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Research Article
 

Antagonistic Activities of Lactic Acid Bacteria Isolated from Nigerian Fermented Dairy Food Against Organisms Implicated in Urinary Tract Infection



B.A. Adeniyi, F.A. Ayeni and S.T. Ogunbanwo
 
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ABSTRACT

Five Lactic Acid Bacteria (LAB) were isolated from different indigenous fermented dairy foods and identified as Lactobacillus fermentum, L. brevis, L. plantarum, Lactococcus lactis and Streptococcus durans. The cell free supernatant of the selected LAB were able to inhibit the growth of all organisms implicated in urinary tract infection (UTI) used in this study. The largest zone of inhibition was produced by Lac. lactis K3 against Staphylococcus saprophyticus UCH 2051. The sensitivity of UTI pathogens to different antibiotics was investigated. The entire Gram-negative pathogens from UTI showed 100% resistance to Colistin, Augmentin, Nalidix acid, Nitrofuranton and Cotrimoxazole while most Gram positive pathogens from UTI were sensitive to different antibiotics used against them. The tested LAB produced various antimicrobial compounds such as organic acid, hydrogen peroxide and diacetyl. The highest yield of lactic acid (1.87 g L-1) was produced by Streptococcus durans K4 while the highest yield of diacetyl (2.324 g L-1) was produced by L. brevis M5 and the highest yield of hydrogen peroxide (0.00544 g L-1) was produced by L. plantarum N2.

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B.A. Adeniyi, F.A. Ayeni and S.T. Ogunbanwo , 2006. Antagonistic Activities of Lactic Acid Bacteria Isolated from Nigerian Fermented Dairy Food Against Organisms Implicated in Urinary Tract Infection. Biotechnology, 5: 183-188.

DOI: 10.3923/biotech.2006.183.188

URL: https://scialert.net/abstract/?doi=biotech.2006.183.188

INTRODUCTION

Urinary tract infections (UTI) are common infectious diseases that can be associated with substantial morbidity and significant expenditures in persons of all ages. Sexually active young women are disproportionately affected, but several other populations, including elderly persons and those undergoing genitourinary instrumentation or catheterization, are also at risk. An estimated 40% of women were reported as having had a UTI at some point in their lives (Kunin, 1994). Worldwide, it is estimated that several million women suffer from UTI annually, many of whom are treated empirically by physicians if their a symptoms suggest acute uncomplicated bacterial cystitis (Gupta et al., 1999; Warren et al.,1999). The annual cost to health care service is Staggering, reaching $2 billion in the United states alone and over $6 billion world wide (Foxman et al., 2000). Escherichia coli is the causative agent in most cases (up to 85%) followed by Staphylococcus saprohyticus and enterococci (Stamm and Hooton, 1993).

The incidence of UTI increases with age. Among school aged girls 1 to 2% are afflicted compared with 2 to 5% of pre-menopausal women and 10 to 15% of post menopausal women (Nicolle, 2000). Typical treatment for this infection is ampicillin or the mixture of trimethoprim and sulfamethanoxazole [TMP/SMX] among others. Unfortunately antibiotics therapy is associated with both side effects and increasing bacterial resistance. Commonly acquired UTI are highly resistant to many antimicrobial agents.

In an effort to decrease the over reliance on antimicrobials, the time has come to carefully explore the therapeutic application of biotherapeutic agents. Lactic Acid Bacteria (LAB) have been associated with production of fermented foods for many centuries. There are some reports on the ability of LAB to inhibit the growth of some pathogens (Sanni et al., 1999; Reid et al., 2001).

So far there is little information on the LAB isolated from Nigerian fermented dairy foods against organisms implicated in UTI. This study therefore reports the antagonistic activities of some selected LAB from Nigerian fermented dairy foods against organism implicated in UTI.

MATERIALS AND METHODS

Collection of samples and bacterial strains: Traditionally prepared fermented dairy foods which are Kidirimu (Yoghurt), Mai-Sanu (Butter), Wara (cheese), Nono (Skimmed Milk) and Whey were randomly collected from local producers in South-Western Nigeria and taken to the laboratory for microbiological analysis. Pure cultures of the isolates were identified using biochemical and sugar fermentation pattern. The UTI pathogens used as indicator organisms were obtained from Medical Microbiology unit, University College Hospital, Ibadan, Nigeria.

Antibiotics susceptibility test: Susceptibility test for each pathogen isolate were performed using disc diffusion (Kirby-Bauer) method to antimicrobial agents according to the National Committee for Clinical Laboratory Standards (NCCLS, 1997) guidelines. The antibiotics sensitivity disc consisting of different antibiotics namely Amoxycillin 25 μg, Cotrimoxazole 25 μg, Nitrofuranton 200 μg, Ciprofloxacin 10 μg, Nallidix acid 30 μg, Gentamycin 10 μg, Ofloxacin 30 μg, Augmentin 30 μg, Colstin 25 μg, Tetracycline 30 μg, Amovin 10 μg, Rapidflox 5 μg, Pelfomed 5 μg, Olfomed 5 μg, Erythromycin 15 μg, Celthiaxone 30 μg and Ampicillin+Cloxacillin 10 μg. were placed on the solidified agar surface. The plates were incubated aerobically for 24 h at 37°C. Resistance was determined according to the reference zone diameter interpretative standards of National Committee for Clinical Laboratory Standards (NCCLS, 1999).

Antibacterial assay: The antagonistic activity of cell free supernatant of selected LAB against the pathogens were done using two different assays, the overlay and agar-well diffusion methods as previously reported (Sanni et al., 1999; Reid et al., 2001). The plates were incubated aerobically at 37°C for 24 h after which they were examined for clear zones around the wells.

Determination of lactic acid, hydrogen peroxide and diacetyl production: For these measurements, the test organisms were grown on MRS broth for 72 h and samples taken at 12 h interval.

Lactic acid: To 25 mL of broth culture of organisms, 3 drops of phenolphthalein were added as indicator. From the burette 0.1 N NaOH was slowly added to the sample until pink colour appears. Each mL of 0.1 NaOH is equivalent to 90.08 mg of lactic acid (AOAC, 1990).

Hydrogen peroxide: Twenty five milliliter of dilute sulphuric acid were added to 25 mL of the broth culture of test organism. Titration was carried out with 0.l N potassium permanganate. Each mL of 0.l N potassium permanganate is equivalent to 1.070 mg of hydrogen peroxide A decolourization of the sample was regarded as end-point (AOAC, 1990).

Table 1: LAB species isolated from different food samples
Image for - Antagonistic Activities of Lactic Acid Bacteria Isolated from Nigerian Fermented Dairy Food Against Organisms Implicated in Urinary Tract Infection

Diacetyl: Twenty five milliliter of broth cultures were transferred into conical flasks and 7.5 mL of hydroxylamine solution were used for the residual titration. The flasks were titrated with 0.l N HCl to a greenish-yellow end point using bromophenol blue as indicator. The equivalence factor of HCl to diacetyl is 21.5 mg (AOAC, 1990).

RESULTS

Twenty strains of LAB were isolated from different fermented dairy foods and identified based on biochemical tests and sugar fermentation pattern as L. brevis, L. fermentum, L. plantarum, Lac. lactis and Streptococcus durans (Table 1). The sensitivity of different UTI pathogens to antibiotics was investigated. The percentage sensitivity/resistance of UTI pathogens to antibiotics were shown in (Fig. 4). All Gram negative pathogens from UTI show 100% resistance to Cotrimoxazole, Nitrofuranton, Nalidix acid, Augmentin, Colstin and Tetracycline while most Gram positive pathogens showed 100% sensitivity to most of the antibiotics used against them (Table 2 and Fig. 4). The cell free supernatants of selected LAB produce antagonistic activities against all UTI pathogens with the largest zone of inhibition produced by Lac. lactis K3 against Staphyloccus saprophyticus (Table 3).

Quantitative estimation of different antimicrobial compounds produced by each LAB species was investigated. Streptococcus durans K4 produced the highest yield of lactic acid (1.874 g L-1) after 36 h of incubation while L. plantarum N2 produced the lowest yield (0.540 g L-1) after 12 h of incubation (Fig. 1). The highest quantity of hydrogen peroxide was produced by L. plantarum N2 (0.00544 g L-1) after 12 h of incubation while the lowest quantity (0.000272 g L-1) was produced by L. brevis M5 after 12 h of incubation (Fig. 2).

Table 2: Antibiotics Sensitivity Test for UTI Pathogens
Image for - Antagonistic Activities of Lactic Acid Bacteria Isolated from Nigerian Fermented Dairy Food Against Organisms Implicated in Urinary Tract Infection
Key: S - Sensitive, VS - Very Sensitive, R - Resistance, NT - Not Tested, Ec - Escherichia coli, Kp - Klebsiella pneumoniae, Kr - Klebsiella rhinoscleromastis, Ss - Staphylococcus saprophyticus, Sa - Staphylococcus aureus

Table 3: Antagonistic activities of selected lactic acid bacteria metabolite against organisms implicated in (UTI) with their zones of inhibition
Image for - Antagonistic Activities of Lactic Acid Bacteria Isolated from Nigerian Fermented Dairy Food Against Organisms Implicated in Urinary Tract Infection
Key: Ec -Escherichia coli, Kp - Klebsiella pneumoniae, Kr - Klebsiella rhinoscleromastis, Ss - Staphylococcus saprophyticus, Sa - Staphylococcus aureus S - Sensitive. R - Resistant. W - Weakly sensitive

The highest quantity of diacetyl (2.324 g L-1) was produced by L. brevis M5 after 72 h of incubation while the lowest quantity (0.547 g L-1) was produced by L. plantarum N2 after 12 h of incubation (Fig. 3).

DISCUSSION

LAB associated with fermentation of indigenous dairy foods were isolated and characterized. Members of the genus have been detected in variety of habitant including fermented foods (Tannock, 1986). Different UTI pathogens; E. coli, K. pneumoniae, K. rhinoscleromastis, S. saprophyticus and S. aureus varies in their sensitivity to different antibiotics. K. pneumoniae UCH 2047 and E. coli UCH 2045 were all resistant to all antibiotics used against them in this study. Isolates from this part of the world have been reported to be more resistant to antibiotics than those from other parts of the world (West and Warner, 1985). This widespread resistance could be attributed to excessive or indiscriminate use of antibiotics in this part of the world (Olukoya et al., 1993) or due to chromosomal resistance (Brooks et al., 1998).

The metabolites of selected LAB have antagonistic activities against all organisms implicated in UTI used in this work. This is in agreement with the work of Reid and Seidenfeld (1997) and Reid et al. (2003) who reported treatment of UTI with LAB. The largest zone of inhibition was produced by Lac. lactis K3 (15 mm) against S. saprophyticus (UCH 2051). It has been demonstrated from this study that E. coli, which is responsible for 85% of UTI in patients are very sensitive to the metabolites of all tested LAB.

Image for - Antagonistic Activities of Lactic Acid Bacteria Isolated from Nigerian Fermented Dairy Food Against Organisms Implicated in Urinary Tract Infection
Fig. 1: Quantitative estimation of lactic acid produced by the test isolates

Image for - Antagonistic Activities of Lactic Acid Bacteria Isolated from Nigerian Fermented Dairy Food Against Organisms Implicated in Urinary Tract Infection
Fig. 2: Quantitative estimation of hydrogen peroxide produced by the test isolates

This is in agreement with the observation of Reid and Seidenfeld (1997). The five UTI pathogens used in this study showed great resistance to antibiotics but they were all sensitive to LAB metabolites. Staphylococcus spp is a common cause of UTI in young women. Brooks et al. (1998) reported rapid development of resistance by Staphylococcus spp. to antimicrobial agents. However, it was sensitive to all LAB metabolites used against them in this study.

The isolated LAB produced antimicrobial compounds to varying degree, the increase in the production of lactic acid with time have been attributed to lowered pH which permit the growth of LAB. The antimicrobial effect of lactic acid is due to undissociated form of the acid which penetrate the membrane and liberate hydrogen ion in the neutral cytoplasm thus leading to inhibition of vital cell functions (Corleh and Brown, 1980). The inhibitory effect of hydrogen peroxide produced by LAB has also been reported. Collins and Aramaki (1980) reported the inhibition of Pseudomonas fragi and S. aureus by hydrogen peroxide produced by certain LAB strains which contribute to their inhibitory activity against other microorganism.

Image for - Antagonistic Activities of Lactic Acid Bacteria Isolated from Nigerian Fermented Dairy Food Against Organisms Implicated in Urinary Tract Infection
Fig. 3: Quantitative estimation of diaetyl produced by the test isolates

Image for - Antagonistic Activities of Lactic Acid Bacteria Isolated from Nigerian Fermented Dairy Food Against Organisms Implicated in Urinary Tract Infection
Fig. 4: Percentage sensitivity and resistance of UTI pathogens to antibiotics

Since there is now an increasing trend in bacterial resistance to antibiotics, Novel approaches are needed in chemotherapy of urinary tract infection. It has been demonstrated from this study that LAB has a high potential for the treatment of UTI with the use of LAB from indigenous fermented dairy foods as probiotic organisms. This is a feasible option for chemotherapy of UTI.

Further work is still going on in our laboratory to ascertain the particular metabolite produced by LAB that actually inhibits the growth of the pathogens associated with urinary tract infection.

ACKNOWLEDGEMENT

BAA acknowledges the Senate Research Grant of University of Ibadan and the technical assistance of the staff of Medical Microbiology unit University College Hospital, Ibadan, Nigeria.

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