection caused by P. aeruginosa.

Keywords

Main Subjects


1. Miller MB and Bassler BL. Quorum sensing in bacteria. Annum Rev Microbiol 2001; 55:165–199.
2.    Whitehead  NA, Barnard AM, Slater H, Simpson NJ Salmond GP. Quorum-sensing in Gram-negative bacteria. FEMS Microbiol Rev 2001; 25: 365–404.
3.    de Kievit TR and Iglewski BH. Bacterial quorum sensing in pathogenic relationships. Infect Immun 2000; 68:4839–4849.
4.    Rumbaugh KP, Griswold JA and  Hamood AN. The role of quorum sensing in the in vivo virulence of Pseudomonas aeruginosa. Microbes Infect 2000; 2:1721–1731.
5.    Bhardwaj S, Bhatia S, Singh S, Franco F Jr. Growing emergence of drug-resistant Pseudomonas aeruginosa and attenuation of its virulence using quorum sensing inhibitors: A critical review. Iran J Basic Med Sci 2022; 24:699-719.
6.    Chernukha MIu, Shaginian IA, Romanova IuM, Maleev GV, Gintsburg AL. The role of “quorum sensing” regulation system in symbiotic interaction of bacteria Burkholderia cepacia and Pseudomonas aeruginosa during mixed infection. Mikrobiol Epidemiol Immunobiol  2006; 4:32-37. 
7.    Husain FM, Ahmad I, Al-Thubiani AS, Abulreesh HH, AlHazza IM, Aqil F. Leaf extracts of Mangifera indica L. Inhibit quorum sensing–regulated production of virulence factors and biofilm in test bacteria. Front Microbiol 2017; 8:727-Last page.
8.    Mantero M, Gramegna A, Pizzamiglio GD’Adda, A.Tarsia P, and Blasi, F. Once daily aerosolised tobramycin in adult patients with cystic fibrosis in the management of Pseudomonas aeruginosa chronic infection. Multidiscip Respir Med 2017; 12:2-Last page. 
9.    Davis R, and Brown PD. Multiple antibiotic resistance index, fitness and virulence potential in respiratory Pseudomonas aeruginosa from Jamaica. J Med Microbiol 2016; 65:261–271. 
10.    Lee K, and Yoon SS. Pseudomonas aeruginosa biofilm, a programmed bacterial life for fitness. J Microbiol Biotechnol 2017; 27:1053–1064.
11.    Mirza Hosseini HK, Hadadi-Fishani M, Morshedi K, and  Khaledi A. Meta-analysis of biofilm formation, antibiotic resistance pattern, and biofilm-related genes in Pseudomonas aeruginosa isolated from clinical samples. Microb. Drug Resist 2020; 26:815–824.
12.    CLSI. Performance standard for antimicrobial susceptibility testing. CLSI Supplement M100, Clinical and Laboratory Standards Institute 2021; 31.
13.    Aboushleib HM, Omar HM, Abozahra R, Elsheredy A, Baraka K. Correlation of quorum sensing and virulence factors in Pseudomonas aeruginosa isolates in Egypt. J Infect Dev Ctries 2015; 9:1091–1099. 
14.    Jazayeri JA, Nguyen, Kotsanas D, Schneiders F, Tan CH, et al. Comparison of virulence factors in Pseudomonas aeruginosa strains isolated from cystic fibrosis patients. J Med Microb Diagn 2016; 5: 242. 
15.    Morihara K, Tsuzuki H, Harada M, Iwata T. Purification of human plasma alpha 1-proteinase inhibitor and its inactivation by Pseudomonas aeruginosa elastase. J Biochem 1984; 95:795-804. 
16.    Nicas TI, Iglewski BH. Production of elastase and other exoproducts by environmental isolates of Pseudomonas aeruginosa. J Clin Microbiol 1986; 23:967‐969.
17.    Rust L, Messing CR, and  Iglewski BH. Elastase assays. Methods Enzymol 1994; 235:554–562.
18.    Brown MRW, Foster JHSA. Simple diagnostic milk medium for Pseudomonas aeruginosa. J Clin Pathol 1970; 23:172-177.
19.    Karatuna O, and Yagci A. Analysis of quorum sensing-dependent virulence factor production and its relationship with antimicrobial susceptibility in Pseudomonas aeruginosa respiratory isolates. Clin Microbiol Infect 2010; 16:1770–1775. 
20.    Gupta P, Gupta RK, and Harjai K. Multiple virulence factors regulated by quorum sensing may help in establishment and colonization of urinary tract by Pseudomonas aeruginosa during experimental urinary tract infection. Indian J Med Microbiol 2013; 31:29-33.
21.    Schmidtchen A, Wolff H, and Hansson C. Differential proteinase expression by Pseudomonas aeruginosa derived from chronic leg ulcers. Acta Derm Venereol 2001; 81:406‐409.
22.    O’Toole GA. Microtiter dish biofilm formation assay. J Vis Exp 2011; 47:2437.
23.    Global burden of bacterial antimicrobial resistance in 2019: A systemic analysis, antimicrobial resistance collaborators, 2022; 399:629-655.
24.    Rojas A,  Palacios-Baena Z, López-Cortés L, Rodríguez-Baño J. Rates, predictors and mortality of community-onset bloodstream infections due to Pseudomonas aeruginosa: Systematic review and meta-analysis. Clin Microbiol Infect 2019; 25:964–970. 
25.    Matta R, Hallit S, Hallit R, Bawab W, Rogues AM, Salameh P. Epidemiology and microbiological profile comparison between community and hospital acquired infections: A multicenter retrospective study in Lebanon. J Infect Public Health 2018; 11:405–411. 
26.    Mancuso G, Midiri A, Gerace E, Biondo C. Bacterial antibiotic resistance: The most critical pathogens. Pathogens 2021; 10:1310.
27.    Afshari A, Pagani L, and Harbarth S. Year in review: Critical Care infection. Crit Care 2012; 16:242. 
28.    Preeti Pachori, Ragini Gothalwal, Puneet Gandhi. Emergence of antibiotic resistance Pseudomonas aeruginosa in intensive care unit; a critical review. Genes Dis 2019; 6:109-119.
29.    Gajdács M. Baráth Z,  Kárpáti K, Szabó D,  Usai D,  Zanetti S. et al. No correlation between biofilm formation, virulence factors, and antibiotic resistance in Pseudomonas aeruginosa: Results from a laboratory-based in vitro study. Antibiotics 2021; 10:1134.
30.    Algun A, Arisoy A, Gunduz T, and Ozbakkaloglu B. The resistance of Pseudomonas aeruginosa strains to flouroquinolone group of antibiotics. Ind J Med Micro 2004; 22:112-114
31.    KM Mohanasoundaram. The antimicrobial resistance pattern in the clinical isolates of Pseudomonas aeruginosa in a tertiary care hospital (A 3 Year Study) 2011;5:491-494.
32.    Ankita N, and Raja R. Detection and antibiotic susceptibility pattern of Pseudomonas aeruginosa Isolates from various clinical samples in tertiary care Hospital, Bhavnagar, Gujarat, Saudi. J Pathol Microbiol 2019; 4:186-194.
33.    Stehling EG, da Silveira WD, Leite and DDS. Study of biological characteristics of Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis and from patients with extra-pulmonary infections. Braz J Infect Dis 2008; 12:86–88.
34.    Stewart PS,  and Costerton  JW. Antibiotic resistance of bacteria in biofilms. Lancet 2001; 358:135–138.
35.    Fowler RC, and Hanson ND. Emergence of carbapenem resistance due to the novel insertion sequence ISPa8 in Pseudomonas aeruginosa. PLoS One 2014; 9:91299.
36.    Peterson E, and Kaur P. Antibiotic resistance mechanisms in bacteria: Relationships between resistance determinants of antibiotic producers, environmental bacteria, and clinical pathogens. Front. Microbiol 2018; 9:2928. 
37.    Abidi SH, Sherwani SK, Siddiqui TR, and Bashir A, Kazmi SU. Drug resistance profile and biofilm forming potential of Pseudomonas aeruginosa isolated from contact lenses in Karachi-Pakistan. BMC Ophthalmol. 2013; 13:57.
38.    Kucers A, Crowe S, Grayson ML, Hoy J eds. The use of antibiotics: A clinical review of antibacterial, antifungal, and antiviral drugs. 5th ed. Oxford: Butterworth Heinemann 1997; 452-457.
39.    Davies J, Wright G. Bacterial resistance to aminoglycoside antibiotics. Trends in Microbiology 1997; 5:234-239.
40.    Poole K. Aminoglycoside resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother 2005; 49:479-487.
41.    Cepas V, López Y, Muñoz E, Rolo D, Ardanuy and C, Martí S, et al. Relationship between biofilm formation and antimicrobial resistance in gram-negative bacteria. Microb Drug Resist 2019; 25:72-79. 
42.    Singhai M, Malik M, Shahid MA, Malik, and R. Goyal. A study on device-related infections with special reference to biofilm production and antibiotic resistance. J Glob Infect Dis 2012; 4:193–198.
43.    Karami P, Mohajeri P, Mashouf RY, Karami M, Yaghoobi MH, and Dastan D et al. Molecular characterization of clinical and environmental Pseudomonas aeruginosa isolated in a burn center. Saudi J Biol Sci 2018; 26:1731–1736. 
44.    Yamani L, Alamri A, Alsultan A, Alfifi, S, Ansari MA, Alnimr A. Inverse correlation between biofilm production efficiency and antimicrobial resistance in clinical isolates of Pseudomonas aeruginosa. Microb Pathog 2021; 157:10498
45.    Lima JLDC, Alves LR,  Jacomé PRLDA, Neto JPB, Maciel MAV et al. Biofilm production by clinical isolates of Pseudomonas aeruginosa and structural changes in LasR protein of isolates non-biofilm-producing. Braz J Infect Dis 2018; 22:129–136. 
46.    Choy MH, Stapleton F, Willcox M, and Zhu H. Comparison of virulence factors in Pseudomonas aeruginosa strains isolated from contact lens and non-contact lens-related keratitis. J Med Microbiol 2008; 57:1539–1546.
47.    Jabalameli F, Mirsalehian A, Khoramian B, Aligholi M, Khoramrooz SS, Asadollahi P. et al. Evaluation of biofilm production and characterization of genes encoding type III secretion system among Pseudomonas aeruginosa isolated from burn patients. Burns 2012; 38:1192–1197.
48.    Behzadi P, Baráth Z, and Gajdács M. It’s not easy being green: A narrative review on the microbiology, virulence and therapeutic prospects of multidrug-resistant Pseudomonas aeruginosa. Antibiotics (Basel). 2021; 10:42. 
49.    Uzunbayir-Akel N, Tekintas Y, Yilmaz FF, Ozturk I, Okeer M, and Aydemir SS. et al M. Effects of disinfectants and ciprofloxacin on quorum sensing genes and biofilm of clinical Pseudomonas aeruginosa isolates. J Infect Public Health 2020; 13:1932–1938, 
50.    Elmouaden C, Laglaoui A, Ennanei L, Bakkali M, Abid M. Virulence genes and antibiotic resistance of Pseudomonas aeruginosa isolated from patients in the Northwestern of Morocco. J Infect Dev Ctries 2019; 13:892–898.
51.    Elnegery AA, Mowafy WK, Zahra TA, Abou El-Khier NT. Study of quorum-sensing LasR and RhlR genes and their dependent virulence factors in Pseudomonas aeruginosa isolates from infected burn wounds. Access Microbiol 2021; 3:000211. 
52.    La Sarre B, and Federle MJ. Exploiting quorum sensing to confuse bacterial pathogens. Microbiol Mol Biol Rev 2013; 77:73-111.
53.    Laxmi M, and Bhat SG. Characterization of pyocyanin with radical scavenging and antibiofilm properties isolated from Pseudomonas aeruginosa strain BTRY1. 3 Biotech. 2016; 6:27.
54.    Khalil MA, Ibrahim Sonbol  F, Mohamed AF, Ali SS. Comparative study of virulence factors among ESβL-producing and nonproducing Pseudomonas aeruginosa clinical isolates. Turk J Med Sci 2015; 45:60-69. 
55.    Zhu H, Bandara R, Conibear TCR, Thuruthyil SJ, Rice SA et al. Pseudomonas aeruginosa with las l quorum sensing deficiency during corneal infection. Invest Ophthalmol Vis Sci 2004; 45:1897-1903.
56.    El-Khashaab TH, Erfan DM, Kamal A, El-Moussely LM, and Ismail DK. Pseudomonas aeruginosa biofilm formation and quorum sensing lasR gene in patients with wound infection. Egypt J Med Microbiol 2016; 25:101–108.
57.    Mahmoud MF, Fathy FM, Gohar MK, Awad AM, Soliman MH. Biofilm formation and quorum sensing lasR gene of Pseudomonas aeruginosa isolated from patients with post-operative wound infections. Europ J Mol Clin Med 2021; 8:2177-2189.
58.    Sabharwal N, Dhall S, Chhibber S, and Harjai K. Molecular detection of virulence genes as markers in Pseudomonas aeruginosa isolated from urinary tract infections. Int J Mol Epidemiol Genet 2014; 5:125-134. 
59.    Cotar AI, Chifiriuc MC, Banu O, and Lazar V. Molecular characterization of virulence patterns in Pseudomonas aeruginosa strains isolated from respiratory and wound samples. Biointerface Res Appl Chem 2013; 3:551-558.
60.    Nikbin VS, Aslani MM, Sharafi Z, Hashemipour M, Shahcheraghi F, Ebrahimipour GH. Molecuclar identification and detection of virulence genes among Pseudomonas aeruginosa isolated from different infectious origins. Iraninan J Microbiol 2012; 4: 118-123.
61.    Wolska K, Szweda P. Genetic features of clini‌cal Pseudomonas aeruginosa strians. Polish J Microbiol 2009; 58:255-260.