Investigating the antibacterial effects of some Lactobacillus, Bifidobacterium and acetobacter strains killed by different methods on Streptococcus mutans and Escherichia coli

Document Type: Original article


1 Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan, Iran

2 Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran


Although there are many health advantages assigned to different live bacteria such as probiotics, some health threatening effects have also been reported. For example, live bacteria can transfer antibiotic resistance genes to other commensal and opportunistic bacteria of gastrointestinal tract. Recently, it was shown that using killed bacteria have some advantages over live ones. In this research, heat, paraformaldehyde and ozone killing methods were used to kill the bacteria. Acetobacter cerevisiae, Lactobacillus acidophilus, Bifidobacterium lactis and traditional vinegar and fermented dairy product (Kumeh) derived bacteria were killed and their antibacterial activity against Streptococcus mutans and Escherichia coli was investigated. To identify the bacteria isolated from the traditional products, 16S rDNA gene was partially sequenced. The gene analysis showed vinegar and Kumeh derived bacteria were Acetobacter pasteurianus and Lactobacillus crustorum (LcK) strains respectively. The S. mutans growth inhibition was detected in the all concentrations of all killed samples. However, generally, E. coli showed more resistant to the killed bacteria than S. mutans and the antibacterial effect of heat-killed bacteria against E. coli was not observed in the all concentrations for some killed bacteria. Among the pathogenic bacteria, S. mutans was the most sensitive one to the killed bacteria with 70% of reduction in its viability. In conclusion, this research showed that different killed bacteria had different effects on other bacteria and the killing method showed an impact on these effects. Overall, paraformaldehyde-killed L.crustorum (LcK) showed the best antibacterial activity against S. mutans; about 70% decrease in bacterial viability.


1. Reid G. The importance of guidelines in the development and application of probiotics. Curr Pharm Des 2005;11:11-16.

2. Gasbarrini G, Bonvicini F, Gramenzi A. Probiotics history. J Clin Gastroenterol 2016; 50:S116-S119.

3. Behnsen J, Deriu E, Sassone-Corsi M, Raffatellu M. Probiotics: properties, examples, and specific applications. Cold Spring Harb Perspect Med 2013;3:211-268.

4. Chapman CM, Gibson GR, Rowland I. Health benefits of probiotics: are mixtures more effective than single strains? Eur J Nutr 2011;50:1-17.

5. Hardy H, Harris J, Lyon E, Beal J, Foey AD. Probiotics, prebiotics and immunomodulation of gut mucosal defences: homeostasis and immunopathology. Nutrients 2013; 5:1869-1912.

6. Sengun IY, Karabiyikli S. Importance of acetic acid bacteria in food industry. Food Control 2011;22:647-656.

7. Haghshenas B, Nami Y, Abdullah N, Radiah D, Rosli R, Khosroushahi AY. Anticancer impacts of potentially probiotic acetic acid bacteria isolated from traditional dairy microbiota. LWT- Food Sci Technol 2015;60:690-697.

8. Lahtinen SJ. Probiotic viability-does it matter? Microb Ecol Health Dis 2012;23:18567.

9. Jakobsen M, Narvhus J. Yeasts and their possible beneficial and negative effects on the quality of dairy products. Int Dairy J 1996;6:755-768.

10. Sharma P, Tomar SK, Goswami P, Sangwan V, Singh R. Antibiotic resistance among commercially available probiotics. Food Res Int 2014;57:176-195.

11. Tynkkynen S, Singh KV, Varmanen P. Vancomycin resistance factor of Lactobacillus rhamnosus GG in relation to enterococcal vancomycin resistance (van) genes. Int J Food Microbiol 1998;41:195-204.

12. Russell AD. Lethal effects of heat on bacterial physiology and structure. Sci Prog 2003; 86:115-137.

13. Isikber AA, Athanassiou CG. The use of ozone gas for the control of insects and micro-organisms in stored products. J Stored Prod Res 2015;64:139-145.

14. Uzun H, Ibanoglu E, Catal H, Ibanoglu S. Effects of ozone on functional properties of proteins. Food Chem 2012;134:647-654.

15. Tiwari B, Brennan CS, Curran T, Gallagher E, Cullen P, O'Donnell C. Application of ozone in grain processing. J Cereal Sci 2010;51:248-255.

16. Kiernan JA. Formaldehyde, formalin, paraformaldehyde and glutaraldehyde: what they are and what they do. Micros Today 2000;8:8-13.

17. Maal KB, Shafiei R, Kabiri N. Production of apricot vinegar using an isolated Acetobacter strain from Iranian apricot. World Acad Sci Eng Technol 2010;4:162-4.

18. Cheng HR, Jiang N. Extremely rapid extraction of DNA from bacteria and yeasts. Biotechnol Lett 2006;28:55-59.

19. Marchesi JR, Sato T, Weightman AJ, Martin TA, Fry JC, Hiom SJ, Wade WG. Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA. Appl Environ Microbiol 1998;64:795-799.

20. Turner S, Pryer KM, Miao VP, Palmer JD. Investigating deep phylogenetic relationships among cyanobacteria and plastids by small subunit rRNA sequence analysis. J  Eukaryotic Microbiol 1999;46:327-338.

21. Dehghanifar S, Keyhanfar M, Emtiazi G. Production and partial purification of thermostable bacteriocins from Bacillus pumilus ZED17 and DFAR8 strains with antifungal activity. Mol Biol Res Commun 2019;8:41-49.

22. Wikler MA. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically: approved standard. CLSI (NCCLS) 2006;26:M7-A7.

23. Fani MM, Kohanteb J, Dayaghi M. Inhibitory activity of garlic (Allium sativum) extract on multidrug-resistant Streptococcus mutans. J Indian Soc Pedod Prev Dent 2007;25:164-168.

24. Hamada S, Slade HD. Biology, immunology, and cariogenicity of Streptococcus mutans. Microbiol Rev 1980;44:331-384.

25. Ahola AJ, Yli-Knuuttila H, Suomalainen T. Poussa T, Ahlström A, Meurman JH, Korpela R. Short-term consumption of probiotic-containing cheese and its effect on dental caries risk factors. Arch Oral Biol 2002;47:799-804.

26. Cildir SK, Germec D, Sandalli N, Ozdemir FI, Arun T, Twetman S, Caglar E. Reduction of salivary mutans streptococci in orthodontic patients during daily consumption of yoghurt containing probiotic bacteria. Eur J Orthod 2009;31:407-411.

27. Caglar E, Cildir SK, Ergeneli S, Sandalli N, Twetman S. Salivary mutans streptococci and lactobacilli levels after ingestion of the probiotic bacterium Lactobacillus reuteri ATCC 55730 by straws or tablets. Acta Odontol Scand 2006;64:314-318.

28. Meurman JH, Antila H, Korhonen A, Salminen S. Effect of Lactobacillus rhamnosus strain GG (ATCC 53103) on the growth of Streptococcus sobrinus in vitro. Eur J Oral Sci 1995; 103:253-258.

29. Prado Acosta M, Mersedes Palomino M, Allievi MC, Sanchez Rivas C, Ruzal SM. Murein hydrolase activity in the surface layer of Lactobacillus acidophilus ATCC 4356. Appl Environ Microbiol 2008;74:7824-7827.

30. Prado-Acosta M, Ruzal SM, Allievi MC, Palomino MM, Sanchez Rivas C. Synergistic effects of the Lactobacillus acidophilus surface layer and nisin on bacterial growth. Appl Environ Microbiol 2010;76:974-977.

31. Schwendicke F, Horb K, Kneist S, Dörfer C, Paris S. Effects of heat-inactivated Bifidobacterium BB12 on cariogenicity of Streptococcus mutans in vitro. Arch Oral Biol 2014;59:1384-1390.

32. Coconnier MH, Liévin V, Bernet-Camard MF, Hudault S, Servin AL. Antibacterial effect of the adhering human Lactobacillus acidophilus strain LB. Antimicrob Agents Chemother 1997;41:1046-1052.

33. Abd El-Gawal IA, El-Sayed EM, El-Zeini HM, Hafez SA, Saleh FA. Antibacterial Activity of Probiotic Yoghurt and Soy-Yoghurt against Escherichia coli and Staphylococcus aureus. J Nutr Food Sci 2014;4:1-6.

34. Spinler JK, Taweechotipatr M, Rognerud CL, Ou CN, Tumwasorn S, Versalovic J. Human-derived probiotic Lactobacillus reuteri demonstrate antimicrobial activities targeting diverse enteric bacterial pathogens. Anaerobe 2008;14:166-171.