Detection of csgA gene in carbapenem-resistant Acinetobacter baumannii strains and targeting with Ocimum sanctum biocompounds

Document Type : Original Article

Authors

1 Department of Microbiology, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences [SIMATS], Saveetha University, P.H.Road, Chennai, Tamilnadu - 600077, India

2 Department of Biotechnology, DG Vaishnav College, Arumbakkam, Chennai – 600 106

3 Department of Microbiology & Blue Lab, [Instead of BRULAC-DRC] Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences [SIMATS], Saveetha University, P.H.Road, Chennai, Tamilnadu - 600077, India

Abstract

Objective(s): Carbapenem-resistant Acinetobacter baumannii (CRAB) is considered highly virulent due to csgA gene-mediated biofilm formation. The present study aimed to target the same gene, employing the antibiofilm effect of Ocimum sanctum (O. sanctum) essential oil compounds among CRAB strains.
Materials and Methods: A semi-quantitative adherent bioassay was performed to detect the biofilm formation in 73 CRAB strains. This was followed by molecular characterization, Polymerase Chain Reaction (PCR) amplification, and csgA gene sequencing. An antibiofilm assay under in vitro conditions, with essential oils of O. sanctum was performed. This was followed with further docking analysis of csgA protein with the selected compounds from the O. sanctum essential oils. A Molinspiration assessment was also done to elicit the drug likeliness of the biocompounds.
Results: The biofilm assay showed 58.9% as high-grade and 31.5% as low-grade biofilm formers, while 9.58% were non-biofilm formers. Molecular characterization of the csgA gene showed 20.54% (15/73) positivity. The strains that were imipenem resistant also showed the csgA gene to be present (100%; 15/15), with 60% (9/15) and 20% (3/15) for meropenem and doripenem resistance respectively. A crystal violet assay for determining cell viability was done in vitro, which gave Minimum biofilm inhibition concentrations of 50% (MBEC50) at 25 µl and 90% (MBEC90) at 50 µl. The docking analysis done in silico showed benzofuran to possess the lowest binding energy and highest hydrogen bond interactions.
Conclusion: The results indicate benzofuran, from the O. sanctum essential oils, to be effective in targeting the csgA gene among CRAB strains. Additionally, validation of these findings through in vivo studies is required.

Keywords

References

1. Gonzalez-Villoria AM, Valverde-Garduno V. Antibiotic-resistant Acinetobacter baumannii increasing success remains a challenge as a nosocomial pathogen. J Pathogens 2016; :1-11.
2. Wang X, Qin LJ. A review on Acinetobacter baumannii. J Acute Dis 2019; 8:16-21.
3. Dijkshoorn L, Nemec A, Seifert H. An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii. Nat Rev Microbiol 2007; 5:939-951.
4. McConnell MJ, Pérez-Ordóñez A, Pérez-Romero P, Valencia R, Lepe JA, Vázquez-Barba I, et al. Quantitative real-time PCR for detection of Acinetobacter baumannii colonization in the hospital environment. J Clin Microbiol 2012; 50:1412-1414. 
5. Anderson GG, Palermo JJ, Schilling JD, Roth R, Heuser J, Hultgren SJ. Intracellular bacterial biofilm-like pods in urinary tract infections. Science 2003; 301:105-107.
6. Priyadharsini JV, Girija AS, Paramasivam A. An insight into the emergence of Acinetobacter baumannii as an oro-dental pathogen and its drug resistance gene profile–an in silico approach. Heliyon 2018; 4:1-18.
7. Smiline Girija AS. CLSI based antibiogram profile and the detection of MDR and XDR strains of Acinetobacter baumannii isolated from urine samples. Med J Islam Repub Iran 2019; 33:3-9.
8. Blanco N, Harris AD, Rock C, Johnson JK, Pineles L, Bonomo RA, et al. Risk factors and outcomes associated with multidrug-resistant Acinetobacter baumannii upon intensive care unit admission. Antimicrobial Agents Chemother 2018; 62:1-7.
9. Akrami F, Namvar AE. Acinetobacter baumannii as nosocomial pathogenic bacteria. Mol Gen Microbiol Virol 2019; 34:84-96.
10. Kikuchi T, Mizunoe Y, Takade A, Naito S, Yoshida SI. Curli fibers are required for development of biofilm architecture in Escherichia coli K‐12 and enhance bacterial adherence to human uroepithelial cells. Microbiol Immunol 2005; 49:875-884.
11. Saleem HG, Seers CA, Sabri AN, Reynolds EC. Dental plaque bacteria with reduced susceptibility to chlorhexidine are multidrug resistant. BMC Microbiol 2016; 16:1-9.
12. DeBenedictis EP, Ma D, Keten S. Structural predictions for curli amyloid fibril subunits CsgA and CsgB. Rsc Adv 2017; 7:48102-48112.
13. Wu L, Wu L, Wang T, Zou Z, Jiang X, Yin Q, Chen S. Use of magnetic nanoparticles for the detection of multidrug-resistant Acinetobacter baumannii. Nanosci Nanotechnol Lett 2018; 10:1165-1171.
14. Bian Z, Brauner A, Li Y, Normark S. Expression of and cytokine activation by Eschevichia coli curi fibers in human sepsis. J Infect Dis 2000; 181:602-612.
15. Hammar MR, Arnqvist A, Bian Z, Olsén A, Normark S. Expression of two csg operons is required for production of fibronectin‐and congo red‐binding curli polymers in Escherichia coli K‐12. Mol Microbiol 1995; 18:661-670.
16. Kwon SO, Gho YS, Lee JC, Kim SI. Proteome analysis of outer membrane vesicles from a clinical Acinetobacter baumannii isolate. FEMS Microbiol Lett 2009; 297:150-156.
17. De Gregorio E, Roscetto E, Iula VD, Martinucci M, Zarrilli R, Di Nocera PP, et al. Development of a real-time PCR assay for the rapid detection of Acinetobacter baumannii from whole blood samples. New Microbiol 2015; 38:251-257.
18. Cevahir N, Demir M, Kaleli I, Gurbuz M, Tikvesli S. Evaluation of biofilm production, gelatinase activity, and mannose-resistant hemagglutination in Acinetobacter baumannii strains. J Microbiol Immunol Infect 2008; 41:513-518.
19. Perov S, Lidor O, Salinas N, Golan N, Tayeb-Fligelman E, Deshmukh M, Willbold D, Landau M. Structural insights into curli CsgA cross-β fibril architecture inspire repurposing of anti-amyloid compounds as antibiofilm agents. PLoS Pathogens 2019; 15:1-31.
20. Robinson LS, Ashman EM, Hultgren SJ, Chapman MR. Secretion of curli fibre subunits is mediated by the outer membrane‐localized CsgG protein. Mol Microbiol 2006; 59:870-881.
21. Obara H, Aikawa N, Hasegawa N, Hori S, Ikeda Y, Kobayashi Y, et al. The role of a real-time PCR technology for rapid detection and identification of bacterial and fungal pathogens in whole-blood samples. J Infect Chemother 2011; 17:327-333.
22. Thummeepak R, Kongthai P, Leungtongkam U, Sitthisak S. Distribution of virulence genes involved in biofilm formation in multi-drug resistant Acinetobacter baumannii clinical isolates. Int Microbiol 2016; 19:121-129.
23. Baliga MS, Jimmy R, Thilakchand KR, Sunitha V, Bhat NR, Saldanha E, et al. Ocimum sanctum L. (holy basil or tulsi) and its phytochemicals in the prevention and treatment of cancer. Nutr Cancer 2013; 65:26-35.
24. Zheljazkov VD, Cantrell CL, Tekwani B, Khan SI. Content, composition, and bioactivity of the essential oils of three basil genotypes as a function of harvesting. J Agric Food Chem 2008; 56:380-385.
25. Bhattacharyya P, Bishayee A. Ocimum sanctum Linn.(tulsi): an ethnomedicinal plant for the prevention and treatment of cancer. Anticancer Drugs 2013; 24:659-666.
26. Kouidhi B, Zmantar T, Hentati H, Bakhrouf A. Cell surface hydrophobicity, biofilm formation, adhesives properties and molecular detection of adhesins genes in Staphylococcus aureus associated to dental caries. Microbial Pathog 2010; 49:14-22.
27. Avila-Novoa MG, Solís-Velázquez OA, Rangel-Lopez DE, González-Gómez JP, Guerrero-Medina PJ, Gutiérrez-Lomelí M. Biofilm formation and detection of fluoroquinolone-and carbapenem-resistant genes in multidrug-resistant Acinetobacter baumannii. Can J Infect Dis Med Microbiol 2019; :1-5.
28. Girija AS S, Priyadharsini J V. Prevalence of Acb and non-Acb complex in elderly population with urinary tract infection (UTI). Acta Clin Belg 2019; 22:1-7.
29. Smiline Girija AS, Vijayashree Priyadharsini J, Arumugam P. CLSI based antibiogram profile and the detection of MDR and XDR strains of Acinetobacter baumannii isolated from urine samples. Med J Islam Repub Iran 2019; 33:11-16.
30. Zeighami H, Valadkhani F, Shapouri R, Samadi E, Haghi F. Virulence characteristics of multidrug resistant biofilm forming Acinetobacter baumannii isolated from intensive care unit patients. BMC Infect Dis 2019; 19:1-9.
31. Stauder M, Papetti A, Mascherpa D, Schito AM, Gazzani G, Pruzzo C, et al. Antiadhesion and antibiofilm activities of high molecular weight coffee components against Streptococcus mutans. J Agric Food Chem 2010; 58:11662-11666.
32. Preuss HG, Echard B, Enig M, Brook I, Elliott TB. Minimum inhibitory concentrations of herbal essential oils and monolaurin for Gram-positive and Gram-negative bacteria. Mol Cell Biochem 2005; 272:29-34.
33.Tariq M, Sirajuddin M, Ali S, Khalid N, Tahir MN, Khan H, Ansari TM. Pharmacological investigations and Petra/Osiris/Molinspiration (POM) analyses of newly synthesized potentially bioactive organotin (IV) carboxylates. J Photochem Photobiol B 2016; 158:174-183.
34. Lipinski CA. Lead-and drug-like compounds: the rule-of-five revolution. Drug Discov Today Technol 2004; 1:337-341.
35. Vanommeslaeghe K, Hatcher E, Acharya C, Kundu S, Zhong S, Shim J, et al. Charmm general force field: A force field for drug‐like molecules compatible with the Charmm all‐atom additive biological force fields. J Comput Chem 2010; 31:671-690.
36. Watnick P, Kolter R. Biofilm, city of microbes. J Bacteriol 2000; 182:2675-2679.
37. Espinal P, Marti S, Vila J. Effect of biofilm formation on the survival of Acinetobacter baumannii on dry surfaces. J Hosp Infect 2012; 80:56-60.
38. Lin LT, Hsu WC, Lin CC. Antiviral natural products and herbal medicines. J Tradit Complement Med 2014; 4:24-35.
39. Davis JS, McMillan M, Swaminathan A, Kelly JA, Piera KE, Baird RW, et al. A 16-year prospective study of community-onset bacteremic Acinetobacter pneumonia: low mortality with appropriate initial empirical antibiotic protocols. Chest 2014; 146:1038-1045.
40. Gualdi L, Tagliabue L, Landini P. Biofilm formation-gene expression relay system in Escherichia coli: modulation of σS-dependent gene expression by the CsgD regulatory protein via σS protein stabilization. J Bacteriol 2007; 189:8034-8043.
41. Al-Kadmy IM, Ali AN, Salman IM, Khazaal SS. Molecular characterization of Acinetobacter baumannii isolated from Iraqi hospital environment. New Microbes New Infect 2018; 21:51-7.
42. Choudhury SS, Bashyam L, Manthapuram N, Bitla P, Kollipara P, Tetali SD. Ocimum sanctum leaf extracts attenuate human monocytic (THP-1) cell activation. J Ethnopharmacol 2014; 154:148-155.
43. Lee JS, Choi CH, Kim JW, Lee JC. Acinetobacter baumannii outer membrane protein A induces dendritic cell death through mitochondrial targeting. J Microbiol 2010; 48:387-392.
44. Choi CH, Lee EY, Lee YC, Park TI, Kim HJ, Hyun SH, et al. Outer membrane protein 38 of Acinetobacter baumannii localizes to the mitochondria and induces apoptosis of epithelial cells. Cell Microbiol 2005; 7:1127-1138.
45. Kalaivani R, Devi VJ, Umarani R, Periyanayagam K, Kumaraguru AK. Antimicrobial activity of some important medicinal plant oils against human pathogens. J Biological Active Prod Nature 2012; 2:30-37.
46. Mirdha BR, Naik SN, Mahapatra SC. Antimicrobial activities of essential oils obtained from fresh and dried leaves of Ocimum sanctum (L.) against enteric bacteria and yeast. Int Symp Med Nutraceutic Plants 756 2007; :267-270.
47. Dey R, De K, Mukherjee R, Ghosh S, Haldar J. Small antibacterial molecules highly active against drug-resistant Staphylococcus aureus. Med Chem Comm. 2019; 10:1907-1915.
48. Burton E, Yakandawala N, LoVetri K, Madhyastha MS. A microplate spectrofluorometric assay for bacterial biofilms. J Ind Microbiol  Biotechnol 2007; 34:1-4.
49. Amin MN, Dewan SM, Noor W, Shahid-Ud-Daula AF. Characterization of chemical groups and determination of total phenolic content and in-vitro anti-oxidant Activities of ethanolic extract of Ocimum sanctum leaves growing in Bangladesh. Eur J Exp Biol 2013; 3:449-454.
50. Hussain EH, Jamil K, Rao M. Hypoglycaemic, hypolipidemic and anti-oxidant properties of tulsi (Ocimum sanctum Linn) on streptozotocin induced diabetes in rats. Indian J Clin Biochem 2001; 16:190-194.
51. Rao YS, Kotakadi VS, Prasad TN, Reddy AV, Gopal DS. Green synthesis and spectral characterization of silver nanoparticles from Lakshmi tulasi (Ocimum sanctum) leaf extract. Spectrochimica Acta A Mol Biomol Spectrosc 2013; 103:156-159.
52. Jiang X, Liu W, Zhang W, Jiang F, Gao Z, Zhuang H, Fu L. Synthesis and antimicrobial evaluation of new benzofuran derivatives. Eur J Med Chem 2011; 46:3526-3530.
53. Barboza JN, da Silva Maia Bezerra Filho C, Silva RO, Medeiros JV, de Sousa DP. An overview on the anti-inflammatory potential and anti-oxidant profile of eugenol. Oxid Med Cell Longev 2018; :1-9.
54. Kenchappa R, Bodke YD, Asha B, Telkar S, Sindhe MA. Synthesis, antimicrobial, and anti-oxidant activity of benzofuran barbitone and benzofuran thiobarbitone derivatives. Med Chem Res 2014; 23:3065-3081.
55. Zhao Y, Sanner MF. FLIPDock: docking flexible ligands into flexible receptors. Proteins Struct Funct Bioinf 2007; 68:726-737.
56. Dhara L, Tripathi A. Antimicrobial activity of eugenol and cinnamaldehyde against extended spectrum beta lactamase producing Enterobacteriaceae by in vitro and molecular docking analysis. Eur J Integr Med 2013; 5:527-536.
57. Hou T, Wang J, Zhang W, Xu X. ADME evaluation in drug discovery. 6. Can oral bioavailability in humans be effectively predicted by simple molecular property-based rules. J Chem Inf Model 2007; 47:460-463.
Iranian Journal of Basic Medical Sciences
Volume 24, Issue 5
May 2021
Pages 690-698

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