Inhibition of Biofilm Formation and Preformed Biofilm in by Resveratrol, Chlorhexidine and Benzalkonium: Modulation of Efflux Pump Activity

Overview

Journal Front Microbiol

Specialty Microbiology

Date 2024 Dec 31

PMID 39736993

Authors

Antonella Migliaccio

Maria Stabile

Maria Triassi

Emmanuelle De

Eliana De Gregorio

Raffaele Zarrilli

Affiliations

Soon will be listed here.

Abstract

Introduction: The persistence of in the contaminated environment is sustained by tolerance to biocides and ability to growth as biofilm. The aim of the study was to analyze the susceptibility of biofilms to chlorhexidine (CHX) and benzalkonium (BZK) biocides and the ability of natural monomeric stilbenoid resveratrol (RV) to modulate the phenomenon.

Methods: Biofilm formation and preformed biofilm were tested by Crystal violet and tetrazolium salt reduction assay, respectively. Analysis of efflux pump (EP) expression during biofilm growth was performed by Real-time RT-PCR assays.

Results: CHX and BZK at ¼ and ½ MICs alone or in combination inhibited biofilm growth of ATCC 19606, 4190, and 3909 strains. RV at 32 mg/L and CHX and BZK at ¼ or ½ MICs showed a synergistic effect and completely inhibited biofilm formation in all strains. Similarly, RV at 32 mg/L and CHX and BZK at ½ MIC significantly inhibited air-liquid biofilm formation of ATCC 19606, 4190 and 3909 strains. The inactivation of AdeB and AdeJ RND EPs in ATCC19606 increased the susceptibility to CHX and BZK alone or in the presence of 32 mg/L RV. Concordantly, carbonyl cyanide m-chlorophenylhydrazine (CCCP) increased the susceptibility to CHX, BZK and RV and dose-dependently inhibited biofilm formation in ATCC 19606, 4190 and 3909 strains. RV at 32 mg/L inhibited basal and CHX-induced EP genes expression, while increased EP gene expression in the presence of BZK during ATCC19606 biofilm growth. In addition, CHX and BZK alone or in combination dose-dependently reduced preformed biofilm of all strains. The combination of RV with CHX and BZK additively decreased minimal biofilm eradicating concentrations in strains.

Conclusion: These results demonstrate that: (i) CHX and BZK alone or in the presence of RV inhibit biofilm growth and preformed biofilm in ; (ii) tolerance to CHX and BZK during biofilm growth is dependent on the activation of AdeB and AdeJ EPs; and (iii) the inhibitory effect of RV on biofilm growth is mediated by the inhibition of EP genes expression in .

References

Weiskirchen S, Weiskirchen R . Resveratrol: How Much Wine Do You Have to Drink to Stay Healthy?. Adv Nutr. 2016; 7(4):706-18. PMC: 4942868. DOI: 10.3945/an.115.011627. View

Ebbensgaard A, Mordhorst H, Overgaard M, Aarestrup F, Hansen E . Dissection of the antimicrobial and hemolytic activity of Cap18: Generation of Cap18 derivatives with enhanced specificity. PLoS One. 2018; 13(5):e0197742. PMC: 5978884. DOI: 10.1371/journal.pone.0197742. View

Rajamohan G, Srinivasan V, Gebreyes W . Molecular and functional characterization of a novel efflux pump, AmvA, mediating antimicrobial and disinfectant resistance in Acinetobacter baumannii. J Antimicrob Chemother. 2010; 65(9):1919-25. DOI: 10.1093/jac/dkq195. View

Dickstein Y, Lellouche J, Ben Dalak Amar M, Schwartz D, Nutman A, Daitch V . Treatment Outcomes of Colistin- and Carbapenem-resistant Acinetobacter baumannii Infections: An Exploratory Subgroup Analysis of a Randomized Clinical Trial. Clin Infect Dis. 2018; 69(5):769-776. DOI: 10.1093/cid/ciy988. View

Giske C, Turnidge J, Canton R, Kahlmeter G . Update from the European Committee on Antimicrobial Susceptibility Testing (EUCAST). J Clin Microbiol. 2021; 60(3):e0027621. PMC: 8925892. DOI: 10.1128/JCM.00276-21. View

Odds F . Synergy, antagonism, and what the chequerboard puts between them. J Antimicrob Chemother. 2003; 52(1):1. DOI: 10.1093/jac/dkg301. View

Esposito A, De Gregorio E, De Fenza M, DAlonzo D, Satawani A, Guaragna A . Expeditious synthesis and preliminary antimicrobial activity of deflazacort and its precursors. RSC Adv. 2022; 9(37):21519-21524. PMC: 9066171. DOI: 10.1039/c9ra03673c. View

Vollaro A, Esposito A, Pia Esposito E, Zarrilli R, Guaragna A, De Gregorio E . PYED-1 Inhibits Biofilm Formation and Disrupts the Preformed Biofilm of . Antibiotics (Basel). 2020; 9(5). PMC: 7277567. DOI: 10.3390/antibiotics9050240. View

Nait Chabane Y, Marti S, Rihouey C, Alexandre S, Hardouin J, Lesouhaitier O . Characterisation of pellicles formed by Acinetobacter baumannii at the air-liquid interface. PLoS One. 2014; 9(10):e111660. PMC: 4216135. DOI: 10.1371/journal.pone.0111660. View

10.

Tucker A, Nowicki E, Boll J, Knauf G, Burdis N, Trent M . Defining gene-phenotype relationships in Acinetobacter baumannii through one-step chromosomal gene inactivation. mBio. 2014; 5(4):e01313-14. PMC: 4128354. DOI: 10.1128/mBio.01313-14. View

11.

Dinu S, Matichescu A, Buzatu R, Marcovici I, Geamantan-Sirbu A, Semenescu A . Insights into the Cytotoxicity and Irritant Potential of Chlorhexidine Digluconate: An In Vitro and In Ovo Safety Screening. Dent J (Basel). 2024; 12(7). PMC: 11276539. DOI: 10.3390/dj12070221. View

12.

Janssen P, Maquelin K, Coopman R, Tjernberg I, Bouvet P, Kersters K . Discrimination of Acinetobacter genomic species by AFLP fingerprinting. Int J Syst Bacteriol. 1997; 47(4):1179-87. DOI: 10.1099/00207713-47-4-1179. View

13.

Di Popolo A, Giannouli M, Triassi M, Brisse S, Zarrilli R . Molecular epidemiological investigation of multidrug-resistant Acinetobacter baumannii strains in four Mediterranean countries with a multilocus sequence typing scheme. Clin Microbiol Infect. 2010; 17(2):197-201. DOI: 10.1111/j.1469-0691.2010.03254.x. View

14.

Lucidi M, Visaggio D, Migliaccio A, Capecchi G, Visca P, Imperi F . Pathogenicity and virulence of : Factors contributing to the fitness in healthcare settings and the infected host. Virulence. 2023; 15(1):2289769. PMC: 10732645. DOI: 10.1080/21505594.2023.2289769. View

15.

Merchel Piovesan Pereira B, Tagkopoulos I . Benzalkonium Chlorides: Uses, Regulatory Status, and Microbial Resistance. Appl Environ Microbiol. 2019; 85(13). PMC: 6581159. DOI: 10.1128/AEM.00377-19. View

16.

Yoon E, Nait Chabane Y, Goussard S, Snesrud E, Courvalin P, De E . Contribution of resistance-nodulation-cell division efflux systems to antibiotic resistance and biofilm formation in Acinetobacter baumannii. mBio. 2015; 6(2). PMC: 4453527. DOI: 10.1128/mBio.00309-15. View

17.

Abuzaid A, Hamouda A, Amyes S . Klebsiella pneumoniae susceptibility to biocides and its association with cepA, qacΔE and qacE efflux pump genes and antibiotic resistance. J Hosp Infect. 2012; 81(2):87-91. DOI: 10.1016/j.jhin.2012.03.003. View

18.

Mattio L, Catinella G, Dallavalle S, Pinto A . Stilbenoids: A Natural Arsenal against Bacterial Pathogens. Antibiotics (Basel). 2020; 9(6). PMC: 7345618. DOI: 10.3390/antibiotics9060336. View

19.

Milstone A, Passaretti C, Perl T . Chlorhexidine: expanding the armamentarium for infection control and prevention. Clin Infect Dis. 2008; 46(2):274-81. DOI: 10.1086/524736. View

20.

Singkham-In U, Higgins P, Wannigama D, Hongsing P, Chatsuwan T . Rescued chlorhexidine activity by resveratrol against carbapenem-resistant Acinetobacter baumannii via down-regulation of AdeB efflux pump. PLoS One. 2020; 15(12):e0243082. PMC: 7710055. DOI: 10.1371/journal.pone.0243082. View