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  • The Internet Journal of Aesthetic and Antiaging Medicine
  • Volume 2
  • Number 1

Original Article

Osmotolerance Studies Of Uropathogenic Escherichia Coli

M Abhilash

Citation

M Abhilash. Osmotolerance Studies Of Uropathogenic Escherichia Coli. The Internet Journal of Aesthetic and Antiaging Medicine. 2008 Volume 2 Number 1.

Abstract


E Coli isolates from urine samples of patients suffering from Urinary Tract Infections caused by E Coli was collected from various private diagnostic centers in Davangere, Karnataka, India. Isolates were cultured and maintained using specific media. Urea and Salinity tolerance of samples was checked by observing bacterial growth in artificial urine with varying urea and salt concentrations. Biochemical assays were carried out to identify virulence factors of Uropathogenic E Coli in samples showing high salt and urea tolerance.

 

Introduction

E. coli is the head of the large bacterial family, Enterobacteriaceae the enteric bacteria, which are facultative anaerobic Gram-negative rods that live in the intestinal tracts of animals in health and disease. The Enterobacteriaceae are among the most important bacteria medically. A number of genera within the family are human intestinal pathogens (e.g. Salmonella, Shigella, Yersinia). Several others are normal colonists of the human gastrointestinal tract (e.g. Escherichia, Enterobacter, Klebsiella), but these bacteria, as well, may occasionally be associated with diseases of humans.

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 E.coli is an ascending infection caused by the strains originating in the intestinal tract, because a high similarity exists between E.coli strains from urine and faeces of infected individuals.

Uropathogenic E. coli cause 90% of the urinary tract infections (UTI) in anatomically-normal, unobstructed urinary tracts. The bacteria colonize from the feces or perineal region and ascend the urinary tract through the bladder. With the aid of specific adhesins they are able to colonize the bladder.

Different virulence factors of E.coli which are thought to have a role in the pathogenesis of Urinary Tract Infections , some of them are O Antigens, K Antigens, Serum resistance, Adhesins, Colicins, Invasins etc .

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 E.coli in great detail and classified them into three groups depending upon their haemagglutinating properties as the MSHA(Mannose Sensitive Haemagglutinating) type ,MRHA(Mannose Resistant Haemagglutinating) type and non-fimbrial haemagglutinin type.There is considerable evidence to support the use of cranberries for the prevention of urinary tract infections (Bodel et al., 1959; Moen, 1962; Sternlieb,1963; Papas et al., 1968; Avorn et al., 1994).

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 .

To assign E.coli from significant bacteriuria cases as ‘uropathogenic’ is not possible because there is no specific identification test. The virulence factors are several and each UTI-pathogen may possess any or all of these virulence factors. From another angle, the primary requirement of E.coli causing UTI is to be able to multiply in urine, under the prevailing urea and salt concentrations. Also, it is now known that they possess osmoregulatory genes that may play a role in countering these conditions. Our objective therefore, was to find out whether the ability of UTI causing E.coli to survive and multiply in urine is a property unique to them and if so, whether this itself could become the test for identifying them as uropathogens.

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 E-coli , it will inhibit the growth of other form of bacteria and supports the growth of only E-coli strains. This procedure was employed for selection of E-coli strains. Strains were labelled for convenience.

Osmotolerance studies

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 E.coli in nutrient broth .2 % of starter culture was inoculated onto various molar concentration of urea and NaCl prepared in nutrient broth. Growth of E Coli in various concentrations (15.625Mm – 1M) of NaCl and urea was noted by measuring absorbance of the samples at 600nm. E.coli K-12 and MTCC –729 were used as positive and negative controls

Biochemical Assays

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

Hemolysin production by E. coli strains can be detected using 2 methods:

  • Tube method and

  • Plate method

  • RBCs of different species which were

  • Human type A (Hu), Sheep (Sp), Guinea-pig (Gp).

Tube method was used for experimentation.

Tube method

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 E.coli K12 colicin sensitive strains were added to 8 ml of this agar gel while it was still molten (450 - 500C). This was mixed thoroughly and overlayed on the surface of 'Chloroform treated' plates. The agar was allowed to set and reincubated at 370C/24 hours. This sensitive strain was examined for zone of inhibition by the test strains. Clear zone of inhibition indicated the production of colicin.

Results

Note:- Samples were labeled with numbers for convenience.

Osmotolerance studies were carried out in Batches

Table 1

Batch 1

Growth of E-coli in various concentration of NaCl noted by measuring the absorbance of the samples at 600nm.

{image:1}

Batch 2

{image:2}

Table 2

Batch 1

Growth of E-coli in various concentration of Urea noted by measuring the absorbance of the samples at 600nm.

{image:3}

Batch 2

{image:4}

E Coli with Samples numbers 522,533,552,553,555,697,702 have shown growth rates even in higher concentration of salt and urea.

Results of Biochemical tests

{image:5}

Conclusion

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 E.coli tested concomitant with increasing concentration of Urea / salt indicating sensitivity. However, we see that they are able to survive at concentrations of Urea which represents normal levels in urine. We believe that this approach warrants further work and would yield a rapid method of differentiating Uropathogenic bacteria.

Acknowledgement

We are thankful to Mrs. Bharathi Sriram, Head of analytical division, GANGAGEN BIOTECHNOLOGY LIMITED, BANGALORE, for her valuable guidance.We would like to thank YASHASWI DIAGNOSTIC CENTRE and POOJA DIAGNOSTIC CENTRE, Davangere, Karnataka, India for helping us by providing E.coli samples to carry out this project.

References

1. Steadman R, Topley N. The virulence of Escherichia coli in urinary tract, Chapter 3
In: Urinary tract infections . 1st Ed. Brumfitt W,Jeremy MT, Hamilton Miller. Eds
(Chapman andHall publication, London) 1998:37-41.
2. Cowan and Steel’s Manual for identification of Medical bacteria, 3rd ed. Eds. Barrow
Gl, Feltham RKA. Eds.(University of Cambridge) 1993.
3. Smith HW. The haemolysins of Escherichia coli. J Pathol Bacteriol 1963;85:197-211.
4. Cavalieri SJ, Bohach GA, Snyder IS. Escherichia coli alpha haemolysin:
characteristics and probable role in pathogenicity. Microbiol Rev 1984;48:326-343.
5. Siegfried L, Marta Kmetova, Hana Puzova, et al. Virulence associated factors in
Escherichia coli strains isolated from children with urinary tract infections. J Med

Microbiol 1994;41:127-152.
6. Benge GR. Bactericidal activity of human serum against strains of Klebsiella from
different sources J Med Microbiol 1988;27:11-15.

Author Information

M. Abhilash, B.E, M.Tech
Department of Biotechnology, The Oxford college of Engineering

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