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Sıkma peynirlerinden izole edilen enterokokların tanımlanmasında MALDI-TOF-MS ve 16S rRNA sekanslamasının karşılaştırılması ve izolatların antibiyotik dirençlikleri ile antimikrobiyal aktivitelerinin belirlenmesi

Year 2020, Volume: 77 Issue: 4, 399 - 412, 01.12.2020

Abstract

Amaç: Bu çalışmanın amacı, sıkma peynirlerden izole edilen enterokokların MALDI-TOF-MS ve 16S rRNA sekans analizleri kullanılarak tanımlanması ve bu izolatların antibiyotik dirençleri ile bazı gıda patojenlerine karşı antibakteriyel etkilerinin değerlendirilmesidir.Yöntem: Sıkma peynirlerden elde edilen 84 Enterococcus izolatının MALDI-TOF-MS yöntemi ile tanımlanması yapılmış ve 16S rRNA bölgeleri çoğaltılarak sekanslanmıştır. İzolatların 14 farklı antibiyotiğe karşı dirençlikleri disk difüzyon yöntemiyle incelenmiştir. Ayrıca izolatların çeşitli gıda patojenlerine karşı gösterdikleri antibakteriyel etkileri agar spot testi ile belirlenmiştir. İstatistiksel değerlendirme için varyans analizi ANOVA, F testi ve antibakteriyel sonuçlar arasındaki korelasyonun tespiti için SPSS 22.0.0 yazılımı SPSS Inc., Chicago, ABD kullanılmışır. Bulgular: Seksen-dört izolat 16S rRNA sekans analizi sonucunda; 33 %39.3 E. faecalis, 29 %34.5 E. faecium, 14 %16.7 E. durans, 4 %4.8 E. gallinarum, 3 %3.5 E. casseliflavus ve 1 %1.2 E. thailandicus olarak tanımlanmıştır. Seksen bir izolat MALDI-TOF-MS yöntemi ile aynı tanımlama sonucunu vermiştir %96.43 . İzolatların tanımlanmasında kullanılan iki tanımlama yönteminin arasında anlamlı bir fark bulunmamıştır p>0.05 . Ancak MALDI-TOF-MS yönteminin daha az iş gücü gerektirmesi, ekonomik ve hızlı olması gibi bazı avantajlarının olduğu görülmüştür. İzolatların %83.3’ünün çoklu antibiyotik direnci taşıdığı tespit edilmiştir. İzolatların nalidiksik asit, oksasilin ve streptomisine karşı dirençliklerinin yüksek olduğu saptanmıştır. E. faecalis suşlarının kullanılan antibiyotiklere karşı E. faecium’dan daha düşük duyarlılık gösterdiği bulunmuştur p

References

  • Giraffa G. Functionality of enterococci in dairy products. Int J Food Microbiol, 2003; 88 (2-3): 215- 2.
  • Gaglio R, Couto N, Marques C, Lopes MDFS, Moschetti G, Pomba C, et al. Evaluation of antimicrobial resistance and virulence of enterococci from equipment surfaces, raw materials, and traditional cheeses. Int J Food Microbiol, 2016; 236: 107-14.
  • Commission Regulation (EC) No 2073/2005 (OJ L338, p1, 22/12/2005 ) of 15 November 2005 on microbiological criteria for foodstuffs. Official Journal of the European Union. 2005; L 338/1-L 338/26.
  • Yuvasen A, Macit E, Dertli E. Microbial species playing roles for the production of traditional kasar cheese during pre-maturation period. LWT, 2018; 91: 406-13.
  • Egan K, Field D, Rea MC, Ross RP, Hill C, Cotter PD. Bacteriocins: novel solutions to age old spore- related problems? Front Microbiol, 2016; 7: 461.
  • Ricci A, Allende A, Bolton D, Chemaly M, Davies R, Girones R, et al. Scientific Opinion on the update of the list of QPS-recommended biological agents intentionally added to food or feed as notified to EFSA. EFSA J, 2017; 15(3):177.
  • Jahan M, Zhanel GG, Sparling R, Holley RA. Horizontal transfer of antibiotic resistance from Enterococcus faecium of fermented meat origin to clinical isolates of E. faecium and Enterococcus faecalis. Int J Food Microbiol, 2015; 199: 78-85.
  • Picon A, Garde S, Avila M, Nunez M. Microbiota dynamics and lactic acid bacteria biodiversity in raw goat milk cheeses. Int Dairy J, 2016; 58: 14-22.
  • Ceylan ZG, Turkoglu H, Dayisoylu KS. The microbiological and chemical quality of sikma cheese produced in Turkey. Pakistan J Nutr, 2003; 2 (2): 95-7.
  • Bareika HA, Sidarenka AV, Novik GI. Starter cultures of lactic acid bacteria for special diet products, Eurobiotech J, 2017; 1(1): 89-90.
  • Carbonnelle E, Grohs P, Jacquier H, Day N, Tenza S, Dewailly, A, et al. Robustness of two MALDI- TOF mass spectrometry systems for bacterial identification, J Microbiol Methods, 2012; 89(2): 133-6.
  • Aydin F, Ardic M. Determination of microbiological and chemical properties of sikma cheeses collected from different provinces. Gıda, 2019; 44(5): 826-6.
  • Ispirli H, Demirbas F, Dertli E. Characterization of functional properties of Enterococcus spp. isolated from Turkish white cheese. LWT, 2017; 75: 358-65.
  • Basic Local Alignment Search Tool (BLAST). National Center for Biotechnology Information (NCBI). http://blast.ncbi.nlm.nih.gov/, Date Accessed: 12.01.2020.
  • Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol, 2016; 33(7): 1870-4.
  • Performance Standards for Antimicrobial Susceptibility Testing. Twenty-sixth Informational Supplement, M100-S26. Wayne: Clinical Laboratory Standards Institute. 2016.
  • Schillinger U, Lucke, FK. Antibacterial activity of Lactobacillus sake isolated from meat. Appl Environ Microbiol, 1989; 55(8): 1901-6.
  • Aras Z. Mikrobiyolojide kullanılan hızlı tanı yöntemleri. Turk Hij Den Biyol Derg, 2011; 68(2): 97-104.
  • Quintilla R, Kolecka A, Casaregola S, Daniel HM, Houbraken J, Kostrzewa M, et al. MALDI-TOF MS as a tool to identify foodborne yeasts and yeast-like fungi. Int J Food Microbiol, 2018; 266, 109-8.
  • Quintela-Baluja M, Böhme K, Fernández-No IC, Morandi S, Alnakip ME, Caamaño-Antelo S, et al. Characterization of different food- isolated Enterococcus strains by MALDI-TOF mass fingerprinting. Electrophoresis, 2013; 34 (15): 2240-50.
  • Kryazhevskikh NA, Loiko NG, Demkina EV, Mulyukin AL, Lebedev AT, Gaponov, AM, et al. Applicability of MALDI mass spectrometry for diagnostics of phase variants in bacterial populations. Microbiol, 2015; 84 (3): 328-46.
  • Sanlibaba P, Senturk E. Prevalence, characterization and antibiotic resistance of enterococci from traditional cheeses in Turkey. Int J Food Prop, 2018; 21(1): 1955-63
  • Russo N, Caggia C, Pino A, Coque TM, Arioli S, Randazzo CL. Enterococcus spp. in ragusano PDO and pecorino siciliano cheese types: a snapshot of their antibiotic resistance distribution. Food Chem Toxicol, 2018; 120: 277-86
  • Britt NS, Potter EM. Clinical epidemiology of vancomycin-resistant Enterococcus gallinarum and Enterococcus casseliflavus bloodstream infections. J Glob Antimicrob Resist, 2016; 5: 57-61
  • Tanasupawat S, Sukontasing S, Lee JS. Enterococcus thailandicus sp. nov, isolated from fermented sausage (‘mum’) in Thailand. Int J Syst Evol Microbiol, 2008; 58(7): 1630-4
  • Karaalioğlu O, Özmen-Togay S, Ay M, Soysal G, Çardak M, Bağcı U, et al. Çiğ balık örneklerinden izole edilen Enterococcus faecium ve Enterococcus faecalis suşlarının gıda güvenliği yönünden bazı özelliklerinin değerlendirilmesi. Turk Hij Den Biyol Derg, 2019; 76(3): 341-52
  • Critically Important Antimicrobials For Human Medicine. Geneva: World Health Organization. 6th ed. 2018. https://apps.who.int/iris/bitstream/ha ndle/10665/312266/9789241515528-eng.pdf, Date Accessed: 07.06.2017
  • Shridhar S, Dhanashree B. Antibiotic susceptibility pattern and biofilm formation in clinical isolates of Enterococcus spp. Interdiscip Perspect Infect Dis, 2019; 1-6
  • Yogurtcu NN, Tuncer Y. Antibiotic susceptibility patterns of Enterococcus strains isolated from Turkish Tulum cheese. Int J Dairy Technol, 2013; 66 (2): 236-42.
  • Diarra MS, Rempel H, Champagne J, Masson L, Pritchard J, Topp E. Distribution of antimicrobial resistance and virulence genes in Enterococcus spp. and characterization of isolates from broiler chickens. Appl Environ Microbiol, 2010; 76 (24): 8033-43.
  • Coleri A, Çökmüş C. Enterokok türlerinde glikopeptid grubu antibiyotiklere direncin moleküler mekanizmaları ve gen aktarım yolları. Turk Hij Den Biyol Derg, 2008; 65 (2): 87-96.
  • Miller WR, Munita JM, Arias CA. Mechanisms of antibiotic resistance in enterococci. Expert Rev Anti Infect Ther, 2014; 12(10): 1221-36.
  • Yuksel FN, Akcelik N, Akcelik M. Incidence of antibiotic resistance and virulence determinants in Enterococcus faecium and Enterococcus faecalis strains, isolated from traditional cheeses in Turkey. Mol Genet Microbiol Virol, 2015; 30 (4): 206-15.
  • Cariolato D, Andrighetto C, Lombardi A. Occurrence of virulence factors and antibiotic resistances in Enterococcus faecalis and Enterococcus faecium collected from dairy and human samples in North Italy. Food Control, 2008; 19 (9): 886-92.
  • Veljovic K, Fira D, Terzic-Vidojevic A, Abriouel H, Galvez A, Topisirovic L. Evaluation of antimicrobial and proteolytic activity of enterococci isolated from fermented products. Eur Food Res Technol, 2009; 230 (1), 63.
  • Masalam B, Maged S, Bahieldin A, Alharbi MG, Al- Masaudi S, Al-Jaouni SK, et al. Isolation, molecular characterization and probiotic potential of lactic acid bacteria in Saudi raw and fermented milk. Evidence-Based Complement Altern Med, 2018; 2018: 7970463.

Comparision of two identification methods: MALDI-TOF-MS & 16s rRNA sequencing in enterococci isolated from raw milk cheeses Sikma cheeses and evaluating their antibiotic susceptibility and antimicrobial activity

Year 2020, Volume: 77 Issue: 4, 399 - 412, 01.12.2020

Abstract

Objective: This study aims to identify enterococci isolated from squeezed cheeses by MALDI-TOF-MS and 16S rRNA sequence analysis and to evaluate antibiotic resistance and antibacterial effects of these isolates against some food pathogens.Methods: Identification of 84 Enterococcus isolates obtained from squeezed cheese was carried out using MALDI-TOF-MS and 16S rRNA gene sequencing. The isolates were tested for resistance to 14 different antibiotics by the disc diffusion method. The antimicrobial effects of isolates against various food pathogens were determined by the agar spot test. SPSS 22.0.0 software SPSS Inc., Chicago, USA was used for the assessment of the correlation between variance analysis ANOVA, F test and antibacterial results.Results: As a result of sequence analysis; 33 39.3% were described to E. faecalis, 29 34.5% to E. faecium, 14 16.7% to E. durans, 4 4.8% to E. gallinarum, 3 3.5% to E. casseliflavus and 1 1.2% to E. thailandicus. Eighty-one isolates gave the same identification result as the MALDI-TOF-MS method 96.43% . The difference between two identification methods has not been found to be statistically significant p>0.05 ; however, MALDI-TOF-MS has some advantages over 16S rRNA sequencing, such as being less labor-intensive, more economical and faster. In total, 83.3% of the strains exhibited multidrug-resistant phenotypes. A high incidence of resistance was detected for nalidixic acid, oxacillin, and streptomycin. E. faecalis isolates were found to show lower sensitivity to antibiotics tested than E. faecium p

References

  • Giraffa G. Functionality of enterococci in dairy products. Int J Food Microbiol, 2003; 88 (2-3): 215- 2.
  • Gaglio R, Couto N, Marques C, Lopes MDFS, Moschetti G, Pomba C, et al. Evaluation of antimicrobial resistance and virulence of enterococci from equipment surfaces, raw materials, and traditional cheeses. Int J Food Microbiol, 2016; 236: 107-14.
  • Commission Regulation (EC) No 2073/2005 (OJ L338, p1, 22/12/2005 ) of 15 November 2005 on microbiological criteria for foodstuffs. Official Journal of the European Union. 2005; L 338/1-L 338/26.
  • Yuvasen A, Macit E, Dertli E. Microbial species playing roles for the production of traditional kasar cheese during pre-maturation period. LWT, 2018; 91: 406-13.
  • Egan K, Field D, Rea MC, Ross RP, Hill C, Cotter PD. Bacteriocins: novel solutions to age old spore- related problems? Front Microbiol, 2016; 7: 461.
  • Ricci A, Allende A, Bolton D, Chemaly M, Davies R, Girones R, et al. Scientific Opinion on the update of the list of QPS-recommended biological agents intentionally added to food or feed as notified to EFSA. EFSA J, 2017; 15(3):177.
  • Jahan M, Zhanel GG, Sparling R, Holley RA. Horizontal transfer of antibiotic resistance from Enterococcus faecium of fermented meat origin to clinical isolates of E. faecium and Enterococcus faecalis. Int J Food Microbiol, 2015; 199: 78-85.
  • Picon A, Garde S, Avila M, Nunez M. Microbiota dynamics and lactic acid bacteria biodiversity in raw goat milk cheeses. Int Dairy J, 2016; 58: 14-22.
  • Ceylan ZG, Turkoglu H, Dayisoylu KS. The microbiological and chemical quality of sikma cheese produced in Turkey. Pakistan J Nutr, 2003; 2 (2): 95-7.
  • Bareika HA, Sidarenka AV, Novik GI. Starter cultures of lactic acid bacteria for special diet products, Eurobiotech J, 2017; 1(1): 89-90.
  • Carbonnelle E, Grohs P, Jacquier H, Day N, Tenza S, Dewailly, A, et al. Robustness of two MALDI- TOF mass spectrometry systems for bacterial identification, J Microbiol Methods, 2012; 89(2): 133-6.
  • Aydin F, Ardic M. Determination of microbiological and chemical properties of sikma cheeses collected from different provinces. Gıda, 2019; 44(5): 826-6.
  • Ispirli H, Demirbas F, Dertli E. Characterization of functional properties of Enterococcus spp. isolated from Turkish white cheese. LWT, 2017; 75: 358-65.
  • Basic Local Alignment Search Tool (BLAST). National Center for Biotechnology Information (NCBI). http://blast.ncbi.nlm.nih.gov/, Date Accessed: 12.01.2020.
  • Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol, 2016; 33(7): 1870-4.
  • Performance Standards for Antimicrobial Susceptibility Testing. Twenty-sixth Informational Supplement, M100-S26. Wayne: Clinical Laboratory Standards Institute. 2016.
  • Schillinger U, Lucke, FK. Antibacterial activity of Lactobacillus sake isolated from meat. Appl Environ Microbiol, 1989; 55(8): 1901-6.
  • Aras Z. Mikrobiyolojide kullanılan hızlı tanı yöntemleri. Turk Hij Den Biyol Derg, 2011; 68(2): 97-104.
  • Quintilla R, Kolecka A, Casaregola S, Daniel HM, Houbraken J, Kostrzewa M, et al. MALDI-TOF MS as a tool to identify foodborne yeasts and yeast-like fungi. Int J Food Microbiol, 2018; 266, 109-8.
  • Quintela-Baluja M, Böhme K, Fernández-No IC, Morandi S, Alnakip ME, Caamaño-Antelo S, et al. Characterization of different food- isolated Enterococcus strains by MALDI-TOF mass fingerprinting. Electrophoresis, 2013; 34 (15): 2240-50.
  • Kryazhevskikh NA, Loiko NG, Demkina EV, Mulyukin AL, Lebedev AT, Gaponov, AM, et al. Applicability of MALDI mass spectrometry for diagnostics of phase variants in bacterial populations. Microbiol, 2015; 84 (3): 328-46.
  • Sanlibaba P, Senturk E. Prevalence, characterization and antibiotic resistance of enterococci from traditional cheeses in Turkey. Int J Food Prop, 2018; 21(1): 1955-63
  • Russo N, Caggia C, Pino A, Coque TM, Arioli S, Randazzo CL. Enterococcus spp. in ragusano PDO and pecorino siciliano cheese types: a snapshot of their antibiotic resistance distribution. Food Chem Toxicol, 2018; 120: 277-86
  • Britt NS, Potter EM. Clinical epidemiology of vancomycin-resistant Enterococcus gallinarum and Enterococcus casseliflavus bloodstream infections. J Glob Antimicrob Resist, 2016; 5: 57-61
  • Tanasupawat S, Sukontasing S, Lee JS. Enterococcus thailandicus sp. nov, isolated from fermented sausage (‘mum’) in Thailand. Int J Syst Evol Microbiol, 2008; 58(7): 1630-4
  • Karaalioğlu O, Özmen-Togay S, Ay M, Soysal G, Çardak M, Bağcı U, et al. Çiğ balık örneklerinden izole edilen Enterococcus faecium ve Enterococcus faecalis suşlarının gıda güvenliği yönünden bazı özelliklerinin değerlendirilmesi. Turk Hij Den Biyol Derg, 2019; 76(3): 341-52
  • Critically Important Antimicrobials For Human Medicine. Geneva: World Health Organization. 6th ed. 2018. https://apps.who.int/iris/bitstream/ha ndle/10665/312266/9789241515528-eng.pdf, Date Accessed: 07.06.2017
  • Shridhar S, Dhanashree B. Antibiotic susceptibility pattern and biofilm formation in clinical isolates of Enterococcus spp. Interdiscip Perspect Infect Dis, 2019; 1-6
  • Yogurtcu NN, Tuncer Y. Antibiotic susceptibility patterns of Enterococcus strains isolated from Turkish Tulum cheese. Int J Dairy Technol, 2013; 66 (2): 236-42.
  • Diarra MS, Rempel H, Champagne J, Masson L, Pritchard J, Topp E. Distribution of antimicrobial resistance and virulence genes in Enterococcus spp. and characterization of isolates from broiler chickens. Appl Environ Microbiol, 2010; 76 (24): 8033-43.
  • Coleri A, Çökmüş C. Enterokok türlerinde glikopeptid grubu antibiyotiklere direncin moleküler mekanizmaları ve gen aktarım yolları. Turk Hij Den Biyol Derg, 2008; 65 (2): 87-96.
  • Miller WR, Munita JM, Arias CA. Mechanisms of antibiotic resistance in enterococci. Expert Rev Anti Infect Ther, 2014; 12(10): 1221-36.
  • Yuksel FN, Akcelik N, Akcelik M. Incidence of antibiotic resistance and virulence determinants in Enterococcus faecium and Enterococcus faecalis strains, isolated from traditional cheeses in Turkey. Mol Genet Microbiol Virol, 2015; 30 (4): 206-15.
  • Cariolato D, Andrighetto C, Lombardi A. Occurrence of virulence factors and antibiotic resistances in Enterococcus faecalis and Enterococcus faecium collected from dairy and human samples in North Italy. Food Control, 2008; 19 (9): 886-92.
  • Veljovic K, Fira D, Terzic-Vidojevic A, Abriouel H, Galvez A, Topisirovic L. Evaluation of antimicrobial and proteolytic activity of enterococci isolated from fermented products. Eur Food Res Technol, 2009; 230 (1), 63.
  • Masalam B, Maged S, Bahieldin A, Alharbi MG, Al- Masaudi S, Al-Jaouni SK, et al. Isolation, molecular characterization and probiotic potential of lactic acid bacteria in Saudi raw and fermented milk. Evidence-Based Complement Altern Med, 2018; 2018: 7970463.
There are 36 citations in total.

Details

Primary Language English
Journal Section Research Article
Authors

Furkan Aydın This is me

Halil İbrahim Kahve This is me

Mustafa Ardıç This is me

İbrahim Çakır This is me

Publication Date December 1, 2020
Published in Issue Year 2020 Volume: 77 Issue: 4

Cite

APA Aydın, F., Kahve, H. İ., Ardıç, M., Çakır, İ. (2020). Comparision of two identification methods: MALDI-TOF-MS & 16s rRNA sequencing in enterococci isolated from raw milk cheeses Sikma cheeses and evaluating their antibiotic susceptibility and antimicrobial activity. Türk Hijyen Ve Deneysel Biyoloji Dergisi, 77(4), 399-412.
AMA Aydın F, Kahve Hİ, Ardıç M, Çakır İ. Comparision of two identification methods: MALDI-TOF-MS & 16s rRNA sequencing in enterococci isolated from raw milk cheeses Sikma cheeses and evaluating their antibiotic susceptibility and antimicrobial activity. Turk Hij Den Biyol Derg. December 2020;77(4):399-412.
Chicago Aydın, Furkan, Halil İbrahim Kahve, Mustafa Ardıç, and İbrahim Çakır. “Comparision of Two Identification Methods: MALDI-TOF-MS & 16s RRNA Sequencing in Enterococci Isolated from Raw Milk Cheeses Sikma Cheeses and Evaluating Their Antibiotic Susceptibility and Antimicrobial Activity”. Türk Hijyen Ve Deneysel Biyoloji Dergisi 77, no. 4 (December 2020): 399-412.
EndNote Aydın F, Kahve Hİ, Ardıç M, Çakır İ (December 1, 2020) Comparision of two identification methods: MALDI-TOF-MS & 16s rRNA sequencing in enterococci isolated from raw milk cheeses Sikma cheeses and evaluating their antibiotic susceptibility and antimicrobial activity. Türk Hijyen ve Deneysel Biyoloji Dergisi 77 4 399–412.
IEEE F. Aydın, H. İ. Kahve, M. Ardıç, and İ. Çakır, “Comparision of two identification methods: MALDI-TOF-MS & 16s rRNA sequencing in enterococci isolated from raw milk cheeses Sikma cheeses and evaluating their antibiotic susceptibility and antimicrobial activity”, Turk Hij Den Biyol Derg, vol. 77, no. 4, pp. 399–412, 2020.
ISNAD Aydın, Furkan et al. “Comparision of Two Identification Methods: MALDI-TOF-MS & 16s RRNA Sequencing in Enterococci Isolated from Raw Milk Cheeses Sikma Cheeses and Evaluating Their Antibiotic Susceptibility and Antimicrobial Activity”. Türk Hijyen ve Deneysel Biyoloji Dergisi 77/4 (December 2020), 399-412.
JAMA Aydın F, Kahve Hİ, Ardıç M, Çakır İ. Comparision of two identification methods: MALDI-TOF-MS & 16s rRNA sequencing in enterococci isolated from raw milk cheeses Sikma cheeses and evaluating their antibiotic susceptibility and antimicrobial activity. Turk Hij Den Biyol Derg. 2020;77:399–412.
MLA Aydın, Furkan et al. “Comparision of Two Identification Methods: MALDI-TOF-MS & 16s RRNA Sequencing in Enterococci Isolated from Raw Milk Cheeses Sikma Cheeses and Evaluating Their Antibiotic Susceptibility and Antimicrobial Activity”. Türk Hijyen Ve Deneysel Biyoloji Dergisi, vol. 77, no. 4, 2020, pp. 399-12.
Vancouver Aydın F, Kahve Hİ, Ardıç M, Çakır İ. Comparision of two identification methods: MALDI-TOF-MS & 16s rRNA sequencing in enterococci isolated from raw milk cheeses Sikma cheeses and evaluating their antibiotic susceptibility and antimicrobial activity. Turk Hij Den Biyol Derg. 2020;77(4):399-412.