M Abhilash. Osmotolerance Studies Of Uropathogenic Escherichia Coli. The Internet Journal of Infectious Diseases. 2008 Volume 7 Number 2.
Of the different organisms known to cause Urinary Tract Infections(UTI), Escherichia coli is the most predominant pathogen being isolated in 70-90% of cases. It has been accepted that UTI caused by
Different virulence factors of
Many studies (Fowler et al 1977, Kallenius et.al 1978) have shown that bacterial adherence is an essential virulence factor in the pathogenesis of community acquired Urinary Tract Infections. Duguid et al (1966) studied fimbriae of
Colicins belong to a general class of natural antimicrobials called bacteriocins. These bacteriocins may be defined as the antimicrobial substances or complexes of antibiotic substances, which are highly specific and are produced by certain strains of intestinal bacteria act upon other related strains i.e. same or related species .
Material and Methods
Urine samples of patients suspected to be suffering from Urinary Tract infection was collected from various private diagnostic centers in Davangere, Karnataka, India. These samples were then swabbed on Mac conkey's media . Media was left for incubation period of 24 hours at 350 C. Since this is a selective media for
Various molar concentration solutions of Urea and NaCl were prepared in nutrient broth and 25 ml of each sample were taken in separate conical flasks. Starter culture of each strain prepared initially with overnight incubation of few colonies of
Following Biochemical assays were carried out to identify certain virulence factors.
Salt Aggregation Test (SAT) for detection of Adhesins
This test was performed as described by lindhal et al. (1981). The test strains have to be grown on CFA-agar plates at 370/18 hrs to enhance the production of fimbrial antigens. 4M Solution of (NH4¬¬)2 SO4 was prepared in 0.002M sodium phosphate (Na2 PO4) buffer solution pH 8.6. Two fold dilutions of ammonium sulphate with sodium phosphate buffer solution was done to obtain 2M, 1M, 0.5M, 0.25M and 0.125 dilutions.
One drop of each dilution over (from 4M, 2M, 1M, 0.5M, 0.25M upto 0.125M) was taken over a clean glass slide. The bacterial growth from the growth of the test strain was taken from CFA agar plate (after incubation at 370 for 18 hours). One drop (50 ul) of each type of 1% PBC was taken separately for MHA and MSHA activity and the growth was emulsified in it and then examined for 1 minute by gently rotating the suspension over the slide. Positive and negative controls were also put simultaneously to compare the results of test strains.E.coli K-12 and MTCC –729 were used as positive and negative controls for the presence and absence of CFAI.
The interpretation of the results was made as follows:
Mannose resistant haemagglutination (MRHA) (If HA occurred in presence of 2% D-mannose).
Mannose sensitive haemagglutination (MSHA) if agglutination of RBCs occurred in absence of mannose but was inhibited in the presence of D-mannose.
No haemagglutination – if there is no agglutination in both.
Hemolysin production by E. coli strains can be detected using 2 methods:
Tube method and
RBCs of different species which were
Human type A (Hu), Sheep (Sp), Guinea-pig (Gp).
Tube method was used for experimentation.
This test was done as described by Feeley and Pittman (1963), with suitable modifications. E.coli test strains were inoculated in the meat extract broth (Difco) (pH 7.4) 0.4 ml per tube. Each test strain was inoculated in the 3 tubes to test against the RBCs from 3 different species. The tubes were incubated at 370C for 24 hrs and 1% suspension of the RBCs (0.4 ml/tube) of different types was added to the respective tubes. These were again incubated at 370C for 2 hrs and examined for the lysis of RBCs. Then without disturbing, the tubes were kept in the cold room at 40C overnight to observe for hot and cold phenomenon and for further lysis of RBCs.
Test for colicin production
This test was performed as described by frederiocq (1951) using colicin agar.
The plates were incubated at 370 C/48 hrs by wrapping the plates in polythene bag. After incubation, the plates were exposed to Chloroform for 10-15 minutes to kill the bacterial growth. The semisolid agar gel (0.5% noble agar) was prepared and 4 drops (0.2 ml) of overnight broth culture of
Growth of E-coli in various concentration of NaCl noted by measuring the absorbance of the samples at 600nm.
Growth of E-coli in various concentration of Urea noted by measuring the absorbance of the samples at 600nm.
Results of Biochemical tests
We have used Urea and salt in the concentration range corresponding to normal physiological levels in our experiments. We have seen a dose-dependent decrease in growth of the
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