Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 26, Num. 1, 2008, pp. 68-70

Indian Journal of Medical Microbiology, Vol. 26, No. 1, January-March, 2008, pp. 68-70

Brief Communication

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

Vagarali MA, Karadesai SG, Patil CS, Metgud SC, Mutnal MB

Department of Microbiology, JN Medical College, Belgaum - 590010, Karnataka
Correspondence Address:Department of Microbiology, JN Medical College, Belgaum - 590010, Karnataka, drmanjulavagrali@yahoo.com

Date of Submission: 02-Sep-2006
Date of Acceptance: 26-Mar-2007

Code Number: mb08015

Abstract

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

Urinary tract is the second most common site of bacterial infection in humans and thus represents a major source of human discomfort. Escherichia 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

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.

Haemagglutination

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

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

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

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