Detection of Cell Surface Hydrophobicity among Uropathogenic isolates of Escherichia coli

 

Gowtham R¹, Dr. Gopinath P^2*

¹Bachelor of Dental Surgery, Saveetha Dental College & Hospital, Chennai, India.

²Senior Lecturer, Department of Microbiology, Saveetha Dental College & Hospital, Chennai, India

 

ABSTRACT:

A number of studies have shown that the hydrophobicity of solid surfaces influences adhesion of bacteria, eukaryotic cell and proteins. On the other hand bacteria and the other microbes including viral particles are evolving many different ways to use the hydrophobicity in order to adhere on to the substrates. A total of 20 clinical isolates of UPEC were subjected for antibiotic sensitivity pattern and cell surface hydrophobicity by salt aggregation method.Most of the strains showed resistant to all the antibiotics tested. 70% of isolates were showed cell surface hydrophobicity positivity. Judicial usage of antibiotics and proper detection of resistant mechanisms operating in the clinical isolates would yield better management of patient’s outcome. Cell surface hydrophobicity might associate with the adhesion of UPEC in both biotic and abiotic surfaces.

 

 

INTRODUCTION:

Escherichia coli are generally non-pathogens and are part of normal micro flora of human and animal intestine. They are genetically heterogeneous in nature. They are 6 different types of E.coli have been recognized such as enterotoxigenic E.coli (ETEC), enteroinvasive E.coli (EIEC), enteropathogenic E.coli  EPEC), enterohaemorrhagic E.coli (EHEC), entero-aggregative E.coli (EAEC), diffuse adhering E.coli (DAEC), out of all ETEC is the most predominant strain reported globally.^[1]

 

Even though, both normal flora and pathogenic strains of E.coli colonizing colon of human being, only strains of  uropathogenic E.coli are able to cause urinary tract infection. The disease spectrums produced by E.coli are sepsis, meningitis, diarrhea, UTI etc.

 

They differ in serotypes about more than 250 serotypes based on O,H and K antigens have been understood. UPEC isolates can exhibit repertoire of virulent determinance that can specifically contribute to their ability to cause specifically contribute to their ability to cause infection in urinary tract of human.^[2]

 

MATERIALS AND METHODS:

Bacterial Isolates:

A total of 20 non repetitive urinary isolates of E.coli were collected from suspected cases of UTI attending Saveetha Medical college, Thandalam. They were processed for a battery of standard biochemical tests and confirmed. Isolates were preserved in semisolid trypticase soy broth stock and stored at 4˚C until further use.

 

Antibiotic Susceptibility Testing:

Antibiotic susceptibility testing was determined for these isolates to routinely used antibiotics such as ampicillin, amoxycillin, ceftazidime, cefotaxime, amikacin, gentamacin, norflaxacin, ciprofloxacin by Kirby Bauer disc diffusion method as per CLSI guidelines. ^[3]

Detection of Cell Surface Hydophobicity:

This was performed by salt aggregation method. Freshly subcultered UPEC isolates were inoculated into 1ml of sterile PBS (pH 6.8) and turbidity were adjusted to McFarland standard 7 to obtain a colony count of 5X10⁹  colonies/ml. Different molar concentrations of ammonium sulfate such as 1M, 1.4M and 2M were prepared. 20µl of PBS was taken in first column of microtitre plate. 20µl of the above mentioned concentrations of ammonium sulfate were placed in each well of other well of microtitre plate. Then 20µl of UPEC suspensions were added to each of these wells. The clumps formed in different molar concentrations of ammonium sulfate was observed using magnify hand lens and scored as 1+, 2+, 3+ and 4+. Isolates were recorded as positive for hydrophobicity when the form aggregation at 1.4M and 2M^.[4]

 

RESULTS:

Sample wise distribution of clinical isolates of E.coli:

Amongst 20 isolates of E.coli, 12/20(60%) were from acute UTI cases and 8/20 (40%) from chronic cases of UTI attending Saveetha Medical college and Hospital.

 

Figure 1 : Sample wise distribution of clinical isolates of E.coli

 

ANTIBIOTIC SUSCEPTIBILITY TESTING:

In our isolates, we have found increased percentage 14/20(70%) of isolates showed sensitivity to amikacin followed by gentamicin, which showed sensitivity of 9/20 (45%). 80-90% of E.coli isolates showed resistance to cephalosporin group of drugs. However, we have observed an increased level of resistance to other routinely used antibiotics. The detailed resistant pattern of E.coli isolates were showed in table 1.

Table1: Results of antibiotic susceptibility pattern of E.coli

 

Figure 2: Representative picture for antibiotic susceptibility testing of E.coli.

 

Results of Cell Surface Hydrophobicity:

Of the 20 UPEC isolates, 14(70%) were found to have cell surface hydrophobicity. Out of 14 positive isolates, 8/14(57.1%) were from acute and 6/14(42.8%) from chronic UTI cases.(Table2)

 

Table2: Results of Cell Surface Hydrophobicity of UPEC

Strain No	Concentrations of Ammonium Sulfate
	1M	1.4M	2M
1	-	-	-
2	-	-	-
3	++	-	-
4	++++	++	+
5	-	-	-
6	++++	++	+
7	++	++	++
8	++++	+	-
9	+	+	+
10	+	+	+
11	++++	++	+
12	++++	+++	+
13	+++	-	-
14	-	-	-
15	+	+	+
16	+++	++	-
17	+++	+	+
18	++++	++	+
19	++++	++++	+++
20	++++	++++	++++

 

Figure 3: Representative Picture Showing Cell Surface Hydrophobicity of UPEC

DISCUSSION:

Hydrophobicity has been associated with pathogenicity, because it is involved in bacterial adhesion to cellular and inert surfaces. We have evaluated the hydrophobicity of UPEC strains by qualitative test by salt aggregation method.

 

Cell surface hydrophobicity study of E.coli isolated from urinary tract infections cases by Siegfried et al. in 1994 showed 81% of isolates were found to be hydrophobic by salt aggregation method. Similar kind of study done by Raksha and coworkers in 2003 from Bangalore showed 26.3% hydrophobicity E. coli isolates.^[4] Similarly, our study showed 70% of UPEC isolates were cell surface hydrophobicity producers.

 

Study conducted by Norouzi et al., in 2010 from Iran found 10.6-66.3% of cell surface hydrophobicity isolates among ESBL producing isolates of P. aeruginosa, whereas 3.8-65.2% of isolates were found to be hydrophobic among non-ESBL producers using microbial adhesion to hydrocarbon method.^[5]

 

CONCLUSION:

The present study reveals high degree of resistance to all antibiotic tested. Drug resistance is on the rise among E.coli isolates causing infections to humans. Proper selection of antibiotics for treatment depends on the results of antibiotic sensitivity test. Therefore, the correct detection of resistance in bacteria is important. Judicial usage of antibiotics and proper antibiotic policy are needed to limit the emergence and spread of drug resistance among E.coli. Virulence factors enable E.coli to colonize specifically the mucosal epithelium and evoke an inflammatory reaction and eventually lead to lower urinary tract to renal cavities and tissue invasion.

 

UPEC isolates are most often associated with urinary catheters. The adhesion of UPEC isolates or strains are mediated by presence of cell surface hydrophobicity. Thus subsequently leads to stubborn biofilm formation which cannot be vanished by antibiotics. It is very apparent that our isolates were also exhibiting cell surface hydrophobicity which might involve in the establishment of UTI.

 

ACKNOWLEDGEMENT:

Authors would like to thank Dr. Jayalakhsmi, Saveetha Medical College, Chennai, for providing the samples to carry out the research.

 

REFERENCES:

1.         Jacobsen SM, Stickler DJ, Mobley HL, Shirtliff ME. Complicated catheter-associated urinary tract infections due to Escherichia coli and Proteus mirabilis. Clin Microbiol Rev. 21; 2008:26-59.

2.         Sarantuya J, Nishi J, Wakimoto N, Erdene S, Nataro JP, Sheikh J. Typical enteroaggregative Escherichia coli is the most prevalent pathotype among E. coli strains causing diarrhea in Mongolian children. J ClinMicrobiol.42;2004:4133-9.

3.         National committee for clinical laboratory standards. Performance standards for antimicrobial disc susceptibility tests. Approved standard M2-A7. National Committee for Clinical Laboratory Standards: Wayne, PA; 2000.

4.         Raksha R, Srinivasa H, Macaden RS. Occurrence and characterization of uropathogenic Escherichia coli in urinary tract infections. Indian J Med Microbiol.21;2003:102-7.

5.          Norouzi F, Mansouri S, Moradi M, Razavi M. Comparison of cell surface hydrophobicity and biofilm formation among ESBL and non ESBL producing P. aeruginosa clinical isolates. Afr J Microbiol Res.4;2010:1143-1147.

 

 

Accepted on 20.07.2016        © RJPT All right reserved

Research J. Pharm. and Tech 2016; 9(11): 1883-1885