Capsular serotyping and molecular characterization of iraqi strains of Klebsiella pneumoniae by multiplex polymerase chain reaction (PCR) and pulsed-field gel electrophoresis (PFGE)
Article Main
Abstract
Typing, especially for pathogenic bacteria, is crucial for tracking outbreaks, monitoring the spread of infectious diseases, and consequently guiding effective infection control measures. the aim of the present work is to conduct molecular typing of Klebsiella pneumoniae strains, with a particular focus on determining the capsular serotype expressed by their genes. Two typing methods were employed to determine the capsular (K) serotype and for molecular characterization of six K. pneumoniae strains (R1, R2, R3, R4, R5, R6). The first method using Multiplex PCR with primers targeting five capsule-associated genes: K1, K2, K5, K20, and K54. The results showed that K2 was the only serotype detected among these six isolates, and indicated by at 641 bp PCR product. The second typing method was Pulsed-Field Gel Electrophoresis (PFGE), and the ApaI restriction enzyme was used to assess genetic relatedness and molecular similarity among the isolates. The results of PFGE revealed distinct patterns, generating 42 DNA fragments per strain, ranging in size from 50 to 850 kb. The PAST software version 5 was adopted to construct a dendrogram based on the PFGE profiles. The analysis showed that two strains shared 95% genetic similarity, indicating a high degree of relatedness, while the other two strains showed only 65% similarity, suggesting significant genetic divergence between them. The novelty of this study is determining at least primarily the most capsular serotype of K. pneumoniae in Mosul, Iraq - a region lacking epidemiological data- by providing baseline data that supports future research in the country.
Article Details
Article Details
Keywords
ApaI restriction enzyme, Capsule-encoding genes Klebsiella pneumoniae, Phylogenetic tree, Typing
References
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Han, H., Zhou, H., Li, H., Gao, Y., Lu, Z., Hu, K. & Xu, B. (2013). Optimization of pulse-field gel electrophoresis for subtyping of Klebsiella pneumoniae. International Journal of Environmental Research and Public Health, 10(7), 2720–2731. doi: 10.3390/ijerph10072720.
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Hashemizadeh Z, Hosseinzadeh Z, Azimzadeh N & Motamedifar M. (2020). Dissemination pattern of multidrug resistant carbapenemase producing Klebsiella pneumoniae isolates using pulsed-field gel electrophoresis in southwestern Iran. Infect Drug Resist.,13:921. doi: 10.2147/IDR.S227955
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Mahamuod R.Y. & Mohammad, G.A. (2026). Isolation and molecular diagnosis of bacteria causing bacteremia, and study their relationship with kidney functions. Rafidain Journal of Science, 35, No. 1, pp. 50-58. doi:10.33899/rsci.v35i1.61987
Mahmood, S. A. & Mohammad, G. A. (2024). Biotyping and genotyping of seven strains of Cutibacterium acnes. Regulatory Mechanisms in Biosystems, 15(3), 548–551. DOI: https://doi.org/10.15421/022476
Maleki, N. S., Babazadeh, F., Arzanlou, M., Teimourpour, R. & Dogaheh, H. P. (2023). Serotyping and molecular profiles of virulence-associated genes among Klebsiella pneumoniae isolates from teaching hospitals of Ardabil, Iran: A cross-sectional study. Health Science Reports, 6(9), Article e1557. doi: 10.1002/hsr2.1557
Mohamed, E. R., Aly, S. A., Halby, H. M., Ahmed, S. H., Zakaria, A. M. & El-Asheer, O. M. (2017). Epidemiological typing of multidrug-resistant Klebsiella pneumoniae causing pediatric ventilator-associated pneumonia in Egypt. Journal of Medical Microbiology, 66(5), 628–634. doi: 10.1099/jmm.0.000473.
Mohammad, G. & Hussein, N. (2021). Detection of some virulence genes and genetic variations of Acinetobacter baumannii. Middle East Journal of Science, 7(1), 64–79. . DOI:10.51477/mejs.899167
Ofek, I. T. Z. H. A. K., Kabha, K. I. S. R. A., Athamna, A. B. E. D., Frankel, G., Wozniak, D. J., Hasty, D. L. & Ohman, D. E. (1993). Genetic exchange of determinants for capsular polysaccharide biosynthesis between Klebsiella pneumoniae strains expressing serotypes K2 and K21a. Infection and immunity, 61(10), 4208-4216. DOI: 10.1128/iai.61.10.4208-4216.1993. PMID: 8104896; PMCID: PMC281146.
Podschun, R., Penner, I. & Ullmann, U. (1992). Interaction of Klebsiella capsule type 7 with human polymorphonuclear leucocytes. Microbial Pathogenesis, 13(5), 371–379. doi: 10.1016/0882-4010(92)90080-8.
Ramadan, A. A. (2022). Bacterial typing methods from past to present: A comprehensive overview. Gene Reports, 29, Article 101675. DOI:10.1016/j.genrep.2022.101675
Rastegar, S., Moradi, M., Kalantar-Neyestanaki, D. & Hosseini-Nave, H. (2019). Virulence factors, capsular serotypes and antimicrobial resistance of hypervirulent Klebsiella pneumoniae and classical Klebsiella pneumoniae in Southeast Iran. Infection & Chemotherapy, 51. doi: 10.3947/ic.2019.0027
Ripabelli G, Tamburro M, Guerrizio G, Fanelli I, Flocco R & Scutellà M,(2018) ; Tracking multidrug-resistant Klebsiella pneumoniae from an Italian hospital: molecular epidemiology and surveillance by PFGE, RAPD and PCR-based resistance genes prevalence. Curr. Microbiol., 75:977-987. doi: 10.1007/s00284-018-1475-3.
Russell, D. L., Uslan, D. Z., Rubin, Z. A., Grogan, T. R. & Martin, E. M. (2018). Multidrug resistant Acinetobacter baumannii: A 15-year trend analysis. Infection Control & Hospital Epidemiology, 39(5), 608–611. doi: 10.1017/ice.2018.52.
Shon, A. S., Bajwa, R. P. & Russo, T. A. (2013). Hypervirulent (hypermucoviscous) Klebsiella pneumoniae: A new and dangerous breed. Virulence, 4(2), 107–118. doi: 10.4161/viru.22718
Tenover, F. C., Arbeit, R. D., Goering, R. V., Mickelsen, P. A., Murray, B. E., Persing, D. H. & Swaminathan, B. (1995). Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: Criteria for bacterial strain typing. Journal of Clinical Microbiology, 33(9), 2233–2239. doi: 10.1128/jcm.33.9.2233-2239.1995.
Tijet, N., Sheth, P. M., Lastovetska, O., Chung, C., Patel, S. N. & Melano, R. G. (2014). Molecular characterization of Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae in Ontario, Canada, 2008–2011. PLoS One, 9(12), Article e116421. doi: 10.1371/journal.pone.0116421.
Turton, J. F., Perry, C., Elgohari, S. & Hampton, C. V. (2010). PCR characterization and typing of Klebsiella pneumoniae using capsular type-specific, variable number tandem repeat and virulence gene targets. Journal of Medical Microbiology, 59(5), 541–547. doi: 10.1099/jmm.0.015198-0.
Yeh, K. M., Kurup, A., Siu, L. K., Koh, Y. L., Fung, C. P., Lin, J. C. & Koh, T. H. (2007). Capsular serotype K1 or K2, rather than magA and rmpA, is a major virulence determinant for Klebsiella pneumoniae liver abscess in Singapore and Taiwan. Journal of Clinical Microbiology, 45(2), 466–471. doi: 10.1128/JCM.01150-06.
Dedeic-Ljubovic, A., Hukic, M., Pfeifer, Y., Witte, W., Padilla, E., López-Ramis, I. & Albertí, S. (2010). Emergence of CTX-M-15 extended-spectrum β-lactamase-producing Klebsiella pneumoniae isolates in Bosnia and Herzegovina. Clinical Microbiology and Infection, 16(2), 152–156. . doi: 10.1111/j.1469-0691.2009.03018.x.
Dendani Chadi, Z. & Arcangioli, M. A. (2023). Pulsed-field gel electrophoresis analysis of bovine associated Staphylococcus aureus: A review. Pathogens, 12(7), Article 966. https://doi.org/10.3390/pathogens12070966
Dos Santos, H. R. M., Argolo, C. S., Argôlo-Filho, R. C. & Loguercio, L. L. (2019). A 16S rDNA PCR-based theoretical to actual delta approach on culturable mock communities revealed severe losses of diversity information. BMC Microbiology, 19(1), 74. https://doi.org/10.1186/s12866-019-1446-2.
Esmaeilnia, M., Saffari, M., Rashki, S., Marzhoseyni, Z., Khaledi, A., Moosavi, G. A. & Alani, B. (2022). Molecular typing of clinical and environmental isolates of Klebsiella pneumoniae producing ESBLs by PFGE. Iranian Journal of Basic Medical Sciences, 25(2), 208–215. doi: 10.22038/IJBMS.2022.58445.12981.
Fung, C. P., Chang, F. Y., Lee, S. C., Hu, B. S., Kuo, B. I., Liu, C. Y. & Siu, L. K. (2002). A global emerging disease of Klebsiella pneumoniae liver abscess: Is serotype K1 an important factor for complicated endophthalmitis? Gut, 50(3), 420–424. doi: 10.1136/gut.50.3.420.
Han, H., Zhou, H., Li, H., Gao, Y., Lu, Z., Hu, K. & Xu, B. (2013). Optimization of pulse-field gel electrophoresis for subtyping of Klebsiella pneumoniae. International Journal of Environmental Research and Public Health, 10(7), 2720–2731. doi: 10.3390/ijerph10072720.
Hasani, A., Soltani, E., Ahangarzadeh Rezaee, M., Pirzadeh, T., Ahangar Oskouee, M., Hasani, A. & Binesh, E. (2020). Serotyping of Klebsiella pneumoniae and its relation with capsule-associated virulence genes, antimicrobial resistance pattern, and clinical infections: A descriptive study in medical practice. Infection and Drug Resistance, 13, 1971–1980. doi: 10.2147/IDR.S243984.
Hashemizadeh Z, Hosseinzadeh Z, Azimzadeh N & Motamedifar M. (2020). Dissemination pattern of multidrug resistant carbapenemase producing Klebsiella pneumoniae isolates using pulsed-field gel electrophoresis in southwestern Iran. Infect Drug Resist.,13:921. doi: 10.2147/IDR.S227955
Khaledi A, Khademi F, Esmaeili D, Esmaeili S-A & Rostami H. (2016); The role of HPaA protein as candidate vaccine against Helicobacter pylori. Der Pharma Chemica., 8:235-237. http// derpharmachemica.com/archive.html
Li W, Raoult D & Fournier P-E. (2009); Bacterial strain typing in the genomic era. FEMS Microbiol. Rev., ;33:892-916. doi: 10.1111/j.1574-6976.2009.00182.x.
Liao, C. H., Huang, Y. T., Lai, C. C., Chang, C. Y., Chu, F. Y., Hsu, M. S. & Hsueh, P. R. (2011). Klebsiella pneumoniae bacteremia and capsular serotypes, Taiwan. Emerging Infectious Diseases, 17(6), 1113–1115. doi: 10.3201/eid/1706.100811.
Mahamuod R.Y. & Mohammad, G.A. (2026). Isolation and molecular diagnosis of bacteria causing bacteremia, and study their relationship with kidney functions. Rafidain Journal of Science, 35, No. 1, pp. 50-58. doi:10.33899/rsci.v35i1.61987
Mahmood, S. A. & Mohammad, G. A. (2024). Biotyping and genotyping of seven strains of Cutibacterium acnes. Regulatory Mechanisms in Biosystems, 15(3), 548–551. DOI: https://doi.org/10.15421/022476
Maleki, N. S., Babazadeh, F., Arzanlou, M., Teimourpour, R. & Dogaheh, H. P. (2023). Serotyping and molecular profiles of virulence-associated genes among Klebsiella pneumoniae isolates from teaching hospitals of Ardabil, Iran: A cross-sectional study. Health Science Reports, 6(9), Article e1557. doi: 10.1002/hsr2.1557
Mohamed, E. R., Aly, S. A., Halby, H. M., Ahmed, S. H., Zakaria, A. M. & El-Asheer, O. M. (2017). Epidemiological typing of multidrug-resistant Klebsiella pneumoniae causing pediatric ventilator-associated pneumonia in Egypt. Journal of Medical Microbiology, 66(5), 628–634. doi: 10.1099/jmm.0.000473.
Mohammad, G. & Hussein, N. (2021). Detection of some virulence genes and genetic variations of Acinetobacter baumannii. Middle East Journal of Science, 7(1), 64–79. . DOI:10.51477/mejs.899167
Ofek, I. T. Z. H. A. K., Kabha, K. I. S. R. A., Athamna, A. B. E. D., Frankel, G., Wozniak, D. J., Hasty, D. L. & Ohman, D. E. (1993). Genetic exchange of determinants for capsular polysaccharide biosynthesis between Klebsiella pneumoniae strains expressing serotypes K2 and K21a. Infection and immunity, 61(10), 4208-4216. DOI: 10.1128/iai.61.10.4208-4216.1993. PMID: 8104896; PMCID: PMC281146.
Podschun, R., Penner, I. & Ullmann, U. (1992). Interaction of Klebsiella capsule type 7 with human polymorphonuclear leucocytes. Microbial Pathogenesis, 13(5), 371–379. doi: 10.1016/0882-4010(92)90080-8.
Ramadan, A. A. (2022). Bacterial typing methods from past to present: A comprehensive overview. Gene Reports, 29, Article 101675. DOI:10.1016/j.genrep.2022.101675
Rastegar, S., Moradi, M., Kalantar-Neyestanaki, D. & Hosseini-Nave, H. (2019). Virulence factors, capsular serotypes and antimicrobial resistance of hypervirulent Klebsiella pneumoniae and classical Klebsiella pneumoniae in Southeast Iran. Infection & Chemotherapy, 51. doi: 10.3947/ic.2019.0027
Ripabelli G, Tamburro M, Guerrizio G, Fanelli I, Flocco R & Scutellà M,(2018) ; Tracking multidrug-resistant Klebsiella pneumoniae from an Italian hospital: molecular epidemiology and surveillance by PFGE, RAPD and PCR-based resistance genes prevalence. Curr. Microbiol., 75:977-987. doi: 10.1007/s00284-018-1475-3.
Russell, D. L., Uslan, D. Z., Rubin, Z. A., Grogan, T. R. & Martin, E. M. (2018). Multidrug resistant Acinetobacter baumannii: A 15-year trend analysis. Infection Control & Hospital Epidemiology, 39(5), 608–611. doi: 10.1017/ice.2018.52.
Shon, A. S., Bajwa, R. P. & Russo, T. A. (2013). Hypervirulent (hypermucoviscous) Klebsiella pneumoniae: A new and dangerous breed. Virulence, 4(2), 107–118. doi: 10.4161/viru.22718
Tenover, F. C., Arbeit, R. D., Goering, R. V., Mickelsen, P. A., Murray, B. E., Persing, D. H. & Swaminathan, B. (1995). Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: Criteria for bacterial strain typing. Journal of Clinical Microbiology, 33(9), 2233–2239. doi: 10.1128/jcm.33.9.2233-2239.1995.
Tijet, N., Sheth, P. M., Lastovetska, O., Chung, C., Patel, S. N. & Melano, R. G. (2014). Molecular characterization of Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae in Ontario, Canada, 2008–2011. PLoS One, 9(12), Article e116421. doi: 10.1371/journal.pone.0116421.
Turton, J. F., Perry, C., Elgohari, S. & Hampton, C. V. (2010). PCR characterization and typing of Klebsiella pneumoniae using capsular type-specific, variable number tandem repeat and virulence gene targets. Journal of Medical Microbiology, 59(5), 541–547. doi: 10.1099/jmm.0.015198-0.
Yeh, K. M., Kurup, A., Siu, L. K., Koh, Y. L., Fung, C. P., Lin, J. C. & Koh, T. H. (2007). Capsular serotype K1 or K2, rather than magA and rmpA, is a major virulence determinant for Klebsiella pneumoniae liver abscess in Singapore and Taiwan. Journal of Clinical Microbiology, 45(2), 466–471. doi: 10.1128/JCM.01150-06.
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Capsular serotyping and molecular characterization of iraqi strains of Klebsiella pneumoniae by multiplex polymerase chain reaction (PCR) and pulsed-field gel electrophoresis (PFGE). (2026). Journal of Applied and Natural Science, 18(2), 1010-1015. https://doi.org/10.31018/jans.v18i2.7519
