تشخیص مولکولی برخی ژن‌های عوامل بیماری‌زا و تشکیل بیوفیلم در جدایه‌های بالینی Yersinia enterocolitica

نوع مقاله : مقاله پژوهشی

نویسندگان

گروه زیست‌شناسی، دانشکده آموزش، دانشگاه القادسیه، عراق.

10.22103/jab.2025.25502.1726

چکیده

هدف: Yersinia enterocolitica یک پاتوژن منتقل‌شده از طریق غذا است که مسئول عفونت‌های گوارشی، به‌ویژه اسهال، می‌باشد که امکان تهاجم به سلول‌های میزبان، فرار از سیستم ایمنی و پایداری در محیط را فراهم می‌کنند. این مطالعه با هدف شناسایی ژن 16S rRNA، ارزیابی ژن‌های مرتبط با بیماری‌زایی (yadA و hreP) و بررسی تشکیل بیوفیلم در جدایه‌های Yersinia enterocolitica از نمونه‌های اسهالی جمع‌آوری‌شده در استان بابل، عراق، انجام شد تا تنوع ژنتیکی و پتانسیل بیماری‌زایی آن‌ها روشن شود.
مواد و روش‌ها: در مجموع 200 نمونه مدفوع از بیماران با سنین مختلف مبتلا به اسهال از بیمارستان‌های مختلف در استان بابل جمع‌آوری شد. جدایه‌ها با استفاده از آزمایش‌های بیوشیمیایی استاندارد و واکنش زنجیره‌ای پلیمراز (PCR) با هدف‌گیری ژن 16S rRNAی Yersinia enterocolitica شناسایی شدند و سپس توالی‌یابی ژن انجام شد. تشخیص مولکولی ژن‌های بیماری‌زایی yadA و hreP با استفاده از پرایمرهای اختصاصی انجام گرفت. تشکیل بیوفیلم از طریق آزمایش‌های کمی ارزیابی شد تا توانایی جدایه‌ها در چسبیدن به سطوح، که نشان‌دهنده پتانسیل پایداری آن‌ها در محیط‌های بالینی است، بررسی شود. داده‌های توالی با استفاده از هم‌ترازی چندگانه توالی و ساخت درخت فیلوژنتیک برای ارزیابی خویشاوندی ژنتیکی تحلیل شدند.
نتایج: تنوع ژنتیکی از طریق توالی‌یابی ژن 16S rRNA ارزیابی شد که نشان‌دهنده حفاظت بالا با شش پلی‌مورفیسم تک‌نوکلئوتیدی (SNP)، عمدتاً انتقالی، در مناطق غیرکدکننده یا ساختاری خنثی بود. جدایه‌های عراقی خویشاوندی نزدیک بین برخی سویه‌ها را نشان دادند، اما در برخی دیگر تنوع فیلوژنتیک مشاهده شد و به سه خوشه اصلی تقسیم شدند: اولین گروه ژنتیکی یکپارچه (FGU)، اولین گروه جدید واگرا (FND) و گروه نهایی منحصربه‌فرد (FU). هم‌ترازی توالی شامل دو توالی عراقی (GenBank: PV628219, PV628221) و 17 توالی مرجع از GenBank بود، با نوکلئوتیدهای حفاظت‌شده با کد رنگی (A: سبز، T: قرمز، G: بنفش و C: آبی) وSNPها برجسته شدند. سویه‌های عراقی شباهت زیادی به توالی مرجع PV628226 داشتند، با SNPهایی در موقعیت‌های 19 و 141 که نشان‌دهنده خویشاوندی نزدیک بود. در مجموع، شش SNP شناسایی شد، به‌طوری‌که سویه‌های PV628219 و PV628221 هر کدام چهار SNP داشتند. تحلیل فیلوژنتیک پروفایل‌های ژنتیکی متنوعی را در میان جدایه‌های Yersinia enterocolitica عراقی تأیید کرد،
نتیجه‌گیری: این مطالعه تنوع ژنتیکی و پتانسیل بیماری‌زایی جدایه‌های Yersinia enterocolitica از استان بابل، عراق را برجسته می‌کند. شناسایی SNPها در ژن 16S rRNA و حضور ژن‌های بیماری‌زایی yadA و hreP در اکثر جدایه‌ها، اساس مولکولی بیماری‌زایی آن‌ها را نشان می‌دهد. تشکیل بیوفیلم مشاهده‌شده، مکانیسمی برای پایداری محیطی را پیشنهاد می‌کند که چالش‌هایی برای کنترل عفونت ایجاد می‌کند.

کلیدواژه‌ها

عنوان مقاله [English]

Molecular detection of some virulence factors genes and biofilm formation in clinical isolates of Yersinia enterocolitica

نویسندگان [English]

  • Baneen Kareem
  • Hadaf Kadhim
Department of Biology, College of Education, University of Al-Qadisiyah, Iraq.
چکیده [English]

Objective
Yersinia enterocolitica is a foodborne pathogen responsible for gastrointestinal infections, notably diarrhea, with its virulence attributed to genetic factors enabling host cell invasion, immune evasion, and environmental persistence. This investigation aimed to detect the 16S rRNA gene, evaluate virulence-related genes (yadA and hreP), and evaluate biofilm formation in Y. enterocolitica isolates from diarrheal samples gathered in Babylon Province, Iraq, to elucidate their genetic diversity and pathogenic potential.

Materials and Methods
A total of 200 stool samples from patients of varying ages with diarrhea were gathered from multiple hospitals across Babylon Province. Isolates were identified applying standard biochemical experiments and polymerase chain reaction (PCR) targeting the 16S rRNA gene of Y. enterocolitica, followed by gene sequencing. Molecular diagnosis of virulence genes yadA and hreP was carried out applying specific primers. Biofilm formation was evaluated through quantitative assays to identify the isolates’ capability to adhere to surfaces, reflecting their potential for persistence in clinical environments. Sequence data were analyzed applying multiple sequence alignment and phylogenetic tree construction to evaluate genetic relatedness.

Results
Genetic diversity was evaluated via 16S rRNA gene sequencing, revealing high conservation with six single nucleotide polymorphisms (SNPs), primarily transitions, located in non-coding or structurally neutral regions. Iraqi isolates demonstrated close relatedness among some strains but phylogenetic diversity in others, clustering into three major clades: First Genetically Unified (FGU), First New Diverging (FND), and Final Unique (FU). Sequence alignment included two Iraqi sequences (GenBank: PV628219, PV628221) and 17 reference sequences from GenBank, with conserved nucleotides color-coded (A: green, T: red, G: purple, C: blue) and SNPs highlighted. Iraqi strains were highly similar to the reference sequence PV628226, with SNPs at positions 19 and 141 denoting close relatedness. In total, six SNPs were identified, with strains PV628219 and PV628221 each exhibiting four SNPs. Phylogenetic analysis affirmed diverse genetic profiles among Iraqi Y. enterocolitica isolates, with some strains closely related and others more divergent. Virulence genes yadA and hreP were detected in 87.5% of isolates (7/8), suggesting meaningful pathogenic potential. Biofilm formation assays revealed that most isolates exhibited moderate to strong biofilm production, denoting their capacity to persist in clinical settings.

Conclusions
This investigation highlights the genetic diversity and pathogenic potential of Y. enterocolitica isolates from Babylon Province, Iraq. The identification of SNPs in the 16S rRNA gene and the presence of virulence genes yadA and hreP in most isolates underscore their molecular basis for pathogenicity.

کلیدواژه‌ها [English]

  • 16S rRNA
  • biofilm formation
  • clinical isolates
  • genetic diversity
  • virulence genes

مراجع

Ahsani, M. R., Mohammadabadi, M. R., & Shamsaddini, M. B. (2010). Clostridium perfringens isolate typing by multiplex PCR. Journal of Venomous Animals and Toxins Including Tropical Diseases, 16(4), 573–578. https://doi.org/10.1590/S1678-91992010000400006
Al-Rawe, A., Al-Jomaily, O., Yousif, Y., Shaban, S., & Suleiman, A. (2023). Comparative Genomics, Phylogenetic and Functional Analysis of Yersinia enterocolitica, a Gastrointestinal Pathogen, with Other Soil-Borne Bacteria Causing Diseases. Mikrobiolohichnyi Zhurnal, 85(5), 31-41. https://doi.org/10.15407/microbiolj85.05.031
 Al-Rudha, A. M., Khalil, N. K., & Al-Taii, N. A. (2021). Evaluation of bacterial contaminants and heavy metals in cow and buffalo raw milk sold in Baghdad governorate. Iraqi Journal of Veterinary Sciences, 35(Supplement III), 101-105. https://doi.org/10.33899/ijvs.2021.131744.1999
Cheng, S., Li, Z., Bai, X., Feng, J., Su, R., Song, L., Yang, H., Zhan, X., Xia, X., Lü, X., & Shi, Ch. (2023). The biochemical characteristics of viable but nonculturable state Yersinia enterocolitica induced by lactic acid stress and its presence in food systems. Food Research International, 170, 113024. https://doi.org/10.1016/j.foodres.2023.113024
Doshi, P., Sivaram, G., & Sievers, C. (2024). An atypical case of Yersinia enterocolitica infection in a patient suspected with ulcerative colitis flare-up. Cureus, 16(2). https://doi.org/10.7759/cureus.53780
Heusipp, G., Young, G. M., & Miller, V. L. (2001). HreP, an in vivo-expressed protease of Yersinia enterocolitica, is a new member of the family of subtilisin/kexin-like proteases. Journal of Bacteriology, 183(12), 3556-3563.‏ https://doi.org/10.1128/JB.183.12.3556-3563.2001
Ioannidis, A., Kyratsa, A., Ioannidou, V., Bersimis, S., & Chatzipanagiotou, S. (2014). Detection of biofilm production of Yersinia enterocolitica strains isolated from infected children and comparative antimicrobial susceptibility of biofilm versus planktonic forms. Molecular Diagnosis & Therapy, 18(3), 309-314. https://doi.org/10.1007/s40291-013-0080-1
Kanan, T. A., & Abdulla, Z. A. (2009). Isolation of Yersinia spp. from cases of diarrhoea in Iraqi infants and children. Eastern Mediterranean Health Journal, 15(2),‏ 276-284. https://pubmed.ncbi.nlm.nih.gov/19554973/
Karlsson, P. A., Tano, E., Jernberg, C., Hickman, R. A., Guy, L., Järhult, J. D., & Wang, H. (2021). Molecular characterization of multidrug-resistant Yersinia enterocolitica from foodborne outbreaks in Sweden. Frontiers in Microbiology, 12, 664665. https://doi.org/10.3389/fmicb.2021.664665
Khabiri, A., Toroghi, R., Mohammadabadi, M., & Tabatabaeizadeh, S. E. (2023). Introduction of a Newcastle disease virus challenge strain (sub-genotype VII.1.1) isolated in Iran. Veterinary Research Forum, 14(4), 221–227. https://doi.org/10.30466/vrf.2022.548152.3373
Khabiri, A., Toroghi, R., Mohammadabadi, M., & Tabatabaeizadeh, S. E. (2025). Whole genome sequencing and phylogenetic relative of a pure virulent Newcastle disease virus isolated from an outbreak in northeast Iran. Letters in Applied Microbiology, 78(4), Article ovaf049. https://doi.org/10.1093/lambio/ovaf049
 Kim, T. J., Young, B. M., & Young, G. M. (2008). Effect of flagellar mutations on Yersinia enterocolitica biofilm formation. Applied and Environmental Microbiology, 74(17), 5466-5474.‏ https://doi.org/10.1128/AEM.00222-08
 Kislichkina, A. A., Sizova, A. A., Skryabin, Y. P., Dentovskaya, S. V., & Anisimov, A. P. (2025). Evaluation of 16S rRNA genes sequences and genome-based analysis for identification of non-pathogenic Yersinia. Frontiers in Microbiology, 15, e1519733. https://doi.org/10.3389/fmicb.2024.1519733
 Kot, B., Piechota, M. A. Ł. G. O. R. Z. A. T. A., Zdunek, E., Borkowska, D., & Binek, T. (2011). Occurrence of virulence genes and biofilm formation in Yersinia enterocolitica isolated from pigs. Bulletin of the Veterinary Institute in Pulawy, 55(2), 187-191.‏ http://bulletin.piwet.pulawy.pl/images/stories/pdf/20112/20112187192.pdf
 Kot, B., Piechota, M., & Jakubiak, K. (2017). Virulence genes and antibiotic resistance of Yersinia enterocolitica strains isolated from children. European Journal of Biological Research, 7(4), 366-373.‏ http://dx.doi.org/10.5281/zenodo.1064835
 Łada, P., Kończyk-Kmiecik, K., & Bancerz-Kisiel, A. (2023). Isolation, characterization and antimicrobial resistance of Yersinia enterocolitica from Polish cattle and their carcasses. BMC Veterinary Research, 19(1), 170. https://doi.org/10.1186/s12917-023-03734-w.
 Lenchenko, E., Lozovoy, D., Strizhakov, A., Vatnikov, Y., Byakhova, V., Kulikov, E., Sturov, N., Kuznetsov, V., Avdotin, V., & Grishin, V. (2019). Features of formation of Yersinia enterocolitica biofilms. Veterinary World, 12(1), 136.‏ https://doi.org/10.14202/vetworld.2019.136-140
Lou, X., Wu, Y., Chen, Z., Zhang, Q., Xiao, X., & Fang, Z. (2025). Novel insights into biofilm formation and the key differentially expressed genes in Yersinia enterocolitica from meat: Implications for food safety and disease prevention. International Journal of Food Microbiology, 426, 110914. https://doi.org/10.1016/j.ijfoodmicro.2024.110914.
Ma, G., Ding, Y., Wu, Q., Zhang, J., Liu, M., Wang, Z., Wang, Z., Wu, S., Yang, X., & Li, Y. (2022). Yersinia enterocolitica-derived outer membrane vesicles inhibit initial stage of biofilm formation. Microorganisms, 10(12), 2357. https://doi.org/10.3390/microorganisms10122357
Meng, J., Bai, J., Xu, J., Huang, C., & Chen, J. (2019). Differential regulation of physiological activities by RcsB and OmpR in Yersinia enterocolitica. FEMS Microbiology Letters, 366(17), https://doi.org/fnz210. 10.1093/femsle/fnz210
 Meuskens, I., Leva-Bueno, J., Millner, P., Schütz, M., Peyman, S. A., & Linke, D. (2022). The trimeric autotransporter adhesin YadA of Yersinia enterocolitica serotype O: 9 binds glycan moieties. Frontiers in Microbiology, 12, 738818.‏ https://doi.org/10.3389/fmicb.2021.738818
Mohammadabadi, M. R., Shaikhaev, G. O., Sulimova, G. E., & Rahman, O. (2004). Detection of bovine leukemia virus proviral DNA in Yaroslavl, Mongolian, and Black Pied cattle by PCR. Cellular and Molecular Biology Letters, 9(4A), 766–768.
Mohammadabadi, M. R., Soflaei, M., Mostafavi, H., & Honarmand, M. (2011). Using PCR for early diagnosis of bovine leukemia virus infection in some native cattle. Genetics and Molecular Research, 10(4), 2658–2663. https://doi.org/10.4238/2011.October.27.2
Mohammadabadi, M., Afsharmanesh, M., Khezri, A., Kheyrodin, H., Babenko, O. I., Borshch, O., Kalashnyk, O., Nechyporenko, O., Afanasenko, V., Slynko, V., & Usenko, S. (2025). Effect of mealworm on GBP4L gene expression in the spleen tissue of Ross broiler chickens. Agricultural Biotechnology Journal, 17(2), 343–360. https://doi.org/10.22103/jab.2025.25277.1714
Mohammadabadi, M., Babenko, O. I., Borshch, O., Kalashnyk, O., Ievstafiieva, Y., & Buchkovska, V. (2024). Measuring the relative expression pattern of the UCP2 gene in different tissues of the Raini Cashmere goat. Agricultural Biotechnology Journal, 16(3), 317–332. https://doi.org/10.22103/jab.2024.24337.1627
 Özdemir, F., & Arslan, S. (2022). Biofilm formation, siderophore production, virulence-associated genes and genetic diversity of Yersinia enterocolitica from food. Journal of the Hellenic Veterinary Medical Society, 73(2), 4077-4084. https://www.openarchives.gr/aggregator-openarchives/edm/JHVMS/000098-26428
Peruzy, M. F., Murru, N., Perugini, A. G., Capuano, F., Delibato, E., Mercogliano, R., Korkeala, H., & Proroga, Y. T. R. (2017). Evaluation of virulence genes in Yersinia enterocolitica strains using SYBR Green real-time PCR. Food Microbiology, 65, 231-235. https://doi.org/10.1016/j.fm.2017.03.004
Schindler, M. K., Schütz, M. S., Mühlenkamp, M. C., Rooijakkers, S. H., Hallström, T., Zipfel, P. F., & Autenrieth, I. B. (2012). Yersinia enterocolitica YadA mediates complement evasion by recruitment and inactivation of C3 products. Journal of Immunology, 189(10), 4900-4908. https://doi.org/10.4049/jimmunol.1201383
 Schutz, M., Weiss, E. M., Schindler, M., Hallstrom, T., Zipfel, P. F., Linke, D., & Autenrieth, I. B. (2010). Trimer stability of YadA is critical for virulence of Yersinia enterocolitica. Infection and Immunity, 78(6), 2677-2690.‏ https://doi.org/10.1128/IAI.01350-09
 Shabana, S. M., Khalil, S. A., & Hegazy, A. E. H. M. (2015). Molecular characterization of Yersinia enterocolitica isolated from chicken meat samples. Alexandria Journal of Veterinary Sciences, 46(1), 124-129.‏ https://doi.org/10.5455/ajvs.184190
Shahdadnejad, N., Mohammadabadi, M. R., & Shamsadini, M. (2016). Typing of Clostridium perfringens isolated from broiler chickens using multiplex PCR. Genetics in the Third Millennium, 14(4), 4368–4374.
 Shoaib, M., Shehzad, A., Raza, H., Niazi, S., Khan, I. M., Akhtar, W., Safdar, W., & Wang, Z. (2019). A comprehensive review on the prevalence, pathogenesis and detection of Yersinia enterocolitica. RSC Advances, 9(70), 41010-41021.‏ https://doi.org/10.1039/c9ra06988g
 Sotohy, S. A., Diab, M. S., Ewida, R. M., Aballah, A., & Eldin, N. K. A. (2024). An investigative study on Yersinia enterocolitica in animals, humans and dried milk in New Valley Governorate, Egypt. BMC Microbiology, 24(1), 395.‏ https://doi.org/10.1186/s12866-024-03527-7
 Tadesse, D. A., Bahnson, P. B., Funk, J. A., Morrow, W. M., Abley, M. J., Ponte, V. A., Thakur, S., Wittum, T., DeGraves, F. J., Rajala-Schultz, P. J., & Gebreyes, W. A. (2013). Yersinia enterocolitica of porcine origin: carriage of virulence genes and genotypic diversity. Foodborne Pathogens and Disease, 10(1), 80-86. https://doi.org/10.1089/fpd.2011.1120
Tian, L., Wu, M., Guo, W., Li, H., Gai, Z., & Gong, G. (2021). Evaluation of the membrane damage mechanism of chlorogenic acid against Yersinia enterocolitica and Enterobacter sakazakii and its application in the preservation of raw pork and skim milk. Molecules, 26(21), 6748. https://doi.org/10.3390/molecules26216748
Ventola, E., Lovari, S., Farneti, S., Finazzi, G., Bilei, S., Owczarek, S., & Delibato, E. (2023). Molecular characterization of Yersinia enterocolitica strains to evaluate virulence associated genes. Annali dell’Istituto Superiore di Sanità, 59(4), 280–285. https://doi.org/10.4415/ANN_23_04_07
Wagner, K., Schilling, J., Fälker, S., Schmidt, M. A., & Heusipp, G. (2009). A regulatory network controls expression of the in vivo-expressed HreP protease of Yersinia enterocolitica. Journal of Bacteriology, 191(5), 1666-1676.‏ https://doi.org/10.1128/JB.01517-08
 Wang, H., Tay, M., Palmer, J., & Flint, S. (2017). Biofilm formation of Yersinia enterocolitica and its persistence following treatment with different sanitation agents. Food Control, 73, 433-437. https://doi.org/10.1016/j.foodcont.2016.08.033
Wang, J., Liu, M., Wang, H., Wu, Q., Ding, Y., Xu, T., Ma, G., Zhong, Y., Zhang, J., & Chen, M. (2021). Occurrence, molecular characterization, and antimicrobial susceptibility of Yersinia enterocolitica isolated from retail food samples in China. LWT, 150, 111876. https://doi.org/10.1016/j.lwt.2021.111876
 Woese, C. R. (1987). Bacterial evolution. Microbiological Reviews, 51(2), 221-271.‏ https://doi.org/10.1128/mr.51.2.221-271.1987
 Younis, G. A., Elkenany, R. M., & Dowidar, H. A. (2021). Virulence genotyping and antimicrobial resistance profiles of Yersinia enterocolitica isolated from meat and meat products in Egypt. Brazilian Journal of Biology Revista Brasleira de Biologia, 81(2), 424-436. https://doi.org/10.1590/1519-6984.229998
 Zheng, H., Sun, Y., Mao, Z., & Jiang, B. (2008). Investigation of virulence genes in clinical isolates of Yersinia enterocolitica. FEMS Immunology & Medical Microbiology, 53(3), 368-374. https://doi.org/10.1111/j.1574-695X.2008.00436.x.
مجله بیوتکنولوژی کشاورزی
دوره 17، شماره 3
شهریور 1404
صفحه 218-201

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