Evaluation of anti-microbial activity of filtrates of Lactobacillus rhamnosus and Saccharomyces boulardii against antibiotic-resistant gram-negative bacteria

  • O. Y. Isayenko I. I. Mechnikov Institute of Microbiology and Immunology of National Academy of Medical Sciences of Ukraine
  • O. V. Knysh I. I. Mechnikov Institute of Microbiology and Immunology of National Academy of Medical Sciences of Ukraine
  • O. V. Kotsar Kharkiv National Medical University
  • T. N. Ryzhkova Kharkiv State University of Food Technology and Trade
  • G. I. Dyukareva Kharkiv State University of Food Technology and Trade
Keywords: lactobacteria; saccharomycetes; disintegrates; metabolites; anti-microbial properties; polyresistant microorganisms.


The article presents the results of the first study on the influence of biologically active substances Lactobacillus rhamnosus GG ATCC 53103 and Saccharomyces boulardii, obtained according to the author`s method, on growth of gram-negative bacteria with broad medical resistance: Pseudomonas aeruginosa PR, Klebsiella pneumoniae PR, Lelliottia amnigena (Enterobacter amnigenus) PR using the spectrophotometric method. Disintegrates of L. rhamnosus GG and S. boulardii were obtained using low-frequency ultrasound processing of suspension of probiotic strains, and metabolites – through cultivation of lactobacteria and saccharomycetes in disintegrates of probiotic microorganisms. To samples of test-cultures with studied filtrates of disintegrates or metabolites we added growth medium and cultivated them (period of monitoring was 5- and 24-hours). Results of the studies were expressed as the percentage of inhibition of increment in polyresistant gram-negative bacteria under the impact of biologically active substances of probiotic microorganisms. Five-hour incubation of test-strains with the studied samples of lactobacteria led to inhibition of their growth properties by 85.6–96.7%. Growth of bacteria under the impact of substances of saccharomycetes was inhibted by 45.1–92.5%. Twenty-four hour exposure of the test-cultures in filtrates of L. rhamnosus GG and S. boulardii caused 100% inhibition of P. aeruginosa and L. amnigena polyresistant strains. Temporal interval of cultivation directly proportionally affected the extent of inhibition of growth of microorganisms: we determined direct correlation dependence within 0.789–0.991. Maximum inhibition of increment of the studied pathogens was observed under the influence of metabolites of lactobacteria, obtained by cultivating primary producers in their disintegrate. We determined a high level of anti-microbial activity of metabolites from L. rhamnosus GG and S. boulardii obtained by cultivation of probiotics in disintegrates against bacteria resistant to a broad range of preparations, which allows us to consider these substances as promising for development of anti-microbial preparations of a new generation against etiologically significant antibiotic-resistant gram-negative microorganisms.


Ali, M. A. E., Janson, J.-C., & Ali, E. (2012). Antimicrobial potential of Saccharomyces boulardii extracts and fractions. Journal of Applied Sciences Research, 8(8), 4537–4543.

Allonsius, C. N., van den Broek, M. F. L., De Boeck, I., Kiekens, S., Oerlemans, E. F. M., Kenn, F., Vandenheuvel, D., Cos, P., Delputte, P., & Lebeer, S. (2017). Interplay between Lactobacillus rhamnosus GG and Candida and the involvement of exopolysaccharides. Microbial Biotechnology, 10(6), 1753–1763.

Al-Malkey, M. K., Munira, C. I., Abo Al-Hur, F. J., Mohammed, S. W., & Nayyefa, H. J. (2017). Antimicrobial effect of probiotic Lactobacillus spp. on Pseudomonas aeruginosa. Journal of Contemporary Medical Sciences, 3(10), 218–223.

Alokomi, H. L., Skytta, E., Saarela, M., Mattila-Sandholm, T., Latva-Kala, K., & Helander, I. M. (2000). Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane. Applied and Environmental Microbiology, 66, 2001–2005.

Ambalam, P. S., Prajapati, J. B., Dave, J. M., Baboo, M. N., Ljungh, Å., & Vyas, B. R. M. (2009). Isolation and characterization of antimicrobial proteins produced by a potential probiotic strain of human Lactobacillus rhamnosus 231 and its effect on selected human pathogens and food spoilage organisms. Microbial Ecology in Health and Disease, 21, 211–220.

Arcilla, M. S., Hattem, J. M., Haverkate, M. R., Bootsma, M. C. J., van Genderen, P. J. J., Goorhuis, A., Grobusch, M. P., Lashof, A. M. O., Molhoek, N., Schultsz, C., Stobberingh, E. E., Verbrugh, H. A., Jong, M. D., Melles, D. C., & Penders, J. (2017). Import and spread of extended-spectrum β-lactamase-producing Enterobacteriaceae by international travellers (COMBAT study): A prospective, multicentre cohort study. Lancet Infectious Diseases, 17(1), 78–85.

Badaoui Najjar, M., Kashtanov, D., & Chikindas, M. L. (2009). Natural Antimicrobials ε-poly-L-lysine and nisin A for control of oral microflora. Probiotics and Antimicrobial Proteins, 1(2), 143.

Bai, A., Weaver, M., Bao, F., Chan, E. D., & Bai, X. (2016). Saccharomyces boulardii produces a factor that inhibits Mycobacterium intracellular burden in human macrophages. Advances in Microbiology, 6(13), 965–974.

Bechinger, B., & Gorr, S. U. (2016). Antimicrobial peptides: Mechanisms of action and resistance. Journal of Dental Research, 96, 3.

Beyer, G., Hiemer-Bau, M., Ziege, S., Edlund, C., Lode, H., & Nord, C. E. (2000). Impact of moxifloxacin versus claritromycin on normal oropharyngeal microflora. European Journal of Clinical Microbiology and Infectious Diseases, 19(7), 548–550.

Brzozowski, B., Bednarski, W., & Gołek, P. (2011). The adhesive capability of two Lactobacillus strains and physicochemical properties of their synthesized biosurfactants. Food Technology and Biotechnology, 49, 177–186.

Buharin, O. V. (1999). Persistencija patogennyh bakterij [Persistence of pathogennic bacteria]. Medicina, Moscow (in Russian).

Daba, H., & Saidi, S. (2015). Detection of bacteriocin-producing lactic acid bacteria from milk in various farms in north-east Algeria by a new procedure. Agronomy Research, 13(4), 907–918.

Dorofeev, J. J., Koljado, E. V., Koljado, V. B., & Beskrovnaja, E. V. (2017). Dinamika smertnosti ot infekcionnyh i parazitarnyh zabolevanij v Altajskom krae [Dynamics of mortality from infectious and parasitic diseases in the Altai Territory]. Medicina v Kuzbasse, 16(4), 91–95 (in Russian).

Ebbensgaard, A., Mordhorst, H., Aarestrup, F. M., & Hansen, E. B. (2018). The role of outer membrane proteins and lipopolysaccharides for the sensitivity of Escherichia coli to antimicrobial peptides. Frontiers in Microbiology, 9, 2153.

El-Halfawy, O. M., & Valvano, M. A. (2013). Chemical communication of antibiotic resistance by a highly resistant subpopulation of bacterial cells. PLoS One, 8(7), e68874.

Forestier, C., Champs, C. D., Vatoux, C., & Joly, B. (2001). Probiotic activities of Lactobacillus casei rhamnosus: In vitro adherence to intestinal cells and antimicrobial properties. Research in Microbiology, 152, 167–173.

Frickmann, H. (2018). Influence of probiotic culture supernatants on in vitro biofilm formation of staphylococci. European Journal of Microbiology and Immunology, 2018, 38.

Gupta, R., & Srivastava, S. (2014). Antifungal effect of antimicrobial peptides (AMPs LR14) derived from Lactobacillus plantarum strain LR/14 and their applications in prevention of grain spoilage. Food Microbiology, 42, 1–7.

Isayenko, O. Y., Knysh, O. V., Babych, Y. M., Ryzhkova, T. N., & Dyukareva, G. I. (2019). Effect of disintegrates and metabolites of Lactobacillus rhamnosus and Saccharomyces boulardii on biofilms of antibiotic resistant conditionally pathogenic and pathogenic bacteria. Regulatory Mechanisms in Biosystems, 10(1), 3–8.

Jakovlev, S. A. (2017). Infekcionnye zabolevanija kak global'naja problema sovremennosti [Infectious diseases as a global problem of our time]. Territorija Nauki, 1, 20–25 (in Russian).

Kaktcham, P. M., Zambou, N. F., Fonteh, A. F., Sieladie, D. V., & Tchouanguep, M. F. (2011). Characterization of bacteriocin produced by Lactobacillus rhamnosus 1K isolated from traditionally fermented milk in the western highlands region of Cameroon. New York Science Journal, 4(8), 121–128.

Kraker, M. E. A., Stewardson, A. J., & Harbarth, S. (2016). Will 10 million people die a year due to antimicrobial resistance by 2050? PLoS Medicine, 13(11), e1002184.

Liévin-Le, M. V., & Servin, A. L. (2014). Anti-infective activities of Lactobacillus strains in the human intestinal microbiota: From probiotics to gastrointestinal anti-infectious biotherapeutic agents. Clinical Microbiology Reviews, 27(2), 167–199.

Lupo, A., Haenni, M., & Madec, J. Y. (2018). Antimicrobial resistance in Acinetobacter spp. and Pseudomonas spp. Microbiology Spectrum, 2018, 6, 3.

Malanovic, N., & Lohner, K. (2016). Gram-positive bacterial cell envelopes: The impact on the activity of antimicrobial peptides. Biochimica et Biophysica Acta, 1858(5), 936–946.

Mančušková, T., Medveďová, A., & Valík, Ľ. (2017). Antimicrobial potential of probiotics in combination with start er lactic acid bacteria. Scientia Agriculturae Bohemica, 48(4), 208–215.

Mirnejad, R., Vahdati, A. R., Rashidiani, J., Erfani, M., & Piranfar, V. (2013). The antimicrobial effect of Lactobacillus casei culture supernatant against multiple drug resistant clinical isolates of Shigella sonnei and Shigella flexneri in vitro. Iranian Red Crescent Medical Journal, 15(2), 122–126.

Mitsakakis, K., Kaman, W. E., Elshout, G., Specht, M., & Hays, J. P. (2018). Challenges in identifying antibiotic resistance targets for point-of-care diagnostics in general practice. Future Microbiology. 13(10), 1157–1164.

Nes, I. F., & Holo, H. (2000). Class II antimicrobial peptides from lactic acid bacteria. Biopolymers, 55(1), 50–61.

Nikaido, H. (2011). Structure and mechanism of RND-type multidrug efflux pumps. Enzymology and Related Areas of Molecular Biology, 77, 1–60.

Nomoto, K. (2005). Prevention of infections by probiotics. Journal of Bioscience and Bioengineering, 100(6), 583–592.

Oliveira, L. C., Silveira, A. M. M., Monteiro, A. S., Dos Santos, V. L., Nicoli, J. R., Azevedo, V. A. C., Soares, S. C., Dias-Souza, M. V., & Nardi R. M. D. (2017). In silico prediction, in vitro antibacterial spectrum, and physicochemical properties of a putative bacteriocin produced by Lactobacillus rhamnosus Strain L156.4. Frontiers in Microbiology, 8, 876.

Östholm, B. Å., Tärnberg, M., Nilsson, M., Nilsson, L. E., Hanberger, H., Hällgren, A., & Southeast Sweden Travel Study Group (2018). Duration of travel-associated faecal colonisation with ESBL-producing Enterobacteriaceae – A one year follow-up study. PLoS One, 13(10), e0205504.

Ponomarev, S. I., & Jakovlev, S. A. (2017). Infekcionnye zabolevanija kak mediko-socialnaja problema [Infectious diseases as a medical and social problem]. Sinergija, 1, 110–119 (in Russian).

Portella, A. C. F., Karp, S., Scheidt, G. N., Woiciechwski, A. L., Parada, J. L., & Soccol, C. R. (2009). Modelling antagonic effect of lactic acid bacteria supernatants on some pathogenic bacteria. Brazilian Archives of Biology and Technology, 52, 29–36.

Ramsamy, Y., Essack, S. Y., Sartorius, B., Patel, M., & Mlisana, K. P. (2018). Antibiotic resistance trends of ESKAPE pathogens in Kwazulu-Natal, South Africa: A five-year retrospective analysis. African Journal of Laboratory Medicine, 7(2), 887.

Richter, M. F., & Hergenrother, P. J. (2019). The challenge of converting Gram-positive-only compounds into broad-spectrum antibiotics. Annals of the New York Academy of Sciences, 1435(1), 18–38.

Sahib, F. H., Aldujaili, N. H., & Alrufae, M. M. (2017). Biosynthesis of silver nanoparticles using Saccharomyces boulardii and study their biological activities. European Journal of Pharmaceutical and Medical Research, 4(9), 65–74.

Sambanthamoorthy, K., Feng, X., Patel, R., Patel, S., & Paranavitana, C. (2014). Antimicrobial and antibiofilm potential of biosurfactants isolated from lactobacilli against multi-drug-resistant pathogens. BMC Microbiology, 14, 197.

Santajit, S., & Indrawattana, N. (2016). Mechanisms of antimicrobial resistance in ESKAPE pathogens. Biomed Research International, 2016, 2475067.

Sarika, A. R., Lipton, A. P., & Aishwarya, M. S. (2010). Bacteriocin production by a new isolate of Lactobacillus rhamnosus GP1 under different culture conditions. Food Science and Technology, 2(5), 291–297.

Stefania, D. M., Miranda, P., Diana, M., Claudia, Z., & Rita, P. (2017). Antibiofilm and antiadhesive activities of different synbiotics. Journal of Probiotics and Health, 5, 182.

Stier, H., & Bischoff, S. (2016). Influence of Saccharomyces boulardii CNCM I-745 on the gut-associated immune system. Clinical and Experimental Gastroenterology, 9, 269–279.

Tsutsui, А., & Suzuki, S. (2018). Japan nosocomial infections surveillance (JANIS): A model of sustainable national antimicrobial resistance surveillance based on hospital diagnostic microbiology laboratories. BMC Health Services Research, 18, 799.

Ursova, N. I. (2013). Aktualnye problemy i nereshennye problemy probiotikoterapii [Relevant problems and unsolved problems of probiotic therapy]. Lechashhij Vrach, 8, 60–65 (in Russian).

Wald, E. R. (2011). Acute otitis media and acute bacterial sinusitis. Clinical Infectious Diseases, 52(4), 277–283.

Wang, Z., Shen, Y., & Haapasalo, M. (2017). Antibiofilm peptides against oral biofilms. Journal of Oral Microbiology, 9(1), 1327308.

How to Cite
Isayenko, O. Y., Knysh, O. V., Kotsar, O. V., Ryzhkova, T. N., & Dyukareva, G. I. (2019). Evaluation of anti-microbial activity of filtrates of Lactobacillus rhamnosus and Saccharomyces boulardii against antibiotic-resistant gram-negative bacteria . Regulatory Mechanisms in Biosystems, 10(2), 245-250. https://doi.org/10.15421/021937

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