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Year : 2009  |  Volume : 52  |  Issue : 1  |  Page : 126-127
Siderophore production by uropathogenic Escherichia coli

Department of Microbiology, Jawaharlal Nehru Medical College, Belgaum, Karnataka, India

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Urinary tract infection (UTI) is one of the most frequently encountered problems in ambulatory medicine. The present study was designed to determine siderophore production as the urovirulence factor of Escherichia coli isolated from the patients of UTI. A total of 160 strains of E. coli isolated from urine of patients with clinically diagnosed UTI were included in the study and 50 fecal isolates of E. coli, siderophore production was seen in 156 (97.5%). In 50 fecal isolates, siderophore production was seen in 2 (4%). Siderophore production has been shown to be more frequent in E. coli from patients with UTI, than in fecal isolates. The results suggest that siderophore production positive strains can be considered as UPEC. Thus, although a great deal has been learned regarding E. coli virulence mechanisms in UTI, much remains to be learned and the practical application of our growing understanding of E. coli virulence factors to the prevention and treatment of UTI has to be continued.

Keywords: Escherichia coli, siderophore, uropathogenic

How to cite this article:
Vagrali MA. Siderophore production by uropathogenic Escherichia coli. Indian J Pathol Microbiol 2009;52:126-7

How to cite this URL:
Vagrali MA. Siderophore production by uropathogenic Escherichia coli. Indian J Pathol Microbiol [serial online] 2009 [cited 2022 Sep 30];52:126-7. Available from:

   Introduction Top

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 to 50%-90% of all uncomplicated urinary tract infections (UTIs). It is now recognized that there are a subset of fecal E. coli which can colonize the periurethral area, enter urinary tract and cause symptomatic disease. These are currently defined as uropathogenic E. coli (UPEC). It has been traditionally described that certain serotypes of E. coli were consistently associated with uropathogenicity and were designated as UPEC. These isolates express chromosomally encoded virulence markers. [1]

E. coli needs iron for aerobic metabolism and multiplication; part of the host response to infection is to reduce the amount of iron available to the invading UPEC by decreasing intestinal iron absorption, synthesizing additional iron proteins and shifting iron from the plasma pool into intracellular storage. Thus, the bacteria face a formidable challenge in meeting their iron needs during infection. In E. coli , the hydroxymate siderophore (aerobactin) is the most effective of the several iron chelation systems employed by the bacteria for iron acquisition.

Two mechanisms of iron uptake in E. coli have been identified - (1) the hydroxymate-type of siderophore (aerobactin) and (2) the catechol-type siderophore (enterochelin).

In E. coli , the hydroxymate siderophore (aerobactin) is the most effective of the several iron chelation systems employed by the bacteria for iron acquisition.

Bacterial siderophores compete for iron with host iron-binding proteins. When bound by the 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 UTI produce siderophore. [2]

The information on the characteristics of E. coli causing UTIs is limited and less studied. Therefore, 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, over a period of one year from October 2002 to September 2003. 160 E. coli strains isolated from urine samples were studied for the detection of siderophore production and 50 fecal isolates were also studied. E. coli was identified as described by Bailey and Scott. The isolates were maintained by inoculating nutrient agar butts and stored at room temperature.

Testing for siderophore production

E. coli strains identified were tested for siderophore production.

Siderophore production assay

This test was carried out by using a method named "chrome azurol sulfonate (CAS) agar diffusion assay." [3] The CAS assay detects color change of CAS-Iron complex form blue to orange after chelation of the bound iron by siderophores. A strong ligand L (e.g., a siderophore) is added to a highly colored iron dye complex; when the iron ligand complex is formed, the release of the free dye is accompanied by a color change.

The CAS agar diffusion assay has the following two principles:

  1. Iron bound to CAS is easily chelated by siderophores to produce a color change from blue to orange and

  2. The principle is like disc diffusion assay, i.e., the siderophores applied to the hole diffuse radially and build a concentration gradient from the centre.

    Fe Dye + L → Fe L + Dye

    Dye → CAS

    L → Siderophore

    Fe → Iron

The CAS agar diffusion assay was performed as follows:

First, 60.5 mg CAS was dissolved in 50 ml deionized water and mixed with 10 ml iron (III) solution (1 mM FeCl3 - 6H 2 O, 10 mM HCl); by stirring, this solution was slowly mixed with 72.9 mg hexadecyltrimethyl ammonium bromide (HDTMA) dissolved in 40 ml water. The resultant dark blue solution was autoclaved and mixed with an autoclaved mixture of 900 ml water, 15 g agar. 30.24 g 1.4-piperazine diethane sulfonic acid (PIPES) and 12 g of solution of 50% (w/v) NaOH to raise the pH to the Pka of PIPES (6.8). The modified CAS agar plate was punched with 2.5-5 mm diameter holes by using a gel puncher. Each hole was filled with 25-35 ml of the broth and Desferal, which was two-fold serially diluted from 2.5 mM. After incubation of the plate at 37°C or room temperature for 4-8 h, orange halo was formed around each hole. The result was taken as positive if there was a color change from blue to orange halo.

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, gentamycin, nalidixic acid, norfloxacin, nitrofurantoin, ciprofloxacin and netilmicin to identify their resistance pattern to the commonly used antibiotics.

   Results Top

Siderophore production

Siderophore production was seen in 156 (97.5%) out of 160 isolates and two of the 50 (4%) among controls [Table 1].

Antibiotic sensitivity pattern of the isolates

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

   Discussion Top

Considering the high degree of morbidity in UTIs, the subject of uropathogenic E. coli (UPEC) is receiving increasing attention. Cell morphology and molecular biology studies have revealed that UPEC expresses siderophore production peculiar to the strains of E. coli causing UTI. 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 siderophore production was significantly more in urinary isolates than in controls. [4]

Siderophore production has been shown to be more frequent in E. coli from patients with UTI than in fecal isolates. [5]

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) is commonly found in isolates from patients with UTI. [4] Acute pyelonephritis isolates had a higher incidence of aerobactin production than asymptomatic bacteriuria isolates. [6]

IreA and IroN are the recently identified siderophore receptors.Molecular epidemiologic evidence from several studies has demonstrated an increased prevalence of IroN among UTI isolates relative to fecal isolates. This evidence suggests that IroN functions as a siderophore receptor and is a urovirulence factor for 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  [PUBMED]  Medknow Journal
2.Sussman M. 'Urinary tract infections' Chapter 32. In: Collier L, Balows A, Sussman M, editors. "Topley and Wilson's Microbiology and Microbial Infections". 9th ed. Vol. 3, Great Britain: Arnold; 1998. p. 601-21.  Back to cited text no. 2    
3.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. 3  [PUBMED]  [FULLTEXT]
4.Johnson. Virulence factors in Escherichia coli urinary tract infection. Clin Microbiol Rev 1991;4:81-113.   Back to cited text no. 4    
5.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. 5  [PUBMED]  
6.Hooton TM, Stamm WC. Diagnosis and treatment of uncomplicated urinary tract infection. Infect Dis Clin North Am 1997;11:551-81.   Back to cited text no. 6    
7.Russo TA, McFadden CD, Carlino-MacDonald UB, Beanan JM, Barnard TJ, Johnson JR. Iron functions as a Siderophore receptor and is a urovirulence factor in an extraintestinal pathogenic isolate of Escherichia coli . Infect Immun 2002;70:7156-60.  Back to cited text no. 7  [PUBMED]  [FULLTEXT]

Correspondence Address:
Manjula A Vagrali
Department of Microbiology, Jawaharlal Nehru Medical College, Belgaum, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.44988

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