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Saudi Journal of Kidney Diseases and Transplantation
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ORIGINAL ARTICLE Table of Contents   
Year : 2009  |  Volume : 20  |  Issue : 4  |  Page : 613-617
The prevalence of virulence genes of E. coli strains isolated from children with urinary tract infection


1 Prof. Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
2 Pediatric Department, Jahrom University of Medical Sciences, Jahrom, Iran

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Date of Web Publication8-Jul-2009
 

   Abstract 

To evaluate the prevalence of virulence genes in E. coli strains isolated from urine samples of children with urinary tract infection(UTI) and their correlation with clinical data, we iso­lated E. coli strains from urine samples of children with UTI during the period of August 2005 - August 2006 and studied them for the presence of the virulence genes by PCR. A total of 96 E. coli strains were isolated. The prevalence of genes, pyelonephritis associated pili (pap genes), S-family adhesions (sfa gene), hemolysin (hly gene), and cytotoxic nercotizing factor type 1 (cnf-1-1 gene) among the isolated strains was 27.1%, 14.6%, 13.5% and 22.9 %, respectively. Pyelonephritis was more prevalent in the cases with positive virulence genes. The results showed significant correlation bet­ween age of the patient and the presence of the genes (P< 0.05). Cnf-1 gene was significantly more common in samples of patients with abnormal finding on the ultrasound of kidneys (P= 0.049). Our study demonstrated higher prevalence of pyelonephritis in the presence of E. coli virulence genes. Detection of the genes in urine samples may help in the management of UTI.

How to cite this article:
Farshad S, Emamghorashi F. The prevalence of virulence genes of E. coli strains isolated from children with urinary tract infection. Saudi J Kidney Dis Transpl 2009;20:613-7

How to cite this URL:
Farshad S, Emamghorashi F. The prevalence of virulence genes of E. coli strains isolated from children with urinary tract infection. Saudi J Kidney Dis Transpl [serial online] 2009 [cited 2014 Jul 29];20:613-7. Available from: http://www.sjkdt.org/text.asp?2009/20/4/613/53250

   Introduction Top


E. coli accounts for as much as 90% of urinary tract infections (UTI) and displays variable vi­rulence properties. [1] Several genes encode uro­virulent factors such as hemolysin (hly gene), cytotoxic nercotizing factor type 1 (cnf-1-1 gene), pyelonephritis associated pili (pap genes) and S-family adhesions (sfa gene). [2] Some genes, such as the hly pap or p fimbriae and cnf-1 1 play an important role in the pathogenesis of E. coli strains. [3],[4],[5]

In the present study, we evaluated the pre­valence of the virulence genes, and their cor­relation with clinical data in uropathogenic E. coli strains isolated from children with UTI.


   Subjects and Methods Top


We studied children with UTI referred to Mo­tahary hospital and clinic, Jahrom, Iran during the period of August 2005 - August 2006.

Diagnosis of UTI was established by clinical symptoms and laboratory investigations (colony count of more than 10 5 colony forming unit/mL in meadstream sampling, 10 4 cfu/mL in urine sampling by catheterization and any count in suprapubic sampling). None of the patients was immune compromised.

Acute pyelonephritis was clinically defined as fever (temperature > 38.5°C), lumbar tenderness, and sepsis in neonate. Cystitis was considered when dysuria and pyuria were present without fever. Data including age, sex, previous history of infection, recent antibiotic usage and history of hospitalization during the previous 28 days were collected on all the patients with commu­nity acquired UTI in questionnaire forms. The exclusion criterion was nosocomial infection, which was defined as infection noted 48 h after admission or within four weeks after a pre­vious discharge.

Isolation of Bacteria

E. coli
strains were isolated from urine samples and identified using standard methods. [6] Urine culture was considered positive if at least 10 5 colony forming unit of E. coli per mL of clean­voided urine were grown in the culture media. The strains were then stored and sub cultured for further analysis as previously described in literature. [7] ,[8]

DNA extraction

E. coli
isolates were grown in Luria Bertani broth at 37°C overnight. Bacteria were then pel­leted from broth, resuspended in sterile distilled water and boiled at 95°C for ten min. After cen­trifugation, the supernatants were stored as DNA template at -20°C until they were used in the PCR.

PCR assay

Detection of pap, sfa, cnf-1, and hly genes was performed by amplifying the genes by PCR. The primers sequences were previously reported in the literature, [9] and obtained from TIB MOL­BIOL Syntheselabor GmbH (Berlin, Germany). Descriptions and sequences of the PCR primers used in this study are shown in [Table 1]. Other enzymes and chemicals were provided by Cinnagen Chemical Company (Tehran, Iran). Am­plification was performed in a thermal cycler (Eppendorf, Germany) according to the methods described by Yamamoto et al. [10] Expected sizes of the amplicons were ascertained by electro­phoresis in 1.5 % agarose gel with an appropriate molecular size marker (100-bp DNA ladder, MBI, Fermentas, Lithuania).


   Statistical Analysis Top


Statistical analysis was performed using SPSS software for Windows, version 11.5 (SPSS). Descriptive analyses were performed for para­metric and non parametric variables. Student 't' test, Chi square, and logistic regression were done for evaluation of variables correlation. P value less than 0.05 was considered as signi­ficant.


   Results Top


A Total of 96 E. coli strains were isolated from 96 (60 female (62.5%) and 36 male 37.5%) children with UTI referred during the study pe­riod. The patients' ages ranged from one month to 14 years with a mean of 21.8 ± 26.9 months. There were 49.2% of cases that were diagnosed as cystitis, and 50.8% as acute pyelonephritis. Pyelonephritis was more prevalent in girls than boys (63.2% vs. 36.4%, P= 0.04). Only 37 pa­tients had ultrasound of the kidneys. Fourteen cases had abnormal findings that included re­flux, UPJ stenosis, multicystic kidney, and single kidney [Table 2].

In polymerase chain reaction analysis [Figure 1], the prevalence of virulence genes ranged from 13.5% for hly to 27.1 % for pap. Of the two studied adhesin coding genes, pap was more prevalent (n=26, 27.1 %) than sfa (n=14, 14.6%), while of the two studied toxin coding genes, cnf-1 was more prevalent (n=22, 22.9 %) than hly (n=13, 13.5 %). Of all isolates, 32 (33.3 %) re­vealed at least one gene and 14 (6.3%) revealed all four genes.

The results showed a significant correlation between the age of the patients and presence of genes. By using Medcalc software, we could define a cut point of 36 months, so the preva­lence of genes pap and sfa was more common in patients older than 36 months (P< 0.05), however, hly was more common in patients younger than 48 months (66.7% vs. 33.3%, (P< 0.05), [Table 2].

Pyelonephritis was diagnosed in 66.7%, 62.5%, 63.6%, and 85.7% of the cases positive for pap, sfa, cnf-1 and hly, respectively. Furthermore, there was a significant correlation between the presence of cnf-1 and abnormal findings on kidney ultrasound; 83.3% of patients with cnf-1 gene in their urine samples had abnormal fin­dings (P= 0.019).


   Discussion Top


In the present study, we found that 27.1%, 22%, 14.6%, and 13.5% of E. coli isolated from urine samples of children with UTI were posi­tive for pap, cnf-1, sfa, and hly genes, respec­tively. Various studies investigated the preva­lence of virulence genes of E. coli isolates from cases of UTI. A study from Spain showed that almost 70% of the urinary strains carried at least one of the target virulence genes. [11] Sil­veira and co workers found prevalence of 38.5%, 15.4%, and 7.7% for the phenotypes of pap +­sfa + - afa + , sfa-related sequences genes positive and pap + - sfa + , respectively, in uropathogenic E. coli strains, while only two cases were po­sitive for cnf-1 gene. [12] Arisoy studied 161 E. coli strains isolated from children with UTI and found that 58.4% was positive for at least one virulence gene; prevalence of genes were found as 22.98%, 9.94%, 6.21%, and 1.24% for pap, cnf-1, sfa, and hly, respectively. [9]

Our results highlight higher frequency of pap compared with the rest of the genes, and this may indicate a crucial role of the virulence ge­nes in E. coli associated UTI. In uropathogenic E. coli, the presence of P fimbriae has been clearly established as a factor that promotes urinary tract colonization and invasion. Bacte­rial adhesion to the bladder mucosa is a critical step for the establishment of E. coli bacteriu­ria. [13] ,[14] Indeed, two genes in the pap class were found to play more important role in the deve­lopment of E. coli bacteremia in patients with UTI than in those with acute cholangitis. [15]

Moreover, an important role of pap adhesion genes in the pathophysiology of pyelonephritis caused by E. coli has been reported in several studies. [16],[17],[18],[19] In our study, pyelonephritis was more prevalent in the cases with virulence gene­positive E. coli isolates; 66.6%, 62.5%, 63.3% and 85.7% of cases that were positive for pap, sfa, cnf-1 and hly, respectively, developed pye­lonephritis. In another study, 25 of 42 cases with pyelonephritis were positive for pap gene, but only 6 of 58 strains isolated from cases with cystitis were positive for this gene. Further­more, in our study, the prevalence of hly and cnf-1 in pyelonephritis was 34% and 21.9%, respectively. [20] Chatal showed that pap operons were found in 79.4% of the pyelonephritis strains, either alone (51.5% of the isolates) or in asso­ciation with either the afa or sfa/foc operon (27.9%). Those observations, which confirmed the high prevalence of pap operons among E. coli strains associated with pyelonephritis, also indicated that the afa and sf a lf oc operons repre­sented uropathogenic determinants per se. [8]

The ability to adhere to epithelial surfaces has been shown to be a prerequisite for E. coli strains to colonize the urinary tract, i.e., to cause UTI in the absence of urological abnormalities. Sur­veys of patients with acute pyelonephritis have suggested that urinary tract abnormalities or medical intervention may allow nonpathogenic organisms' access to the kidneys. [2] Accordingly, some investigators have proposed that the ab­sence of adhesive properties in a pyelonephritis isolate could be used as an indicator of a po­ssible underlying anatomic abnormality or me­dical illness, warranting further investigation of patients. [21] In our study, the prevalence of pap and sfa was lower in patients with abnormal findings on kidney ultrasound, but the preva­lence of cnf-1 was significantly higher in these patients (P= 0.019).

In conclusion, our study found higher preva­lence of pyelonephritis in the presence of viru­lence genes (pap, sfa, hly and cnf-1) of uro­pathogenic E. coli strains. Detection of the genes in urine samples may help predict incidence of pyelonephritis.


   Acknowledgement Top


This work was supported by research grant #83-14 from Professor Alborzi Clinical Micro­biology Research Center, Shiraz University of Medical Sciences. The authors would like to thank Mr. Mehdi Kalani and Mrs. Marzieh Hos­seini for their assistance in bacterial isolation, identification, purification and molecular diag­nosis.

 
   References Top

1.Kunin CM. Urinary tract infections: detection, prevention, and management. 5th ed. Baltimore Md: Williams & Wilkins;1997.  Back to cited text no. 1    
2.Johnson JR. Virulence factors in Escherichia coli urinary tract infection. Clin Microbiol Rev 1991;4:80-128.  Back to cited text no. 2  [PUBMED]  [FULLTEXT]
3.Soto M, Jimenez de Anta JM, Vila J. Quinolones Induce Partial or Total Loss of Pathogenicity Islands in Uropathogenic Escherichia coli by SOS-Dependent or Independent Pathways, Respectively. Antimicrob Agents Chemother 2006;50: 649-53.  Back to cited text no. 3    
4.Donnenberg MS, Welch RA. Virulence determi­nants of uropathogenic Escherichia coli. In: Mobley HLT, Warren JW, eds. Urinary Tract Infections: Molecular Pathogenesis and Clinical Management. J Am Soc Microbiol 1996:135-74.  Back to cited text no. 4    
5.5.Orskov I, Orskov F. Esherichia coli in extra­intestinal infections. J Hyg 1978;95: 551-75.  Back to cited text no. 5    
6.Farmer JJ. Enterobacteriaceae: introduction and identification. In Manual of clinical micro­biology. 7th ed. Washington, ASM Press, 1999.  Back to cited text no. 6    
7.Blanco M, Blanco JE, Alonso MP, et al. Detec­tion of pap, sfa and afa adhesion encoding operons in uropathogenic Escherichia coli strains: relationship with expression of adhesins and production of toxins. Res Microbiol 1997; 148: 745-55.  Back to cited text no. 7  [PUBMED]  [FULLTEXT]
8.Le Bouguenec C, Archambaud M, Labigne A. Rapid and specific detection of the pap, afa, and sfa adhesion encoding operons in uropathogenic Escherichia coli strains by polymerrase chain reaction. J clin Microbiol 1992;30:1189-93.  Back to cited text no. 8  [PUBMED]  [FULLTEXT]
9.Arisoy M, Aysev D, Ekim M, et al. Detection of virulence factors of Escherichia coli from children by multiplex polymerase chain reac­tion. Int J Clin Pract 2006;60:170-3.  Back to cited text no. 9  [PUBMED]  [FULLTEXT]
10.Yamamoto S, Terai A, Yuri K, Kurazono H, Takeda Y, Yoshida O. Detection of uroviru­lence factors in Esherichia coli by multiplex polymerase chain reaction. FEMS Immunol Med Microbiol 1995;12:85-90.  Back to cited text no. 10  [PUBMED]  [FULLTEXT]
11.National Institute of Research and Develop­ment for Microbiology and Immunology. Com­parison of genomic profiles of Escherichia coli isolates from urinary tract infections. Roum Arch Microbiol Immunol 2003; 62:137-54.  Back to cited text no. 11    
12.Silveira WD, Benetti F, Lancelotti M, Ferreira A, Solferini VN, Brocchi M. Biological and Genetic Characteristics of Uropathogenic Escherichia coli Strains. Rev Inst Med Trop 2001;43:345-50.  Back to cited text no. 12    
13.Wullt B. The role of P fimbriae for Escherichia coli establishment and mucosal inflammation in the human urinary tract. Int J Antimicrob Agents 2003;21(6):605-21.  Back to cited text no. 13    
14.Wullt B, Bergsten G, Connell H, et al. P fimbriae trigger mucosal responses to Escherichia coli in the human urinary tract. Cell Microbiol 2001;3:255-64.  Back to cited text no. 14  [PUBMED]  [FULLTEXT]
15.Wang MC, Tseng CC, Chen CY, Wu JJ, Huang JJ. The role of bacterial virulence and host factors in patients with Escherichia coli bacteremia who have acute cholangitis or upper urinary tract infection. Clin Infect Dis 2002; 35:1161-6.  Back to cited text no. 15  [PUBMED]  [FULLTEXT]
16.Gander RM, Thomas VL, Forland M. Man nose resistant hemagglutination and P recaptor recognition of uropathogenic Escherichia coli isolated from adult patients. J Infect Dis 1985; 151:508-13.  Back to cited text no. 16  [PUBMED]  
17.Norgren M, Baga M, Tennent JM, Normark S. Nucleotide sequence, regulation and functional analysis of the pap C gene required for cell surface localization of Pap pili of uropathogenic Escherichia coli. Mol Microbiol 1987;1:169-78.  Back to cited text no. 17    
18.O'Hanley P, Low D, Romero I, et al. Gal-Gal binding and hemolysin phenotypes and geno­types associated with uropathogenic Escherichia coli. N Engl J Med 1985;15:414-20.  Back to cited text no. 18    
19.Westerlund B, Kuusela P, Risteli J, et al. The O75X adhesin of uropathogenic Escherichia coli is a type IV collagen-binding protein. Mol Microbiol. 1989;3:329-37.  Back to cited text no. 19    
20.Vila J, Simon K, Ruiz J. Are Quinolone Resistant Uropathogenic Escherichia coli Less Virulent? J Infect Dis 2002;186:1039-42.  Back to cited text no. 20    
21.Lomberg H, Hellstrom M, Jodal U, Leffler H, Lincoln K, Svanborg-Eden C. Virulence­associated traits in Escherichia coli causing first and recurrent episodes of urinary tract infection in children with or without vesicoureteral reflux. J Infect Dis 1984;150:561-9.  Back to cited text no. 21    

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Correspondence Address:
Fatemeh Emamghorashi
Pediatric Department, Jahrom University of Medical Sciences, Jahrom
Iran
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    Abstract
    Introduction
    Subjects and Methods
    Statistical Analysis
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    Discussion
    Acknowledgement
    References
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