Assessment of antibiotic resistance patterns in two potential probiotic bacteria, Lactococcus lactis subsp. cremoris NABRII64 and Lactococcus lactis subsp. cremoris NABRII66 isolated from rainbow trout (Oncorhynchus mykiss) intestine

Document Type : Research Paper

Authors

1 M.Sc. in Aquaculture, Fisheries Department, Faculty of Natural Resources, University of Guilan, Sowmeh Sara, Iran

2 Assistant Professor in North Region Branch, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education and Extension Organization (AREEO), Rasht, Iran

3 Technician in Genomics Department, North Region Branch, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education and Extension Organization (AREEO), Rasht, Iran

4 Assistant Professor in Fisheries Department, Faculty of Natural Resources, University of Guilan, Sowmeh Sara, Iran

Abstract

This study aimed to investigate the phenotypic and genotypic patterns of antibiotic resistance in two potential probiotic lactic acid bacteria, Lactococcus lactis subsp. cremoris (NABRII64 and NABRII66) isolated from the rainbow trout intestine. The phenotypic susceptibility pattern of the strains was studied based on the Minimum Inhibitory Concentration (MIC) of eight most commonly used antibiotics in medicine and veterinary including ampicillin, kanamycin, gentamicin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol. After comparing the MICs with standard values recommended by European Food Safety Authority (EFSA), the nature of phenotypic resistance observed in bacterial strains was investigated by polymerase chain reaction (PCR) through plasmid DNA extraction. The results of the phenotypic evaluation indicated the tetracycline resistance in both bacterial strains (MIC<4mg/L). Genotyping of antibiotic resistance genes including tet (S), tet (L), tet (M), tet (O), tet (W) and tet (K) indicated the presence of tet (S) and tet (M) resistance genes in plasmid DNA of both bacterial strains. These results exhibited the acquired resistance and the presence of two tetracycline resistance genes in the plasmid DNA of two bacterial strains, NABRII64 and NABRII66. However, further studies are required to understand the nature of the acquired resistance mechanism in the future.

Keywords


مرتضائی ف.، رویان م.، علاف نویریان ح. و باباخانی آ. 1397. بررسی پتانسیل پروبیوتیکی باکتری‌های اسید لاکتیک جداسازی شده از روده ماهی قزل‌آلای رنگین‌کمان (Oncorhynchus mykiss, Walbaum) علیه باکتری Streptococcus iniae. مجله علوم آبزی‌پروری، 6(3): 54-43.
 Aarestrup F.M., Agerso Y., Gerner-Smidt P., Madsen M. and Jensen L.B. 2000. Comparison of antimicrobial resistance phenotypes and resistance genes in Enterococcus faecalis and Enterococcus faecium from humans in the community, broilers, and pigs in Denmark. Diagnostic Microbiology and Infectious Disease, 37: 127–37.
Akinbowale O.L., Peng H. and Barton M.D. 2007. Diversity of tetracycline resistance genes in bacteria from aquaculture sources in Australia. Journal of Applied Microbiology, 103: 2016 –2025.
Alonso A., Sanchez P. and Martinez J.L. 2001. Environmental selection of antibiotic resistance genes. Environmental Microbiology, 3: 1–9.
Aminov R.I., Garrigues-Jeanjean N. and Mackie R.I. 2001. Molecular ecology of tetracycline resistance: Development and validation of primers for detection of tetracycline resistance genes encoding ribosomal protection proteins. Applied and Environmental Microbiology, 67: 22–32.
Ammor M.S., Florez A.B., Van Hoek A.H., Clara G., Aarts H.J., Margolles A. and Mayo B. 2008. Molecular characterization of intrinsic and acquired antibiotic resistance in lactic acid bacteria and bifidobacteria. Journal of Molecular Microbiology and Biotechnology, 14: 6–15.
Araujo C., Munoz-Atienza E., Ramirez M., Poeta P., Igrejas G., Hernandez P.E., Herranz C. and Cintas L.M. 2015. Safety assessment, genetic relatedness and bacteriocin activity of potential probiotic Lactococcus lactis strains from rainbow trout (Oncorhynchus mykiss, Walbaum) and rearing environment. European Food Research and Technology, 241: 647–662.
Asfie M., Yoshijima T. and Sugita H. 2003. Characterization of the goldfish fecal microflora by the fluorescent in situ hybridization method. Fisheries Research, 69: 21–26.
Beaber J.W., Hochhut B. and Waldor M.K. 2004. SOS response promotes horizontal dissemination of antibiotic resistance genes. Nature, 427: 72–74.
Cai Y., Suyanandana P., Saman P. and Benno Y. 1999. Classification and characterization of lactic acid bacteria isolated from the intestines of common carp and freshwater prawns. Journal of General and Applied Microbiology, 45: 177–184.
Chopra I. and Roberts M. 2001. Tetracycline antibiotics: Mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiology and Molecular Biology Reviews, 65: 232–260.
Clewell D.B., Flannagan S.E. and Jaworski D.D. 1995. Unconstrained bacterial promiscuity: The Tn916-Tn1545 family of conjugative transposons. Trends in Microbiology, 3: 229–236.
CLSI 2005. Performance standards for antimicrobial susceptibility testing. 15th Informational Supplement, CLSI/NCCLS M100-S15. CLSI, Wayne, Pa, from https://clsi.org/standards/products/microbiology/documents/m100.
D'costa V.M., McGrann K.M., Hughes D.W. and Wright G.D. 2006. Sampling the antibiotic resistome. Science, 311: 374–377.
De Vuyst L. and Leroy F. 2007. Bacteriocins from lactic acid bacteria: Production, purification and food applications. Journal of Molecular Microbiology and Biotechnology, 13: 194–199.
Devirgiliis C., Barile S. and Perozzi G. 2011. Antibiotic resistance determinants in the interplay between food and gut microbiota. Genes and Nutrition, 6: 275–284.
EFSA 2012. Panel on additives and products or substances used in animal feed, guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance. EFSA Journal, 10: 1–10.
FEEDAP. 2008. EFSA panel on additives and products or substances used in animal feed technical guidance on the update of the criteria used in the assessment of bacterial resistance to antibiotics of human or veterinary importance. EFSA Journal, 732: 1–15.
Fajardo A. and Martinez J.L. 2008. Antibiotics as signals that trigger specific bacterial responses. Current Opinion in Microbiology, 11: 161–167.
FAO/WHO 2006. Probiotics in Food. Health and Nutritional Properties and Guidelines for Evaluation, in FAO Food and Nutrition Paper, Italy. 56P.
Feliciello I. and Chinali G. 1993. A modified alkaline lysis method for the preparation of highly purified plasmid DNA from Escherichia coli. Analytical Biochemistry, 212: 394–401.
Florez A.B., Danielsen M., Korhonen J., Zycka J., Von Wright A., Bardowski J. and Mayo B. 2007. Antibiotic survey of Lactococcus lactis strains to six antibiotics by Etest, and establishment of new susceptibility-resistance cut-off values. Journal of Dairy Research, 74: 262–268.
Guglielmetti E., Korhonen J.M. Heikkinen J., Morelli L. and Von Wright A. 2009. Transfer of plasmid-mediated resistance to tetracycline in pathogenic bacteria from fish and aquaculture environments. FEMS Microbiology Letters, 293: 28–34.
Kim S.R., Nonaka L. and Suzuki S. 2004. Occurrence of tetracycline resistance genes tet (M) and tet (S) in bacteria from marine aquaculture sites. FEMS Microbiology Letters, 237: 147–156.
Klare I., Konstabel C., Muller-Bertling S., Reissbrodt R., Huys G., Vancanneyt M., Swings J., Goossens H. and Witte W. 2005. Evaluation of new broth media for microdilution antibiotic susceptibility testing of Lactobacilli, Pediococci, Lactococci, and Bifidobacteria. Applied Environmental Microbiology, 71: 8982–8986.
Lampkowska J., Feld L., Monaghan A., Toomey N., Schjorring S., Jacobsen B., Van Der Voet H., Andersen S.R., Bolton D., Aarts H. and Krogfelt K.A. 2008. A standardized conjugation protocol to asses antibiotic resistance transfer between lactococcal species. International Journal of Food Microbiology, 127: 172–175.
Lyons P.P., Turnbull J.F., Dawson K.A. and Crumlish M. 2017. Exploring the microbial diversity of the distal lumen and mucosa of farmed rainbow trout Oncorhynchus mykiss (Walbaum) using next generation sequencing (NGS). Aquaculture Research, 48: 77–91.
Martinez J.L. 2003. Recent advances on antibiotic resistance genes. Recent Advances in Marine Biotechnology, 10: 13–32.
McMurry L.M. and Levy S.B. 2000. Tetracycline resistance in gram positive bacteria. P: 660–677. In: Fischetti V.A., Novick R.P., Ferretti J.J. Portnoy D.A. and Rood J.I. (Eds.). Gram-positive Pathogens. American Society for Microbiology, USA.
Miranda C.D., Godoy F.A. and Lee M. 2018. Current status of the use of antibiotics and their antimicrobial resistance in the Chilean salmon farms. Frontiers in Microbiology, 9: 1284, from https://doi.org/10.3389/fmicb.2018.01284.
Nguyen T.L., Park C.I. and Kim D.H. 2017. Improved growth rate and disease resistance in olive flounder, Paralichthys olivaceus, by probiotic Lactococcus lactis WFLU12 isolated from wild marine fish. Aquaculture, 471: 113–120.
Perreten V., Schwarz F., Cresta L., Boeglin M., Dasen G. and Teuber M. 1997. Antibiotic resistance spread in food. Nature, 389: 801–802.
Petersen A. and Dalsgaard A. 2003. Species composition and antimicrobial resistance genes of Enterococcus spp., isolated from integrated and traditional fish farms in Thailand. Environmental Microbiology, 5: 395–402.
Preethi C., Thumu S.C.R. and Halami P.M. 2017. Occurrence and distribution of multiple antibiotic-resistant Enterococcus and Lactobacillus spp. from Indian poultry: In vivo transferability of their erythromycin, tetracycline and vancomycin resistance. Annals of Microbiology, 67: 395–404.
Roberts M.C. 1996. Tetracycline resistance determinants: Mechanisms of action, regulation of expression, genetic mobility, and distribution. FEMS Microbiology Reviews, 19: 1–24.
Sorum H. 2006. Antimicrobial drug resistance in fish pathogens. P: 213–238. In: Aarestrup F.M. (Ed.). Antimicrobial Resistance in Bacteria of Animal Origin. American Society for Microbiology Press, USA.
Tirado M.C., Clarke R., Jaykus L.A., McQuatters-Gollop A. and Frank J.M. 2010. Climate change and food safety: A review. Food Research International, 43: 1745–1765.
Van Reenen C.A. and Dicks L.M. 2011. Horizontal gene transfer amongst probiotic lactic acid bacteria and other intestinal microbiota: What are the possibilities? A review. Archives of Microbiology, 193: 157–168.
Zhang X.X., Zhang T. and Fang H.H. 2009. Antibiotic resistance genes in water environment. Applied Microbiology and Biotechnology, 82: 397–414.