Inhibitory
Effects of Lactobacillus Species Against Human Pathogens
Shanthi V.*, Jemima Florence Borgia, Bhavani S. and Sathya M.
P.G. Department of
Zoology, Jayaraj Annapackiam
College for Women (Autonomous), Periyakulam – 625
601, Tamilnadu.
ABSTRACT:
The influence of Lactobacillus strains (L.fermentum, L.brevis, L.plantarum, L.curvatus, L.vulgaricus, L.casei, L.acidophilus and
L.lactis) on the inactivation of human pathogens
(Staphylococcus aerians, Salmonella typhi, S.paratyphi,
Klebsiella oxytoca,
Pseudomonas aeruginosa , Vibrio parahaemolyticus, Proteus mirabilis, Lactobacillus vulgaris, Vibrio cholerae and Shigella flexneri) were
investigated. Strong antibacterial activities were exhibted
on the plates inoculated in the Shigella flexneri by L.curvatus, L.plantarum and Proteus
mirabilis and Pseudomonas aeruginosa by L.vulgaricus. L.plantarum showed broad spectrum of antibacterial activity
against all tested pathogens. A weak inhibitory effect was detected in L.brevis on Vibrio parahaemolyticus
and L.fermantum
on L.vulgaris
and S.typhi.
KEYWORDS: Lactobacillus, test pathogens,
antibacterial
INTRODUCTION:
Probiotics can be defined as a food (feed) or drug containing
live microbes that when ingested is expected to give beneficial physiologic
effects to the host animal through microbial actions (Ishibashi
and Yamazabi, 2001).Lactic bacteria commonly used for
probiotioc food production are also a research
subject of environmental hygiene studies (Ligocka and
Paluszak,2005) Lactic acid bacteria are characterized by their tolerance in
which other bacteria are unable to grow and thereby ensuring food safety.
Widely documented antagonistic properties of these microorganisms in relation
to many dangerous pathogens can be used in technologies aiming at the
limitation or total elimination of pathogenic microflora
from animal faces or sewage sludge intended for land application. Lactobacillus
strains are able to inhibit the growth of all strains of anaerobic gastro
intestinal pathogens (Roberfroid, 2002). The trasity lactic acid bacteria in the gastrointestinal tract
are capable of delivering enzymes and other substances into the intestine which
possibly help to control intestinal flora (Collins et al., 1999). Lactobacilli and other commensal
bacteria provide the protection of the host against possible colonization by
the pathogenic bacteria (Reid et al.,
1998) by inhibiting the pathogen cell association and invasion (Chauviere et al.,
1992; Coconier, et
al., 1992; Bujankova, et al., 2004). Lactobacilli are believed to interfere with pathogen
by different mechanisms, e.g. by the production of antimicrobial compounds,
such as lactic acid, dihydrogen peroxide or bacteriocin like substances. Some of the Lactobacillus strains
co aggregate with pathogenic bacteria (Kmei’t and Lucchini, 1999). The aim of this study was to select
potential probiotics, which possessed antibacterial
activity against certain pathogens, from gastrointestinal tracts of marine fish
available in a tropical marine environment.
MATERIAL
AND METHODS:
Eight strains of lactic bacteria of the genus
Lactobacillus (L.fermentum,
L.brevis, L.plantarum, L.curvatus, L.vulgaricus, L.acidophilus, L.casei, and L.lactis) were
isolated from the gastrointestinal tract of marine fishes using MRS broth and
confirmed in the department of Microbiology, CAS in Marine Biology, Portonovo).The identification of isolates were performed
according to the criteria of Bergey’s manual of
determinative bacteriology.
The monocultured test
pathogens such as Staphylococcus aerians (Sa), Salmonella
typhi (St), Pseudomonas
aeruginosa (Pa), Vibrio
parahaenmolytica(Vp), Proteus mirabilis (Pm), Lactobacillus vulgaricus
(Lv), Vibrio cholerae (Vc), Shigella flexneri (Sf) were obtained from the basic Biomedical sciences of Bharathidasan University, Trichy.
Lactobacillus species strains were inoculated in MRS at
37°C for 16 to 18 hours. Activated cultures were centrifuged at 4000 rpm for 15
minutes and the clear supernatants were sterilized by filtration (0.45 µl) to
obtain cell free filtrates. The test pathogens (bacteria) were streaked on the
Petri dishes in 20ml of nutrient agar solidified; the dishes were stored in a
refrigerator for 2 hours. Wells (6mm) were then made and filled using 0.1ml of
cells free filtrate of Lactobacillus. The inoculated plates were incubated at
37°C for 24 hours and the diameter of the zone was measured with calipers. The
measurements recorded were the radius from the edge of the zone to the edge of
the well.
The studies were conducted to assess the inhibitory
potential of Lactobacillus towards tested pathogens after 24 hours. The results
obtained were analyzed statistically.
RESULTS
AND DISCUSSION:
The agar well diffusion method was able to detect the
inhibitory effect of the different Lactobacillus tested against the human
pathogens. Three of the eight Lactobacillus spp. [L. plantarum (21±1 mm) and L. casei (14.3
±3.05 mm)] tested, showed maximum inhibitory effect against Shigella flexneri and two of the eight Lactobacillus
spp. [L. acidophilus (18.3±1.52 mm), L. fermentation (19.5±9.53 mm)], showed
strong inhibitory action against Vibrio parahaemolyticus (Table 1 and 2)
The antagonistic activity of lactic acid bacteria
towards Shigella flexneri was
confirmed by Bernet et al., (1997). L.casei after 5 hours caused a clear inhibition of the
growth of Shigella flexneri (Forestoer, et al.,
2001)
L.plantarum showed the broadest range of inhibitory
action. This is agreeing by the results of Kelly et al., (1996) and Nowroozi et al., (2004).
In the present study the inhibitory effect of L.acidphilus
strongly antagonistic to pathogenic bacteria Pseudomonas aeruginosa, Vibrio parahaemolyticus
and Proteus mirabilis. Similar result
were obtained by Tuomola et al., (1998); Ouwenland et al., (2001); Fornandez
et al., (2003). It was shown
previously that L.acidophilus inhibited the growth of pathogenic
bacteria such as C.perfringens,
E.coli, Salmonella enteritis (Collins et al., 1980).
Among all the tested Lactobacillus spp. L.vulgaricus
showed high inhibitory effect (23.66±2.08 mm) against Proteus mirabils.
Tomas et al.,
(2003) reported that Klebsiella species was inhibited
by the vaginal Lactobacilli. Similar results were obtained by Lactobacilli
species against human pathogen in Klebsiella species.
Many of the Lactobacillus strains showed weak
inhibition of some of the pathogenic strains. Results of this investigation
showed that the presence of different strains of Lactobacillus in gut of marine
fish exhibits variable levels of inhibitory effect against human pathogens. The
studies of Fernandez et al., (2003)
in turn, seem to prove that the activity of antagonistic strains may be varied
in relation to different pathogenic microorganism.
Lactobacillus
offer potential as an alternative to antimicrobial as a means of controlling
pathogens. The probiotic strains are expected to fulfill several health promoting characters.
ZONE INHIBITION OF STAPHYLOCOCCUS
AERIANS AGAINST LACTO BACILLUS BY
AGAR WELL DIFFUSION METHOD
a.
L.plantarum, b. L.curvatus,
n. L.acidophilus,
c. L.casei,
d. L.lactis
ZONE INHIBITION OF SALMONELLA TYPHI AGAINST LACTOBACILLUS
BY AGAR WELL DIFFUSION METHOD
f.
L.brevis, g. L.curvatus, n.L.fermentume,
h. L.casei, i. L.plantarum,
ZONE INHIBITION OF SALMONELLA
PARATYPHI AGAINST LACTO BACILLUS BY AGAR WELL DIFFUSION METHOD
g. L.vulgaricus, k.
L.plantarum, l. L.lactis,
m. L.curvatus
ZONE INHIBITION OF SHIGELLA FLEXNERI AGAINST LACTOBACILLUS
BY AGAR WELL DIFFUSION METHOD
f.
L.fermentum, h. L.casei, g. L.plantarum,
i. L.curvatus
ZONE INHIBITION OF PROTEUS MIRABILIS AGAINST LACTOBACILLUS
BY AGAR WELL DIFFUSION METHOD
f.L.curvatus, i. L.lactis, g. L.acidophilus, h.
L.vulgaricus
ZONE INHIBITION OF SALMONELLA
PARATYPHI AGAINST LACTO BACILLUS BY AGAR WELL DIFFUSION METHOD
f.L.vulgaricus, g.L.plantarum, h.L.fermentum,
e.L.casei, i.L.acidophilus
Table 1: DIAMETER
OF INHIBITION ZONE (mm) CAUSED BY ANTIMICROBIAL ACTIVITY OF LACTOBACILLUS STRAINS AGAINST TEST
MICROORGANISM. EACH VALUE REPRESENTS MEAN ±SD
|
Lactobacillus strains |
Staphylococcus
aerians |
Salmonella
typhi |
Salmonella paratyphi |
Klebsiella oxytoca |
Pseudomonas
aeruginosa |
|
L.fermentum |
4± 0.1 |
3.8±0.77 |
5.2±0.3 |
4.06±0.60 |
5.6±0.95 |
|
L.brevis |
4.93±0.86 |
4.03±0.95 |
3.9±0.95 |
6.2±0.49 |
4.66±0.75 |
|
L.plantarum |
7.76±0.92 |
14.3±2.08 |
13±9.53 |
7±1.32 |
17.3±5.68 |
|
L.curvatus |
7.4±1.27 |
2.96±1.67 |
18±3.60 |
12.66±2.51 |
5.66±1.05 |
|
L.vulgaricus |
6.7±1.6 |
15±2 |
7.3±1.1 |
7.8±0.7 |
22±2.18 |
|
L.casei |
7.6±1.55 |
10.6±1.21 |
7.2±1.11 |
5±1.61 |
5.5±0.94 |
|
L.acidophilus |
15±3 |
17.6±4.93 |
9.8±0.15 |
14±2 |
16±2.64 |
|
L.lactis |
7.7±1.15 |
17±1 |
15±4.58 |
6.6±1.30 |
14±10.44 |
Table 2: DIAMETER
OF INHIBITION ZONE (mm) CAUSED BY ANTIMICROBIAL ACTIVITY OF LACTOBACILLUS
STRAINS AGAINST TEST MICROORGANISM. EACH VALUE REPRESENTS MEAN ±SD
|
Lactobacillus strains |
Vibrio parahaemolyticas |
Proteus
mirabilis |
Lactobacillus
vulgaris |
Vibrio cholerae |
Shilgella flexneri |
|
L.fermentum |
19.5± 9.53 |
5.8±0.77 |
3.26±0.92 |
6±0.7 |
13±2 |
|
L.brevis |
3.03±0.90 |
4.43±0.76 |
6.1±4.61 |
16±1 |
9.2±0.52 |
|
L.plantarum |
16.6±2.51 |
14±4.35 |
17±1 |
14.33±3.05 |
21±1.2 |
|
L.curvatus |
5.53±1.011 |
6.86±0.85 |
8.46±1.27 |
9.5±3.89 |
24±1 |
|
L.vulgaricus |
9.66±8.06 |
23.66±2.08 |
6.63±1.60 |
15.33±1.52 |
4.9±2 |
|
L.casei |
9±1.19 |
9.1±1.21 |
8.4±0.30 |
10.2±0.68 |
14.3±3.05 |
|
L.acidophilus |
18.3±1.52 |
13.6±4.16 |
9.8±1.57 |
11.3±7.37 |
17.3±3.21 |
|
L.lactis |
7.7±1.01 |
7.7±0.86 |
16±6.24 |
16.23±2.51 |
24±1 |
REFERENCE
1. Bernet-Cemard M. F., Lieven
V., Brassart D., Nesser J.
R., Sercin A. L., Hudualt
S. The human Lactobacillus acidophillus
strain LAI secretes a nonbacteriocin antibaterical substances(s) active in vitro and in vivo. Appl Environ Microbiol 1997. 63.
2747-2753.
2. Chauviere G., Coconnier M.H., Kerneis
S., Fourniaat J., Servin
A.L., Adhesion
f human Lactobacillus acidophilus
strain L.B to human enterocyte - like Caco - 2 Cells. J.Gen.Microbiol.
138, 1689-1696 (1992).
3. Collins,
E. –B. and Aramaki, K. 1980: Production of hydrogen peroxide by Lactobacillus acidophilus J. Dairy Sci.,
63, 353 – 357.
4. Collins M, Glenn D, Gibson R, (1999). Probiotics, prebiotics
and symbiotics: Approaches for modulating the
microbial ecology of the gut. American J Clin Nutri, 69 (5): 1052s-57s.
5. Fernandez
M.F., Boris S., Barbes C. Probiotic properties of
human lactobacilli strains to be used in gastrointestinal tract. J. Appl Microbiol 2003. 94, 449-455.
6. Forestier C., De Champs C., Vatoux
C., Joly B. Probiotic
activities of Lactobacillus casei rhamnosius In vivo
adherence to intestinal cells and antimicrobial properties Res Microbiol 2001. 152, 167-173.
7. Ishibashi, N. and S.Yamazaki, 2001. Probiotica and safety. Am.J.Clin.Nutr.,
73: 465 – 470.
8. Kmet V., Lucchini F., Aggregation of sow Lactobacilli with diarrheagenic
Escherichia colli, J.Vet.
Med. B 46, 683-687 (1999).
9. Ligocka A., Paluszak Z., Capability of lactic acid
bacteria to inhibit pathogens sewage sludge subjected to biotechnological
processes. Bull Vet Inst pulawy 2005, 49, 23-27.
10. Reid G., The scientific basis for probiotic
strains of Lactobacillus, Minivreview. Appl Environ Microbiol 1999, 65, 3763-3766.,
11. Roberfroid MB (2002). Prebiotics and probiotics :
are they functional foods? Am J Clin Nutr, 71 (6 Suppl) : 16925-7S;
discussion 1688S-90S.
12. Tomas, M. –S. –J., Brue, E. and Nader – Macias, M. –E. (2003): Comparison of the growth and
hydrogen peroxide production by vaginal probiotic
lactobacilli under different culture conditions. Am. J. Obstet. Gynecol., 188,
35 – 44.
Received on 13.04.2011
Accepted on 24.05.2011
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Research Journal of Pharmacognosy and
Phytochemistry. 3(4): July- August 2011, 174-177