Molecular mechanism of antimicrobial activity of chlorhexidine against carbapenem-resistant Acinetobacter baumannii

Autoři: Deepika Biswas aff001;  Monalisa Tiwari aff001;  Vishvanath Tiwari aff001
Působiště autorů: Department of Biochemistry, Central University of Rajasthan, Ajmer, India aff001
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article


Acinetobacter baumannii causes hospital-acquired infections, especially in those with impaired immune function. Biocides or disinfectants are widely used antibacterial agents used to eradicate the effect of A. baumannii on inanimate objects and health care environments. In the current study, the antimicrobial activity of chlorhexidine has been investigated against carbapenem-resistant (RS-307, RS-7434, RS-6694, and RS-122), and sensitive (ATCC-19606 and RS-10953) strains of A. baumannii. We have determined growth kinetics, antimicrobial susceptibility, ROS production, lipid peroxidation, cell viability using flow cytometry assay (FACS), and membrane integrity by scanning electron microscope (SEM). The effect of chlorhexidine on the bacterial membrane has also been investigated using Fourier transform infrared (FTIR) spectroscopy. The present study showed that 32μg/ml chlorhexidine treatment results in the decreased bacterial growth, CFU count and cell viability. The antibacterial activity of chlorhexidine is due to the elevated ROS production and higher lipid peroxidation. These biochemical changes result in the membrane damage and alteration in the membrane proteins, phospholipids, carbohydrates, nucleic acids as evident from the FTIR and SEM data. Therefore, chlorhexidine has the potential to be used in the hospital setups to remove the spread of A. baumannii.

Klíčová slova:

Acinetobacter baumannii – Antibacterials – Flow cytometry – Lipid peroxidation – Nosocomial infections – Nucleic acids – Phospholipids – Scanning electron microscopy


1. Falah F, Shokoohizadeh L, Adabi M (2019) Molecular identification and genotyping of Acinetobacter baumannii isolated from burn patients by PCR and ERIC-PCR. Scars, burns & healing 5: 1–7.

2. Agard MJ, Ozer EA, Morris AR, Piseaux R, Hauser AR (2019) A Genomic Approach to Identify Klebsiella pneumoniae and Acinetobacter baumannii Strains with Enhanced Competitive Fitness in the Lungs During Multi-Strain Pneumonia. Infection and Immunity 87(6):e00871. doi: 10.1128/IAI.00871-18 30936161

3. Sousa C, Silva L, Grosso F, Lopes J, Peixe L (2014) Development of a FTIR-ATR based model for typing clinically relevant Acinetobacter baumannii clones belonging to ST98, ST103, ST208 and ST218. J Photochem Photobiol B 133: 108–114. doi: 10.1016/j.jphotobiol.2014.02.015 24727403

4. Al-Anazi KA, Al-Jasser AM (2014) Infections Caused by Acinetobacter baumannii in Recipients of Hematopoietic Stem Cell Transplantation. Front Oncol 4: 186. doi: 10.3389/fonc.2014.00186 25072028

5. Tiwari V, Vashistt J, Kapil A, Moganty RR (2012) Comparative proteomics of inner membrane fraction from carbapenem-resistant Acinetobacter baumannii with a reference strain. PLoS One 7: e39451. doi: 10.1371/journal.pone.0039451 22761799

6. Zhang L, Lou J, Zhang W, Wu C, Jin Z (2018) Bacteria killing in ICU associated infections: antibacterial nanosheets as disinfectant. RSC Advances 8: 278–283.

7. Davies GE, Francis J, Martin AR, Rose FL, Swain G (1954) 1:6-Di-4'-chlorophenyldiguanidohexane (hibitane); laboratory investigation of a new antibacterial agent of high potency. Br J Pharmacol Chemother 9: 192–196. doi: 10.1111/j.1476-5381.1954.tb00840.x 13172429

8. Sompornrattanaphan M, Kreetapirom P, Srinoulprasert Y, Kanistanon D, Klinniyom A, Wongsa C, et al. (2019) Severe anaphylaxis after pelvic examination: a case report of dual latex and chlorhexidine allergies. Allergy, Asthma & Clinical Immunology 15: 19.

9. Johnson J, Suwantarat N, Colantuoni E, Ross TL, Aucott SW, Carroll KC, et al. (2019) The impact of chlorhexidine gluconate bathing on skin bacterial burden of neonates admitted to the Neonatal Intensive Care Unit. J Perinatol 39: 63–71. doi: 10.1038/s41372-018-0231-7 30237474

10. Saleem HG, Seers CA, Sabri AN, Reynolds EC (2016) Dental plaque bacteria with reduced susceptibility to chlorhexidine are multidrug resistant. BMC Microbiol 16: 214. doi: 10.1186/s12866-016-0833-1 27629863

11. Hardy K, Sunnucks K, Gil H, Shabir S, Trampari E, Hawkey P, et al. (2018) Increased Usage of Antiseptics Is Associated with Reduced Susceptibility in Clinical Isolates of Staphylococcus aureus. MBio 9.

12. Raut U, Narang P, Mendiratta DK, Narang R, Deotale V (2008) Evaluation of rapid MTT tube method for detection of drug susceptibility of Mycobacterium tuberculosis to rifampicin and isoniazid. Indian J Med Microbiol 26: 222–227. 18695318

13. Stevens MG, Olsen SC (1993) Comparative analysis of using MTT and XTT in colorimetric assays for quantitating bovine neutrophil bactericidal activity. J Immunol Methods 157: 225–231. doi: 10.1016/0022-1759(93)90091-k 8423367

14. Grela E, Kozlowska J, Grabowiecka A (2018) Current methodology of MTT assay in bacteria—A review. Acta Histochem 120: 303–311. doi: 10.1016/j.acthis.2018.03.007 29606555

15. Mshana RN, Tadesse G, Abate G, Miorner H (1998) Use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide for rapid detection of rifampin-resistant Mycobacterium tuberculosis. J Clin Microbiol 36: 1214–1219. 9574679

16. Haase H, Jordan L, Keitel L, Keil C, Mahltig B (2017) Comparison of methods for determining the effectiveness of antibacterial functionalized textiles. PLoS One 12: e0188304. doi: 10.1371/journal.pone.0188304 29161306

17. Yalcin HT, Ozen MO, Gocmen B, Nalbantsoy A (2014) Effect of Ottoman Viper (Montivipera xanthina (Gray, 1849)) Venom on Various Cancer Cells and on Microorganisms. Cytotechnology 66: 87–94. doi: 10.1007/s10616-013-9540-z 23381026

18. Zhao X, Drlica K (2014) Reactive oxygen species and the bacterial response to lethal stress. Curr Opin Microbiol 21: 1–6. doi: 10.1016/j.mib.2014.06.008 25078317

19. Mageshwaran T, Ebenezar AR, Madhanamadhubala M, Kavitha S, Mahalaxmi S (2012) Counteraction of reactive oxygen species and determination of antibacterial efficacy of proanthocyanidin and lycopene when mixed with calcium hydroxide and chlorhexidine mixture: An in vitro comparative study. J Conserv Dent 15: 337–341. doi: 10.4103/0972-0707.101893 23112480

20. Yeung SY, Huang CS, Chan CP, Lin CP, Lin HN, Lee PH, et al. (2007) Antioxidant and pro-oxidant properties of chlorhexidine and its interaction with calcium hydroxide solutions. Int Endod J 40: 837–844. doi: 10.1111/j.1365-2591.2007.01271.x 17877724

21. Trastoy R, Manso T, Fernandez-Garcia L, Blasco L, Ambroa A, Perez Del Molino ML, et al. (2018) Mechanisms of Bacterial Tolerance and Persistence in the Gastrointestinal and Respiratory Environments. Clin Microbiol Rev 31:e00023–18. doi: 10.1128/CMR.00023-18 30068737

22. Biswas D, Tiwari M, Tiwari V (2018) Comparative mechanism based study on disinfectants against multidrug-resistant Acinetobacter baumannii. J Cell Biochem 119:10314–10326. doi: 10.1002/jcb.27373 30145822

23. Arakha M, Pal S, Samantarrai D, Panigrahi TK, Mallick BC, Pramanik K, et al. (2015) Antimicrobial activity of iron oxide nanoparticle upon modulation of nanoparticle-bacteria interface. Sci Rep 5: 14813. doi: 10.1038/srep14813 26437582

24. Bankier C, Cheong Y, Mahalingam S, Edirisinghe M, Ren G, Cloutman-Green E, et al. (2018) A comparison of methods to assess the antimicrobial activity of nanoparticle combinations on bacterial cells. PLOS ONE 13: e0192093. doi: 10.1371/journal.pone.0192093 29390022

25. Booyens J, Thantsha MS (2014) Fourier transform infra-red spectroscopy and flow cytometric assessment of the antibacterial mechanism of action of aqueous extract of garlic (Allium sativum) against selected probiotic Bifidobacterium strains. BMC Complement Altern Med 14: 289. doi: 10.1186/1472-6882-14-289 25099661

26. Golding CG, Lamboo LL, Beniac DR, Booth TF (2016) The scanning electron microscope in microbiology and diagnosis of infectious disease. Sci Rep 6: 26516. doi: 10.1038/srep26516 27212232

27. O’Brien-Simpson NM, Pantarat N, Attard TJ, Walsh KA, Reynolds EC (2016) A Rapid and Quantitative Flow Cytometry Method for the Analysis of Membrane Disruptive Antimicrobial Activity. PLoS One 11: e0151694. doi: 10.1371/journal.pone.0151694 26986223

28. Gui S, Li R, Feng Y, Wang S (2014) Transmission electron microscopic morphological study and flow cytometric viability assessment of Acinetobacter baumannii susceptible to Musca domestica cecropin. ScientificWorldJournal 2014: 657536. doi: 10.1155/2014/657536 24883421

29. Sousa C, Silva L, Grosso F, Nemec A, Lopes J, Peixe L, et al. (2014) Discrimination of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex species by Fourier transform infrared spectroscopy. Eur J Clin Microbiol Infect Dis 33: 1345–1353. doi: 10.1007/s10096-014-2078-y 24563251

30. Sousa C, Grosso F, Meirinhos-Soares L, Peixe L, Lopes J (2014) Identification of carbapenem-resistant Acinetobacter baumannii clones using infrared spectroscopy. J Biophotonics 7: 287–294. doi: 10.1002/jbio.201200075 23192934

31. Tang M, McEwen GD, Wu Y, Miller CD, Zhou A (2013) Characterization and analysis of mycobacteria and Gram-negative bacteria and co-culture mixtures by Raman microspectroscopy, FTIR, and atomic force microscopy. Anal Bioanal Chem 405: 1577–1591. doi: 10.1007/s00216-012-6556-8 23196750

32. Talari ACS, Martinez MAG, Movasaghi Z, Rehman S, Rehman IU (2017) Advances in Fourier transform infrared (FTIR) spectroscopy of biological tissues. Applied Spectroscopy Reviews 52: 456–506.

33. Aboualizadeh E, Bumah VV, Masson-Meyers DS, Eells JT, Hirschmugl CJ, Enwemeka CS (2017) Understanding the antimicrobial activity of selected disinfectants against methicillin-resistant Staphylococcus aureus (MRSA). PLoS One 12: e0186375. doi: 10.1371/journal.pone.0186375 29036196

34. Tiwari V, Rajeswari MR (2013) Effect of Iron Availability on the Survival of Carbapenem-Resistant Acinetobacter baumannii: a Proteomic Approach. Journal of Proteomics & Bioinformatics 06: 125–131.

35. Tiwari V, Moganty RR (2014) Conformational stability of OXA-51 beta-lactamase explains its role in carbapenem resistance of Acinetobacter baumannii. J Biomol Struct Dyn 32: 1406–1420. doi: 10.1080/07391102.2013.819789 23879430

36. Tiwari V, Kapil A, Moganty RR (2012) Carbapenem-hydrolyzing oxacillinase in high resistant strains of Acinetobacter baumannii isolated from India. Microb Pathog 53: 81–86. doi: 10.1016/j.micpath.2012.05.004 22610043

37. Tiwari V, Rajeswari MR, Tiwari M (2019) Proteomic analysis of iron-regulated membrane proteins identify FhuE receptor as a target to inhibit siderophore-mediated iron acquisition in Acinetobacter baumannii. International Journal of Biological Macromolecules 125: 1156–1167. doi: 10.1016/j.ijbiomac.2018.12.173 30579900

38. Tiwari V, Mishra N, Gadani K, Solanki PS, Shah NA, Tiwari M (2018) Mechanism of Anti-bacterial Activity of Zinc Oxide Nanoparticle Against Carbapenem-Resistant Acinetobacter baumannii. Front Microbiol 9: 1218. doi: 10.3389/fmicb.2018.01218 29928271

39. Tiwari V, Tiwari M, Solanki V (2017) Polyvinylpyrrolidone-Capped Silver Nanoparticle Inhibits Infection of Carbapenem-Resistant Strain of Acinetobacter baumannii in the Human Pulmonary Epithelial Cell. Front Immunol 8: 973. doi: 10.3389/fimmu.2017.00973 28861082

40. Tiwari V, Tiwari M, Biswas D (2018) Rationale and design of an inhibitor of RecA protein as an inhibitor of Acinetobacter baumannii. J Antibiot (Tokyo) 71: 522–534.

41. Tiwari V, Tiwari M (2015) Phosphoproteomics as an emerging weapon to develop new antibiotics against carbapenem resistant strain of Acinetobacter baumannii. Journal of Proteomics 112: 336–338. doi: 10.1016/j.jprot.2014.09.008 25252118

42. Harbarth S, Tuan Soh S, Horner C, Wilcox MH (2014) Is reduced susceptibility to disinfectants and antiseptics a risk in healthcare settings? A point/counterpoint review. J Hosp Infect 87: 194–202. doi: 10.1016/j.jhin.2014.04.012 24996517

43. Ye J Jr., Huang C-T, Shie S-S, Huang P-Y, Su LH, Chiu CH, et al. (2010) Multidrug Resistant Acinetobacter baumannii: Risk Factors for Appearance of Imipenem Resistant Strains on Patients Formerly with Susceptible Strains. PLOS ONE 5: e9947. doi: 10.1371/journal.pone.0009947 20369056

44. Boucher HW, Talbot GH, Bradley JS, Edwards JE, Gilbert D, Rice LB, et al. (2009) Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. Clin Infect Dis 48: 1–12. doi: 10.1086/595011 19035777

45. Sepkowitz KA (2002) Antibiotic prophylaxis in patients receiving hematopoietic stem cell transplant. Bone Marrow Transplant 29: 367–371. doi: 10.1038/sj.bmt.1703366 11919724

46. Sanchez-Vizuete P, Orgaz B, Aymerich S, Le Coq D, Briandet R (2015) Pathogens protection against the action of disinfectants in multispecies biofilms. Front Microbiol 6: 705. doi: 10.3389/fmicb.2015.00705 26236291

47. Simon AY, Ihejirika KA, Ogunkoya AB, Hassan AZ, Adawa DAY, Adeyanju JB (2007) Comparison of the antimicrobial efficacy oftopical antiseptic creams on canine wounds(preliminary communication). Bulgarian Journal of Veterinary Medicine 10: 273–281.

48. Septimus E, Huang SS, Septimus E, Kleinman K, Moody J, Hickok J, et al. (2019) Chlorhexidine versus routine bathing to prevent multidrug-resistant organisms and all-cause bloodstream infections in general medical and surgical units (ABATE Infection trial): a cluster-randomised trial. Lancet 393: 1205–1215. doi: 10.1016/S0140-6736(18)32593-5 30850112

49. Brynildsen MP, Winkler JA, Spina CS, MacDonald IC, Collins JJ (2013) Potentiating antibacterial activity by predictably enhancing endogenous microbial ROS production. Nat Biotechnol 31: 160–165. doi: 10.1038/nbt.2458 23292609

50. Pischel D, Buchbinder JH, Sundmacher K, Lavrik IN, Flassig RJ (2018) A guide to automated apoptosis detection: How to make sense of imaging flow cytometry data. PLOS ONE 13: e0197208. doi: 10.1371/journal.pone.0197208 29768460

51. Lotfollahi M, Berisha S, Daeinejad D, Mayerich D (2019) Digital Staining of High-Definition Fourier Transform Infrared (FT-IR) Images Using Deep Learning. Applied Spectroscopy 73: 556–564. doi: 10.1177/0003702818819857 30657342

Článek vyšel v časopise


2019 Číslo 10
Nejčtenější tento týden