Evaluation of antimicrobial and antibiotic resistance properties of microbial community in a traditional cheese

Document Type : Complete scientific research article

Author

Assistant Professor, Department of Food Science and Technology, Bu‐Ali Sina University, Hamedan, Iran

Abstract

Background and objectives: The presence of antibiotic-resistant genes in local cheeses is not desirable due to the risk of gene migration, increased resistance to antibiotics in consumers, and therefore the effectiveness of related drugs during the treatment period. Recently, the studies showed that the demand of local cheeses made from raw milk have increased. However, there is concern that consumption of these cheeses could increase the risk of antibiotic-resistant‌ genes transfer‌ through bacteria especially‌ Enterococcus spp to consumers. Of course, traditional fermented products may contain intrinsic microbial flora with‌ unique useful properties, including the production of‌ antimicrobial metabolites and resistance to phages, which is very important for the dairy industry. The ability of bacteriocin production by the microbial flora that responsible for fermentation of cheeses made from raw milk is very important because it prevents the growth of pathogenic bacteria. Therefore, the aims of this study were the evaluation of the presence of antibiotic-resistant microbial flora of local cheeses made from raw milk and the investigation of the useful properties such as the presence of its antimicrobial metabolites.
Materials and methods: Cheese made from raw milk was produced based on a traditional recipe. For isolation of Enterococcus spp., KAA agar was used, followed by incubation for 48 h at 37°C. The serial dilution of cheese was carried out until the final dilution of 10-8 in ringer. For isolation of Enterococcus spp., a 100 µl of diluted sample was cultured on KAA agar, then incubated for 48 h under anaerobic conditions at 37°C. The catalase-negative and Gram-positive isolates were phenotypically distinguished at genus level using physiological tests )growth at salt concentrations 6.5% and 18%, pH 9.6 and 4.4, temperatures 10°C and 45°C and gas production. After phenotypic detection of isolates at genus level, culture-dependent characterization through PCR and subsequently sequencing was carried out. DNA extracted from cheese was proliferated and sequenced by NGS technique. The metagenomics data were processed for phage resistance, antibiotic resistance, bacteriocin and antioxidant components production. Furthermore, antibiotic resistance of isolates and antimicrobial properties of cell free supernatant (CFS) from cheese were evaluated.
Results: A total of 20 bacteria were isolated from cheese and molecularly identified as Enterococcus malodoratous (60%), Enterococcus faecalis (5%), Enterococcus duran (5%), Enterococcus spp. (30%). Also, the metagenomic analysis showed that Enterococcal community of cheese include Enterococcus malodoratous (72%), Enterococcus faecalis (6%), Enterococcus italicus (13%), Enterococcus spp. (10%) and its microbiome is resistance to ‌-antibiotics ‌and bacteriophages and has the production potential of antioxidant compounds and antimicrobial compounds such as plantaricin. Moreover, laboratory tests confirmed ‌‌antibiotics resistance and antimicrobial properties of isolates.
Conclusion: This study showed that some local cheeses can be responsible for transferring antibiotic-resistant genes to humans and consumption of these cheeses should be limited.

Keywords

References

  1. 1.Trejo-González, L., Gutiérrez-Carrillo, A. E., Rodríguez-Hernández, A. I., del Rocío López-Cuellar, M. and Chavarría-Hernández, N. 2021. Bacteriocins Produced by LAB Isolated from Cheeses within the Period 2009–2021: a Review. Probiotics and Antimicrobial Proteins. 1-14.

    2.Darvishi, F. 2017. Screening of New Microorganisms and Their Useful Genes: From Traditional Methods to Metagenomics. Iranian journal of biology. 1: 1. 43-51.

    3.Russoa, N., Caggiaa, C., Pinoa, A., Coqueb, T.M., and Ariolie, S. 2018. Enterococcus spp. in Ragusano PDO and Pecorino Siciliano cheese types: A snapshot of their antibiotic resistance distribution. Food and Chemical Toxicology. 120. 277-286.

    4.Kamilaria, E., Dimitrios, A., Papademasa, P., Kamilarisb, A. and Tsaltas, D. 2020. Characterizing Halloumi cheese's bacterial communities through metagenomic analysis.  LWT - Food Science and Technology. 126:109298.

    5.Ruiz-Barba, J.L., Maldonado, A. and Jiménez-Díaz, R. 2005. Small-scale total DNA extraction from bacteria and yeast for PCR applications. Analytical biochemistry. 347: 2. 333-5.

    6.Jamet, E., Akary, E., Poisson, M.A., Chamba, J.F., Bertrand, X. and Serror, P. 2012. Prevalence and characterization of antibiotic resistant Enterococcus faecalis in French cheeses. Food Microbiology. 31.191-198.

    7.Flórez, A.B., Ammor, M.S., and Mayo, B. 2008. Identification of tet (M) in two Lactococcus lactis strains isolated from a Spanish traditional starter-free cheese made of raw milk and conjugative transfer of tetracycline resistance to lactococci and enterococci. International Journal of Food Microbiology. 121. 189-194.

    1. Delgado, S., Fracchetti, F., Mayo, B. and Torriani, S. 2011. Development and validation of a multiplex PCR-based DNA microarray hybridisation method for detecting bacterial antibiotic resistance genes in cheese. International dairy Journal. 21: 3.149-157.
    2. Meng, F., Zhu, X., Zhao, H., Nie, T., Lu, F. Lu, L. and Lu, Y. 2021. A class III bacteriocin with broad-spectrum antibacterial activity from Lactobacillus acidophilus NX2-6 and its preservation in milk and cheese. Food Control. 107597.

    10.Harrigan, W. 1998. Laboratory methods in food microbiology. Gulf Professional Publishing.

    11.Cardinal, M.J., Meghrous, J., Lacroix, C. and Simard, R.E. 1997. Isolation of Lactococcus lactis strains producing inhibitory activity against Listeria. Food Biotechnology. 11: 2.129-146.

    12.Lahtinen, S., Ouwehand, A.C., Salminen, S. and von Wright, A. (Eds.). 2011. Lactic acid bacteria: microbiological and functional aspects. Crc Press.

    13.Bulut, Ç. 2003. Isolation and molecular characterization of lactic acid bacteria from cheese (Master's thesis, İzmir Institute of Technology).

    14.Cortés-Zavaleta, O., López-Malo, A., Hernández-Mendoza, A. and García, H.S. 2014. Antifungal activity of lactobacilli and its relationship with 3-phenyllactic acid production. International journal of food microbiology. 173. 30-35.

    16.Kumar, P., Mishra, S., Kumar, A. and Sharma, A.K. 2016. Antifungal efficacy of plant essential oils against stored grain fungi of Fusarium sp. Journal of Food Science and Technology. 53. 3725-3734.

    17.Mousavi, S.H. and Kafili, T. 2020. Antibiotic Resistance of Lactic Acid Bacteria Isolated from Traditional Raw Milk Taleshi Cheese. Journal of Innovation in Food Science and Technology. 11: 4. 103-114.

    18.Fox, P.F., Guinee, T.P., Cogan, T.M. and McSweeney, P.L. 2017. Microbiology of cheese ripening. In Fundamentals of cheese science. Springer, Boston, MA. 333-390.

    19.Tabatabaei, M. and Pourmazaheri, H. 2012. Metagenomics and its application in identification of genetic diversity of microbial ecosystems. Modern genetic Journal. 7: 4. 313-324.

    20.Andrighetto, C., Knijff, E.D.O., Lombardi, A., Torriani, S., Vancanneyt, M., Kersters, K., Swings, J. and Dellaglio, F. 2001. Phenotypic and genetic diversity of enterococci isolated from Italian cheeses. Journal of Dairy Research. 68: 2. 303-316.

    21.Hajikhani, R., Darilmaz, D.O., Yuksekdag, Z.N. and Beyatli, Y. 2021. Assessment of some metabolic activities and potential probiotic properties of eight Enterococcus bacteria isolated from white cheese microbiota. Antonie van Leeuwenhoek. 1-16.

    1. Donowitz, G.R., and Mandell, G.L. 1988. Beta-lactam antibiotics. New England Journal of Medicine. 318: 7. 419-426.

    24.Bahrami, A., and Davati, N. 2021. Analysis of the microbial community in an Iranian local cheese. Iranian Food Science and Technology Research Journal. 17: 4. 631-645.

    25.Dubikova, K., Pristaš, P. and Javorský, P. 2015. Occurrence of abortive infection systems and phage resistance in lactic acid bacteria isolated from bryndza ewes' cheese. Journal of Food & Nutrition Research. 54: 1.

    26.Virtanen, T., Pihlanto, A., Akkanen, S. and Korhonen, H. 2007. Development of antioxidant activity in milk whey during fermentation with lactic acid bacteria. Journal of applied microbiology. 102: 1. 106-115.

    27.Gupta, A., Mann, B., Kumar, R. and Sangwan, R.B. 2009. Antioxidant activity of Cheddar cheeses at different stages of ripening. International Journal of Dairy Technology. 62: 3. 339-347.

Food Processing and Preservation Journal
Volume 14, Issue 2
December 2022
Pages 131-146

Files

Share

How to cite

Statistics