Volume 4, Issue 3, September 2019, Page: 72-86
In Vitro Antimicrobial Characterization of Lactobacillus Isolates Towards Their Use as Probiotic Alternatives to Antibiotic Growth Promoters
Raoul Emeric Guetiya Wadoum, Department of Biochemistry, University of Dschang, Dschang, Cameroon
Fonteh Anyangwe Florence, Department of Animal Production, University of Dschang, Dschang, Cameroon
Kaktcham Pierre Marie, Department of Biochemistry, University of Dschang, Dschang, Cameroon
Ulrich Landry Bemmo Kamdem, Department of Biochemistry, University of Dschang, Dschang, Cameroon
Chancel Hector Momo Kenfack, Department of Biochemistry, University of Dschang, Dschang, Cameroon
Foko Kouam Edith-Marius, Department of Biochemistry, University of Dschang, Dschang, Cameroon
Nathalie Nzekwa, Evangelical, University of Cameroon, Bandjoun, Cameroun
Evina Horpa, Evangelical, University of Cameroon, Bandjoun, Cameroun
Vittorio Colizzi, Evangelical, University of Cameroon, Bandjoun, Cameroun
François Zambou Ngoufack, Department of Biochemistry, University of Dschang, Dschang, Cameroon
Received: Jun. 16, 2019;       Accepted: Jul. 16, 2019;       Published: Aug. 5, 2019
DOI: 10.11648/j.ijmb.20190403.13      View  22      Downloads  11
In the present study, the probiotic potential of Lactobacillus isolates selected from fecal samples of farmyard chickens and ducks was scientifically validated for their use as alternatives to antibiotics in poultry. A total of 129 Lactobacillus isolates were characterized of which four produced inhibitory substances with antimicrobial activities. They were further identified on the basis of their carbohydrate fermentation profile and High-Resolution Melting analysis as Lactobacillus paracasei MW-37CGZ, Lactobacillus paracasei MW-38CGZ, Lactobacillus plantarum MW-48CGZ and Lactobacillus plantarum MW-18CGZ. The obtained results revealed that L. plantarum MW-18CGZ and L. paracasei MW-37CGZ showed strong antagonistic activities against human (nine) and zoonotic pathogens (eleven). The antimicrobial substance produced by L. plantarum MW-18CGZ was found to be proteinaceous, thus indicating that this substance may belong to a group of potent antimicrobial peptides produced by some microorganisms including lactic acid bacteria (LAB). Both viable and non-viable cells of the four isolates demonstrated good hydrophobicity in xylene with L. plantarum MW-48CGZ exhibiting higher hydrophobicity than other isolates (77.64±5.18%). They were susceptible to chloramphenicol, clindamycin, ampicilin and erythromycin with Minimum Inhibitory Concentration (MIC) below cut-off values established by the European Food Safety Authority (EFSA). Among the four Lactobacillus, L. plantarum MW-18CGZ and L. paracasei MW-37CGZ displayed high autoaggregation and coaggregation towards pathogens and all isolates survived in low-pH, high bile salt concentrations and none exhibited virulent factors. According to the obtained results, L. plantarum MW-18CGZ and L. paracasei MW-37CGZ could be considered as future biotherapeutic substitutes for antibiotics to reduce antibiotic residues in food derived from poultry as well as the generation and spread of antibiotic resistance.
Lactobacillus, Probiotics, Antimicrobial Activity, Antibiotics Resistance, Public Health
To cite this article
Raoul Emeric Guetiya Wadoum, Fonteh Anyangwe Florence, Kaktcham Pierre Marie, Ulrich Landry Bemmo Kamdem, Chancel Hector Momo Kenfack, Foko Kouam Edith-Marius, Nathalie Nzekwa, Evina Horpa, Vittorio Colizzi, François Zambou Ngoufack, In Vitro Antimicrobial Characterization of Lactobacillus Isolates Towards Their Use as Probiotic Alternatives to Antibiotic Growth Promoters, International Journal of Microbiology and Biotechnology. Vol. 4, No. 3, 2019, pp. 72-86. doi: 10.11648/j.ijmb.20190403.13
Copyright © 2019 Authors retain the copyright of this article.
This article is an open access article distributed under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
G. Cheng, H. Hao, S. Xie, X. Wang, M. Dai, L. Huang and Z. Yuan, “Antibiotic alternatives: the substitution of antibiotics in animal husbandry”. Frontiers in Microbiology, no. 5, 2014, pp. 217.
V. Economou and P. Gousia, “Agriculture and food animals as a source of antimicrobial-resistant bacteria”. Infection and Drug Resistance, no. 8, 2015, pp. 49-61.
R. E. W. Guetiya, N. F. Zambou, F. F. Anyangwe, J. R. Njimou, M. M. Coman, M. C. Verdenelli, C. Cecchini, S. Silvi, C. Orpianesi, A. Cresci, and V. Colizzi, “Abusive use of antibiotics in poultry farming in Cameroon and the public health implications”. Bri Poultry Sci, no. 57, 2016, pp. 483-493.
FAO/WHO, “Guidelines for the evaluation of probiotics in food, Report of a Joint FAO/WHO Working Group on Drafting Guidelines for the Evaluation of Probiotics in Food”. Food and Agriculture Organization/World Health Organization http://ftp.fao.org/es/esn/food/wgreport2.pdf, 2002, (Accessed 26.01.18).
M. H. Helland, T. Wicklund and J. A. Narvhus, “Growth and metabolism of selected strains of probiotic bacteria in maize porridge with added malted barley”. International Journal of Food Microbiology, no. 91, 2004, pp. 305-313.
L. N. Tatsadjieu, K. S. Tanedjeu, and C. M. F. Mbofung, “Impact de l’utilisation des antibiotiques sur la sensibilité des bactéries pathogènes de poules dans la ville de Ngaoundéré”. Cameroon Journal of Experimental Biology, no. 5, 2009, pp. 52–61.
D. V. Sieladie, N. F. Zambou, P. M. Kaktcham, A. Cresci, and F. Fonteh, “Probiotic properties of Lactobacilli strains isolated from raw cow milk in the Western Highlands of Cameroon”. Innov Rom Food Biotechnol, no. 9, 2011, pp. 12-28.
P. M. Kaktcham, N. F. Zambou, F. M. Tchouanguep, M. El-Soda, and M. I. Choudhary, “Antimicrobial and Safety Properties of Lactobacilli Isolated from two Cameroonian Traditional Fermented Foods”. Scientia Pharmaceutica, no. 80, 2012, pp. 189-203.
P. M. Kaktcham, J. B. Temgoua, F. N. Zambou, G. Diaz-Ruiz, C. Wacher, and M. L. Pérez-Chabela, “In Vitro Evaluation of the Probiotic and Safety Properties of Bacteriocinogenic and Non-Bacteriocinogenic Lactic Acid Bacteria from the Intestines of Nile Tilapia and Common Carp for Their Use as Probiotics in Aquaculture”. Probiotics & Antimicro. Prot., no. 10, 2018, pp. 98-109.
C. Kilkenny, W. Browne, I. C. Cuthill, M. Emerson, and D. G. Altman, “Animal research: reporting in vivo experiments-the ARRIVE guidelines”. Journal of Cerebral Blood Flow & Metabolism, no. 31, 2011, pp. 991–993.
P. Shokryazdan, C. Sieo, R. Kalavathy, J. Liang, N. Alitheen, Jahromi, and Y. Ho, “Probiotic Potential of Lactobacillus Strains with Antimicrobial Activity against Some Human Pathogenic Strains”. BioMed Research International, 2014, pp. 16.
D. Pelinescu, E. Sasarman, M. Chifiriuc, I. Stoica, A. Nohita, I. Avram, F. Serbancea, and T. Dimov, “Isolation and identification of some Lactobacillus and Enterococcus strains by a polyphasic taxonomical approach”. Romanian Biotechnological Letters, no. 14, 2009, pp. 4225-423.
N. F. Zambou, P. M. Kaktcham, F. F. Anyangwe, R. E. W. Guetiya and D. V. Sieladie, “Effects of Inclusion of Two Probiotic Strains Isolated From “Sha’a”, a Maize-Based Traditionally Fermented Beverage on Lipid Metabolism of Rabbits fed a Cholesterol-Enriched Diet”. International Journal of Animal and Veterinary Advances, no. 5, 2013, pp. 87-97.
U. Schillinger, C. Guigas, and W. Holzapfel, “In vitro adherence and other properties of Lactobacilli used in probiotic yoghurt like products”. International Dairy Journal, no. 15, 2005, pp. 1289–1297.
J. Mathara, U. Schillinger, C. Guigas, C. Franz, P. Kutima, S. Mbugua, H. Shin, and Holzapfel, “Functional characteristics of Lactobacillus spp. from traditional Maasai fermented milk products in Kenya”. International Journal of Food Microbiology, no. 126 2008, pp. 57–64.
M. PM. Collado, J. Meriluot, and S. Salminen, “Adhesion and aggregation properties of probiotic and pathogen strains”. Eur Food Res Technol, no. 226, 2008, pp. 1065–1073.
R. Reniero, P. Cocconcelli, V. Bottazzi, and L. Morelli, “High frequency of conjugation in Lactobacillus mediated by an aggregation-promoting factor”. J Gen Microbiol, no. 138, 1992, pp. 763–768.
P. Handley, D. Harty, J. Wyatt, C. Brown, J. Doran, and A. Gibbs, “A comparison of the adhesion, coaggregation and cell-surface hydrophobicity properties of fibrillar and fimbriate strains of Streptococcus salivarius”. J Gen Microbiol, no. 133, 1987, pp. 3207–3217.
R. Toure, E. Kheadr, C. Lacroix, O. Moroni, and I. Fliss, “Production of antibacterial substances by bifidobacterial isolates from infant stool active against Listeria monocytogenes”. Journal of Applied Microbiology, no. 95, 2003, pp. 1058–1069.
EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), G. Rychen, G. Aquilina, G. Azimonti, V. Bampidis, M. L. Bastos, G. Bories, A. Chesson, P. Cocconcelli, G. Flachowsky, J. Gropp, B. Kolar, M. Kouba, M. Lopez-Alonso, S. Lopez, A. Mantovani, B. Mayo, F. Ramos, M. Saarela, R. Villa, R. Wallace, P. Wester, B. Glandorf, L. Herman, S. Karenlampi, J. Aguilera, M. Anguita, R. Brozzi, and J. Galobart, “Guidance on the characterisation of microorganisms used as feed additives or as production organisms”. EFSA Journal, no. 16, 2018, pp. 5206. https://doi.org/10.2903/j.efsa.
P. Gerhardt, R. Murray, R. Costilow, E. Nester, W. Wood, N. Krieg, and G. Phillips, “Manual of methods for general bacteriology”. American society for Microbilogy, 1981.
W. F. Harrigan, and M. McCance, “Laboratory Methods”. Food and Dairy Microbiology, 1976, pp. 12-15.
X. Guo, J. Kim, H. Nam, S. Park, and M. Kim, “Screening lactic acid bacteria from swine origins for multistrain probiotics based on in vitro functional properties”. Anaerobe, no. 16, 2010, pp. 321-326.
D. H. Tambekar, and S. A. Bhutada, (2010) “An evaluation of probiotic potential of Lactobacillus sp. from milk of domestic animals and commercially available probiotic preparations in prevention of enteric bacterial infections”. Recent Res. Sci. Technol, no. 2, 2010, pp. 82–88.
The FAO/OIE/WHO Collaboration - A Tripartite Concept Note. “Sharing responsibilities and coordinating global activities to address health risks at the animal-human-ecosystems interfaces” .https://www.who.int/foodsafety/zoonoses/final_concept_note_Hanoi.pdf, ( Accessed 06.03.19).
Suskovic J, Blazenka K, Beganovi J, Pavunc AL, Habjani K, Matosic S (2010) Antimicrobial activity-the most important property of probiotic and starter lactic acid bacteria. Food Technology and Biotechnology 48: 296–307.
Centers for Disease Control and Prevention, “Salmonella surveillance: annual tabulation summaries”. Available at: https://www.cdc.gov/nationalsurveillance/salmonella-surveillance.html, (Accessed 14.01.18).
A. Abdel-Daim, N. Hassouna, M. Hafez, M. Ashor, and M. Aboulwafa, “Antagonistic Activity of Lactobacillus Isolates against Salmonella typhi In Vitro”. BioMed Research International, 2013, pp. 12.
D. Saulnier, J. Spinler, G. Gibson, and J. Versalovic, “Mechanisms of probiosis and prebiosis: considerations for enhanced functional foods”. Current Opinion in Biotechnology no. 20, 2009, pp. 135–141.
P. Li, Q. Gu, and Q. Zhou, “Complete genome sequence of Lactobacillus plantarum LZ206, a potential probiotic strain with antimicrobial activity against food-borne pathogenic microorganisms”. Journal of Biotechnology, no. 238, 2016, pp. 52-55.
M. P. Zacharof, and Lovitt RW, “Bacteriocins produced by lactic acid bacteria: A review article”. APCBEE Procedia, no. 2, 2012, pp. 50-56.
L. Poppi, J. Rivaldi, T. Coutinho, C. Astolfi-Ferreira, F. Piantino, and I. Mancilha, “Effect of Lactobacillus sp. isolates supernatant on Escherichia coli O157: H7 enhances the role of organic acids production as a factor for pathogen control”. Pesquisa Veterinária Brasileira, no. 35, 2015, pp. 353-359.
R. Wasfi, O. Abd El‐Rahman, M. Zafer, and H. Ashour, “Probiotic Lactobacillus sp. inhibit growth, biofilm formation and gene expression of caries‐inducing Streptococcus mutans”. Journal of Cellular and Molecular Medicine, no. 22, 2018, pp. 1972-1983.
P. Koll, R. Mandar, H. Marcotte, E. Leibur, M. Mikelsaar, and L. Hammarstrom, “Characterization of oral lactobacilli as potential probiotics for oral health”. Oral Microbiology and Immunology, no. 23, 2008, pp. 139-147.
M. Ehrmann, P. Kurzak, J. Bauer, and R. Vogel, “Characterization of lactobacilli towards their use as probiotic adjuncts in poultry”. Journal of Applied Microbiology, no. 92, 2002, pp. 966-975.
K. Mourad, and K. Nour-Eddine, “In vitro preselection criteria for probiotic Lactobacillus plantarum strains of fermented olives origin. Int. J. Probiot. Prebiot, no. 1, 2006, pp. 27-32.
W. P. Charteris, P. M. Kelly, L. Morelli, and Collins, “Development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species in the upper human gastrointestinal tract”. Journal of Applied Microbiology, no. 84, 1998, pp. 759-768.
R. Campana, S. van Hemert, and W. Baffone, “Strain-specific probiotic properties of lactic acid bacteria and their interference with human intestinal pathogens invasion”. Gut Pathogens, no. 9, 2017, pp. 12.
M. Chen, H. Tang, and C. Chiang, “Effects of heat, cold, acid and bile salt adaptations on the stress tolerance and protein expression of kefir-isolated probiotic Lactobacillus kefiranofaciens M1”. Food Microbiol, no. 66, 2017, pp. 20-27.
M. Shehata, S. El Sohaimy, M. El-Sahn, and M. Youssef, “Screening of isolated potential probiotic lactic acid bacteria for cholesterol lowering property and bile salt hydrolase activity”. Annals of Agricultural Sciences, no. 61, 2016, pp. 65-75.
F. Armas, C. Camperio, and C. Marianelli, “In Vitro Assessment of the Probiotic Potential of Lactococcus lactis LMG 7930 against Ruminant Mastitis-Causing Pathogens”. PLoS ONE, no. 12, 2017, pp. 125-129.
F. Hamadi, H. Latrache, A. El Ghmari, M. Ellouali, M. Mabrrouki, and N. Kouider, “Effect of pH and ionic strength on hydrophobicity and electron donor and acceptor characteristics of Escherichia coli and Staphylococcus aureus”. Ann Microbiol, no. 54, 2004, pp. 213-225.
B. Del Re, B. Sgorbati, M. Miglioli, and D. Palenzona, “Adhesion, autoaggregation and hydrophobicity of 13 strains of Bifidobacterium longum”. Lett Appl Microbiol, no. 31, 2000, pp. 438–442.
M. Espeche, M. Otero, F. Sesma, and M. Nader-Macias, “Screening of surface properties and antagonistic substances production by lactic acid bacteria isolated from the mammary gland of healthy and mastitic cows”. Vet Microbiol, no. 135, 2009, pp. 346-357.
M. Espeche, M. Pellegrino, I. Frola, A. Larriestra, C. Bogni, and M. Nader-Macías, “Lactic acid bacteria from raw milk as potentially beneficial strains to prevent bovine mastitis”. Anaerobe, no. 18, 2012, pp. 103-109.
H. Xu, S. Jeong, Y. Lee, and J. Ahn, “Assessment of cell surface properties and adhesion potential of selected probiotic strains”. Lett Appl Microbiol, no. 49, 2009, pp. 434-442.
M. Ammor, A. Flórez, A. van Hoek, C. de Los Reyes-Gavilán, H. Aarts, A. Margolles, and B. Mayo, “Molecular characterization of intrinsic and acquired antibiotic resistance in lactic acid bacteria and bifidobacterial”. J Mol Microbiol Biotechnol, no. 14, 2008, pp. 6-15.
S. Ammor, B. Florez, and B. Mayo, “Antibiotic resistance in non-enterococcal lactic acid bacteria and bifidobacterial”. Food Microbiol, no. 24, 2007, pp. 559-570.
O. Cataloluk, and B. Gogebakan, “Presence of drug resistance in intestinal lactobacilli of dairy and human origin in Turkey”. FEMS Microbiol Lett, no. 236, 2004, pp. 7-12.
C. Lin, Z. Fung, L. Wu, and T. Chung, “Molecular characterization of a plasmid-borne (pTC82) chloramphenicol resistance determinant (cat-TC) from Lactobacillus reuteri G4”. Plasmid, no. 36, 1996, pp. 116-124.
M. Feichtinger, S. Mayrhofer, W. Kneifel, and J. Konrad, “Tetracycline Resistance Patterns of Lactobacillus buchneri Group Strains”. Journal of Food Protection, no. 79, 2016, pp. 1741-1747.
A. Pavunc, B. Kos, J. Beganović, K. Uroić, D. Bučan, and J. Šušković, “Antibiotic susceptibility and antimicrobial activity of autochthonous starter cultures as safety parameters for fresh cheese production”. Mljekarstvo časopis za unaprjeđenje proizvodnje i prerade mlijeka, no. 63, 2013, pp. 34-39.
S. Mayrhofer, K. Domig, C. Mair, U. Zitz, G. Huys, and W, Kneifel, “Comparison of broth microdilution, Etest, and agar disk diffusion methods for antimicrobial susceptibility testing of Lactobacillus acidophilus group members”. Applied and Environmental Microbiology, no. 74, 2008, pp. 3745-3748.
J. Korhonen, A. van Hoek, and Saarela, “Antimicrobial susceptibility of Lactobacillus rhamnosus”. Beneficial Microbes, no. 1, 2010, pp. 75-80.
P. Thakkar, H. Modi, and J. Prajapati, “Isolation, characterization and safety assessment of lactic acid bacterial isolates from fermented food products”. Int. J. Curr. Microbiol. App. Sci, no. 4, 2015, pp. 713-725.
E. Könönen, and W. Wade, “Propionibacterium, Lactobacillus, Actinomyces, and Other Non-Spore-Forming Anaerobic Gram-Positive Rods”. Clinical Microbiology, no. 9, 2007, pp. 872-888.
M. Boyd, M. Antonio, and S. Hillier, “Comparison of API 50 CH strips to whole-chromosomal DNA probes for identification of Lactobacillus species”. J Clin Microbiol, no. 43, 2005, pp. 5309–5311.
E. Nagy, M. Petterson, and P. Mardh, “Antibiosis between bacteria isolated from the vagina of women with and without signs of bacterial vaginosis”. Apmis, no. 99, 1991, pp. 739-744.
M. Alvarez-Olmos, M. Barousse, L. Rajan, B. Van Der Pol, D. Fortenberry, D. Orr, and P. Fidel, “Vaginal lactobacilli in adolescents - presence and relationship to local and systemic immunity, and to bacterial vaginosis”. Sex Transm Dis, no. 31, 2004, pp. 393-400.
N. Gautam, and S. Sharma, “Characterization of bacteriocin producer Lactobacillus brevis as potential probiotic strain”. J. Microbiol. Biotechnol. Food Sci, no. 5, 2015, pp. 216-220.
S. Handa, and N. Sharma, “Evaluation of health benefits of lassi (Buttermilk): A traditional non-alcoholic beverage of northern India”. J. Innov. Biol, no. 3, 2016, pp. 297–301.
N. Sharma, A. Gupta, and S. Handa, “An exploration of rich microbial diversity of rare traditional functional foods of Trans Himalayan state of India with proven additional probiotic effect”. Int. J. Curr. Microbiol. Appl. Sci, no. 3, 2014, pp. 99-1014.
Browse journals by subject