Antagonistic Effects on Enteropathogens and Plasmid Analysis of Lactobacilli Isolated from Fermented Dairy Products
In order to reduce the incidence of
foodborne disease which constitutes a major public health problem, we
are focussing our attention on the exploitation of indigenous food grade
lactic acid bacteria to improve the safety and hygiene of fermented products.
In this study we isolated lactobacilli from four fermented dairy products
and screened them for the production of antimicrobial substances by eliminating
the effects of organic acids and hydrogen peroxide. Three foodborne bacterial
pathogens, enterotoxigenic Escherichia coli (ETEC), Salmonella
typhimurium and Listeria monocytogenes were used as indicators.
Of the twelve Lactobacillus isolates obtained, six showed antagonistic
effects against at least one of the pathogens. Three of the twelve isolates
(one with detectable antimicrobial activity and two without) were found
to harbour plasmids. Plasmid elimination by curing with ethidium bromide
revealed no association between the antimicrobial activity and the plasmids.
With these results it may be assumed that the antimicrobial effects of
these isolates on the pathogens investigated were determined by their
chromosomes. In conclusion we have identified indigenous strains of food
grade lactobacilli with antimicrobial activity against clinically important
Fermented products are a significant part of many indigenous
diets. In Africa many of the products result from lactic acid fermentation
and Lactobacillus species have been reported to be the dominant
species (Molin, 2001; Sanni et al., 2002). The indigenous fermentation
processes are natural or spontaneous i.e., attributable to chance inocula
from the environment, the vessels used and/or the microbial flora on the
substrates. Coupled with improper food handling, poor food hygiene and
sanitation, these fermented products are exposed to spoilage organisms
and pathogens which are foodborne disease risk factors (WHO, 1999). Illnesses
arising from contaminated foods are major, global public health problems
(King et al., 2000; D`Souza et al., 2004). In England and
Wales, the number of cases annually is estimated to be above two million
resulting in over twenty thousand hospitalizations and above seven hundred
mortalities (Adak et al., 2002). The trend in the rest of the developed
world is probably similar (Roucourt et al., 2003).The situation
is worse in the developing world. Acute diarrhoea from infectious agents
is the commonest single factor responsible for infectious disease morbidity
and mortality worldwide and millions of children lose their lives annually
to diarrhoea, over three million in the developing world alone, while
many more suffer the impairment of nutritional status resulting from frequent
diarrhoea episodes (Ribeiro, 2000; Sheth and Dwivedi, 2006; McFarland
et al., 2006).
There are many obstacles hindering the control and treatment
of infectious diseases. These include a marked increase in antibiotics
resistant pathogens, emerging infectious diseases and a rise in the population
of immunocompromised people with an accompanying rise in opportunistic
infections. These problems cause a marked increase in antibiotics usage
which is a key factor in the development of resistance and selection of
resistant strains (Cristino, 1999; Moura et al., 2001). As long
as antibiotics are used resistance will develop, though the rate of development
and the population of organisms with resistance may vary (Cristino, 1999;
The possibility of microbial resistance exceeding the
present antibiotics development capabilities exists (Rolfe, 2000). Thus
there is an urgent need to develop alternatives to antibiotics usage.
In the case of diseases caused by foodborne pathogens, a logical approach
will be to reduce or eliminate the routes of food contamination. With
fermented products, the use of protective cultures with antagonistic activity
against undesired microbes in the fermentation will help to improve the
processes as well as the safety and hygiene of the products.
The world over, lactic acid bacteria are finding increasing
use in the prevention, control and treatment of diseases and health maintenance
(Reid, 1999; Anurada and Rajeshwari, 2005; Ishida-Fujii et al.,
2007). However, In Africa, we are yet to fully explore the potentials
of indigenous strains for health purposes.
This study is part of continuing efforts to explore the
potentials of our indigenous microbial flora in developing fermented products
with improved safety and benefits beyond nutritional provisions (Osuntoki
et al., 1999, 2007). In the present study, we isolated indigenous
Lactobacillus species from fermented dairy products and evaluated
the inhibitory action against some clinically important enteropathogens.
The essence was to identify candidate strains for use as protective cultures.
The indigenous fermented dairy products were chosen because they are not
subjected to pasteurisation or other heating processes that kill viable
organisms and may denature labile antimicrobial substances present. Additionally,
this eliminates the problem of acceptability, affordability and accessibility
of potential products to the primary target populace expected to benefit
from the research. The second objective was to determine the contribution
of plasmids to the antagonistic activity.
MATERIALS AND METHODS
Sources of organisms: The Lactobacillus
isolates were obtained from four Nigerian fermented dairy products; wara
(an indigenous soft unripened cheese produced by adding leaves extracts
of Calotropsis procera to whole cow milk), nunu (an indigenous
yoghurt produced from skimmed cow milk) and two unpasteurised commercially
available yoghurt. The indicator organisms; enterotoxigenic Escherichia
coli (ETEC), Salmonella typhimurium and Listeria monocytogenes
were clinical isolates from the culture collection of The Nigerian Institute
for Medical Research.
Isolation and identification of lactobacilli:
Samples taken from the products under aseptic conditions were serially
diluted with 0.1% peptone water and plated on sterile MRS agar (De Man
et al., 1960). The plates were incubated at 37 °C for 48 h
under microaerophilic conditions. To ensure purity, isolates were randomly
picked and reinoculated on fresh sterile MRS agar (Oxoid, UK) plates under
the previously stated conditions. Identification of the isolates were
done based on the Bergey`s manual of systematic bacteriology (Sneath,
1986) using these criteria: Gram stain reaction, microscopic and macroscopic
morphological examination, absence of catalase and oxidase production,
absence of spores and fermentation of different carbon sources.
Antimicrobial activity assay: The Lactobacillus
isolate to be screened for activity was grown overnight in MRS broth and
spotted onto the surface of sterile Bacteriocin Screening Medium (BSM)
described by Tichaczek et al. (1992) which excludes inhibitory
activity caused by organic acids and hydrogen peroxide (H2O2)
by low sugar content and buffering and contains catalase to degrade the
hydrogen peroxide. The plate was incubated at 37 °C for 24 h under
microaerophilic conditions. The indicator organism, grown overnight in
brain heart infusion broth, was inoculated into brain heart infusion agar,
Oxoid and used to overlay the overnight culture of the Lactobacillus
isolate. The plate was incubated at 37 °C overnight (16 h) and observed
for clear halos around the Lactobacillus colony indicating inhibition
of the growth of the pathogen. Sterile bacteriocin screening agar was
used as negative control.
Plasmid screening: The lactobacilli isolated were
screened for plasmids using the plasmid DNA isolation technique described
by Zhou et al. (1990). The plasmid sizes were estimated by running
the plasmid preparation alongside molecular mass markers prepared from
Escherichia coli strain V517 (Macrina et al., 1978) on agarose
Agarose gel electrophoresis: Horizontal electrophoresis
was carried out on 0.8% agarose gel prepared in running buffer (TBE, in
mmol L-1: Tris 89, boric acid 89 EDTA 2; pH 8.0) at 100 v for
1 h. Stained in 0.5 mg L-1 ethidium bromide (EtBr).
Plasmid curing: The isolates found to harbour
plasmids were treated with EtBr (20-40 mg L-1) as previously
described by Osuntoki et al. (2003). After which serial dilutions
of the treated organisms were made and plated on fresh MRS agar to obtain
colonies. Plasmid screening and the antimicrobial activity assay were
repeated for each colony as described earlier in this study in order to
detect cured organisms, mutants with altered activities and to correlate
antagonism with plasmid possession.
Lactobacillus isolation: A total of twelve
lactobacilli were isolated from the four fermented dairy products employed.
The isolates were 3 strains of L. acidophilus, 3 strains of
||Lactobacillus species isolated from the fermented
|*: Unpasteurised commercial yoghurt 1, #: Unpasteurised
commercial yoghurt 2
L. jensenii, 2 strains each of L. brevis
and L. fermentum and 1 strain each of L. casei and L.
salivarium (Table 1).
Antimicrobial activity: Six of the twelve isolates
(five from indigenous products and one from a commercial yoghurt) showed
antimicrobial activity by inhibiting the growth of an indicator organism
on BSM. Four of the isolates inhibited the growth of L. monocytogenes
while S. typhimurium and ETEC were inhibited by two organisms each.
Inhibitory action on L. monocytogenes was shown by isolates from
wara only, with the highest activity (as seen from the zone of inhibition)
shown by a strain of L. casei and the least by a strain of L.
jensenii. Two of the isolates inhibited two of three indicator pathogens.
These were a strain of L. casei with activity against L. monocytogenes
and S. typhimurium. This organism had a higher activity on Listeria.
The other was a strain of L. jensenii which in addition to inhibiting
L. monocytogenes also inhibited ETEC, with a more pronounced action
on ETEC. The only isolate from the commercial yoghurt with antagonistic
action on the pathogens was a strain of L. salivarius which inhibited
ETEC though with a lower activity than the L. jensenii described
above. The spectrum of activity of the lactobacilli from the dairy products
against the selected pathogens are shown on Table 2.
Plasmid screening: Plasmids were detected in three
of the isolates, one with detectable antimicrobial activity and two without.
The isolates harboured small plasmids between 3.8 and 5.5 kb in size (Fig.
1, 2, Table 3).
Plasmid curing and inhibitory activity: Treatment
of the plasmid bearing isolates with EtBr successfully cured them. However,
the loss of these plasmids did not alter the antagonistic activity of
||Agarose gel electrophoresis of plasmid DNA: 1: LAA5,
2: LAA11, 3 LAA9, 4: LAA6, 5: LAA2, 6: 7: Markers, 8: LAA10, 9: LAA3,
10: LAA4, 11: LAA8, Chr- band of chromosomal DNA. The sizes (kb) of
the molar mass markers are indicated on the right
||Agarose gel electrophoresis of plasmid DNA: 1: LAA7,
2: LAA12, 3: LAA1, 4,5: Markers, Chr- band of chromosomal DNA. The
sizes (kb) of the molar mass markers are indicated on the right
||Inhibitory activity of Lactobacillus isolates
against selected foodborne pathogens
|+: Inhibitory activity observed, -: No detectable inhibitory
activity. Values in bracket show the mean zones of inhibition ±
SD in mm for triplicates measurements
||Plasmids profiles of the lactobacilli isolates from
the fermented dairy products
The present study observed growth inhibitory activity
in some strains of Lactobacillus isolated from fermented dairy
products though a screening medium which eliminates antimicrobial effects
due to pH and H2O2 activity was used. This reveals
the production of antimicrobial substances by these strains. Lei and Jacobsen
(2004) reported that antimicrobial activity by lactic acid bacteria from
African fermented foods in many previous studies were due to the production
of lactic acid and low pH. Hydrogen peroxide (H2O2)
has also been reported to enact antimicrobial effects (Pericone et
al., 2000; Batdorj et al., 2007). The virulence potential of
pathogens is reduced by inhibitory or antagonistic activity. Thus the
antimicrobials producing isolates may prevent or reduce the extent of
food contamination and the gastrointestinal tract infections caused by
The results of this study are quite significant because
the pathogens inhibited by the lactobacilli are of clinical importance.
Reports implicate ETEC and S. typhimurium as being amongst the
major agents responsible for the high morbidity and death in GIT infections
(Ljungh, 1999; King et al., 2000). L. monocytogenes is
responsible for sporadic epidemics. It is credited with a high case mortality
rate and the highest rate of hospitalization, above 90%, for known foodborne
pathogens. Certain populations; pregnant women, their foetuses and immunocompromised
people are particularly vulnerable (Vazquez-Boland et al., 2001;
Jemmi and Stephen, 2006; Gandhi and Chikindas, 2007). The contemporary
means of management of the diseases caused by these pathogens include
antibiotics therapy which is a selector for resistant strains. Present
study therefore shows a potential application of indigenous strains of
lactobacilli in ameliorating a major global health problem and reducing
Only one out of the five isolates from the non pasteurised
commercial yoghurt showed inhibitory activity while five out of the seven
isolates from the indigenous products were active. Though the precise
reasons are not known, it may be as a result of the wild strains` adaptation
to survival and competition with other microbes under the non sterile
processes of natural fermentation.
The antimicrobials produced were not associated with
plasmids. Plasmid curing which resulted in the loss of the plasmids harboured
by some of the isolates did not alter the pattern of inhibitory activity.
In addition, no plasmids were found in most of the lactobacilli with antimicrobial
activity. These are suggestive of the antimicrobial activities being chromosomally
mediated. Chromosomal location may be an advantage in the exploitation
of these Lactobacillus strains because plasmid losses can occur
due to selection pressure, plasmid instability or plasmids incompatibility.
In conclusion, we have isolated and identified lactobacilli
strains from indigenous fermented products with antimicrobial effects
on some clinically important foodborne bacterial pathogens. This reveals
potential applications of indigenous Lactobacillus strains as protective
cultures for the improvement of the microbial safety of fermented foods
and reduction in the incidence of illnesses arising from food contamination
which is a major global public health concern.
Adak, G.K., S.M. Long and S.J. O'Brien, 2002.
Intestinal infection: Trends in indigenous foodborne disease and deaths, England and Wales: 1992 to 2000. Gut, 51: 832-841.CrossRef | Direct Link |
Anderson, R.M., 1999.
The pandemic of antibiotic resistance. Nat. Med., 5: 147-149.
Anuradha, S. and K. Rajeshwari, 2005.
Probiotics in health and disease. J. Indian Acad. Clin. Med., 6: 67-72.Direct Link |
Batdorj, B., V. Trinetta, M. Dalgalarrondo, H. Prevost and X. Dousset et al
Isolation, taxonomic identification and hydrogen peroxide production by Lactobacillus delbrueckii
subsp. lactis T31, isolated from Mongolian yoghurt: Inhibitory activity on food-borne pathogens. J. Applied Microbiol., 103: 584-593.CrossRef | Direct Link |
Cristino, J.M., 1999.
Correlation between consumption of antimicrobials in humans and development of resistance in bacteria. Int. J. Antimicrob. Agents, 12: 99-202.
D’Souza, R.M., N.G. Becker, G. Hall and K.B.A. Moodie, 2004.
Does ambient temperature affect foodborne disease? Epidemiology, 15: 86-92.Direct Link |
Gandhi, M. and M.L. Chikindas, 2007. Listeria
: A foodborne pathogen that knows how to survive. Int. J. Food Microbiol., 113: 1-15.CrossRef | Direct Link |
Ishida-Fujii, K., R. Sato, S. Goto, X.P. Yang, S. Hirano and M. Sato, 2007.
Prevention of pathogenic Escherichia coli
infection in mice and stimulation of macrophage activation in rats by oral administration of probiotic Lactobacillus casei
I-5. Biosci. Biotechnol. Biochem., 71: 866-873.Direct Link |
Jemmi, T. and R. Stephen, 2006. Listeria monocytogenes
: Food-borne pathogen and hygiene indicator. Rev. Sci. Tech., 25: 571-580.Direct Link |
King, J.C., R.E. Black, M.P. Doyle, K.L. Fritsche and B.H. Halbrook et al
Foodborne illnesses and nutritional status: A statement from an American society for nutritional sciences working group. J. Nutr., 130: 2613-2617.Direct Link |
Lei, V. and M. Jakobsen, 2004.
Microbiological characterization and probiotic potential of koko
and koko sour water
, African spontaneously fermented millet porridge and drink. J. Applied Microbiol., 96: 384-397.CrossRef | Direct Link |
Ljungh, A., 1999.
Bacterial infections of the small intestine and colon. Curr. Opin. Gastroenterol., 15: 43-43.Direct Link |
Macrina, F.L., D.J. Kopecko, K.R. Jones, D.J. Ayers and S.M. McCowen, 1978.
A multiple plasmid-containing Escherichia coli
strain: Convenient source of size reference plasmid molecules. Plasmid, 1: 417-420.CrossRef | PubMed | Direct Link |
McFarland, L.V., G.W. Elmer and M. McFarland, 2006.
Meta-analysis of probiotics for the prevention and treatment of acute pediatric diarrhea. Int. J. Probiotics Prebiotics, 1: 63-76.Direct Link |
Molin, G., 2001.
Probiotics in foods not containing milk or milk constituents, with special reference to Lactobacillus plantarum
299v. Am. J. Clin. Nutr., 73: 380s-385s.Direct Link |
Moura, L.N., E. Neumann, L.Q. Vieira and J.R. Nicoli, 2001.
Protection by Lactobacillus acidophilus
UFV-H2B20 against experimental oral infection with Salmonella enterica
subsp. Enterica ser. typhimurium
in gnotobiotic and conventional mice. Braz. J. Microbiol., 32: 66-69.Direct Link |
Osuntoki, A.A., D.K. Olukoya, G.O. Gbenle and E.A. Omonigbehin, 1999.
Isolation of Lactobacillus
species with antifungal activity from Nigerian fermented foods. Niger. Quart. J. Hosp. Med., 9: 314-316.CrossRef | Direct Link |
Osuntoki, A.A., G.O. Gbenle and D.K. Olukoya, 2003.
Evidence for chromosomal determination of fungicidal activity in strains of Lactobacillus brevis
and Lactobacillus fermentum
isolated from fermented foods. Folia Microbiol., 48: 56-58.Direct Link |
Osuntoki, A.A., E.A. Omonigbehin, S.O. Gbenebitse and U.E. Mojiminiyi, 2007.
Prevention of salt induced hypertension in rats by oral administration of Lactobacillus acidophilus
yoghurt. Nig. Food J., 25: 184-189.Direct Link |
Pericone, C.D., K. Overweg, P.W.M. Hermans and J.N. Weiser, 2000.
Inhibitory and bactericidal effects of hydrogen peroxide production by Streptococcus pneumoniae
on other inhabitants of the upper respiratory tract. Infect. Immun., 68: 3990-3997.Direct Link |
Reid, G., 1999.
The scientific basis for probiotic strains of Lactobacillus
. Applied Environ. Microbiol., 65: 3763-3766.Direct Link |
Ribeiro, H. Jr., 2000.
Diarrheal disease in a developing nation. Am. J. Gastroenterol., 95: S14-S15.Direct Link |
Rolfe, R.D., 2000.
The role of probiotic cultures in the control of gastrointestinal health. J. Nutr., 130: 396S-402S.Direct Link |
Roucourt, J., G. Moy, K. Vierk and J. Schlundt, 2003.
The Present State of Foodborne Disease in OECD Countries. World Health Organization, Geneva, Switzerland
Sanni, A.I., J. Morlon-Guyot and J.P. Guyot, 2002.
New efficient amylase-producing strains of Lactobacillus plantarum
and L. fermentum
isolated from different Nigerian traditional fermented foods. Int. J. Food Microbiol., 72: 53-62.Direct Link |
Sheth, M. and R. Dwivedi, 2006.
Complementary foods associated with diarrhea. Indian J. Pediatr., 73: 61-64.CrossRef | PubMed | Direct Link |
Sneath, P.H.A., N.S. Mair, M.E. Sharpe and J.G. Holt, 1986.
Bergey's Manual of Systematic Bacteriology. Vol. 2, 1st Edn., Williams and Wilkins Co., Baltimore
Tichaczek, P.S., J.N. Nissen-Meyer, I.F. Nes, R.F. Vogel and W.P. Hammes, 1992.
Characterization of the bacteriocins curvacin A from Lactobacillus curvatus
LTH1174 and sakacin P from L. sake
LTH673. Syst. Applied Microbiol., 15: 460-468.CrossRef | Direct Link |
Vazquez-Boland, J.A., M. Kuhn, P. Berche, T. Chakraborty and G. Dominguez- Bernal et al
., 2001. Listeria
pathogenesis and molecular virulence determinants. Clin. Microbiol. Rev., 14: 584-640.Direct Link |
WHO (World Health Organization), 1999.
Removing Obstacles to Healthy Development-World Health Organization Report on Infectious Diseases. WHO, Geneva, Switzerland
Zhou, C., Y. Yang and A.Y. Jong, 1990.
Mini-prep in ten minutes. Biotechniques, 8: 172-173.PubMed |
De Man, J.C., M. Rogosa and M.E. Sharpe, 1960.
A medium for the cultivation of Lactobacilli
. J. Applied Bacteriol., 23: 130-135.CrossRef | Direct Link |