Gıda örneklerinden izole edilen Enterococcus türlerinin çeşitli virülans özellikleri, biyofilm oluşumu ve antibiyotik dirençliliklerinin belirlenmesi

Year 2021, Issue: 28, 924 - 932, 30.11.2021

Tuğçe Gürkan Meryem Burcu Külahcı , Sumru Çıtak

https://doi.org/10.31590/ejosat.1012135

Cited By: 3

Abstract

Çalışmamızda Eylül -Aralık 2020 tarihlerinde 20 çiğ süt, 20 peynir, 20 çiğ tavuk eti, ve 20 çiğ et olmak üzere 80 adet gıda ürününden izole edilen Enterococcus izolatlarının tanımlanması için temel biyokimyasal testler ve identifikasyon işlemi MALDI-TOF Kütle Spektrofotometresi ile yapılarak 60 izolattan 42 (%70) Enterococcus faecalis ve 18 (%30) Enterococcus faecium türü izole edilmiştir. E.faecalis ve E.faecium türlerinin Kirby-Bauer disk difüzyon metoduyla antibiyotik direnç profilleri, lipaz, jelatinaz, DNaz aktiviteleri, hemolitik aktivitesi, S tabakası varlığı ve biyofilm aktiviteleri incelenmiştir. E.faecalis türlerinin en fazla duyarlı olduğu antibiyotikler %100 kloramfenikol, streptomisin, vankomisin ve teikoplanin, %97,6 gentamisin ve norfloksasin, %95,2 ampisilin, %92,8 siprofloksasin olarak saptanmıştır. En fazla dirençlilik gösterdiği antibiyotik %90,4 tetrasiklindir. E.faecium türlerinin en fazla duyarlı olduğu antibiyotikler %100 ampisilin, kloramfenikol, streptomisin, gentamisin, vankomisin ve teikoplanin, % 77,7 siprofloksasin, %72,2 norfloksasindir. En fazla dirençlilik gösterdiği antibiyotik %61,1 tetrasiklindir. Lipaz ve jelatinaz aktiviteleri tümünde negatif, DNaz aktiviteleri ise tümünde pozitif sonuç vermiştir. S tabakası varlığı E.faecalis’te %83,3 ve E.faecium’da %16,6 bulunmuştur. Biyofilm aktiviteleri Kongo Red Agar’da 60 adet Enterococcus spp. türü bakterinin 2‘si (%3.3) kuvvetli, 22‘si (%36.6) orta kuvvetli, 36’sı (%60) zayıf biyofilm pozitif üretmiştir. Mikroplak yönteminde E.faecalis’te 32’si (%53,3) zayıf, 8’i (%13,3) orta kuvvetli, 2’si (%3,3) kuvvetli biyofilm üretmiştir. E.faecium’da 17’si (%28,3) zayıf, 1’i (%1,6) orta kuvvetli biyofilm üretmiştir. Kuvvetli biyofilm üreten Enterococcus türleri saptanmamıştır.

Keywords

Gıda, Enterococcus, Virülans özellikler, Virülans genler, Biyofilm oluşumu, Antibiyotik dirençliliği, Disk difüzyon.

Determination of various virulence properties and biofilm formation antibiotic resistance of enterococcus species isolated from food samples

Abstract

In our study, basic biochemical tests and identification process for the identification of Enterococcus isolates isolated from 80 food products, including 20 raw milk, 20 cheese, 20 raw chicken meat, and 20 raw meat, between September and December 2020, were performed with MALDI-TOF Mass Spectrophotometer and 42 of 60 isolates were identified. (70%) Enterococcus faecalis and 18 (30%) Enterococcus faecium species were isolated. Antibiotic resistance profiles, lipase, gelatinase, DNase activities, hemolytic activity, presence of S layer and biofilm activities of E.faecalis and E.faecium species were investigated by Kirby-Bauer disc diffusion method. The antibiotics to which E.faecalis species were most susceptible were found to be 100% chloramphenicol, streptomycin, vancomycin and teicoplanin, 97.6% gentamicin and norfloxacin, 95.2% ampicillin, 92.8% ciprofloxacin. The antibiotic with the highest resistance is 90.4% tetracycline. The antibiotics to which E.faecium species are most susceptible are 100% ampicillin, chloramphenicol, streptomycin, gentamicin, vancomycin and teicoplanin, 77.7% ciprofloxacin, 72.2% norfloxacin. The antibiotic with the highest resistance is 61.1% tetracycline. Lipase and gelatinase activities were negative in all, and DNase activities were positive in all. Presence of S layer was found 83.3% in E.faecalis and 16.6% in E.faecium. Biofilm activities of 60 Enterococcus spp. on Congo Red Agar. Of the bacteria, 2 (3.3%) were strong, 22 (36.6%) were medium strong, and 36 (60%) were weak biofilm positive. In the microplate method, 32 (53.3%) weak, 8 (13.3%) medium-strong, 2 (3.3%) strong biofilms were produced in E.faecalis. In E.faecium, 17 (28.3%) produced weak biofilms and 1 (1.6%) produced medium-strength biofilm. Strong biofilm-producing Enterococcus species were not detected.

Keywords

Food, Enterococcus species, Enterococcus, Enterococci, Virulence determinants, Virulence genes, Biofilm formation, Antibiotic resistance, Disc diffusion.

References

• Franz, C. M., Muscholl-Silberhorn, A. B., Yousif, N. M., Vancanneyt, M., Swings, J., & Holzapfel, W. H. (2001). Incidence of virulence factors and antibiotic resistance among enterococci isolated from food. Applied and environmental Microbiology, 67(9), 4385-4389.
• Oryaşın, E. (2008). Çeşitli çevresel kaynaklardan izole edilen enterokokların disk difüzyon yöntemi ile antibiyotik duyarlılıklarının tespiti (Doctoral dissertation, Adnan Menderes Üniversitesi).
• Klein, G. (2003). Taxonomy, ecology and antibiotic resistance of enterococci from food and the gastro-intestinal tract. International journal of food microbiology, 88(2-3), 123-131.
• İşleroğlu, H., Yıldırım, Z., & Yıldırım, M. (2008). Identification and izolation of lactic acid bacterium having antimicrobial activity from traditionaly produced cheese. Journal of the Agricultural Faculty of Gaziosmanpaşa University.
• Khan, H., Flint, S., & Yu, P. L. (2010). Enterocins in food preservation. International journal of food microbiology, 141(1-2), 1-10.
• Toğay, S. Ö., & Temiz, A. (2011). Gıda kaynaklı enterokokların gıda ve insan sağlığı yönünden önemi. Gıda, 36(5), 303-310.
• Yu, M. K., Kim, M. A., Rosa, V., Hwang, Y. C., Del Fabbro, M., Sohn, W. J., & Min, K. S. (2019). Role of extracellular DNA in Enterococcus faecalis biofilm formation and its susceptibility to sodium hypochlorite. Journal of Applied Oral Science, 27.
• Igbinosa, I. H., Beshiru, A., Egharevba, N. E., & Igbinosa, E. O. (2020). Distribution of Enterobacteria in Ready-to-Eat Food in Cafeterias and Retail Food Outlets in Benin City: Public Health Implications. Journal of Community Medicine and Primary Health Care, 32(2), 80-94.
• Mohamed, J. A., & Huang, D. B. (2007). Biofilm formation by enterococci. Journal of medical microbiology, 56(12), 1581-1588.
• Bollinger, N., Hassett, D. J., Iglewski, B. H., Costerton, J. W., & McDermott, T. R. (2001). Gene expression in Pseudomonas aeruginosa: evidence of iron override effects on quorum sensing and biofilm-specific gene regulation. Journal of bacteriology, 183(6), 1990-1996.
• Lewis, K. (2001). Riddle of biofilm resistance. Antimicrobial agents and chemotherapy, 45(4), 999-1007.
• Kodeksi, T. G. (2011). Türk Gıda Kodeksi Mikrobiyolojik Kriterler Yönetmeliği. RG, 29(2011), 28157.
• Wayne, P. A. (2011). Clinical and laboratory standards institute. Performance standards for antimicrobial susceptibility testing.
• Semedo, T., Almeida Santos, M., Martins, P., Silva Lopes, M. F., Figueiredo Marques, J. J., Tenreiro, R., & Barreto Crespo, M. T. (2003). Comparative study using type strains and clinical and food isolates to examine hemolytic activity and occurrence of the cyl operon in enterococci. Journal of Clinical Microbiology, 41(6), 2569-2576.
• Jeong, J. Y., Jo, Y. H., Kim, S. B., Liu, Q., Lee, J. W., Mo, E. J., ... & Lee, M. K. (2015). Pancreatic lipase inhibitory constituents from Morus alba leaves and optimization for extraction conditions. Bioorganic & medicinal chemistry letters, 25(11), 2269-2274.
• Kanemitsu, K., Nishino, T., Kunishima, H., Okamura, N., Takemura, H., Yamamoto, H., & Kaku, M. (2001). Quantitative determination of gelatinase activity among enterococci. Journal of microbiological methods, 47(1), 11-16.
• Omar, N. B., Castro, A., Lucas, R., Abriouel, H., Yousif, N. M., Franz, C. M., ... & Gálvez, A. (2004). Functional and safety aspects of enterococci isolated from different Spanish foods. Systematic and Applied Microbiology, 27(1), 118-130.
• Bernoth, E. M. (1990). Autoagglutination, growth on tryptone‐soy‐Coomassieagar, outer membrane protein patterns and virulence of Aeromonas salmonicida strains. Journal of Fish Diseases, 13(2), 145-155.
• Stepanović, S., Vuković, D., Hola, V., Bonaventura, G. D., Djukić, S., Ćirković, I., & Ruzicka, F. (2007). Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci. Apmis, 115(8), 891-899.
• Moreno, M. F., Sarantinopoulos, P., Tsakalidou, E., & De Vuyst, L. (2006). The role and application of enterococci in food and health. International journal of food microbiology, 106(1), 1-24.
• Ben Braïek, O., & Smaoui, S. (2019). Enterococci: between emerging pathogens and potential probiotics. BioMed Research International, 2019.
• Ghosh, A., & Zurek, L. (2015). Antibiotic resistance in Enterococci: A food safety perspective. In Antimicrobial Resistance and Food Safety (pp. 155-180). Academic Press.
• Hanchi, H., Mottawea, W., Sebei, K., & Hammami, R. (2018). The genus Enterococcus: between probiotic potential and safety concerns—an update. Frontiers in microbiology, 9, 1791.
• Oruc, O., Ceti̇n, O., Darilmaz, D. O., & Yüsekdag, Z. N. (2021). Determination of the biosafety of potential probiotic Enterococcus faecalis and Enterococcus faecium strains isolated from traditional white cheeses. LWT, 148, 111741.
• Pavia, M., Nobile, C. G., Salpietro, L., & Angelillo, I. F. (2000). Vancomycin resistance and antibiotic susceptibility of enterococci in raw meat. Journal of food protection, 63(7), 912-915.
• Gelsomino, R., Vancanneyt, M., Cogan, T. M., Condon, S., & Swings, J. (2002). Source of enterococci in a farmhouse raw-milk cheese. Applied and Environmental Microbiology, 68(7), 3560-3565.
• Saavedra, L., Taranto, M. P., Sesma, F., & de Valdez, G. F. (2003). Homemade traditional cheeses for the isolation of probiotic Enterococcus faecium strains. International journal of food microbiology, 88(2-3), 241-245.
• G., Suzzi, M., Caruso, F., Gardini, A., Lombardi, L., Vannini, M. E., Guerzoni and M. T., Lanorte, “A survey of the enterococci isolated from an artisanal Italian goat's cheese (semicotto caprino),” Journal of Applied Microbiology, vol. 89 pp. 2, 267-274, 2000.
• Devriese, L. A., Pot, B., Van Damme, L., Kersters, K., & Haesebrouck, F. (1995). Identification of Enterococcus species isolated from foods of animal origin. International journal of food microbiology, 26(2), 187-197.
• Litopoulou‐Tzanetaki, E. (1990). Changes in numbers and kinds of lactic acid bacteria during ripening of Kefalotyri cheese. Journal of Food Science, 55(1), 111-113.
• Çitak, S., Yucel, N., & Orhan, S. (2004). Antibiotic resistance and incidence of Enterococcus species in Turkish white cheese. International Journal of Dairy Technology, 57(1), 27-31.
• Citak, S., Yucel, N., & Mendi, A. (2005). Antibiotic resistance of enterococcal isolates in raw milk. Journal of food processing and preservation, 29(3‐4), 183-195.
• Jett, B. D., Huycke, M. M., & Gilmore, M. S. (1994). Virulence of enterococci. Clinical microbiology reviews, 7(4), 462-478.
• Giraffa, G. (2002). Enterococci from foods. FEMS microbiology reviews, 26(2), 163-171.
• Sanlibaba, P., & Senturk, E. (2018). Prevalence, characterization and antibiotic resistance of enterococci from traditional cheeses in Turkey. International Journal of Food Properties, 21(1), 1955-1963.
• Hugas, M., Garriga, M., & Aymerich, M. T. (2003). Functionalty of enterococci in meat products. International journal of food microbiology, 88(2-3), 223-233.
• Hummel, A., Holzapfel, W. H., & Franz, C. M. (2007). Characterisation and transfer of antibiotic resistance genes from enterococci isolated from food. Systematic and applied microbiology, 30(1), 1-7.
• Theilacker, C., Sanchez‐Carballo, P., Toma, I., Fabretti, F., Sava, I., Kropec, A., ... & Huebner, J. (2009). Glycolipids are involved in biofilm accumulation and prolonged bacteraemia in Enterococcus faecalis. Molecular microbiology, 71(4), 1055-1069.
• Carlos, A. R., Semedo‐Lemsaddek, T., Barreto‐Crespo, M. T., & Tenreiro, R. (2010). Transcriptional analysis of virulence‐related genes in enterococci from distinct origins. Journal of applied microbiology, 108(5), 1563-1575.
• İspirli, H., Demirbaş, F., & Dertli, E. (2017). Characterization of functional properties of Enterococcus spp. isolated from Turkish white cheese. LWT, 75, 358-365.
• Lauková, A., Focková, V., & Pogány Simonová, M. (2021). Enterococcal Species Associated with Slovak Raw Goat Milk, Their Safety and Susceptibility to Lantibiotics and Durancin ED26E/7. Processes, 9(4), 681.
• İlhan, G. Ü. N., & Ekinci, F. Y. (2009). Biyofilmler: yüzeylerdeki mikrobiyal yaşam. Gıda, 34(3), 165-173.
• Di Rosa, R., Creti, R., Venditti, M., D'Amelio, R., Arciola, C. R., Montanaro, L., & Baldassarri, L. (2006). Relationship between biofilm formation, the enterococcal surface protein (Esp) and gelatinase in clinical isolates of Enterococcus faecalis and Enterococcus faecium. FEMS microbiology letters, 256(1), 145-150.