Yersinia ruckeri İzolatlarında Biyofilm Oluşumunun ve Bazı Virulens Faktörlerinin Belirlenmesi

Yıl 2023, , 81 - 88, 20.06.2023

Esra Demirbaş , Cansu Aktaş , Volkan Kaydu , Enescan Aksoy , İlker Hancı Ünver Oğuzhan Tekay , Meltem Özer Ertan Emek Onuk

https://doi.org/10.35864/evmd.1239732

Öz

Çalışmada gökkuşağı alabalıklarından izole edilen 20 adet Yersinia ruckeri izolatının biyofilm oluşturma yeteneği ve bazı virülens faktörleri fenotipik olarak incelendi. İzolatların biyofilm oluşturma yeteneklerinin belirlenmesinde Kongo Red Agar (CRA), Modifiye Tüp Aderans (Christensen) ve Mikroplak yöntemleri kullanıldı. İzolatların hareket özelliği, hemolitik aktivitesi, lipaz aktivitesi ve proteaz aktivitesi fenotipik metotlar ile ortaya konuldu. CRA ve Christensen yöntemleri ile izolatlar biyofilm oluşturmazken mikroplak yönteminde izolatların büyük bir kısmının zayıf düzeyde biyofilm oluşturduğu saptandı. İzolatların 13’ünün hareketli ve lipaz aktivitesine sahip olduğu belirlenirken, 14 izolatın ise proteaz aktivitesine sahip olduğu belirlendi. Ek olarak izolatların tamamının nonhemolitik olduğu saptandı. Çalışma sonuçlarına göre sadece hareket ve lipaz aktivitesi arasında istatiksel olarak anlamlı, pozitif yönlü, güçlü bir korelasyon saptandı. Mikroorganizmaların patojenitesi virulens faktörleri ve biyofilm oluşumu ile bağlantılı bir süreçtir. Dolayısıyla Y. ruckeri izolatlarının virulens faktörlerinin ve biyofilm oluşumlarının periyodik olarak izlenmesi, Y. ruckeri enfeksiyonlarının önlenmesi veya tedavi edilmesinde anti-biyofilm terapilerinin geliştirilmesi gibi yeni stratejilerin oluşturulmasına önemli katkılar sağlayacaktır.

Anahtar Kelimeler

Akuakültür, Biyofilm, Virulens faktörleri, Yersinia ruckeri

Determination of Biofilm Formation and Some Virulence Factors in Yersinia ruckeri Isolates

Öz

In the current study, biofilm forming ability and some virulence factors of 20 Yersinia ruckeri isolates obtained from rainbow trout were examined phenotypically. The Congo Red Agar (CRA), Modified Tube Aderans (Christensen) and Microplate methods were used to determine the biofilm forming ability of these isolates. The motility characteristics, hemolytic activity, lipase activity and protease activity of the isolates were determined by phenotypic tests. It was determined that the majority of the isolates produced a weak biofilm when examined using the microplate method, despite the fact that isolates did not produce biofilms when evaluated using the CRA and Christensen methods. While 14 isolates were discovered to have protease activity, 13 of the isolates were determined to be motile and to have lipase activity. All of the isolates were also determined to be nonhemolytic. According to the study's results, a statistically significant, positive and strong correlation was found only between motility and lipase activity. The pathogenicity of microorganisms is associated with virulence factors and biofilm formation. Therefore routine monitoring of the virulence factors and biofilm formation of Y. ruckeri isolates will significantly contribute to the establishment of novel techniques such as the development of anti-biofilm therapies for prevention or treatment of Y. ruckeri infections.

Anahtar Kelimeler

Yersinia ruckeri, Aquaculture, Biofilm, Virulence factors

Kaynakça

• Akışoğlu Ö, Engin D, Sarıçam S, Müştak HK, Şener B, ve Hasçelik G. (2019). Kistik fibrozisli ve kistik fibrozisi olmayan hasta grubunda Burkholderia türlerinin multilokus sekans analizi, biyofilm oluşturma, antibiyotik duyarlılık ve sinerji testleri. Mikrobiyol Bul. 53(1), 22-36.
• Altun S, Onuk EE, Çiftci A, Duman M, & Büyükekiz AG. (2013). Determination of phenotypic, serotypic and genetic diversity and antibiotyping of Yersinia ruckeri isolated from rainbow trout. Kafkas Unıv Vet Fak Derg. 19 (2): 225-232. (2013)
• Altinok I, Capkin E, & Boran H. (2016). Comparison of molecular and biochemical heterogeneity of Yersinia ruckeri strains isolated from Turkey and the USA. Aquaculture. 450, 80-88.
• Arias CR, Olivares-Fuster O, Hayden K, Shoemaker CA, Grizzle JM, & Klesius PH. (2007). First report of Yersinia ruckeri biotype 2 in the USA. J Aquat Anim Health. 19(1), 35-40.
• Atshan SS, Shamsudin MN, Thian Lung LT, Sekawi Z, Ghaznavi-Rad E, & Pei Pei C. (2012). Comparative characterisation of genotypically different clones of MRSA in the production of biofilms. J Biomed Biotechnol. 2012.
• Banu A, Kabbin J, Anand M. (2011). Extraintestinal infections due to Escherichia coli: an emerging issue. J Clin Diag Res. 5:486–90.
• Bell, M. (2001). Biofilms: a clinical perspective. Curr Infect Dis Rep. 3: 483–486.
• Busch RA, & Lingg AJ. (1975). Establishment of an asymptomatic carrier state infection of enteric redmouth disease in rainbow trout (Salmo gairdneri). J Fish Res Board Can. 32(12), 2429-2432.
• Cai W, & Arias CR. (2017). Biofilm formation on aquaculture substrates by selected bacterial fish pathogens. J Aquat Anim. 29(2), 95-104.
• Calvez S, Gantelet H, Blanc G, Douet DG, & Daniel P. (2014). Yersinia ruckeri biotypes 1 and 2 in France: presence and antibiotic susceptibility. Dis Aquat Org. 109(2), 117-126.
• Chenia HY, & Duma S. (2017). Characterization of virulence, cell surface characteristics and biofilm‐forming ability of Aeromonas spp. isolates from fish and sea water. J Fish Dis. 40(3), 339-350.
• Christensen GD, Simpson WA, Younger JJ, Baddour LM., Barrett FF, Melton DM, & Beachey EH. (1985). Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. J Clin Microbiol. 22(6), 996-1006.
• Ciftci A, Findik A, Onuk EE, & Savasan S. (2009). Detection of methicillin resistance and slime factor production of Staphylococcus aureus in bovine mastitis. Braz J Microbiol. 40, 254-261.
• Costerton J, Stewart P & Greenberg E. (1999). Bacterial biofilms: a common cause of persistent infections. Science. 284(5418), 1318-1322.
• Coquet L, Cosette P, Junter GA, Beucher E, Saiter JM, Jouenne T. (2002a). Adhesion of Yersinia ruckeri to fish farm materials: influence of cell and material surface properties. Colloids Surf B:Biointerfaces. 26(4), 373–378 . Coquet L, Cosette P, Quillet L, Petit F, Junter G, Jouenne T. (2002b). Occurrence and phenotypic characterization of Yersinia ruckeri strains with biofilm-forming capacity in a rainbow trout farm. Appl Environ Microbiol. 68(2), 470–475.
• Çagırgan H, Yüreklitürk O. First isolation of Yersinia ruckeri from rainbow trout in Turkey. Fifth International Conference, Disease of Fish and Shellfish Book of Abstracts 1991;131.
• Duman M, Altun S, Cengiz M, Saticioglu IB, Buyukekiz AG, & Sahinturk P. (2017). Genotyping and antimicrobial resistance genes of Yersinia ruckeri isolates from rainbow trout farms. Dis Aquat Org. 125(1), 31-44.
• Filik F & Kubilay A. (2019a). Bazı bakteriyel balık patojenlerinde biyofilm oluşumunun farklı in vitro metodlarla tespiti. Acta Aquat Turc. 15(3), 378-390.
• Filik N & Kubilay A. (2019b). Yersinia ruckeri suşlarında çevreyi algılama sistemi ve yönetimindeki virülens faktörlerinin araştırılması. Acta Aquat Turc. 15(3), 391-403.
• Fouz B, Zarza C, & Amaro C. (2006). First description of non‐motile Yersinia ruckeri serovar I strains causing disease in rainbow trout, Oncorhynchus mykiss (Walbaum), cultured in Spain. J Fish Dis. 29(6), 339-346.
• Gajdács M, Baráth Z, Kárpáti K, Szabó D, Usai D, Zanetti S, & Donadu MG. (2021). No correlation between biofilm formation, virulence factors, and antibiotic resistance in Pseudomonas aeruginosa: results from a laboratory-based in vitro study. Antibiotics. 10(9), 1134.
• Guijarro J A, García-Torrico AI, Cascales D, & Méndez J. (2018). The infection process of Yersinia ruckeri: reviewing the pieces of the Jigsaw puzzle. Front Cell Infect Microbiol. 8, 218.
• Hassan A, Usman J, Kaleem F, Omair M, Khalid A, & Iqbal M. (2011). Evaluation of different detection methods of biofilm formation in the clinical isolates. Braz J Infect Dis. 15, 305-311.
• Horne MT, Barnes AC. (1999). Enteric Red Mouth Diseases (Yersinia ruckeri) In: Fish Diseases and Disorders Volume 3. Viral, Bacterial and Fungal Infections. Ed.: P.T.K. Woo, D.W. Bruno, CAB International, New York, USA. p.455-479.
• Igbinosa IH, Beshiru A, Odjadjare EE, Ateba CN, & Igbinosa EO. (2017). Pathogenic potentials of Aeromonas species isolated from aquaculture and abattoir environments. Microb Pathog. 107, 185-192.
• Kumar D, Kumar L, Nagar S, Raina C, Parshad R, & Gupta VK. (2012). Screening, isolation and production of lipase/esterase producing Bacillus sp. strain DVL2 and its potential evaluation in esterification and resolution reactions. Arch Appl Sci Res. 4(4), 1763-1770.
• Kumar G, Menanteau-Ledouble S, Saleh M, & El-Matbouli M. (2015). Yersinia ruckeri, the causative agent of enteric redmouth disease in fish. Vet Res. 46(1), 1-10.
• Lavender HF, Jagnow JR, Clegg S. (2004). Biofilm formation in vitro and virulence in vivo of mutants of Klebsiella pneumoniae. Infect Immun. 72: 4888–4890.
• Marma CC, Ahmed S, Akhter F, Keya SA, Ullah P, & Barua S. (2022). Detection of Biofilm Producing Uropathogenic Bacteria and Their Antibiotic Sensitivity Pattern. Sch J App Med Sci. 8, 1244-1251.
• Mohamed A, Rajaa AM, Khalid Z, Fouad M, & Naima R. (2016). Comparison of three methods for the detection of biofilm formation by clinical isolates of Staphylococcus aureus isolated in Casablanca. Int J Sci Res. 5(10), 1156-9.
• Moreau E, Thomas T, Brevet M, Thorin C, Fournel C, & Calvez S. (2019). Mutations involved in the emergence of Yersinia ruckeri biotype 2 in France. Transbound Emerg Dis. 66(3), 1387-1394.
• Onuk EE, Çiftçi A, Fındık A, Çiftçi G, Altun S, Balta F, Özer S & Çoban AY. (2011). Phenotypic and molecular characterization of Yersinia ruckeri isolates from Rainbow Trout (Oncorhynchus mykiss, Walbaum, 1792) in Turkey. Berl Munch Tierarztl Wonchenschr. 124 (7-8): 320-328.
• Öztürk RÇ, & Altınok İ. (2014). Bacterial and viral fish diseases in Turkey. Turkish J Fish Aquat Sci. 14(1). Pajdak‐Czaus J, Platt‐Samoraj A, Szweda W, Siwicki AK, & Terech‐Majewska E. (2019). Yersinia ruckeri-A threat not only to rainbow trout. Aquac Res. 50(11), 3083-3096.
• Rather MA, Gupta K, Bardhan P, Borah M, Sarkar A, Eldiehy KS, ... & Mandal M. (2021). Microbial biofilm: A matter of grave concern for human health and food industry. J Basic Microbiol. 61(5), 380-395.
• Ríos‐Castillo AG, Thompson KD, Adams A, Marín de Mateo M, & Rodríguez‐Jerez, JJ. (2018). Biofilm formation of Flavobacterium psychrophilum on various substrates. Aquac Res. 49(12), 3830-3837.
• Romalde JL, & Toranzo AE. (1993). Pathological activities of Yersinia ruckeri, the enteric redmouth (ERM) bacterium. FEMS Microbiol Lett. 112(3), 291-299.
• Romalde JL, Magarinos B, Barja JL, & Toranzo AE. (1993). Antigenic and molecular characterization of Yersinia ruckeri proposal for a new intraspecies classification. Syst Appl Microbiol. 16(3), 411-419.
• Saffari F, Dalfardi MS., Mansouri S, & Ahmadrajabi R. (2017). Survey for correlation between biofilm formation and virulence determinants in a collection of pathogenic and fecal Enterococcus faecalis isolates. Infect Chemother. 49(3), 176-183.
• Satpathy S, Sen SK, Pattanaik S, & Raut S. (2016). Review on bacterial biofilm: an universal cause of contamination. Biocatal Agric Biotechnol. 7, 56-66.
• Soto E, Coleman D, Yazdi Z, Purcell SL, Camus A, & Fast MD. (2021). Analysis of the white sturgeon (Acipenser transmontanus) immune response during immunostimulation and Veronaea botryosa infection. Comparative Biochemistry and Physiology Part D: Genomics and Proteomics. 40, 100879.
• Stepanović S, Vuković D, Hola V, Bonaventura GD, 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.
• Temel A, & Eraç B. (2018). Bakteriyel biyofilmler: Saptama yöntemleri ve antibiyotik direncindeki rolü. Turk Mikrobiyol Cemiy Derg. 48(1), 1-13.
• Tinsley JW, Lyndon AR, & Austin B. (2011). Antigenic and cross‐protection studies of biotype 1 and biotype 2 isolates of Yersinia ruckeri in rainbow trout, Oncorhynchus mykiss (Walbaum). J Appl Microbiol. 111(1), 8-16.
• Tobback E, Decostere A, Hermans K, Haesebrouck F, & Chiers K. (2007). Yersinia ruckeri infections in salmonid fish. J Fish Dis. 30(5), 257-268.
• Torabi Delshad S, Soltanian S, Sharifiyazdi H, & Bossier P. (2019). Effect of catecholamine stress hormones (dopamine and norepinephrine) on growth, swimming motility, biofilm formation and virulence factors of Yersinia ruckeri in vitro and an in vivo evaluation in rainbow trout. J Fish Dis. 42(4), 477-487.
• Wrobel A, Leo JC, & Linke D. (2019). Overcoming fish defences: the virulence factors of Yersinia ruckeri. Genes, 10(9), 700.
• Wrobel A, Saragliadis A, Pérez‐Ortega J, Sittman C, Göttig S, Liskiewicz K, ... & Linke D. (2020). The inverse autotransporters of Yersinia ruckeri, YrInv and YrIlm, contribute to biofilm formation and virulence. Environ Microbiol. 22(7), 2939-2955.
• Yang Q, Anh ND, Bossier P, & Defoirdt T. (2014). Norepinephrine and dopamine increase motility, biofilm formation, and virulence of Vibrio harveyi. Front Microbiol. 5, 584.