ASSESSMENT OF BIOFILM FORMATION, QUORUM SENSING AND MAJOR CARBAPENEMASE GENES OF PSEUDOMONAS AERUGINOSA STRAINS ISOLATED FROM CATTLE CARCASSES

Yıl 2024, Cilt: 49 Sayı: 4, 730 - 740, 14.08.2024

Tolga Uyanık , Ayşegül Bölükbaş , Ali Gücükoğlu , Özgür Çadırcı , Merve Gizem Sezener

https://doi.org/10.15237/gida.GD24043

Öz

This study was carried out to investigate the biofilm formation capacity of carbapenem resistant P. aeruginosa strains isolated from cattle carcasses by using qualitative and quantitative methods, and also to determine the presence of major carbapenemase and quorum sensing genes. In the study, 13 P. aeruginosa strains, each isolated from a different cattle carcass surface and confirmed by molecular methods, were used as materials. Determination of carbapenem resistance was carried out by disk diffusion method. The presence of blaKPC, blaOXA-48, blaNDM, blaVIM, and blaIMP genes was investigated by PCR. Biofilm production was determined by qualitative and quantitative methods. In order to detect quorum sensing systems, the presence of lasI, lasR, rhlI, and rhlR genes was investigated by PCR. In the study, all P. aeruginosa strains were found to be resistant to at least one of the carbapenems tested. Carbapenemase genes could not be detected in any isolate. All isolates showing carbapenem resistance were found to be quantitatively strong biofilm producers. According to PCR analyses, 12 of the 13 isolates analyzed (92.3%) were found to contain all lasI, lasR, rhlI and rhlR genes. It was determined that one isolate (7.69%) contained only lasR and rhlR genes. With this study, it was determined that carbapenem resistance and biofilm production in P. aeruginosa strains of animal origin are related.

Anahtar Kelimeler

Pseudomonas aeruginosa, quorum sensing, biofilm, carbapenem, carcass, slaughterhouse

SIĞIR KARKASLARINDAN İZOLE EDİLEN PSEUDOMONAS AERUGINOSA SUŞLARININ BİYOFİLM OLUŞTURMA ÖZELLİKLERİ İLE QUORUM SENSING VE MAJOR KARBAPENEMAZ GENLERİNİN ARAŞTIRILMASI

Öz

Bu çalışma mezbahalardaki sığır karkaslarından izole edilen ve fenotipik olarak karbapenem direnci gösteren P. aeruginosa suşlarının biyofilm oluşturma kapasitelerini kalitatif ve kantitatif yöntemlerle araştırmak, karbapenem direncinden sorumlu major karbapenemaz ve ayrıca quorum sensing (lasI, lasR, rhlI ve rhlR) genlerinin varlığı belirlemek amacıyla gerçekleştirilmiştir. Çalışmada her biri farklı bir sığır karkas yüzeyinden izole edilen ve moleküler yöntemlerle doğrulanmış 13 P. aeruginosa suşu materyal olarak kullanılmıştır. Çalışmada tüm P. aeruginosa suşlarının test edilen karbapenemlerden en az birine karşı dirençli olduğu belirlenmiştir. Karbapenemaz genleri (blaKPC, blaOXA-48, blaNDM, blaVIM ve blaIMP) hiçbir izolatta tespit edilememiştir. Karbapenem direnci gösteren tüm izolatların kantitatif olarak güçlü biyofilm üreticisi olduğu saptanmıştır. PCR analizleri doğrultusunda analiz edilen 13 izolattan 12’sinin (%92.3) lasI, lasR, rhlI ve rhlR genlerinin tümünü içerdiği tespit edilmiştir. Bir izolatın (%7.69) sadece lasR ve rhlR genlerini içerdiği belirlenmiştir. Yapılan bu çalışma ile hayvansal orjinli P. aeruginosa suşlarında karbapenem direncinin ve biyofilm üretiminin birbirleriyle ilişki içerisinde olduğu belirlenmiştir. Halk sağlığının korunması açısından çiğ etlerden kaynaklanabilecek kontaminasyona karşı dikkatli olunması tavsiye edilmektedir.

Anahtar Kelimeler

Pseudomonas aeruginosa, quorum sensing, biyofilm, karbapenem, karkas, mezbaha

Etik Beyan

Çalışmanın etik kurul izni gerektirmeyen çalışmalar arasında yer aldığını beyan ederiz.

Kaynakça

• Abidi, S.H., Sherwani, S.K., Siddiqui, T.R., Bashir, A., Kazmi, S.U. (2013). Drug resistance profile and biofilm forming potential of Pseudomonas aeruginosa isolated from contact lenses in Karachi-Pakistan. BMC Ophthalmology, 13: 1-6.
• Akyol, N., Gündoğ, D.A., Özkaya, Y., Güngör, C., Onmaz, N.E. (2023). Kasap dükkanları ve şarküterilerde gıda ile temas eden yüzeylerden elde edilen Staphylococcus aureus izolatlarında biyofilm üretiminin fenotipik ve genotipik karakterizasyonu. Erciyes Üniversitesi Veteriner Fakültesi Dergisi, 20(3): 198-205.
• Botta, C., Franciosa, I., Coisson, J., Ferrocino, I., Colasanto, A., Arlorio, M., Cocolin, L., Rantsiou, K. (2023). Beef carcass microbiota after slaughtering and primary cooling: A metataxonomic assessment to infer contamination drivers. Food Research International, 174: 113466.
• Cayci, Y.T., Biyik, I., Birinci, A. (2022). VIM, NDM, IMP, GES, SPM, GIM, SIM metallobetalactamases in carbapenem-resistant Pseudomonas aeruginosa isolates from a Turkish university hospital. Journal of Archives in Military Medicine, 10(1): e118712.
• CDC 2022. COVID-19: U.S. impact on antimicrobial resistance, special report 2022. https://www.cdc.gov/drugresistance/pdf/covid19-impact-report-508.pdf (Erişim tarihi: 23.04.2024)
• Cho, H.H., Kwon, K.C., Kim, S., Park, Y., Koo, S.H. (2018). Association between biofilm formation and antimicrobial resistance in carbapenem-resistant Pseudomonas aeruginosa. Annals of Clinical and Laboratory Science, 48(3): 363-368.
• Choy, M.H., Stapleton, F., Willcox, M.D., Zhu, H. (2008). Comparison of virulence factors in Pseudomonas aeruginosa strains isolated from contact lens-and non-contact lens-related keratitis. Journal of Medical Microbiology, 57(12): 1539-1546.
• Christensen, G.D., Simpson, W.A., Bisno, A.L., Beachey, E.H. (1982). Adherence of slime-producing strains of Staphylococcus epidermidis to smooth surfaces. Infection and Immunity, 37(1): 318-326.
• De Kievit, T. (2009). Quorum sensing in Pseudomonas aeruginosa biofilms. Environmental Microbiology, 11(2), 279-288.
• De Vos, D., Lim Jr, A., Pirnay, J.P., Struelens, M., Vandenvelde, C., Duinslaeger, L., Vanderkelen, A., Cornelis, P. (1997). Direct detection and identification of Pseudomonas aeruginosa in clinical samples such as skin biopsy specimens and expectorations by multiplex PCR based on two outer membrane lipoprotein genes, oprI and oprL. Journal of Clinical Microbiology, 35(6): 1295-1299.
• El-Mahdy, R., El-Kannishy, G. (2019). Virulence factors of carbapenem-resistant Pseudomonas aeruginosa in hospital-acquired infections in Mansoura, Egypt. Infection and Drug Resistance, 12: 3455-3461.
• El Askary, S.A. (2020). Relation between biofilm formation, quorum sensing genes (Rh1I-LasI) and carbapenem resistance among Pseudomonas aeruginosa clinical isolates. Egyptian Journal of Medical Microbiology, 29(2): 1-9.
• Endimiani, A., Carias, L.L., Hujer, A.M., Bethel, C.R., Hujer, K.M., Perez, F., Hutton, R.A., Fox, W.R., Hall, G.S., Jacobs, M.R. (2008). Presence of plasmid-mediated quinolone resistance in Klebsiella pneumoniae isolates possessing blaKPC in the United States. Antimicrobial Agents and Chemotherapy, 52(7): 2680-2682.
• EUCAST (2024). Breakpoint tables for interpretation of MICs and zone diameters. Version 14.0, 2024. http://www.eucast.org (Erişim tarihi: 23.04.2024)
• Faisal, A.J., Ali, M.R., Said, L.A. (2020). Co-existence of lasI, rhI, and Pseudomonas quinolone signal quorum-sensing genes in clinical Pseudomonas aeruginosa isolates. International Journal of Drug Delivery Technology, 10: 338-343.
• Freeman, D., Falkiner, F., Keane, C. (1989). New method for detecting slime production by coagulase negative staphylococci. Journal of Clinical Pathology, 42(8): 872-874.
• Gonçalves, I.R., Dantas, R.C.C., Ferreira, M.L., Batistão, D.W.D.F., Gontijo-Filho, P.P., Ribas, R.M. (2017). Carbapenem-resistant Pseudomonas aeruginosa: association with virulence genes and biofilm formation. Brazilian Journal of Microbiology, 48: 211-217.
• Gröbner, S., Linke, D., Schütz, W., Fladerer, C., Madlung, J., Autenrieth, I.B., Witte, W., Pfeifer, Y. (2009). Emergence of carbapenem-non-susceptible extended-spectrum β-lactamase-producing Klebsiella pneumoniae isolates at the university hospital of Tübingen, Germany. Journal of Medical Microbiology, 58(7): 912-922.
• Gündoğ, D.A., Güngör, G., Güngör, C., Onmaz, N.E., Gönülalan, Z. (2023). Çeşitli gıda kaynaklı patojenik bakterilerin biyofilm oluşturma yeteneklerinin tespitinde kongo kırmızısı agar’ın etkinliğinin değerlendirilmesi. Bozok Veterinary Sciences, 4(1): 18-26.
• Güngör, C., Gündoğ, D.A., Onmaz, N.E. (2023). Mezbaha ortamından izole edilen Enterococcus faecalis izolatlarının biyofilm oluşturma kapasitesi ve biyofilm ile ilişkili virülans genlerin varlığı. Bozok Veterinary Sciences, 4(1): 12-17.
• Gürlük, N., Koluman, A., Kahraman, T. (2022). Gıda işletmelerinde biyofilm sorunu ve gümüş nanopartikül uygulamaları. Aydın Gastronomy, 6(1): 51-63.
• Halat, D.H., Moubareck, C.A. (2020). The current burden of carbapenemases: review of significant properties and dissemination among gram-negative bacteria. Antibiotics, 9(4): 186.
• Heidari, R., Sheikh, F.A., Hashemzadeh, M., Farshadzadeh, Z., Salmanzadeh, S., Saki, M. (2022). Antibiotic resistance, biofilm production ability and genetic diversity of carbapenem-resistant Pseudomonas aeruginosa strains isolated from nosocomial infections in southwestern Iran. Molecular Biology Reports, 49(5): 3811-3822.
• Hemati, S., Azizi-Jalilian, F., Pakzad, I., Taherikalani, M., Maleki, A., Karimi, S., Monjezei, A., Mahdavi, Z., Fadavi, M.R., Sayehmiri, K. (2014). The correlation between the presence of quorum sensing, toxin-antitoxin system genes and MIC values with ability of biofilm formation in clinical isolates of Pseudomonas aeruginosa. Iranian Journal of Microbiology, 6(3): 133.
• Hujer, K.M., Hujer, A.M., Hulten, E.A., Bajaksouzian, S., Adams, J.M., Donskey, C.J., Ecker, D.J., Massire, C., Eshoo, M.W., Sampath, R. (2006). Analysis of antibiotic resistance genes in multidrug-resistant Acinetobacter sp. isolates from military and civilian patients treated at the Walter Reed Army Medical Center. Antimicrobial Agents and Chemotherapy, 50(12): 4114-4123.
• Karami, P., Khaledi, A., Mashoof, R.Y., Yaghoobi, M.H., Karami, M., Dastan, D., Alikhani, M.Y. (2020). The correlation between biofilm formation capability and antibiotic resistance pattern in Pseudomonas aeruginosa. Gene Reports, 18: 100561.
• Karatuna, O., Yagci, A. (2010). Analysis of quorum sensing-dependent virulence factor production and its relationship with antimicrobial susceptibility in Pseudomonas aeruginosa respiratory isolates. Clinical Microbiology and Infection, 16(12): 1770-1775.
• Lee, J., Zhang, L. (2015). The hierarchy quorum sensing network in Pseudomonas aeruginosa. Protein & Cell, 6(1): 26-41.
• Li, X., Gu, N., Huang, T.Y., Zhong, F., Peng, G. (2023). Pseudomonas aeruginosa: a typical biofilm forming pathogen and an emerging but underestimated pathogen in food processing. Frontiers in Microbiology, 13: 1114199.
• McAfee, A.J., McSorley, E.M., Cuskelly, G.J, Moss, B.W., Wallace, J.M., Bonham, M.P., Fearon, A.M. (2010). Red meat consumption: an overview of the risks and benefits. Meat Science, 84(1), 1-13.
• McSharry, S., Koolman, L., Whyte, P., Bolton, D. (2021). The microbiology of beef from carcass chilling through primal storage to retail steaks. Current Research in Food Science, 4: 150-162.
• Meletis, G., Exindari, M., Vavatsi, N., Sofianou, D., & Diza, E. (2012). Mechanisms responsible for the emergence of carbapenem resistance in Pseudomonas aeruginosa. Hippokratia, 16(4), 303. Miller, M.B., & Bassler, B.L. (2001). Quorum sensing in bacteria. Annual Reviews in Microbiology, 55(1): 165-199. Ng, W.L., Bassler, B.L. (2009). Bacterial quorum-sensing network architectures. Annual Review of Genetics, 43: 197-222.
• Ochoa, S.A., López-Montiel, F., Escalona, G., Cruz-Córdova, A., Dávila, L.B., López-Martínez, B., Jiménez-Tapia, Y., Giono, S., Eslava, C., Hernández-Castro, R. (2013). Pathogenic characteristics of Pseudomonas aeruginosa strains resistant to carbapenems associated with biofilm formation. Boletín Medico del Hospital Infantil de México, 70(2): 136-150.
• Perez, L.R.R., Machado, A.B.M.P., Barth, A.L. (2013). The presence of quorum-sensing genes in Pseudomonas isolates infecting cystic fibrosis and non-cystic fibrosis patients. Current Microbiology, 66: 418-420. Poirel, L., Potron, A., Nordmann, P. (2012). OXA-48-like carbapenemases: the phantom menace. Journal of Antimicrobial Chemotherapy, 67(7): 1597-1606.
• Poirel, L., Walsh, T.R., Cuvillier, V., Nordmann, P. (2011). Multiplex PCR for detection of acquired carbapenemase genes. Diagnostic Microbiology and Infectious Disease, 70(1): 119-123.
• Reynolds, D., Kollef, M. (2021). The epidemiology and pathogenesis and treatment of Pseudomonas aeruginosa infections: an update. Drugs, 81(18): 2117-2131.
• Rutherford, S.T., Bassler, B.L. (2012). Bacterial quorum sensing: its role in virulence and possibilities for its control. Cold Spring Harbor Perspectives in Medicine, 2(11): a012427.
• 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.
• TEPGE (2023). Durum ve tahmin kırmızı et 2023. https://arastirma.tarimorman.gov.tr/ (Erişim tarihi: 23.04.2024)
• USDA (2023). 2023 U.S. livestock and poultry outlook. https://www.usda.gov/sites/default/ files/documents/2023aof-Choe.pdf (Erişim tarihi: 23.04.2024)
• Uzunbayır-Akel, N., Tekintaş, Y., Yılmaz, F.F., Öztürk, İ., Okeer, M., Aydemir, S.Ş., Çilli, F. F., Hoşgör-Limoncu, M. (2019). Klinik Pseudomonas aeruginosa izolatlarının virülans özellikleri ve epidemiyolojik ilişkisi. Türk Hijyen ve Deneysel Biyoloji Dergisi, 76(4): 395-404.
• Viducic, D., Murakami, K., Amoh, T., Ono, T., Miyake, Y. (2017). Role of the interplay between quorum sensing regulator VqsR and the Pseudomonas quinolone signal in mediating carbapenem tolerance in Pseudomonas aeruginosa. Research in Microbiology, 168(5): 450-460.
• Wang, X., Gao, K., Chen, C., Zhang, C., Zhou, C., Song, Y., Guo, W. (2023). Prevalence of the virulence genes and their correlation with carbapenem resistance amongst the Pseudomonas aeruginosa strains isolated from a tertiary hospital in China. Antonie van Leeuwenhoek, 116(12): 1395-1406.
• Wyness, L. (2016). The role of red meat in the diet: nutrition and health benefits. Proceedings of the Nutrition Society, 75(3): 227-232.