The effect of Lactobacillus reuteri cell free supernatant on growth and biofilm formation of Paenibacillus larvae

Document Type : Full paper (Original article)

Authors

1 Ph.D. Student in Microbiology, Department of Microbiology, Faculty of Biological Sciences, Alzahra University, Vanak Village, Tehran, Iran

2 Department of Microbiology, Faculty of Biological Sciences, Alzahra University, Vanak Village, Tehran, Iran

3 Nuclear Agricultural Research School, Nuclear Science and Technology Research Institute, Karaj, Iran

4 Department of Biotechnology, Faculty of Biological Sciences, Alzahra University, Vanak Village, Tehran, Iran

Abstract

Background: Paenibacillus larvae is the etiological agent of American foulbrood (AFB) disease, the most lethal disease in honeybee (Apis mellifera) larvae. Aims: The aim of the present work was to study the antimicrobial effect of cell free supernatant (CFS) of probiotics on an Iranian isolate’s biofilm formation. Methods: A local strain was identified by 16S rRNA sequencing. The antibacterial effect of some probiotics was evaluated through drop plate method, minimum inhibitory concentrations (MIC), minimum bactericidal concentrations (MBC) and time-kill assay. The biofilm formation ability of P. larvae and the inhibition of biofilm formation by CFS were studied by microplate and scanning electron microscopy (SEM). The nature of the secondary metabolites in CFS was examined by microscale optical density assay (MODA). Results: Alignment of the results of P. larvae KB10 (GenBank accession number MH000685.1) 16S rRNA with the database revealed more than 97% identity with P. larvae. The most antibacterial effect was observed in the CFS of Lactobacillus reuteri ATCC23272 with 12.75 ± 3.2 mm for zone of inhibition (ZOI) at 1000 µL/ml for MIC and MBC. Time-kill assay revealed that CFS eliminated 1.5 × 108 CFU/ml P. larvae KB10 at 2 h of exposure. Microtitre plate and SEM results revealed that CFS (at sub-MIC concentration) was able to inhibit biofilm formation by P. larvae. The results of MODA assay showed that antimicrobial activity were related to the production of organic acids. Conclusion: Cell free supernatant from L. reuteri ATCC 23272 had inhibitory effects on P. larvae KB10 growth and biofilm production due to its acidic nature. The obtained results can be used for antibiotic substitution in AFB control and treatment.

Keywords

References

Alippi, AM; López, AC; Reynaldi, FJ; Grasso, DH and Aguilar, OM (2007). Evidence for plasmid-mediated tetracycline resistance in Paenibacillus larvae, the causal agent of American Foulbrood (AFB) disease in honeybees. Vet. Microbiol., 125: 290-303. http://doi.org/10.1016/j. vetmic.2007.05.018.
Ayoub, ZN; Saeed, AY and Vandame, J (2013). Detection of American foulbrood disease in the apiaries of Duhok province, Kurdistan region, Iraq. IOSR-J. Agri. Vet. Sci., 6: 18-21.
Bajpai, VK; Han, JH; Rather, IA; Park, C; Lim, J; Paek, WK; Lee, JS; Yoon, JI and Park, YH (2016). Characterization and antibacterial potential of lactic acid bacterium Pediococcus pentosaceus 4I1 isolated from freshwater fish Zacco koreanus. Front. Microbiol., 7: 1-15. http://doi.org/10.3389/fmicb.2016.02037.
Balouiri, M; Sadiki, M and Ibnsouda, SK (2016). Methods for in vitro evaluating antimicrobial activity: a review. J. Pharmaceut. Anal., 6: 71-79. http://doi.org/10.1016/j.jpha. 2015.11.005.
Cai, W; De La Fuente, L and Arias, CR (2013). Biofilm formation by the fish pathogen flavobacterium columnare: development and parameters affecting surface attachment. Appl. Environ. Microbiol., 79: 5633-5642. http://doi.org/ 10.1128/AEM.01192-13.
Chen, L; Bromberger, PD; Nieuwenhuiys, G and Hatti-Kaul, R (2016). Redox balance in Lactobacillus reuteri DSM20016: roles of iron-dependent alcohol dehydrogenases in glucose/glycerol metabolism. PLoS One. 11: 1-20. http://doi.org/10.1371/journal.pone. 0168107.
Cleusix, V; Lacroix, C; Vollenweider, S; Duboux, M and Le Blay, G (2007). Inhibitory activity spectrum of reuterin produced by Lactobacillus reuteri against intestinal bacteria. BMC Microbiol., 7: 1-9. http://doi.org/10.1186/ 1471-2180-7-101.
Cornman, RS; Lopez, D and Evans, JD (2013). Transcriptional response of honey bee larvae infected with the bacterial pathogen Paenibacillus larvae. PloS One. 8: e65424. http://doi.org/10.1371/journal.pone.0065424.
Dasari, S; Shouri, RND; Wudayagiri, R and Valluru, L (2014). Antimicrobial activity of Lactobacillus against microbial flora of cervicovaginal infections. Asian Pac. J. Trop. Dis., 4: 18-24. http://doi.org/10.1016/S2222-1808 (14)60307-8.
Dhanani, AS and Bagchi, T (2013). Lactobacillus plantarum CS24.2 prevents Escherichia coli adhesion to HT-29 cells and also down-regulates enteropathogen-induced tumor necrosis factor-α and interleukin-8 expression. Microbiol. Immunol., 57: 309-315. http://doi.org/10.1111/1348-0421. 12038.
Dingman, DW and Stahly, DP (1983). Medium promoting sporulation of Bacillus larvae and metabolism of medium components. Appl. Environ. Microbiol., 46: 860-869.
Fang, K; Jin, X and Hong, SH (2018). Probiotic Escherichia coli inhibits biofilm formation of pathogenic E. coli via extracellular activity of DegP. Sci. Rep., 8: 1-12. http://doi. org/10.1038/s41598-018-23180-1.
Garrett, TR; Bhakoo, M and Zhang, Z (2008). Bacterial adhesion and biofilms on surfaces. Prog. Nat. Sci. Mater., 18: 1049-1056. http://doi.org/10.1016/j.pnsc.2008.04.001.
Gómez, NC; Ramiro, JMP; Quecan, BXV and de Melo Franco, BDG (2016). Use of potential probiotic lactic acid bacteria (LAB) biofilms for the control of Listeria monocytogenes, Salmonella typhimurium, and Escherichia coli O157:H7 biofilms formation. Front. Microbiol., 7: 1-15. http://doi.org/10.3389/fmicb.2016.00863.
Hamdi, C; Essanaa, J; Sansonno, L; Crotti, E; Abdi, K; Barbouche, N; Balloi, A; Gonella, E; Alma, A; Daffonchio, D; Boudabous, A and Cherif, A (2013). Genetic and biochemical diversity of Paenibacillus larvae isolated from tunisian infected honey bee broods. BioMed. Res. Int., 2013: 479893. http://doi.org/10.1155/2013/ 479893.
Jaouani, I; Abbassi, MS; Alessandria, V; Bouraoui, J; Ben Salem, R; Kilani, H; Mansouri, R; Messadi, L and Cocolin, L (2014). High inhibition of Paenibacillus larvae and Listeria monocytogenes by Enterococcus isolated from different sources in tunisia and identification of their bacteriocin genes. Lett. Appl. Microbio., 59: 17-25. http://doi.org/10.1111/lam.12239.
Joo, HS and Otto, M (2012). Molecular basis of in vivo biofilm formation by bacterial pathogens. Chem. Biol., 19: 1503-1513. http://doi.org/10.1016/j.chembiol.2012.10.022.
Lash, BW; Tami, H and Gouama, H (2005). Detection and partial charachterization of a broad range bacteriocin produced by Lactobacillus plantarm ATCC8014. Food Microbiol., 22: 199-204. http://doi.org/10.1016 J.FM.2004. 03.006.
Mao, DP; Zhou, Q; Chen, CY and Quan, ZX (2012). Coverage evaluation of universal bacterial primers using the metagenomic datasets. BMC Microbiol., 12: 66. http://doi.org/10.1186/1471-2180-12-66.
Morita, H; Toh, H and Fukuda, S (2008). Comparative genome analysis of Lactobacillus reuteri and Lactobacillus fermentum reveal a genomic Island for Reuterin and Cobalamin producion. DNA Res., 15: 151-161. http://doi. org/10.1093/dnares/dsn009.
Mudroňová, D; Rumanovská, K; Toporčák, J; Nemcová, R; Gancarčíková, S and Hajdučková, V (2011). Selection of probiotic Lactobacilli designed for the prevention of American Foubrood. Folia Vet., 4: 127-132.
Pehrson, M; Mancilha, IM and Pereira, C (2015). Antimicrobial activity of probiotic Lactobacillus strains towards gram-negative enteropathogens. Eur. Inte. J. Sci.
Technol., 4: 136-149.
Poppinga, L; Janesch, B; Fünfhaus, A; Sekot, G; Garcia-Gonzalez, E; Hertlein, G; Hedtke, K and Schäffer, CGE (2012). Identification and functional analysis of the S-layer protein SplA of Paenibacillus larvae, the causative agent of American Foulbrood of honey bees. PLoS Path., 8. http://doi.org/10.1371/journal.ppat.1002716.
Vahedi Shahandashti, R; Kasra Kermanshahi, R and Ghadam, P (2016). The inhibitory effect of bacteriocin produced by Lactobacillus acidophilus ATCC 4356 and Lactobacillus plantarum ATCC 8014 on planktonic cells and biofilms of Serratia marcescens. Turkish J. Med. Sci., 46: 1188-1196. http://doi.org/10.3906/sag-1505-51.
Wei, Q and Ma, LZ (2013). Biofilm matrix and its regulation in Pseudomonas aeruginosa. Int. J. Mol. Sci., 14: 20983-21005. http://doi.org/10.3390/ijms141020983.
Wojnicz, D and Tichaczek-Goska, D (2013). Effect of sub-minimum inhibitory concentrations of ciprofloxacin, amikacin and colistin on biofilm formation and virulence factors of Escherichia coli planktonic and biofilm forms isolated from human urine. Brazilian J. Microbiol., 44: 259-265. http://doi.org/10.1590/S1517-83822013000100037.
Zamani, H; Rahdar, S; Garakoui, SR; Afsah Sahebi, A and Jafari, H (2017). Antibiofilm potential of Lactobacillus plantarum spp. cell free supernatant (CFS) against multidrug resistant bacterial pathogens. Pharmac. Biomed. Res., 3: 39-44.

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