Identification and antibioticresistance of lactic acid bacteria isolated from the gastrointestinal tract of a rabbit
DOI:
https://doi.org/10.15587/2519-8025.2019.169077Keywords:
lactic acid bacteria, probiotics, rabbits, antibiotic resistance, identification, polymerase chain reactionAbstract
The probiotic preparations, used in rabbit breeding, contain bacteria, isolated from various econiches. Therefore, these drugs are universal and recommended for different types of animals. One of the ways to increase the effectiveness of probiotics, used in breeding rabbits is the selection of strains of biologically active representatives of the obligate microflora of the gastrointestinal tract of these animals.
Purpose. Identify lactic acid bacteria, isolated from the gastrointestinal tract of a rabbit, promising for the creation of probiotic preparations. Investigate antibiotic resistance of the most biologically active isolates.
Methods. The identification of lactic acid bacteria to the genus Lactobacillus was carried out according to generally accepted methods. Molecular genetic identification was performed using a polymerase chain reaction. For the most promising strains, the sensitivity to antibiotics was studied using the disco-diffusion method and the minimum inhibitory concentration of antibiotics, which completely retarded the growth of bacteria.
Results. 250 bacterial isolates, isolated from the gastrointestinal tract of rabbits were identified to the Lactobacillus genus, 10 most active of which were selected for the further work. It has been established, that 40% of selected isolates of lactic acid bacteria are capable of fermenting arabinose, dulcite; 50% beckoning; 70% sorbitol; 80% - mannose, fructose; 90% - lactose; 100% - galactose, maltose, sucrase, glucose; 20% - raffinose. Molecular genetic studies have established that the isolate Lactobacillus sp. 13/2 does not have genes typical for L. acidophilus and L. helveticus species. It has been found, that the lowest value of the minimum inhibitory concentration of antibiotics of the studied isolates was revealed to prolonged-action penicillins, which act bactericidally, disrupting the synthesis of the bacterial cell wall. The highest is for ampicillin, which is active against gram-positive bacteria that are affected by benzylpenicillin. The results indicate the absence of acquired antibiotic resistance. However, isolate L-13/2 showed resistance to oxacillin, kanamycin, streptomycin, nalidixic acid.
Findings. According to a complex of morphological, cultural, and physiological and biochemical properties, the ten most active isolates of lactic acid bacteria, isolated from the gastrointestinal tract of rabbits, of 250 ones are previously assigned to phylogenetic groups: L. acidophilus, L. lactis, L. casei, L. plantarum. For use the selected isolate Lactobacillus sp. 13/2 as a basis for the probiotic preparation, it is necessary to use additional identification methods to determine the species.
It has been established, that the studied isolates as a whole did not exhibit antibiotic resistance. Perspective isolate Lactobacillus sp. 13/2 was resistant to some antibiotics. Therefore, there is a need to study in greater detail the antibiotic resistance of the isolate under study in order to exclude the possibility of horizontal transfer of resistance genes
References
- Kovalchuk, I., Yashchuk, I. V. (2016). Suchasnyi stan ta perspektyvy rozvytku haluzi krolivnytstva v Ukraini [The modern state and prospects of development of industry of the rabbit breeding are in Ukraine]. Technology of production and processing of livestock products, 5, 24–29.
- Aksenov, Ye. O. (2016). Rozvitok krolivnictva v Ukrayini ta sviti [Development of rabbit meat in Ukraine and the world]. Scientific and technical bulletin, 116, 15–21.
- Stephen, W. B., Stephen, M. G., Dean, H. P. (2016). Pathology of laboratory rodents and rabbits. Chichester: John Wiley & Sons, 372.
- Antimicrobial resistance (2017). WHO. Available at: http://apps.who.int/gb/ebwha/pdf_files/wha70/a70_12-en.pdf
- WHO Global Strategy for the Containment of Antimicrobial Resistance (2001). WHO. Available at: https://www.who.int/drugresistance/WHO_Global_Strategy_English.pdf
- Khachatourians, G. G. (1998). Agricultural use of antibiotics and the evolution and transfer of antibiotic-resistant bacteria. Canadian Medical Association Journal, 159, 1129–1136.
- Kalachniuk, G. I. (1996). Probiotiki u tvarinnictvi [Probiotics in livestock breeding]. Animal husbandry of Ukraine, 5, 16–18.
- Veterinary informational and analytical resource of Ukraine. Available at: https://vet.in.ua/
- Bernbom, N., Norrung, B., Saadbye, P., Molbak, L., Vogensen, F. K., Licht, T. R. (2006). Comparison of methods and animal models commonly used for investigation of fecal microbiota: Effects of time, host and gender. Journal of Microbiological Methods, 66 (1), 87–95. doi: http://doi.org/10.1016/j.mimet.2005.10.014
- Tarakanov, B. V. (2006). Metodyi issledovaniya mikrofloryi pischevaritelnogo trakta selskohozyaystvennyih zhivotnyih i ptitsyi [Methods for studying the microflora of the digestive tract of farm animals and poultry]. Moscow: Nauchnyiy mir, 188.
- Pokhilko, Yu. M., Kravchenko, N. O., Bozhok, L. V., Ageev, V. O., Dmitruk, O. M. (2015). Osoblivosti kishkovogo mikrobocenozu molodnyaku kroliv za riznih tipiv godivli [Features of intestinal micrococoenosis of young rabbits for different types of feeding]. Agricultural Microbiology, 22, 48–52.
- Pohilko, Yu. M., Kravchenko, N. O. (2016). Vidilennya iz travnoyi sistemi kroliv molochnokislih bakterii, perspektivnih dlya stvorennya probiotichnih preparativ [Isolation from the digestive system of rabbits lactic acid bacteria, promising for the creation of probiotic preparations]. Bioresources and nature management, 8 (5-6), 63–66.
- Pohilko, Y. M., Kravchenko, N. O. (2017). Resistance of bacteria of the genus lactobacillus to the metabolites of the digestive system. Microbiology&Biotechnology, 2 (38), 101–111. doi: http://doi.org/10.18524/2307-4663.2017.2(38).105019
- Pohilko, Y. M., Kravchenko, N. O. (2018). Recovery and correction of the balance of microbiota of the gastrointestinal tract of rabbits, disabled as resulting from the use of antibiotics. Biological Resources and Nature Managment, 10 (3-4), 19–31. doi: http://doi.org/10.31548/bio2018.03.003
- Pohilko, Y. M., Kravchenko, N. O. (2018). Probiotic properties of bacteria of Lactobacillus genus isolated from the gastrointestinal tract of rabbits. Studia Biologica, 12 (1), 35–46. doi: http://doi.org/10.30970/sbi.1201.535
- De Vos, P., Garrity, G. M., Jones, D., Krieg, N. R., Ludwig, W., Rainey, F. A. et. al. (2009). Bergey’s manual of systematic bacteriology. Vol. 3. New York: Springer, 1422.
- Egorov, N. S. (1986). Basics of Antibiotic Teaching. Moscow: Vysshaia shkola, 448.
- Dec, M., Wernicki, A., Puchalski, A., Urban-Chmiel, R. (2015). Antibiotic susceptibility ofLactobacillusstrains isolated from domestic geese. British Poultry Science, 56 (4), 416–424. doi: http://doi.org/10.1080/00071668.2015.1058919
- Makarova, K., Slesarev, A., Wolf, Y., Sorokin, A., Mirkin, B., Koonin, E. et. al. (2006). Comparative genomics of the lactic acid bacteria. Proceedings of the National Academy of Sciences, 103 (42), 15611–15616. doi: http://doi.org/10.1073/pnas.0607117103
- Oren, A., Garrity, G. M. (2018). Notification that new names of prokaryotes, new combinations, and new taxonomic opinions have appeared in volume 68, part 1, of the IJSEM. International Journal of Systematic and Evolutionary Microbiology, 68 (4), 979–981. doi: http://doi.org/10.1099/ijsem.0.002597
- Marhamatizadeh, M. H., Sayyadi, S. (2019). Mining of lactic acid bacteria from traditional yogurt (Mast) of Iran for possible industrial probiotic use. Italian Journal of Animal Science, 18 (1), 663–667. doi: http://doi.org/10.1080/1828051x.2018.1552541
- Wang, Y., Ryu, B. H., Yoo, W., Lee, C. W., Kim, K. K., Lee, J. H., Kim, T. D. (2018). Identification, characterization, immobilization, and mutational analysis of a novel acetylesterase with industrial potential ( La AcE) from Lactobacillus acidophilus. Biochimica et Biophysica Acta (BBA) – General Subjects, 1862 (1), 197–210. doi: http://doi.org/10.1016/j.bbagen.2017.10.008
- Tamminen, M., Joutsjoki, T., Sjoblom, M., Joutsen, M., Palva, A., Ryhanen, E.-L., Joutsjoki, V. (2004). Screening of lactic acid bacteria from fermented vegetables by carbohydrate profiling and PCR-ELISA. Letters in Applied Microbiology, 39 (5), 439–444. doi: http://doi.org/10.1111/j.1472-765x.2004.01607.x
- Tilsala-Timisjärvi, A., Alatossava, T. (1997). Development of oligonucleotide primers from the 16S-23S rRNA intergenic sequences for identifying different dairy and probiotic lactic acid bacteria by PCR. International Journal of Food Microbiology, 35 (1), 49–56. doi: http://doi.org/10.1016/s0168-1605(97)88066-x
- Fortina, M. G., Ricci, G., Mora, D., Parini, C., Manachini, P. L. (2001). Specific identification ofLactobacillus helveticusby PCR withpepC,pepN andhtrA targeted primers. FEMS Microbiology Letters, 198 (1), 85–89. doi: http://doi.org/10.1111/j.1574-6968.2001.tb10623.x
- Ashraf, R., Shah, N. (2011). Antibiotic resistance of probiotic organisms and safety of probiotic dairy products. International Food Research Journal, 18 (3), 837–853.
- Hughes, P., Heritage, J. (2004). Antibiotic growth-promoters in food animals. FAO Animal Production and Health Paper, 129–152.
- Shenderov, B. A. (2001). Probiotiki i funktsionalnoye pitaniye [Probiotics and functional foods]. Moscow: Grants, 288.
- Charteris, W. P., Kelly, P. M., Morelli, L., Collins, J. K. (2001). Gradient Diffusion Antibiotic Susceptibility Testing of Potentially Probiotic Lactobacilli. Journal of Food Protection, 64 (12), 2007–2014. doi: http://doi.org/10.4315/0362-028x-64.12.2007
- Shah, N. P. (2000). Probiotic Bacteria: Selective Enumeration and Survival in Dairy Foods. Journal of Dairy Science, 83 (4), 894–907. doi: http://doi.org/10.3168/jds.s0022-0302(00)74953-8
- Nekrasova, L. S., Svyta, V. M., Hlushkevych, T. H., Tomchuk, V. V., Zherebko, N. M., Pokas, O. V. (Eds.) (2007). Vyznachennia chutlyvosti mikroorhanizmiv do antybakterialnykh preparative [Determination of the sensitivity of microorganisms to antibacterial drugs]. Kyiv: MOZ Ukrainy, 74.
Downloads
Published
How to Cite
Issue
Section
License
Copyright (c) 2019 Yury Pohilko, Natalya Kravchenko
This work is licensed under a Creative Commons Attribution 4.0 International License.
Our journal abides by the Creative Commons CC BY copyright rights and permissions for open access journals.
Authors, who are published in this journal, agree to the following conditions:
1. The authors reserve the right to authorship of the work and pass the first publication right of this work to the journal under the terms of a Creative Commons CC BY, which allows others to freely distribute the published research with the obligatory reference to the authors of the original work and the first publication of the work in this journal.
2. The authors have the right to conclude separate supplement agreements that relate to non-exclusive work distribution in the form in which it has been published by the journal (for example, to upload the work to the online storage of the journal or publish it as part of a monograph), provided that the reference to the first publication of the work in this journal is included.