CC BY
89
52
• ••
Известия ДГПУ, №3, 2015
МЕДИЦИНСКИЕ НАУКИ
УДК 579.61
GENOTYPIC DETECTION OF FIMH VIRULENCE GENE IN UROPATHOGENIC ESCHERICHIA COU (UPEC) ISOLATED
FROM URINARY TRACT INFECTION
ОПРЕДЕЛЕНИЕ ГЕНА ВИРУЛЕНТНОСТИ FIMH УРОПАТОГЕННЫХ ШТАММОВ ESCHERICHIA COLI,
ВЫДЕЛЕННЫХ ПРИ ИНФЕКЦИЯХ МОЧЕВЫВОДЯЩИХ ПУТЕЙ
© 2015 Аль-Баяти Б. М.1, Глинская Е. В.1, Нечаева О. В.2, Лунева Л. О.2
Саратовский государственный университет, Саратовский государственный медицинский университет
© 2015 Al-Bayati B. M.1, Glinskaya E. V.1, Nechaeva O. V.2, Luneva I. O.2
1Saratov State University, 2Saratov State Medical University
Резюме. В статье приводятся результаты определения наличия гена вирулентности FimH уропатогенных штаммов Escherichia coli, выделенных из мочи пациентов с инфекциями мочевыводящих путей при помощи полимеразной цепной реакции (ПЦР). Из 325 протестированных образцов в 200 был обнаружен рост патогенных микроорганизмов, среди которых превалировала Escherichia coli - 55,5%. Ген вирулентности FimH (амплифицированный определенными праймерами и имеющий сигнал в области 446 пар оснований [п.о.]) был выявлен у 70% выделенных уропатогенных штаммов Escherichia coli.
Abstract. This study was performed to detect the presence of type 1 fimbriae (FimH) virulence gene among uropathogenic strains Escherichia coli (UPEC) recovered from urine of patients with UTIs by polymerase chain reaction technique (PCR). Of the 325 tested specimens, 200 revealed growth of pathogens, and among which Escherichia coli (E. coli) were the most prevalent, 55.5%. The FimH virulence gene (amplified using specific primers and showed a band about 446 bp) was found in 70% of UPEC isolates.
Rezjume. V stat’e privodjatsja rezul'taty opredelenija nalichija gena virulentnosti FimH uropatogen-nyh shtammov Escherichia coli, vydelennyh iz mochi pacientov s infekcijami mochevyvodjashhih putej pri pomoshhi polimeraznoj cepnoj reakcii (PCR). Iz 325 protestiro-vannyh obrazcov v 200 byl ob-naruzhen rost patogennyh mikroorganizmov, sredi kotoryh pre-valirovala Escherichia coli - 55,5%. Gen virulentnosti FimH (amplificirovannyj opredelennymi prajmerami i imejushhij signal v oblasti 446 par osnovanij [p.o.]) byl vyjavlen u 70% vydelennyh uropatogennyh shtammov Escherichia coli.
Ключевые слова: уропатогенные штаммы, Escherichia coli, FimH, гены вирулентности. Keywords: Genotypic detection of E. coli virulence genes, E. coli and FimH by PCR.
Kljuchevyeslova: uropatogennye shtammy, Escherichia coli, FimH, geny virulentnosti.
Urinary tract infection is considered as the most commonly diagnosed and treated bacterial infection. Usually, bladder and urethra are prevalent sites of infection. Women are more prone to have UTIs than men. In general En-
terobacteriaceae and in particular E. coli are the most common bacterial causative agents, responsible for 50-75% of all UTIs in both community and healthcare facilities [2, 9]. The strains of UPEC possess special virulence fac-
Естественные и точные науки
• ••
53
tors, including: adhesions (type 1 fimbriae, P fimbriae, curli fimbriae, afimbrial adhesion, and flagellum), aerobactins, hemolysins, and cytotoxic necrotizing factor-1 (CNF-1) [7], which are important in the attachment and colonization of the urogenital tract, extraintestinal survival, and creation of cytopathic effects [20, 15].
The attachment of UPEC to uroepithelial cells, which is mediated by small proteins placed at the tips of the bacterial fimbriae known as «bacterial ligands» is considered as an essential step for the beginning and development of UTI. These ligands bind to the host cell wall and work as receptors, permit the bacteria to resist the mechanical elimination through the flow of urine and bladder emptying, and increase bacterial persistence [7, 14]. Among the adhesions produced by UPEC, is type 1 fimbria [16]. FimH (adhesive subunit of type 1 fimbriae) is a major determinant, possess a high harmony with urinary tract receptors, and thus, it is fundamental for the recognition, attachment, and colonization to the urinary tract [19].
Due to the little information about the FimH virulence factor and its role in UTI, in Russia, this study was conducted to determine the prevalence of this gene among UPEC isolates from urine specimens of patients with UTI.
A total of 325 midstream urine specimens were collected from patients in different ages (6-76) years, with clinical symptoms suspected to be UTI. Investigation of urine specimens (including initial screening, cultivation, and identification of isolates) was done as prescribed by [10]. The Antimicrobial susceptibility test was carried out by Kirby-Bauer's technique on Muller-Hinton agar as prescribed by [6]. The tested antibiotics were selected as recommended by [6].
The isolates of E. coli were inoculated in Luria Bertani broth media and cultured for 24 hrs. The growth were suspended in sterile distilled water, and then incubated at 95 °C for 10 min. After centrifugation, the supernatant was stored at -20 °C as a template DNA stock [8].
The detection of FimH virulence gene was done by PCR. Primers sequences were previously reported and obtained from Integrated DNA Technologies Company (Belgium) [12], which are shown in Table 1. Amplification was performed in a thermocycler (Eppendorf, Germany) as mentioned by [22]. The reaction mixture included an initial denaturation for 5 min at 94 °C, consisted of 30 cycles of 94 eC for one min, specific annealing temperature 63 °C for one min, 68 eC for three min, and a
final extension at 72 eC for seven min [17]. The thermocycler reaction conditions were calculated on the basis of the annealing temperature and the length of the product size. Amplified DNA was analyzed on (0.8-1%) agarose gel by electrophoresis. The phylogenetic group to which E. coli strains belonged was determined by a PCR based method, as described by [5].
Table 1
Descriptions and sequences of the PCR primers
Primer Oligonucleotide sequence Size of amplicons
F CTG ATG GGC TGG TCG
FimH GTA AAT 446 bp
R TGC ACA TTC CCT GCA
GTC A
The current study demonstrates the prevalence of FimH virulence factor among UPEC isolated from urine specimens of patients with UTI. Of the 325 tested specimens, 200 revealed a growth of pathogens, and among which the most prevalent were E. coli 111 (55.5%). Such isolation rate was also documented in other studies [3, 18]. This may be due to the fact that UPEC affecting the urinary tract possess a variety of virulence factors that facilitate their recognition, attachment, and colonization to the anatomically normal urinary tract [3, 19].
The distribution of FimH virulence factor and the PCR amplification were shown in Table 2 and Figure 1. Seventy (70%) out of 100 tested UPEC isolates demonstrate a positive existence of this gene. This finding was in agreement with other studies [21, 1, 11] reported such predominance of this factor among the studied virulence factors of UPEC strains, with a detection rate ranged (68100%), and thus it can play an important role in the pathogenesis of UTI. The prevalence of FimH gene was also detected in other strains of E. coli. Studies done in 2011 and 2012 have been found that FimH virulence gene was existed among 97% of E. coli isolates with and without K1 antigen, in 97% of non O157:H7 E. coli strains, and in 100% of O157:H7 E. coli strains [13, 4]
Table 2
Distribution of FimH virulence gene among UPEC isolates
Virulence Positive isolates Total E. coli
gene No. (%) isolates No. (%)
FimH 70 (70) 100 (100)
54
• ••
Известия ДГПУ, №3, 2015
Fig. 1. The PCR amplification of the FimH virulence gene
We can conclude that type 1 fimbria is present among UPEC as well as other strains of E. coli. The high binding ability of FimH virulence gene could be resulted in increasing the bacterial binding to the target cells and increas-
ing the pathogenicity of E. coli. Thus, FimH could be used to design vaccine for prevention of E. coli infections by blocking the bacterial attachment and colonization.
References
I. Abass S K., Ali M R., and Authman S H. Isolation of multi antibiotics resistance E. coli from UTI and the
detection of PapC and fimH virulence genes by Polymerase chain reaction technique // Diyala J. Pure Sciences. 2014. Vol. 10, No. 1. P. 112-127. 2. Agarwal J., Srivastava S., and Singh M. Pathogenomics of Uro-pathogenic Escherichia coli // Indian J. Medical Microbiology. 2012. Vol. 30, No. 2. P. 141-149. 3. Al-
Mayahi F. S. A., and Al-Mohana A. M. Incidence of Extended-Spectrum p-lactamases ESBLs Producing Escherichia coli in Patients with Urinary Tract Infection // J. Al-Qadisiyah Pure Science. 2014. Vol. 19, No. 2. P. 1-55. 4. Biscola F. T., Abe C. M., and Guth B. E. Determination of Adhesin Gene Sequences in, and Biofilm Formation by, O157 and non-O157 Shiga toxin-producing E. coli Strains isolated from Different Sources // Applied Environmental Microbiology. 2011. Vol. 77, No. 7. P. 2201-2208. 5. Clermont O., Bonacorsi S., and Bingen E. Rapid and simple determination of the E. coli phylogenetic group // Applied Environment Microbiology. 2000. Vol. 66. P. 4555-4558. 6. Clinical and Laboratory Standards Institute (CLSI). Perfor-
mance Standards for Antimicrobial Susceptibility Testing. 22nd Informational Supplements Update // CLSI Document M100-S22 U. Pennsylvania, USA. 2012. Available from www.clsi.org. 7. Emody L., Kerenyi M.,
and Nagy G. Virulence Factors of Uropathogenic Escherichia coli // International J. Antimicrobial Agents. 2003. Vol. 22, Suppl 2. P. 29-33. 8. Farshad S., Emamghoraishi F., and Japoni A. Association of Virulent
Genes Hly, SFA, CNF-1 and Pap with Antibiotic Sensitivity in E. coli Strains Isolated from Children with Community-Acquired UTI // Iranian Red Crescent Medical J. 2010. Vol. 12, No. 1. P. 33-37. 9. Gupta K.,
Hooton T M., et al. International Clinical Practice Guidelines for the Treatment of Acute Uncomplicated Cystitis and Pyelonephritis in Women: A 2010 Update by the Infectious Diseases Society of America and the European Society for Microbiology and Infectious Diseases // Clinical Infectious Diseases. 2011. Vol. 52, No. 5. P. e103-e120. 10. Health Protection Agency. Investigation of Urine. UK Standards for Microbiology Investigations. 2012. B 41, Issue 7.1. P. 1-41. Available from http://www.hpa.org.uk/SMI/pdf. 11. Hojati Z., Zamanzad B., Hashemzadeh M., Molaie R., and Gholipour A. Detection of FimH Gene in Uropathogenic E. coli Strains isolated From Patients with Urinary Tract Infection // Jundishapur J. Microbiology. 2015. Vol. 8, No. 2. P. e17520 (1-4). 12. Integrated DNA Technologies. Specification Sheet: Sequences of Primer 1 Forward and Reverse. Integrated DNA Technologies, BVBA, Interleuvenlaan 12 A, B-3001, Leuven, Belgium. 2015. www.IDTDNA.com 13. Kaczmarek A., Budzynska A., and Gospodarek E. Prevalence of Genes Encoding Virulence Factors among Escherichia coli with K1 antigen and non-K1 E. coli strains //
J. Medical Microbiology. 2012. Vol. 61, Pt 10. P. 1360-1365. 14. Le Bouguenec C. Adhesins and Invasions of Pathogenic Escherichia coli // International J. Medical Microbiology. 2005. Vol. 295, No. 6-7. P. 471-
Естественные и точные науки
• ••
55
478. 15. Mladin C., Usein C.R., Chifiriuc M., Palade A., Slavu C.L., et al. Genetic analysis of virulence and
pathogenicity features of UPEC isolated from patients with Neurogenic bladder // Romanian Biotechnological Letters. 2009. Vol. 14, No. 6. P. 4906-4911. 16. Oliveira F. A., Paludo K. S., Arend L. N., Farah S. M.,
Pedrosa F. O., et al. Virulence characteristics and antimicrobial susceptibility of uropathogenic E. coli strains. Genetic and Molecular Research. 2011. Vol 10, No. 4. P. 4114-4125. 17. Ribeiro Tiba M., Yano T., and Silva Leite D. Genotypic Characterization of Virulence Factors in Escherichia coli Strains from Patients with Cystitis. Revista do Instituto de Medicina Tropical de Sao Paulo // 2008. Vol. 50, No. 5. P. 255-260. 18. Sharma A., Prakash M.R., Veena M., Singh R.E., et al. ESBL-A Continuous Diagnosis Challenge to Clinical Microbiology Laboratories // International J. Applied Biology and Pharmaceutical Technology. 2012. Vol. 3, No. 2. P. 9-15. 19. Sokurenko E. V., Feldgarden M., Trintchina E., Weissman S.J., Avagyan S., Chat-topadhyay S., et al. Selection Footprint in the FimH Adhesin Shows Pathoadaptive Niche Differentiation in Escherichia coli // Molecular Biology and Evolution. 2004. Vol. 21, No. 7. P. 1373-1383.
20. Soutourina O.A. and Bertin P.N. Regulation Cascade of Flagellar Expression in Gram-negative Bacteria
// FEMS Microbiology Reviews. 2003, 27 (4): P. 505-523. 21. Tarchouna. M.; Ferjani. A.; Ben-Selma. W.;
and Boukadida. J. Distribution of Uropathogenic Virulence Genes in Escherichia coli isolated from Patients with UTI // International J. Infectious Diseases. 2013. Vol. 17, No. 6. P. e450-e453. 22. Yamamoto S.,
Nakano M., and Terai A. The Presence of the Virulence Island Containing the usp Gene in Uropathogenic Escherichia coli is Associated with UTI in an Experimental Mouse Model // J. Urology. 2001. Vol. 165. P. 1347-1351.
Literatura
Technol-ogies, BVBA, Interleuvenlaan 12 A, B-3001, Leuven, Belgium. 2015. www.IDTDNA.com 13. Kacz-marek A., Budzynska A., and Gospodarek E. Prevalence of Genes Encoding Virulence Factors among Escherichia coli with K1 antigen and non-K1 E. coli strains // J. Medical Microbiology. 2012. Vol. 61, Pt 10. P. 1360-1365. 14. Le Bouguenec C. Adhesins and Invasions of Pathogenic Escherichia coli // International J. Medical Microbiology. 2005. Vol. 295, No. 6-7. P. 471-478. 15. Mladin C., Usein C.R., Chifiriuc M.,
Palade A., Slavu C.L., et al. Genetic analysis of virulence and pathogenicity fea-tures of UPEC isolated from patients with Neurogenic bladder // Romanian Biotechnological Letters. 2009. Vol. 14, No. 6. P. 49064911. 16. Oliveira F. A., Paludo K. S., Arend L. N., Farah S. M., Pedrosa F. O., et al. Virulence characteristics and antimicrobial susceptibility of uropathogenic E. coli strains. Ge-netic and Molecular Research. 2011. Vol 10, No. 4. P. 4114-4125. 17. Ribeiro Tiba M., Yano T., and Sil-va Leite D. Genotypic Characterization of Virulence Factors in Escherichia coli Strains from Patients with Cystitis. Revista do Instituto de Medicina Tropical de Sao Paulo // 2008. Vol. 50, No. 5. P. 255-260. 18. Sharma A., Prakash M.R., Veena M., Singh R.E., et al. ESBL-A Continuous Diagnosis Challenge to Clini-cal Microbiology Laboratories // International J. Applied Biology and Pharmaceutical Technology. 2012. Vol. 3, No. 2. P. 9-15. 19. Sokurenko E. V., Feldgarden M., Trintchina E., Weissman S.J., Avagyan S., Chat-topadhyay S., et al. Selection Footprint in the FimH Adhesin Shows Pathoadaptive Niche Differentiation in Escherichia coli // Molecular Biology and Evolution. 2004. Vol. 21, No. 7. P. 1373-1383. 20. Soutourina O.A. and Bertin P.N. Regulation Cascade of Flagellar Expression in Gram-negative Bacteria // FEMS Microbiology Reviews. 2003, 27 (4): P. 505-523.
21. Tarchouna. M.; Ferjani. A.; Ben-Selma. W.; and Boukadida. J. Distribution of Uropathogenic Virulence Genes in Escherichia coli isolated from Patients with UTI // International J. Infectious Diseases. 2013. Vol. 17, No. 6. P. e450-e453. 22. Yamamoto S., Nakano M., and Terai A. The Presence of the Virulence Island Containing the usp Gene in Uropathogenic Escherichia coli is Associated with UTI in an Experimental Mouse Model // J. Urology. 2001. Vol. 165. P. 1347-1351.
Статья поступила в редакцию 03.11.2015 г.
