Influence of subinhibitory-concentration (sub-MIC) Cefetoxime on biofilm formation. SEM study of ESBL-producing Salmonella typhi

Objectives: In the present study, we have analyzed ESBL-producing S. typhi’s capability in forming a significant amount of biofilm on plastic and glass surface, and the influence of cefetoxime on biofilm development at subinhibitory (Sub-MIC) concentration. Methods: Nine strains of cefetoxime-mediated ESBL-producing S. typhi were used in the study. S. typhi formed biofilm on plastic and glass materials; it was demonstrated using micro titre plate (MTP) and standard test tube methods. Comparative study of the influence of cefetoxime on biofilm formation in its MIC (128 µg/ml) and at sub-MIC (64 µg/ml) was demonstrated by microtitre plate method. The biofilm production was observed in SEM images, statistical analysis (ANOVA) showed significant increase in cell surface and volume due to the influence of Cefetoxime. Results: Of the nine selected isolates, two S. typhi strains, namely BST 51 and BST 130, produced relatively strong biofilm in the presence of cefetoxime at sub-MIC level (64 µg/ml), comparatively weak biofilm formation at MIC level (128 µg/ml). Typical morphological changes were observed in cefetoxime-resistant strains, S. typhi BST 51 and BST 130, in comparison to cefetoxime-sensitive strain S. typhi BST 63 used as a control. We found an increase in surface and volume of a cell in response to cefetoxime and statistical data (ANOVA) proved that resistant strains were significantly different from control strains. Conclusion: The above study clearly shows that cefetoxime at sub-MIC level efficiently induces biofilm formation and promotes changes in morphology of the cell. J Microbiol Infect Dis 2017; 7(2): 67-75 

___

  • 1. Rowe B, Ward LR, Threlfall EJ. Multidrug-resistant Salmonella typhi: a worldwide epidemic. Clin Infect Dis 1997; 24:S106-109.
  • 2. Le TA, Lejay-Collin M, Grimont PA, et al. Endemic, an epidemic clone of Salmonella enterica serovar Typhi harboring a single multidrug-resistant plasmid in Vietnam between 1995 and 2002. J Clin Microbiol 2004; 42:3094- 3099.
  • 3. Gautam V, Gupta NK, Chaudhary U, Arora DR. Sensitivity pattern of Salmonella serotypes in Northern India. Braz J Infect Dis 2002; 6:281-287.
  • 4. Thong KL, Bhutta ZA, Pang T.Multidrug-resistant strains of Salmonella enterica serotype Typhi are genetically homogenous and coexist with antibiotic-sensitive strains as distinct, independent clones. Int J Infect Dis 2000; 4:194-197.
  • 5. Bharat MP, Janak K, Rajan KD, Shyam KM, Prem KK, Tuladhar NR. Multidrug-resistant and extended-spectrum beta-lactamase (ESBL)- producing Salmonella enterica (serotypes Typhi and Paratyphi A) from blood isolates in Nepal: surveillance of resistance and a search for newer alternatives. Int J Infect Dis 2006; 10:434-438.
  • 6. Donlan RM. Biofilms: microbial life on surfaces. Emerg Infect Dis.2002; 8:881–890.
  • 7. Mah TF, O’Toole GA. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol.2001; 9:34–39.
  • 8. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science.1999; 284:1318–1322.
  • 9. Wojnicz D, Jankowski S. Effects of subinhibitory concentrations of Amikacin and Ciprofloxacin on the hydrophobicity and adherence to epithelial cells of uropathogenic Escherichia coli strains. Int J Antimicrob Agents.2007; 29:700–704.
  • 10. Wang Y, Rotman ER, Schoemaker NB, Salyers AA. Translational control of tetracycline resistance and conjugation in the Bacteroides conjugative transposon CTnDOT. J Bacteriol.2005; 18:2673– 2680.
  • 11. Schroeder R, Waldisch C, Wank H. Modulation of RNAFmai function by aminoglycoside antibiotics. EMBO J 2000; 19:1-9.
  • 12. Majtán J1, Majtánová L, Xu M, Majtán V. In vitro effect of subinhibitory concentrations of antibiotics on biofilm formation by clinical strains of Salmonella enterica serovar Typhimurium isolated in Slovakia. J Appl Microbiol 2007; 104:1294- 1301.
  • 13. Marne TJ, Costerton JW. Scanning and transmission electron microscopy of in situ bacterial colonization of intravenous and intraarterial catheters. J Clin Microbiol 1984; 19:687- 693.
  • 14. Drieux L, Brossier F, Sougakoff W, Jarlier V. Phenotypic detection of extended-spectrum blactamase production in Enterobacteriaceae: review and bench guide. Clin Microbiol Infect 2008; 14 (Suppl 1):90-103.
  • 15. Clinical Laboratory Standard Institute M100-S22, Vol 32, No 3; January 2012.
  • 16. Andrews JM. Determination of minimum inhibitory concentration. J Antimicrob Chemother 2001; 48: suppl S1, 5-16
  • 17. Christensen GD, Simpson WA, Bisno AL, Beachey EH. Adherence of slime producing strains of Staphylococcus epidermidis to smooth surfaces. Infect Immun 1982; 37:318-326.
  • 18. Stepanovic S, Cirkovic I, Mijac M, Svabic-Vlahovic M. Influence of the incubation temperature, atmosphere, and dynamic conditions on biofilm formation by Salmonella spp. Food Microbiol 2003; 20:339–343.
  • 19. Stepanovic S, Cirkovic I, Ranin L, Svabic-Vlahovic M. Biofilm formation by Salmonella spp. and Listeria monocytogenes on plastic surface. Lett Appl Microbiol 2004; 38:428-432.
  • 20. Peters G, Locci R, Pulverer G. Adherence and growth of coagulase-negative staphylococci on surfaces of intravenous catheters. J Infect Dis 1982; 146 (4):479–482.
  • 21. Soumya EA, Saad KI, Hassan L, Fatima H. Scanning Electron Microscopy (SEM) and Environmental SEM: Suitable Tools for Study of Adhesion Stage and Biofilm Formation, Scanning Electron Microscopy, Dr. Viacheslav Kazmiruk (Ed.), ISBN.2012;978-953-51-0092-8.
  • 22. Guo D, Wang L, Lu C. In vitro biofilm forming potential of Streptococcus suis isolated from human and swine in China. Braz J Microbiol 2012; 43 (3):993-1004.
  • 23. The World Health Report: Fighting disease, fostering development. Geneva: World Health Organization, 1996.
  • 24. Crump JA, Luby SP, Mintz ED.The global burden of typhoid fever. Bull World Health Organ 2004; 82:346-353.
  • 25. WHO/UNICEF Joint Monitoring Programme for water supply and sanitation. Global Water supply and sanitation assessment 2000 report. Geneva: WHO/UNICEF; 2000.
  • 26. Black RE, Levine MM, Ferreccio C, et al. Efficacy of one or two doses of Ty21a Salmonella typhi vaccine in enteric coated capsules in a controlled field trial. Vaccine 1990; 8:81-84.
  • 27. Levine MM, Ferreccio C, Cryz S, Ortiz E. Comparison of enteric coated capsules and liquid formulation of Ty21a typhoid vaccine in randomised controlled field trial. Lancet 1990 336:891-894.
  • 28. Wahdan MH, Serie C, Cersier Y, Sallam S, Germainer R. A controlled field trial of live Salmonella typhi Ty21a oral vaccine against typhoid: three years results. J Infect Dis 1982; 145:292.5.
  • 29. Ashcroft MT, Singh B, Nicholson CC, Ritchie JM, Sobryan E, Williams F. A seven-year field trial of two typhoid vaccines in Guyana. Lancet 1967; 2:1056-1059.
  • 30. Chuttani CS, Prakash K, Gupta P, Grover V, Kumar A. Controlled field trial of high dose oral killed typhoid vaccine in India. Bull World Health Organ 1977; 55:643-644.
  • 31. Hejfec LB, Levina LA, Kuzminova ML, Salmin LV, Salvina AM, Vasileva AV. Controlled field trials of paratyphoid B vaccine and evaluation of the effectiveness of a single administration of typhoid vaccine. Bull World Health Org 1968; 38:907-915.
  • 32. Fey PD, Safranek TJ, Rupp ME, et al. Ceftriaxone-resistant salmonella infection acquired by a child from cattle. N Engl J Med.2000; 342:1242–1249.
  • 33. Winokur PL, Brueggemann A, DeSalvo DL, et al. Animal and human multidrug-resistant, cephalosporin-resistant salmonella isolates expressing a plasmid-mediated CMY-2 AmpC beta-lactamase. Antimicrob Agents Chemother 2000; 44:2777–2783.
  • 34. Szych J, Cieslik A, Paciorek J, Kaluzewski S. Antibiotic resistant in Salmonella enterica subspecies enterica strains isolated in Poland in the 1998-1999 periods. Med Dosw Mikrobiol 2001; 53:17-29.
  • 35. Hood SK, Zottola EA. Adherence to stainless steel by food borne microorganisms during growth in model food systems. Int J Food Microbiol 1997; 37:145–153.
  • 36. Momba MN, Kaleni P. The growth and survival of indicator microorganisms on the surfaces of household containers used for the storage of drinking water in rural communities of South Africa. Water Res 2002; 36:3023–3028.
  • 37. Kim SH, Wei CI. Biofilm formation by multidrugresistant Salmonella enterica serotype typhimurium phage type DT104 and other pathogens. J Food Prot 2007; 70:22–29.
  • 38. Gottfredsson M, Erlendsdottir H, Sigfusson A, Gudmundsson S. Characteristics and dynamics of bacterial populations during post antibiotic effect determined by flow cytometry. Antimicrob Agents Chemother 1998; 42:1005–1011.
  • 39. Rachid S, Ohlsen K, Witte W, Hacker J, Ziebuhr W. Effect of subinhibitory antibiotic concentrations on polysaccharide intercellular adhesion expression in biofilm forming Staphylococcus epidermidis. Antimicrob Agents Chemother 2000; 44:3357-3363.
  • 40. Hoffman LR, D'Argenio DA, MacCoss MJ, Zhang Z, Jones RA, Miller SI. Aminoglycoside antibiotics induce bacterial biofilm formation. Nature 2005; 436 (7054):1171-1175.
  • 41. Boehm A, Steiner S, Zaehringer F, et al. Second messenger signalling governs Escherichia coli biofilm induction upon ribosomal stress. Mol Microbiol 2009; 72(6):1500-1516.
  • 42. Anderl JN, Zahller J, Roe F, Stewart PS. Role of nutrient limitation and stationary-phase existence in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin. Antimicrob.Agents Chemother.2003; 47:1251–1256.
  • 43. Haddadin RN, Saleh S, Al-Adham IS, Buultjens TE, Collier PJ. The effect of subminimal inhibitory concentrations of antibiotics on virulence factors expressed by Staphylococcus aureus biofilms. J Appl Microbiol 2010; 108 (4):1281-1291.