Investigation of Biofilm Formation Abilities of Escherichia coli, Klebsiella pneumoniae, and Salmonella enterica Isolates from Broiler Chicken Flocks

Öz

Bacteria use biofilm formation as a critical survival strategy to increase their resistance to environmental stress factors and sanitation procedures, which means that it also plays a key role in the persistence of foodborne pathogens in poultry environments. This study investigated the biofilm formation abilities of Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), and Salmonella enterica (S. enterica) isolates obtained from broiler chicken flocks. A total of 90 isolates were tested for biofilm formation ability, comprising 30 isolates from each species. Biofilm formation ability of the isolates was quantitatively examined by the microtiter plate test. The analysis indicated that all isolates (100.0%, 90/90) were biofilm producers, of which 95.6% (86/90) and 4.4% (4/90) were weak and moderate biofilm producers, respectively. No isolates were strong biofilm producers. All E. coli isolates (100.0%, 30/30) were weak biofilm producers. The prevalence of weak biofilm producers (93.3%, 28/30) and moderate biofilm producers (6.7%, 2/30) was similar for both K. pneumoniae and S. enterica isolates. The mean optical density of K. pneumoniae isolates (0.094) was higher than those of the E. coli and S. enterica isolates (0.079 and 0.090, respectively). To the best of knowledge, this study is the first study to investigate biofilm formation ability of E. coli and K. pneumoniae isolates originated from broiler chicken flocks in Türkiye. It was revealed a high prevalence of biofilm formation among the analyzed enteric pathogens in broiler chicken flocks. High biofilm-forming potential increases bacterial persistence in poultry environments, thereby complicating sanitation measures and increasing the risk of foodborne transmission. The present study emphasizes the need for improved control strategies against biofilm-forming bacteria in poultry production systems to reduce the threat to public health.

Anahtar Kelimeler

• Biofilm
• E. coli
• K. pneumoniae
• Microtiter plate method
• Poultry
• S. enterica.

Broyler Tavuk Sürülerinden İzole Edilen Escherichia coli, Klebsiella pneumoniae ve Salmonella enterica İzolatlarının Biyofilm Oluşturma Kapasitelerinin Araştırılması

Öz

Bakteriler, çevresel stres faktörlerine ve sanitasyon uygulamalarına karşı dirençlerini artırmak amacıyla biyofilm oluşumunu hayatta kalma stratejisi olarak kullanmaktadır. Bu durum, biyofilm oluşumunun, kanatlı hayvan yetiştiriciliğinde gıda kaynaklı patojenlerin kalıcılığında kritik bir rol oynamasına neden olmaktadır. Bu çalışmada, broyler tavuk sürülerinden elde edilen Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae) ve Salmonella enterica (S. enterica) izolatlarının biyofilm oluşturma kapasiteleri araştırılmıştır. Her türden 30’ar adet olmak üzere toplam 90 adet izolat, biyofilm oluşturma kapasiteleri açısından değerlendirilmiştir. İzolatların biyofilm oluşturma kapasiteleri, mikrotitre plak testi ile kantitatif olarak incelenmiştir. Yapılan analizler, tüm izolatların (%100,0, 90/90) biyofilm oluşturduğunu göstermiştir. Bu izolatların %95,6’sı (86/90) zayıf, %4,4’ü (4/90) ise orta düzeyde biyofilm üreticisi olarak sınıflandırılmıştır. Güçlü biyofilm üreticisi izolat tespit edilmemiştir. Tüm E. coli izolatları (%100,0, 30/30) zayıf biyofilm üreticisi olarak sınıflandırılmıştır. K. pneumoniae ve S. enterica izolatlarında zayıf (%93,3, 28/30) ve orta düzeyde (%6,7, 2/30) biyofilm üreticisi olma oranları benzer bulunmuştur. K. pneumoniae izolatlarının ortalama optik yoğunluğu (0,094), E. coli ve S. enterica izolatlarına (sırasıyla 0,079 ve 0,090) göre daha yüksek bulunmuştur. Bilinen kadarıyla bu çalışma, Türkiye'deki etlik piliç sürülerinden elde edilen E. coli ve K. pneumoniae izolatlarının biyofilm oluşturma kapasitesini araştıran ilk çalışmadır. Broyler tavuk sürülerinde analiz edilen enterik patojenler arasında biyofilm oluşumunun yüksek yaygınlıkta olduğu ortaya konmuştur. Yüksek biyofilm oluşturma potansiyeli, bakterilerin kümes ortamlarında kalıcılığını artırarak hijyen uygulamalarını zorlaştırmakta ve gıda kaynaklı bulaşma riskini artırmaktadır. Bu çalışma, halk sağlığına yönelik tehditlerin azaltılması amacıyla, kanatlı üretim sistemlerinde biyofilm oluşturan bakterileri hedefleyen gelişmiş kontrol stratejilerinin geliştirilmesi gerektiğini göstermektedir.

Anahtar Kelimeler

• Biyofilm
• E. coli
• Kanatlı
• K. pneumoniae
• Mikrotitre plak yöntemi

Kaynakça

1. Akinola SA, Tshimpamba ME, Mwanza M, Ateba CN (2020). Biofilm production potential of Salmonella serovars isolated from chickens in North West Province, South Africa. Pol J Microbiol, 69 (4), 427-439.
2. Al-Marri T, Al-Marri A, Al-Zanbaqi R, Ajmi AA, Fayez M (2021). Multidrug resistance, biofilm formation, and virulence genes of Escherichia coli from backyard poultry farms. Vet World, 14 (11), 2869-2877.
3. Ashwath P, Bhavyashree C, Gatty AM, Kavitha GM, Sannejal AD (2022). Presence of extended spectrum beta lactamase, virulence genes and resistance determinants in biofilm forming Klebsiella pneumoniae isolated from food sources: A potent risk to the consumers. JPAM, 16 (3), 2099-2109.
4. Barilli E, Vismarra A, Frascolla V, Rega M, Bacci C (2020). Escherichia coli strains isolated from retail meat products: Evaluation of biofilm formation ability, antibiotic resistance, and phylogenetic group analysis. J Food Prot, 83 (2), 233-240.
5. Benameur Q, Gervasi T, Giarratana F, et al. (2021). Virulence, antimicrobial resistance and biofilm production of Escherichia coli isolates from healthy broiler chickens in Western Algeria. Antibiotics, 10, 1157.
6. Chaves AC, Boa Ventura PV, Pereira MS, et al. (2024). Preliminary snapshot reveals a relationship between multidrug-resistance and biofilm production among enterobacteriaceae isolated from fecal samples of farm-raised poultry in ceara, Brazil. Microb Pathog, 193, 106778.
7. Chylkova T, Cadena M, Ferreiro A, Pitesky M (2017). Susceptibility of Salmonella biofilm and planktonic bacteria to common disinfectant agents used in poultry processing. J Food Prot, 80 (7), 1072-1079.
8. European Commission (2015). Commission Regulation (EC) No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs. Off J Eur Union L, 338, 1-26.

9. Corcoran M, Morris D, De Lappe N, et al. (2014). Commonly used disinfectants fail to eradicate Salmonella enterica biofilms from food contact surface materials. Appl Environ Microbiol, 80 (4), 1507-1514.
10. Dishan A, Hizlisoy H, Barel M, et al. (2023). Biofilm formation, antibiotic resistance and genotyping of Shiga toxin-producing Escherichia coli isolated from retail chicken meats. Br Poult Sci, 64 (1), 63-73.
11. EFSA (2024). The European Union One Health 2023 Zoonoses report. EFSA J, 22 (12), e9106.
12. Flemming HC, Wuertz S (2019). Bacteria and archaea on Earth and their abundance in biofilms. Nat Rev Microbiol, 17 (4), 247-260.
13. Grakh K, Mittal D, Prakash A, Jindal N (2022). Characterization and antimicrobial susceptibility of biofilm-producing avian pathogenic Escherichia coli from broiler chickens and their environment in India. Vet Res Commun, 46 (2), 537-548.
14. Grimont PAD, Weill FX (2007). Antigenic formulae of the Salmonella serovars. 9th ed. Paris, France: WHO Collaborating Centre for Reference and Research on Salmonella, Institute Pasteur, 1-166.
15. Gunathilaka GADKK, Dewasmika WAPM, Sandaruwan UM, et al. (2024). Biofilm-forming ability, antibiotic resistance and phylogeny of Escherichia coli isolated from extra intestinal infections of humans, dogs, and chickens. Comp Immunol Microbiol Infect Dis, 105, 102123.
16. Gundogan N, Külahcı MB, Yavas ES (2019). Amino acid decarboxylase activity, biofilm formation and antibiotic resistance of gram-negative bacteria isolated from marine fish, calf meat and chicken. J Food Saf Food Qual, 70 (4), 99–110.
17. Hacioglu O, Turkyilmaz S (2024). Biofilm production potential of Salmonella Enteritidis and Salmonella Infantis serovars isolated from broiler flocks. Isr J Vet Med, 79 (3), 1-8.
18. Hassan A, Usman J, Kaleem F, et al. (2011). Evaluation of different detection methods of biofilm formation in the clinical isolates. Braz J Infect Dis, 15 (4), 305-311.
19. Ibrahim AN, Khalefa HS, Mubarak ST (2023). Residual contamination and biofilm formation by opportunistic pathogens Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa in poultry houses isolated from drinking water systems, fans and floors. EJVS, 54, 1041-1057.
20. Karaca B, Akcelik N, Akcelik M (2013). Biofilm-producing abilities of Salmonella strains isolated from Türkiye. Biologia, 68 (1), 1-10.
21. Khalefa HS, Arafa AA, Hamza D, El-Razik KAA, Ahmed Z (2025). Emerging biofilm formation and disinfectant susceptibility of ESBL-producing Klebsiella pneumoniae. Sci Rep, 15 (1), 1599.
22. Kulac Ö, Baskan C, Kosar N, et al. (2024). Klebsiella pneumoniae clinical isolates: extended spectrum β‑lactamase production, biofilm formation, and virulence factors. Biologia, 79: 3209–3217.
23. Kus, H Arslan U, Türk Daği H, et al. (2017). Investigation of various virulence factors of Klebsiella pneumoniae strains isolated from nosocomial infections. Mikrobiyol Bul, 51 (4), 329-339.
24. Lamas A, Regal P, Vazquez B, et al. (2018). Salmonella and Campylobacter biofilm formation: a comparative assessment from farm to fork. J Sci Food Agric, 98 (11), 4014-4032.
25. Liu X, Yao H, Zhao X, Ge C (2023). Biofilm formation and control of foodborne pathogenic bacteria. Molecules, 28 (6), 1-19.
26. Makhrmash JH, Al-Aidy SR, Qaddoori BH (2022). Investigation of biofilm virulence genes prevalence in Klebsiella pneumoniae isolated from the urinary tract infections. Arch Razi Inst 77 (4), 1421-1427.
27. Ohashi I, Kobayashi S, Tamamura-Andoh Y, Arai N, Takamatsu D (2022). Disinfectant resistance of Salmonella in in vitro contaminated poultry house models and investigation of efficient disinfection methods using these models. J Vet Med Sci, 84 (12), 1633-1644.
28. Oyardi O, Hacioglu M, Yilmaz FN, et al. (2023). Antibiotic susceptibility and biofilm formation of multi-drug resistant Gram-negative bacteria. Istanbul J Pharm, 53 (1), 45-50.
29. Rodrigues SV, Laviniki V, Borges KA, et al. (2019). Biofilm formation by avian pathogenic Escherichia coli is not related to in vivo pathogenicity. Curr Microbiol, 76 (2), 194-199.
30. Saha O, Basri R, Hossain MA, Sultana M (2023). Characterization of multidrug and heavy metal resistance of carbapenemases producing Klebsiella pneumoniae from poultry samples in Bangladesh. Vet Med Sci, 9 (4), 1685-1701.
31. Saha U, Jadhav SV, Pathak KN, Saroj SD (2024). Screening of Klebsiella pneumoniae isolates reveals the spread of strong biofilm formers and class 1 integrons. J Appl Microbiol, 135 (11), lxae275.
32. Seifi K, Kazemian H, Heidari H, et al. (2016). Evaluation of biofilm formation among Klebsiella pneumoniae isolates and molecular characterization by ERIC-PCR. Jundishapur J Microbiol, 9 (1), e30682.
33. Shen X, Yin L, Zhang A, et al. (2023). Prevalence and characterization of Salmonella isolated from chickens in Anhui, China. Pathogens, 12, 465.
34. Sivaranjani M, McCarthy MC, Sniatynski MK, et al. (2022). Biofilm formation and antimicrobial susceptibility of E. coli associated with colibacillosis outbreaks in broiler chickens from Saskatchewan. Front Microbiol, 13, 841516.
35. Stahlhut SG, Struve C, Krogfelt KA, Reisner A (2012). Biofilm formation of Klebsiella pneumoniae on urethral catheters requires either type 1 or type 3 fimbriae. FEMS Immunol Med Microbiol, 65 (2), 350-359.
36. Stepanovic S, Vukovic D, Dakic I, Savic B, Svabic-Vlahovic M (2000). A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods, 40 (2), 175-179.
37. Subhasinghe I, Bandara H, Karunarathna H, et al (2025). Antimicrobial resistance patterns and biofilms resurgence ability of Escherichia coli associated with commercial layer chicken farms in Sri Lanka. Vet Microbiol, 302, 110422.
38. Wingender J, Flemming HC (2011). Biofilms in drinking water and their role as reservoir for pathogens. Int J Hyg Environ, 214, 417-423.
39. Yin W, Wang Y, Liu L, He J (2019). Biofilms: The microbial "Protective Clothing" in extreme environments. Int J Mol Sci, 20, 3423.