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Year : 2008  |  Volume : 26  |  Issue : 1  |  Page : 68-70

Haemagglutination and siderophore production as the urovirulence markers of uropathogenic Escherichia coli

Department of Microbiology, JN Medical College, Belgaum - 590010, Karnataka, India

Date of Submission02-Sep-2006
Date of Acceptance26-Mar-2007

Correspondence Address:
M A Vagarali
Department of Microbiology, JN Medical College, Belgaum - 590010, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0255-0857.38863

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 ~ Abstract 

A total of 160 strains of Escherichia coli isolated from urine of patients with clinically diagnosed urinary tract infection were included in the study and 50 faecal isolates of E. coli were studied. They were studied for virulence factors, namely mannose-resistant and mannose-sensitive haemagglutination (MRHA, MSHA) and siderophore production.Among 160 urinary isolates of E. coli , 40 (25%) showed MRHA, siderophore production was seen in 156 (97.5%). In 50 faecal isolates, two (4%) were MRHA, four (8%) MSHA and siderophore production in two (4%). The results suggest that MRHA and siderophore production positive strains can be considered as UPEC.

Keywords: E. coli mannose-resistant haemagglutination, siderophore uropathogenic

How to cite this article:
Vagarali M A, Karadesai S G, Patil C S, Metgud S C, Mutnal M B. Haemagglutination and siderophore production as the urovirulence markers of uropathogenic Escherichia coli. Indian J Med Microbiol 2008;26:68-70

How to cite this URL:
Vagarali M A, Karadesai S G, Patil C S, Metgud S C, Mutnal M B. Haemagglutination and siderophore production as the urovirulence markers of uropathogenic Escherichia coli. Indian J Med Microbiol [serial online] 2008 [cited 2020 Sep 30];26:68-70. Available from:

Urinary tract is the second most common site of bacterial infection in humans and thus represents a major source of human discomfort.  Escherichia More Details coli is the most frequently isolated urinary pathogen, which accounts for 50 to 90% of all uncomplicated urinary tract infections. It is now recognized that there are subsets of faecal E. coli , which can colonize periurethral area, enter urinary tract and cause symptomatic disease. These are currently defined as uropathogenic E. coli . [1] It has been traditionally described that certain serotypes of E. coli were consistently associated with uropathogenicity and were designated as uropathogenic E. coli . These isolates express chromosomally encoded virulence markers.

In the late 1970s, it was recognized for the first time that E. coli strains causing urinary tract infections typically agglutinate human erythrocytes despite the presence of mannose and this was mediated mainly by fimbriae. [2] The virulence factors include different adhesins, hemolysin production and siderophore production. Fimbriae mediate the ability of E. coli to adhere to the uroepithelium, thereby resisting elimination by the flow of urine. Adhesion is therefore considered to be an important step in the pathogenesis of UTI. [3]

Bacterial siderophores compete for iron with host iron binding proteins. When bound by siderophore, the iron is taken up by special bacterial surface receptors and can be utilized by the pathogen, many strains of E. coli associated with urinary tract infection produce siderophore. [4]

The information on the characteristics of E. coli causing urinary tract infections is limited and less studied. So, the present study was designed to determine the urovirulence factors of E. coli isolated from the patients of UTI and to study their antimicrobial susceptibility pattern.

 ~ Materials and Methods Top

The study was conducted in the Department of Microbiology, J. N. Medical College, Belgaum, from October 2002 to September 2003. One hundred and sixty E. coli strains isolated from urine samples and 50 faecal isolates were studied for the detection of virulence markers of E. coli . Escherichia coli were identified as described by Bailey and Scott. The isolates were maintained by inoculating nutrient agar butts and stored at room temperature and tested for haemagglutination and siderophore production.


The haemagglutination was detected by clumping of erythrocytes by fimbriae of bacteria in the presence of d-mannose. This test was carried out as per the direct bacterial haemagglutination test - slide method and mannose-sensitive and mannose-resistant haemagglutination tests. [5] The strains of E. coli were inoculated into 1% nutrient broth and incubated at 37 C for 48 hours for full fimbriation. A panel of red blood cells was selected by obtaining blood from guinea-pig, sheep and human (blood group 'O'). The red blood cells were then washed thrice in normal saline and made up to a 3% suspension in fresh saline. They were used immediately or within a week when stored at 3-5 C. The slide haemagglutination test was carried out on a multiple-concavity slide. One drop of the RBC suspension was added to a drop of the broth culture and slide was rocked to and fro at room temperature for 5 minutes. Presence of clumping was taken as positive for haemagglutination. Mannose-sensitive haemagglutination was detected by the absence of haemagglutination in a parallel set of test in which a drop of 2% w/v d-mannose was added to the red cells and a drop of broth culture. Mannose-resistant haemagglutination was detected by the presence of haemagglutination of 3% 'O' group human RBC in the presence of 2% mannose.

Siderophore production assay

This test was carried out by using a method named 'chrome azurol sulphonate (CAS) agar diffusion assay. [6] The chrome aurol sulphonate (CAS) assay detects colour change of CAS-Iron complex from blue to orange after chelation of the bound iron by siderophores. A strong ligand 'L' (e.g., a siderophore) is added to a highly coloured iron dye complex; when the iron ligand complex is formed, the release of the free dye is accompanied by a colour change.

The result was taken as positive if there was a colour change from blue to orange halo [Figure - 1].

Antibiotic sensitivity testing

Antibiotic sensitivity testing was performed for all the isolates of E. coli by Kirby Bauer's disc diffusion method like ampicillin, cotrimoxazole, gentamicin, nalidixic acid, norfloxacin, nitrofurantoin, ciprofloxacin, netilmicin to identify their resistance pattern to the commonly used antibiotics.

 ~ Results Top

A total of 40 (25%) among 160 isolates from cases and 2 (4%) out of 50 controls showed mannose-resistant haemagglutination (MRHA). There was a significant difference in MRHA between cases and controls.

Fifty-five (34.38%) from cases and four (8%) from controls showed mannose-sensitive haemagglutination (MSHA) [Table - 1].

Siderophore production was seen in 156 (97.5%) among 160 cases and 2 (4%) among controls [Table - 2].

Out of 160 isolates of E. coli , 150 (93.75%) were susceptible to nitrofurantoin followed by netilmicin 149 (93.13%), and 40 (25%) were sensitive to ampicillin.

 ~ Discussion Top

Considering the high degree of morbidity in urinary tract infections, the subject of uropathogenic E. coli (UPEC) is receiving increasing attention. Cell morphology and molecular biology studies have revealed that uropathogenic E. coli express fimbriae and siderophore production peculiar to the strains of E. coli causing urinary tract infection. Hence, it is important to identify UPEC isolates in the urinary samples. [1]

The occurrence of virulence factors in UPEC strains strengthens the concept of association of UPEC with urinary pathogenicity. UPEC with virulence factors were significantly more in urinary isolates than in controls. Haemagglutination is mediated by fimbriae. MRHA can be mediated by P-fimbriae and X, FIC, DR fimbriae. Thus, MRHA-positive strains can be considered as UPEC most likely having P-fimbriae. [1],[7]

Virulence determinants such as P-fimbriae, siderophore production have been shown to be more frequent in E. coli from patients with UTI than in faecal isolates. [8]

Type I fimbriae, which bind to a mannose-containing receptor, are found in most E. coli urinary isolates. [9] The expression of type I fimbriae is indicated by MSHA. [1]

In E. coli , the hydroxymate siderophore (aerobactin) is the most effective of the several iron chelation systems employed by the bacteria for iron acquisition. The siderophore (aerobactin) and P-fimbriae are commonly found together in isolates from patients with UTI. [7]

In view of the emerging drug resistance among UPEC, therapy should be advocated as far as possible after culture and sensitivity has been performed. This would not only help in the proper treatment of the patients, but would also discourage the indiscriminate use of the antibiotics and prevent further development of bacterial drug resistance.

 ~ References Top

1.Raksha R, Srinivasa H, Macaden RS. Occurrence and characterization of uropathogenic Escherichia coli in urinary tract infections. Indian J Med Microbiol 2003;21:102-7.  Back to cited text no. 1    
2.Johnson JR. Virulence factors in Escherichia coli urinary tract infection. Clin Microbiol Rev 1991;4:81-128.  Back to cited text no. 2    
3.Struve C, Krogfelt KA. In vivo detection of Escherichia coli type-1 fimbrial expression and phase variation during experimental urinary tract infection. Microbiology 1999; 145:2683-90.  Back to cited text no. 3    
4.Sussman M. Urinary tract infections, Chapter 32. In : Topley and Wilson's Microbiology and Microbial Infections, 9 th ed. Collier L, Balows A, editors. Vol.3, Arnold: Great Britain; 1998. p. 601-21.  Back to cited text no. 4    
5.Duguid JP, Clegg S, Wilson MJ. The fimbrial and non-fimbrial haemagglutinins of Escherichia coli . J Med Microbiol 1979;12:213-27.  Back to cited text no. 5    
6.Shin SH, Lim Y, Lee SE, Yang NW, Rhee JH. CAS agar diffusion assay for the measurement of Siderophores in biological fluids. J Microbiol Methods 2001;44:89-95.  Back to cited text no. 6    
7.Johnson JR. Virulence factors in Escherichia coli urinary tract infection. Clin Microbiol Rev 1991;4:80-128.  Back to cited text no. 7    
8.Stapleton A, Moseley S, Stamm WE. Urovirulence determinants in Escherichia coli isolates causing First episode and recurrent cystitis in women. J Infect Dis 1991;163:773-9.  Back to cited text no. 8    
9.Tseng CC, Wu JJ, Liu HL, Sung JM, Huang JJ. Roles of host and bacterial virulence factors in the development of upper urinary tract infection caused by Escherichia coli . Am J Kidney Dis 2002;39:744-52.  Back to cited text no. 9    


  [Figure - 1]

  [Table - 1], [Table - 2]

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