Synergistic Effect Of Bacteriocin-Like Inhibitory Substances Produced By Vaginal Lactobacillus Against Group B Streptococci

Project and Seminar Material for Microbiology

Project and Seminar Material for Microbiology


The vaginal normal flora of a healthy woman is dominated by species of Lactobacillus. The balance between the vaginal flora is maintained by the protective antagonizing compounds produced by Lactobacillus. Group B Streptococci (GBS) is an opportunistic organism usually carried asymptomatically by pregnant women but cause severe neonatal infections. Antibiotics chemotherapy poses a lot of threat especially to pregnant women. Thus, this study was aimed at evaluating the antimicrobial activity of bacteriocin-like inhibitory substance produced by vaginal Lactobacillus of pregnant women on GBS. Identification of isolates was by microscopy, cultural characteristics and biochemical tests.

Lactobacillus isolates were tested for their ability to inhibit GBS using agar diffusion method. Partial purification was done using ammonium sulphate precipitation and dialysis. The result revealed that of the 24 Lactobacilli isolates obtained, 7 strains were bacteriocin producers. Bacteriocin-like inhibitorysubstances (BLIS) from the seven strains inhibited the growth of GBS. Partially purified extracts of Bacteriocin-like substances showed more inhibitory activity than the crude extract. The mean diameter of crude BLIS (16.9 mm) produced by isolate R5 was the highest, while isolate L2 had the lowest activity at 12.1mm. The partially purified BLIS from isolate R5 had the highest activity at 18.5 mm while L2 had the lowest activity at 14.2 mm. BLIS showed inhibition of the test organism at pH 5.5, 6.0, 7.0, 7.5 and 8.0 and at temperatures of 30°C – 100°C for 10 mins except for BLIS from isolate L4 t hat lost its activity at 100°C.

The zones of inhibition were strongest at 30°C for all the is olates. Generally, their activities decreased with an increase in temperature although there was no significant reduction. At 30°C, inhibitory activity of isolate L6 was 15.0 mm and 13.4 mm; R6 was 17.4 mm and 16.7 mm; R5 was 17.4 mm and 15.4 mm; L3 was 14.0 mm and 12.8 mm; Rl was 14.3 mm and 12.0 mm; L3 was 16.0 mm and 14.8 mm; L4 was 16.1 mm and 15.3 mm for the partially and crude BLIS respectively. Crude BLIS produced by isolate L6, R6, L2, L3 and L4 showed strongest activity at pH 5.5. Isolate R5 and Rl showed strongest activity at pH 6.0. At pH 5.5, the partially purified BLIS produced by isolate L6, R6, L2 and L3 showed the strongest inhibition; isolate Rl and L3 was strongest at pH 7.0.

The combined effect of BLIS produced by isolate R5 and R6 was 19.06 mm and 21.07 mm for crude and partially purified BLIS respectively; were stronger than the individual effect. The combined effect was strongest at pH 6.0 with 21.20 mm. At temperature 30°C – 100°C; the inhibitory effects were relatively stable. Hence the study showed that BLIS produced from the isolates have potential of being used as probiotics and antibiotics for the prevention of Group B Streptococcicolonization in pregnant women and subsequent reduction of neonatal infections.

Chapter One

Introduction And Literature Review

1.1 Introduction

Bacteriocins are defined as ribosomally synthesized antimicrobial peptides which are generally active against microorganisms closely related to the producer strain (Turovskiy et al., 2009). The term bacteriocin-like substance is applied to antagonistic substances that arenot completely defined or do not fit the typical criteria of bacteriocins. They have been reported to inhibit a wide range of both Gram-positive and Gram-negative bacteria as well as fungi (Ocanaet al., 1999). They function mainly by creating pores across bacterial cell membrane.

Lactobacillus species are non-pathogenic Gram positive rods that are predominantlyisolated from the vagina of healthy premenopausal women (McClelland et al., 2009). In this environment, they exert a protective effect against pathogenic microorganisms by using different mechanisms such as production of antimicrobial agents, which include organic acids, hydrogen peroxide, and bacteriocins (Ocana et al., 1999).

The protective role of Lactobacilli in the vagina becomes evident when their concentrations drop, which may be as a result of the use of antibiotics or in an immunocompromised host, in which case the hydrogen ion concentration (pH) of their environment tends to increase. This, in turn, favors colonization by intestinal bacteria and the overgrowth of microorganisms indigenous to the vagina but usually present in low numbers. These include the etiological agents of bacterial vaginosis, yeast vaginosis, trichomoniasis, and the potential pathogen Streptococcus agalactiae (Rebeca et al., 2008).

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