INTRODUCTION

Serum resistant is appears to be an important virulence factor for the pathogenicity of different gram negative bacteria. There are several mechanisms by which the bacteria can resist them from complement mediated killing such as early release of membrane attack complex mediated by lipopolysaccharide (1), insertion of membrane attack complex into non-lethal targets of the bacterial cell envelope by certain outer membrane protein (OMP) (2), interference of certain capsular polysaccharides using alternative pathway complement activation (3).

The ability of microorganism to combat with human system by two mechanisms. The first is a specific immunity and the second is natural or non-specific resistance which does not occur for every invading microorganisms. (4) These two host defense mechanisms are present in every healthy hosts. However, they may differ among individuals depending on various known and unidentified factors.

The variation of host immunity in human may occur through different known factors such as age, sex, genetic, pregnancy, environment and nutrition. (5)

One of these nonspecific mechanism occur in serum of healthy human is bactericidal activity against certain bacteria. This immunity may act among various defenses, in relation with phagocytes and complement to prevent bacteremia from those species.(6) This bactericidal activity is mainly against some gram-negative bacteria (7). This effect is arrested when serum has been incubated for 30 minutes at 56°C. Gram-positive bacteria generally are less sensitive than gram-negative bacteria to this effect. (8). Hence, we therefore planned to study the serum bactericidal activity of pooled serum of our healthy individuals against E. coli.

MATERIALS AND METHOD:

Bacterial Isolates:

A total of 20 non repetitive clinical isolates of E. coli were collected from the Saveetha Medical College, Chennai. They were processed by battery of standard biochemical tests and confirmed. Isolates were preserved in semisolid Trypticase soy broth stock and stored at 4ºC until further use.

Collection of Human Serum:

5mL of blood were collected from healthy human volunteers who had not undergone any antimicrobial treatment for the past 1 month. The collected blood samples were incubated at 37ºC for 1 hour then 4ºC till further use. Blood were centrifuged and sera were collected aseptically, pooled and kept at -20ºC. (9)

Serum Bactericidal Test:

Isolates of E. coli were grown in trypticase soy broth (TSB) for overnight incubation and cell density was adjusted to1x10⁸cfu/mL. 100µL of cell suspension was mixed with 900µL of pooled serum. Immediately 100µL of the samples were removed and inoculated on nutrient agar plates. Similarly, 100µL of samples were removed at 2 hours, 3 hours and 24 hours durations and inoculated on nutrient agar plates. All plates were incubated at 37ºC for 24 hours and results were recorded.

RESULTS:

Sample wise distribution of clinical isolates of E. coli:

Of the 20 clinical isolates of E.coli, 12/20 (60%) were from urine, 4/20 (20%) from stool, 3/20 (15%) and 1/20 (5%) were from wound swab and pus respectively. Figure 1 depicts the sample wise distribution of clinical isolates of E.coli.

Figure 1: Sample Wise Distribution of Clinical Isolates of E.coli Serum Bactericidal Activity:

Serum bactericidal activity of human sera against 20 clinical isolates of E. coli were determined by subjecting the isolates with pooled human sera in different time intervals of incubation such as 0 minute, 2 hours, 3 hours and 24 hours. All clinical isolates of E. coli were resistant at 0 minute interval. 12/20 (60%) clinical isolates of E. coli were sensitive at 2 hours of incubation. 6/20 (30%)E. coli isolates were sensitive at 3 hours of incubation. 2/20 (10%) were found to be resistant even after 24 hours of incubation.

Figure 2:Time kill assay of pooled human sera against E. coli

DISCUSSION:

Resistance to the bacterial activity of human serum is an important factor in bacterial virulence (10). Results obtained in the present study showed 2/20 (10%) of clinical isolates of E.coli exhibited resistance to normal human serum. Valvano et al., (1992) and Russo et al., (1993) reported that bacteremia due to gram negative bacteria are found to be more resistant to normal human serum and phagocytosis than bacteria causing other types of infections (10,11). Valvanoet al., in 1992 reported a significant correlation between the degrees of resistance of gram negative bacteria recovered from septicemia to the lytic activity of complement and their ability to invade and survive in human fluids. The participation of some bacterial components such as outer membrane protein (OMP), capsular polysaccharide (CPS) and lipopolysaccharide LPS) in this property has been found to be protective (10,11).

CONCLUSION:

As suggested by different authors in different studies, the serum bactericidal activity is attributed by the effect of OMP, CPS and LPS of bacteria. It is understood that in this study also any of these component of bacteria might have involved in showing bactericidal activity. For understanding the better knowledge about the serum bactericidal activity more sample size with in depth study have to be carried out.

ACKNOWLEDGMENT:

We thank Dr. Kalyani, Professor and Head of the Department of Microbiology, Saveetha Medical College, Chennai for kindly providing the clinical isolates to carry out our research work fruitfully.

REFERENCES:

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Received on 13.06.2016 Modified on 22.06.2016

Research J. Pharm. and Tech 2016; 9(10):1588-1590.

DOI: 10.5958/0974-360X.2016.00313.9