Evidence of significant synergism between antibiotics and the antipsychotic, antimicrobial drug flupenthixol

  • L. Jeyaseeli
  • A. Dasgupta
  • S. G. Dastidar
  • J. Molnar
  • L. Amaral


Previously, the antipsychotic, non-antibiotic compound flupenthixol dihydrochloride (Fp) was shown to exhibit distinct in vitro antibacterial activity against Gram-positive and Gram-negative bacteria and to significantly protect Swiss albino mice challenged with a known mouse virulent salmonella. The present study was designed to ascertain whether this drug could efficiently augment the action of an antibiotic or a non-antibiotic when tested in combination. A total of 12 bacterial strains belonging to various genera were selected for this study and were sensitive to the antibiotics penicillin (Pc), ampicillin, chloramphenicol, tetracycline, streptomycin, gentamicin, erythromycin, ciprofloxacin, and to the non-antibiotics methdilazine, triflupromazine, promethazine, and Fp. Pronounced and statistically significant synergism (p < 0.01) was observed when Fp was combined with Pc following the disc diffusion assay system. With the help of the checkerboard method, the fractional inhibitory concentration (FIC) index of this pair was found to be 0.375, confirming synergism. This pair of Fp+ Pc was then subjected to in vivo experiments in mice challenged with Salmonella enterica serovar Typhimurium NCTC 74. Statistical analysis of the mouse protection test suggested that this combination was highly synergistic (p < 0.001, Chi-squared analysis). Fp also revealed augmentation of its antimicrobial property when combined with streptomycin, gentamicin, ciprofloxacin, and the non-antibiotic methdilazine. The results of this study may provide alternatives for the therapy of problematic infections such as those associated with Salmonella spp.


Minimum Inhibitory Concentration Lacidipine Test Bacterium Fractional Inhibitory Concentration Disc Diffusion Assay 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



L. Amaral was supported by the BCC grant SFRH/BCC/51099/2010 provided by the Fundação para a Ciência e a Tecnologia (FCT) of Portugal and PTDC/SAU-FCF/102807/2008 provided by the UPMM.


  1. 1.
    Pagès JM, Amaral L, Fanning S (2011) An original deal for new molecule: reversal of efflux pump activity, a rational strategy to combat gram-negative resistant bacteria. Curr Med Chem 18:2969–2680PubMedCrossRefGoogle Scholar
  2. 2.
    Machado L, Spengler G, Evaristo M, Handzlik J, Molnár J, Viveiros M, Kiec-Kononowicz K, Amaral L (2011) Biological activity of twenty three hydantoin derivatives on intrinsic efflux pump system of Salmonella enterica Enteritidis NCTC 13349. In Vivo 25:769–772PubMedGoogle Scholar
  3. 3.
    Takács D, Cerca P, Riedl Z, Hajós G, Molnár J, Viveiros M, Couto I, Amaral L (2011) Evaluation of forty new phenothiazine derivatives for activity against intrinsic efflux pump systems of reference Escherichia coli, Salmonella Enteritidis, Enterococcus faecalis and Staphylococcus aureus strains. In Vivo 25:719–724PubMedGoogle Scholar
  4. 4.
    Dasgupta A, Dastidar SG, Shiratki Y, Motohashi N (2004) Antibacterial activity of artificial phenothiazines and isoflavones from plants. In: Bioactive heterocycles, VI, vol 15. Springer, Berlin Heidelberg, pp 67–132Google Scholar
  5. 5.
    Dasgupta A, Jeyaseeli L, Dutta NK, Mazumdar K, Karak P, Dastidar SG, Motohashi N, Shirataki Y (2007) Studies on the antimicrobial potential of the cardiovascular drug lacidipine. In Vivo 21:847–850PubMedGoogle Scholar
  6. 6.
    Levy SB, Marshall B (2004) Antibacterial resistance worldwide: cause, challenges and responses. Nat Med 10 [Suppl]:S122–S129PubMedCrossRefGoogle Scholar
  7. 7.
    Martinez JL, Baquero F, Anderson DI (2007) Predicting antibiotic resistance. Nat Rev Microbiol 5:958–965PubMedCrossRefGoogle Scholar
  8. 8.
    Bhatawadekar S, Chattopadhyay A (2010) Quinpristin-dalfopristin resistance among methicillin-resistant strains of Staphylococcus. Indian J Pharmacol 42:56PubMedCrossRefGoogle Scholar
  9. 9.
    Howe RA, Monk F, Wootton M, Wlash TR, Enright MC (2004) Vancomycin susceptibility within methicillin resistant Staphylococcus aureus lineages. Emerg Infect Dis 10:855–857PubMedGoogle Scholar
  10. 10.
    Dastidar SG, Chaudhury A, Annadurai S, Roy S, Mookerjee M, Chakrabarty AN (1995) In vitro and in vivo antimicrobial action of fluphenazine. J Chemother 7:201–206PubMedGoogle Scholar
  11. 11.
    Dastidar SG, Debnath S, Mazumdar K, Ganguly K, Chakrabarty AN (2004) Triflupromazine: a microbicide non-antibiotic compound. Acta Microbiol Immun Hung 51:75–83Google Scholar
  12. 12.
    Jeyaseeli L, Dasgupta A, Kumar KA, Mazumdar K, Dutta NK, Dastidar SG (2006) Antimicrobial potentiality of thioxanthene fluphenthixol through extensive in vitro and in vivo experiments. Int J Antimicrob Agents 27:58–62PubMedCrossRefGoogle Scholar
  13. 13.
    Kristiansen JE (1992) The antimicrobial activity of non-antibiotics. APMIS 100:7–14Google Scholar
  14. 14.
    Mazumdar R, Ganguly K, Dastidar SG, Chakrabarty AN (2001) Trifluoperazine: a broad-spectrum bactericide especially active on staphylococci and vibrios. Int J Antimicrob Agents 18:403–406CrossRefGoogle Scholar
  15. 15.
    Keyzer H, Eckert GM, Forrest IS, Gupta RR, Gutmann F, Molnar J (eds) (1992) Thiazines and structurally related compounds. Krieger, Malabar, pp 197–202Google Scholar
  16. 16.
    Annadurai S, Guhathakurta A, Sa B, Dastidar SG, Ray R, Chakrabarty AN (2002) Experimental studies on synergism between aminoglycosides and the antimicrobial anti-inflammatory agent diclofenac sodium. J Chemother 14:47–53PubMedGoogle Scholar
  17. 17.
    Asok Kumar K, Mazumdar K, Dutta NK, Karak P, Dastidar SG, Ray R (2004) Evaluation of synergism between the aminoglycoside antibiotic streptomycin and the cardiovascular agent amlodipine. Biol Pharm Bull 27:1116–1120PubMedCrossRefGoogle Scholar
  18. 18.
    Basu LR, Mazumdar K, Dutta NK, Karak P, Dastidar SG (2005) Antibacterial property of the antipsychotic agent prochlorperazine, and its synergism with methdilazine. Microbiol Res 160:95–100CrossRefGoogle Scholar
  19. 19.
    Dasgupta A, Chaki S, Mukherjee S, Jeyaseeli L, Mazumdar K, Dutta NK, Dastidar SG (2010) Experimental analyses of synergistic combinations of antibiotics with a recently recognized antibacterial agent, lacidipine. Eur J Clin Microbiol Infect Dis 29:239–243PubMedCrossRefGoogle Scholar
  20. 20.
    Amaral L, Kristiansen JE, Lorian V (1992) Synergistic effect of chlorpromazine on the activity of some antibiotics. J Antimicrob Chemother 30:556–558PubMedCrossRefGoogle Scholar
  21. 21.
    Kristiansen JE, Amaral L (1997) The potential management of resistant infections with non-antibiotics. J Antimicrobial Chemother 40:319–327CrossRefGoogle Scholar
  22. 22.
    Collee FG, Miles RS, Watt B (1996) Mackie & McCartney’s practical medical microbiology, 14th edn. Churchill Livingstone, New York, pp 131–150Google Scholar
  23. 23.
    McFarland J (1907) Nephelometer: an instrument for estimating the number of bacteria in suspensions. J Am Med Assoc 14:1176–1178CrossRefGoogle Scholar
  24. 24.
    Clinical and Laboratory Standards Institute (2009) Methods for dilution antimicrobial susceptibility testing of bacteria that grow aerobically, 7th edn. Approved standard M7-A7.2009. Clinical and Laboratory Standards Institute, Wayne, PAGoogle Scholar
  25. 25.
    Clinical and Laboratory Standards Institute (2009) Performance Standards for antimicrobial disk susceptibility tests, 10th edn. Approved standard M02-A10.2009. Clinical and Laboratory Standards Institute, Wayne, PAGoogle Scholar
  26. 26.
    Miles RS, Amyes SGB (1996) Laboratory control of antimicrobial therapy. In: Collee JG, Fraser AG, Marmino BP, Simons A (eds) Mackie & McCartney’s practical medical microbiology. Churchill Livingstone, New York, pp 151–177Google Scholar
  27. 27.
    Eliopoulos GM, Moellering RC Jr (1996) Antimicrobial combinations. In: Lorian V (ed) Antibiotics in laboratory medicine, 4th edn. Williams & Wilkins, Baltimore, MD, pp 432–492Google Scholar
  28. 28.
    American Society for Microbiology (1995) Instruction to authors. Antimicrob Agents Chemother 39:i–xivGoogle Scholar
  29. 29.
    Mitruka BM, Rawnsle HM, Vadehra DV (eds) (1976) In: Animals for medical research. Wiley, New York, pp 145–150Google Scholar
  30. 30.
    Dasgupta A, Mukherjee S, Chaki S, Dastidar SG, Hendricks O, Christensen JB, Kristiansen JE, Amaral L (2010) Thioridazine protects the mouse from a virulent infection by Salmonella enterica Serovar Typhimurium 74. Int J Antimicrob Agents 35:174–176PubMedCrossRefGoogle Scholar
  31. 31.
    Mishra L (ed) (2006) Drugs today; ready reckoner of current medical formulations. Lorina, New DelhiGoogle Scholar
  32. 32.
    Crossland J (1980) Lewis’s pharmacology. Churchill Livingstone, London. pp 834–835Google Scholar
  33. 33.
    Mazumdar K, Dutta NK, Kumar KA, Dastidar SG (2005) An in vitro and in vivo synergism between tetracycline and the cardiovascular agent oxyfedrine HCl against common bacterial strains. Biol Pharm Bull 28:713–717PubMedCrossRefGoogle Scholar
  34. 34.
    Amaral L, Fanning S, Pagès JM (2011) Efflux pumps of gram-negative bacteria: genetic responses to stress and the modulation of their activity by pH, inhibitors, and phenothiazines. Adv Enzymol Relat Areas Mol Biol 77:61–108PubMedCrossRefGoogle Scholar
  35. 35.
    Rodrigues L, Aínsa JA, Amaral L, Viveiros M (2011) Inhibition of drug efflux in mycobacteria with phenothiazines and other putative efflux inhibitors. Recent Pat Antiinfect Drug Discov 6:118–127PubMedCrossRefGoogle Scholar
  36. 36.
    Jin J, Zhang JY, Guo N, Sheng H, Li L, Liang JC, Wang XL, Li Y, Liu MY, Wu XP, Yu L (2010) Farnesol, a potential efflux pump inhibitor in Mycobacterium smegmatis. Molecules 15:7750–7762PubMedCrossRefGoogle Scholar
  37. 37.
    Kristiansen JE, Thomsen VF, Martins A, Viveiros M, Amaral L (2010) Non-antibiotics reverse resistance of bacteria to antibiotics. In Vivo 24:751–754PubMedGoogle Scholar
  38. 38.
    Amaral L, Martins A, Molnar J, Kristiansen JE, Martins M, Viveiros M, Rodrigues L, Spengler G, Couto I, Ramos J, Dastidar S, Fanning S, McCusker M, Pages JM (2010) Phenothiazines, bacterial efflux pumps and targeting the macrophage for enhanced killing of intracellular XDRTB. In Vivo 24:409–424PubMedGoogle Scholar
  39. 39.
    Dutta NK, Mehra S, Kaushal D (2010) A Mycobacterium tuberculosis sigma factor network responds to cell-envelope damage by the promising anti-mycobacterial thioridazine. PLoS One 5:e10069PubMedCrossRefGoogle Scholar
  40. 40.
    Rodrigues L, Sampaio D, Couto I, Machado D, Kern WV, Amaral L, Viveiros M (2009) The role of efflux pumps in macrolide resistance in Mycobacterium avium complex. Int J Antimicrob Agents 34:529–533PubMedCrossRefGoogle Scholar
  41. 41.
    Spengler G, Martins A, Schelz Z, Rodrigues L, Aagaard L, Martins M, Costa SS, Couto I, Viveiros M, Fanning S, Kristiansen JE, Molnar J, Amaral L (2009) Characterization of intrinsic efflux activity of Enterococcus faecalis ATCC29212 by a semi-automated ethidium bromide method. In Vivo 23:81–87PubMedGoogle Scholar
  42. 42.
    Kvist M, Hancock V, Klemm P (2008) Inactivation of efflux pumps abolishes bacterial biofilm formation. Appl Environ Microbiol 74:7376–7382PubMedCrossRefGoogle Scholar
  43. 43.
    Sabatini S, Kaatz GW, Rossolini GM, Brandini D, Fravolini A (2008) From phenothiazine to 3-phenyl-1,4-benzothiazine derivatives as inhibitors of the Staphylococcus aureus NorA multidrug efflux pump. J Med Chem 51:4321–4330PubMedCrossRefGoogle Scholar
  44. 44.
    Couto I, Costa SS, Viveiros M, Martins M, Amaral L (2008) Efflux-mediated response of Staphylococcus aureus exposed to ethidium bromide. J Antimicrob Chemother 62:504–513PubMedCrossRefGoogle Scholar
  45. 45.
    Tegos GP, Masago K, Aziz F, Higginbotham A, Stermitz FR, Hamblin MR (2008) Inhibitors of bacterial multidrug efflux pumps potentiate antimicrobial photoinactivation. Antimicrob Agents Chemother 52:3202–3209PubMedCrossRefGoogle Scholar
  46. 46.
    Lehtinen J, Lilius EM (2007) Promethazine renders Escherichia coli susceptible to penicillin G: real-time measurement of bacterial susceptibility by fluoro-luminometry. Int J Antimicrob Agents 30:44–51PubMedCrossRefGoogle Scholar
  47. 47.
    Kristiansen JE, Hendricks O, Delvin T, Butterworth TS, Aagaard L, Christensen JB, Flores VC, Keyzer H (2007) Reversal of resistance in microorganisms by help of non-antibiotics. J Antimicrob Chemother 59:1271–1279PubMedCrossRefGoogle Scholar
  48. 48.
    Chan YY, Ong YM, Chua KL (2007) Synergistic interaction between phenothiazines and antimicrobial agents against Burkholderia pseudomallei. Antimicrob Agents Chemother 51:623–630PubMedCrossRefGoogle Scholar
  49. 49.
    Kristiansen MM, Leandro C, Ordway D, Martins M, Viveiros M, Pacheco T, Molnar J, Kristiansen JE, Amaral L (2006) Thioridazine reduces resistance of methicillin-resistant staphylococcus aureus by inhibiting a reserpine-sensitive efflux pump. In Vivo 20:361–366PubMedGoogle Scholar
  50. 50.
    Ramón-García S, Martín C, Aínsa JA, De Rossi E (2006) Characterization of tetracycline resistance mediated by the efflux pump Tap from Mycobacterium fortuitum. J Antimicrob Chemother 57:252–259PubMedCrossRefGoogle Scholar
  51. 51.
    Marquez B (2005) Bacterial efflux systems and efflux pumps inhibitors. Biochimie 87:1137–1147PubMedCrossRefGoogle Scholar
  52. 52.
    Amaral L, Viveiros M, Molnar J (2004) Antimicrobial activity of phenothiazines. In Vivo 18:725–731. ReviewPubMedGoogle Scholar
  53. 53.
    Hendricks O, Butterworth TS, Kristiansen JE (2003) The in-vitro antimicrobial effect of non-antibiotics and putative inhibitors of efflux pumps on Pseudomonas aeruginosa and Staphylococcus aureus. Int J Antimicrob Agents 22:262–264PubMedCrossRefGoogle Scholar
  54. 54.
    Gunn JS (2008) The Salmonella PmrAB regulon: lipopolysaccharide modifications, antimicrobial peptide resistance and more. Trends Microbiol 16:284–290PubMedCrossRefGoogle Scholar
  55. 55.
    Moreau A, Le Vee M, Jouan E, Parmentier Y, Fardel O (2011) Drug transporter expression in human macrophages. Fundam Clin Pharmacol. doi: 0.1111/j.1472-8206.2010.00913.x
  56. 56.
    Lemaire S, Tulkens PM, Van Bambeke F (2010) Cellular pharmacokinetics of the novel biaryloxazolidinone radezolid in phagocytic cells: studies with macrophages and polymorphonuclear neutrophils. Antimicrob Agents Chemother 54:2540–2548PubMedCrossRefGoogle Scholar
  57. 57.
    Lemaire S, Kosowska-Shick K, Appelbaum PC, Verween G, Tulkens PM, Van Bambeke F (2010) Cellular pharmacodynamics of the novel biaryloxazolidinone radezolid: studies with infected phagocytic and nonphagocytic cells, using Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, and Legionella pneumophila. Antimicrob Agents Chemother 54:2549–2559PubMedCrossRefGoogle Scholar
  58. 58.
    Baudoux P, Lemaire S, Denis O, Tulkens PM, Van Bambeke F, Glupczynski Y (2010) Activity of quinupristin/dalfopristin against extracellular and intracellular Staphylococcus aureus with various resistance phenotypes. J Antimicrob Chemother 65:1228–1236PubMedCrossRefGoogle Scholar
  59. 59.
    Kodavanti UP, Lockard VG, Mehendale HM (1990) In vivo toxicity and pulmonary effects of promazine and chlorpromazine in rats. J Biochem Toxicol 5:245–251PubMedCrossRefGoogle Scholar
  60. 60.
    Dijkstra J, van Galen M, Scherphof G (1985) Effects of (dihydro)cytochalasin B, colchicine, monensin and trifluoperazine on uptake and processing of liposomes by Kupffer cells in culture. Biochim Biophys Acta 845:34–42PubMedCrossRefGoogle Scholar
  61. 61.
    Drenckhahn D, Kleine L, Lüllmann-Rauch R (1976) Lysosomal alterations in cultured macrophages exposed to anorexigenic and psychotropic drugs. Lab Invest 35:116–123PubMedGoogle Scholar
  62. 62.
    Molnár J, Haszon I, Bodrogi T, Martonyi E, Turi S (1990) Synergistic effect of promethazine with gentamycin in frequently recurring pyelonephritis. Int Urol Nephrol 22:405–411PubMedCrossRefGoogle Scholar
  63. 63.
    Amaral L, Viveiros M, Molnar J, Kristiansen JE (2011) Effective therapy with the neuroleptic thioridazine as an adjunct to second line of defence drugs, and the potential that thioridazine offers for new patents that cover a variety of “New Uses”. Recent Pat Antiinfect Drug Discov 6:84–87PubMedCrossRefGoogle Scholar
  64. 64.
    Amaral L, Boeree MJ, Gillespie SH, Udwadia ZF, van Soolingen D (2010) Thioridazine cures extensively drug-resistant tuberculosis (XDR-TB) and the need for global trials is now! Int J Antimicrob Agents 35:524–526PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • L. Jeyaseeli
    • 1
  • A. Dasgupta
    • 1
    • 5
  • S. G. Dastidar
    • 2
  • J. Molnar
    • 3
  • L. Amaral
    • 4
  1. 1.Division of Microbiology, Department of Pharmaceutical TechnologyJadavpur UniversityKolkataIndia
  2. 2.Department of MicrobiologyHerbicure Healthcare Bio-Herbal Research FoundationPailan, KolkataIndia
  3. 3.Institute of Medical Microbiology and Immunobiology, Faculty of MedicineUniversity of SzegedSzegedHungary
  4. 4.Grupo de Micobactérias, Unidade de Microbiologia Médica, UPMM, Instituto de Higiene e Medicina TropicalUniversidade Nova de LisboaLisboaPortugal
  5. 5.Laboratory of Molecular Biology & ImmunologyNational Institute on AgingBaltimoreUSA

Personalised recommendations