You are on page 1of 7

Anaerobe 33 (2015) 117e123

Contents lists available at ScienceDirect

journal homepage:

Clinical microbiology

Characterization of Lactobacillus isolated from dairy samples

for probiotic properties
Ashwani Kumar, Dinesh Kumar*
School of Bioengineering and Food Technology, Shoolini University of Biotechnology and Management Sciences, Solan 173212, H.P., India

a r t i c l e i n f o a b s t r a c t

Article history: In the present study twelve Lactobacillus isolates (LBS 1-LBS 12) were characterized for probiotic prop-
Received 21 November 2014 erties. Out of the twelve, eight isolates (LBS 1e6, 8 and 11) were bile resistant (survival > 50% at 0.3% bile
Received in revised form salt w/v) and five isolates (LBS 1, 2, 5, 6 and 11) were found acid pH value resistant (survival > 50% at pH
3 March 2015
3). All twelve isolates inhibited the growth of Staphylococcus aureus whereas isolate LBS 2 also inhibited
Accepted 10 March 2015
Available online 12 March 2015
the growth of Escherichia coli and Salmonella typhimurium. Antibiotic susceptibility testing of isolates was
also performed and isolate LBS 2 was selected for further study based on its broad spectrum effect in
clinical pathogen inhibition. LBS 2 was characterized phenotypically at Institute of Microbial Technology
Lactobacillus characterization
(IMTECH), Chandigarh, India and was confirmed as Lactobacillus rhamnosus by 16S rDNA sequencing and
Isolate LBS 2 subsequent analysis using BLAST. The gene sequence was deposited in GenBank with accession number
16S rDNA amplification KJ562858. Scanning electron microscopy (SEM) study was used to study in vitro epithelial cell adherence
Epithelial cell adherence and bile salt effect on isolate LBS 2. Epithelial cells adherence assay showed positive results and surface
SEM roughness of LBS 2 increased with increase in bile salt (0.15e0.45% w/v).
© 2015 Elsevier Ltd. All rights reserved.

1. Introduction cholesterol level [11,12]. There is an increase in use of probiotic

bacteria in variety of food products viz. yoghurt, cheese, drinks and
Probiotics are live microorganisms which, when administered in dietary supplements based on their therapeutic benefits [13].
adequate amounts, confer a health benefit on the host [1]. Most Keeping in view the importance of probiotics, the present study is
probiotic microorganism belongs to the lactic acid bacteria (LAB) endeavored to screen twelve previously reported Lactobacillus
viz. Lactobacillus spp., Enterococcus spp. and Bifidobacterium [2]. isolates (8 from household curd and 4 from household milk) [14] for
Most of the species of the genus Lactobacillus are part of human and the probiotic properties viz. resistance against bile salt, acid pH
animal commensal intestinal flora [3] and consists of a physiolog- values, antibiotics, pathogenic microbe inhibition and select a po-
ically and genetically diverse group of rod-shaped, Gram-positive, tential isolate for further investigation.
non-pigmented, non-spore forming [4], catalase negative, micro-
aerophilic to anaerobic LAB [5] with widespread use in fermented
2. Materials and methods
food production [6]. These microorganisms are also called friendly
bacteria and are generally recognized as safe (GRAS) microorgan-
2.1. Bacterial strains
isms [7]. The lactobacilli isolated from dairy products have long
history of safe use [1] and are widely used as starter cultures in the
Thirty dairy samples (household milk and curd) were collected
food industry viz. fermented milk, alcoholic beverages, sourdough
from local area of Solan, H.P., India. Serial dilutions of 1 ml dairy
and silage preparation [8]. Several health benefits associated with
samples were prepared in peptone water and then 100 ml sample
the consumption of probiotic bacteria include; controlling intesti-
from different dilutions were spread over the solidified MRS (de
nal infections, improving lactose utilization, lowering blood
Man, Rogosa and Sharpe) medium (HiMedia, India) and incubated
ammonia level [9,10], influencing immune system and low serum
at 37  C for 24e48 h under anaerobic conditions for the isolation
of Lactobacillus. Pure cultures of isolated colonies were obtained
after re-streaking on MRS agar plate. Each culture was studied for
* Corresponding author. morphological investigation (type of colony, color, margin,
E-mail address: (D. Kumar). elevation, opacity and presence of pigment) and Lactobacillus
1075-9964/© 2015 Elsevier Ltd. All rights reserved.
118 A. Kumar, D. Kumar / Anaerobe 33 (2015) 117e123

specific biochemical tests (motility test, endospore test, catalase substances. All MHA plates were incubated at 37  C for 24 h and the
and sugar (glucose) fermentation test) as reported in our earlier zone of inhibition was measured using Hi antibiotic zone scale. The
publication [14] and twelve selected Lactobacillus isolates were inhibition zone diameter of 10 mm and above was considered as
given code as LBS 1-LBS 12 [8 from household curd (LBS 1,2,4,5,7, positive antimicrobial effect.
9,10,12) and 4 from household milk (LBS 3,6,8,11)] and used in the
present study. 2.2.3. Antibiotic susceptibility test
For the antimicrobial activity clinical isolates of Staphylococcus Antibiotic susceptibility of all isolates was determined by the
aureus, Escherichia coli and Salmonella typhimurium were obtained standard disc diffusion method on MHA plates [19]. After adjusting
from the Post Graduate Institute of Medical Education and Research the turbidity standard of bacterial inoculum (107e108 CFU/ml) a
(PGIMER), Chandigarh, India and maintained on Nutrient agar sterile cotton swab was dipped into each inoculum and swab
medium (HiMedia, India) at 4  C after 24 h growth at 37  C. streaked over the entire surface of respective MHA plates. Now
different antibiotic discs (vancomycin, amoxycillin, amikacin, gen-
2.2. Characterization of isolates for probiotic properties tamycin, tetracycline, chloramphenicol, co-trimoxazole and cipro-
floxacin) were placed on these test plates with the help of sterile
All twelve Lactobacillus isolates were screened for their resis- forceps and incubated at 37  C for 24 h and the inhibition zone
tance against bile salt, acid pH values, antibiotics and ability to diameter was measured with Hi antibiotic zone scale.
inhibit selected microbial pathogens. Inoculums of each isolates The isolate LBS 2 was used for the further investigation viz.
was prepared using standard protocol M7-A7- CLSI (Chemical phenotypic and genotypic characterization and scanning electron
Laboratory Standard Institute) [15] in test tubes containing 5 ml of microscopy (SEM) study to see in vitro cell adherence assay and
broth medium and incubated at 37  C for 24 h. The turbidity of effect of bile salt on its morphology by surface roughness study.
inoculum was maintained with 0.5 McFarland standards, contain-
ing 107e108 CFU/ml. 2.3. Phenotypic and genotypic characterization of LBS 2

2.2.1. Bile and acid tolerance The phenotypic characterization of isolate LBS 2 was performed
All isolates were investigated for bile salt tolerance following the on the basis of its morphological (colony morphology, Gram's re-
method of Vinderola and Reinheimer [16]. Briefly, 0.2 ml of each action, spore formation and motility), biochemical (methyl red,
isolate inoculum suspension (107e108 CFU/ml) was added to 10 ml citrate utilization, Voges Proskaeur, casein, starch and urea hydro-
of MRS broth containing different concentrations of bile salt lysis, nitrate reduction, H2S production, cytochrome oxidase, argi-
(0.15e0.45% w/v) and MRS broth without bile salt as control and nine dihydrolysis, lysine decarboxylase and indole test) and
incubated at 37  C for 24 h. Optical density (OD), was recorded at physiological characteristics (growth at varying temperature
560 nm after incubation and compared with the control. Lactoba- 25e42  C, pH 5e9 and 2.5e5% (w/v) NaCl) at Institute of Microbial
cillus isolates showing resistance more than 50% at 0.3% (w/v) bile Technology (IMTECH), Chandigarh, India [20,21]. The isolate LBS 2
salt were considered as bile resistant as this is the minimum range was further identified by 16S rDNA gene sequencing. Briefly,
for being a probiotic culture. genomic DNA was extracted by the standard chloroform-isoamyl
The ability of isolates to tolerate the acid pH values was deter- alcohol method as described by Sambrook et al. [22]. PCR amplifi-
mined as described by Sieladie et al. [17]. Briefly, 1 ml of inoculum cation of the 16S rDNA was performed using the following forward
(107e108 CFU/ml) of the each isolate was transferred into 10 ml of and reverse primers: 8F: 50 AGA GTT TGA TCC TGG CTC AG 30 and
MRS broth at pH 2, 3 and 5 respectively and OD was taken after 24 h 1492R: 50 ACG GCT ACC TTG TTA CGA CTT 30 . The polymerase chain
incubation at 37  C against the control (pH 7.0). Isolates showing reaction (PCR) mixture consisted of 7.50 mL of DNase-RNase free
resistance more than 50% at pH 3 were considered acid tolerant water, 12.50 mL (1) of 2 PCR master mix, 1.0 mL (10 pmole) of each
strains. primer and 30 ng of DNA template. The PCR reaction was performed
The percentage resistance in both cases (bile/acid pH values) in 25 mL volumes for 30 cycles at 95  C, 30 s at 52  C, and 1 min at
was calculated as: 72  C with additional extension for 10 min at 72  C. Amplified DNA

Increment of OD in MRS broth with bile salt=pH 2; 3; 5

% Resistance ¼  100
Increment of OD in MRS broth without bile salt =at pH 7

was examined by electrophoresis in 1% agarose with 5 mL aliquots of

PCR product using non-amplified DNA as a negative control. The
2.2.2. Antimicrobial activity 16S rDNA PCR product was purified by QIAquick Gel Extraction Kit
Antimicrobial activity of the isolates against pathogenic bacteria (Qiagen, India). The purified PCR product was sequenced (forward
viz. E. coli, S. aureus and S. typhimurium was determined by standard and reverse sequence) by Xcelris lab, Ahmadabad, India. In the
well diffusion method using Mueller Hinton Agar (MHA) plates present study only the forward sequence was used. The sequence
[18]. The 100 ml inoculum (107e108 CFU/ml) of each bacterium was was compared with the nucleotide database from the NCBI Gen-
spread on MHA plates. Wells were made in each inoculated plate Bank using the BLAST program. A similarity of >98% to the 16S
and filled with 30 ml of cell free supernatant of Lactobacillus isolates rDNA sequence of the reference Lactobacillus rhamnosus was used
obtained after 24 h growth in MRS broth and centrifugation of the as a criterion for the identification. The obtained 16S rDNA
inoculums at 4000 rpm for 10 min. Ciprofloxacin (30 mg/ml) was sequence was deposited in the NCBI GenBank database. A phylo-
used as positive control and MRS broth was used as negative con- genetic tree was generated from the alignment of the deposited
trol. All supernatant broths were neutralized to pH 6.5 to maintain sequence by the Neighbor-joining method using Sea-View version
that the inhibition is not due to lactic acid but by antimicrobial 4 [23].
A. Kumar, D. Kumar / Anaerobe 33 (2015) 117e123 119

2.4. In vitro epithelial cells adherence assay cultural and biochemical characteristics, 12 bacterial isolates (8
from curd and 4 from milk) were considered to belong to the genus
The in vitro adherence of LBS 2 cells to epithelial cells was Lactobacillus as reported in our previous publication [14] and these
studied using rat ileum as a model. Epithelial cells were prepared were further characterized for the probiotic potential and the re-
according to the method described by Annika et al. [24]. Small sults are discussed in the following sections.
segment of rat ileum was opened and washed thrice with sterilized
phosphate buffer saline (PBS) (0.1 mol/l, pH 7.2). It was further held 3.1. Characterization of isolates for probiotic properties
in PBS and incubated at 4  C for 30 min to remove the surface
mucus and then washed thrice with PBS. Epithelial cells were 3.1.1. Bile and acid tolerance
scrapped into sterilized PBS. One ml of bacterial inoculum Bile salt resistance is one of the criteria for any microbial strain
(107e108 CFU/ml) was mixed with 1 ml of the cell suspension of to be used as a probiotic culture. The presence of bile in the in-
epithelial cells. The mixture was incubated at 37  C for 30 min. The testine affects the viability of LAB. All twelve isolates were grown in
in vitro adhesion of LBS 2 to epithelial cells was observed using FEI different concentrations of bile salt and results showed that only
Nova Nanosem 450, Scanning Electron Microscope. eight isolates were resistant at 0.3% (w/v) of bile salt i.e. percentage
of resistance 50%. Maximum resistance was observed with LBS 2
2.5. Effect of bile salt on morphology of LBS 2 (76.88%, 72.36%, 68.62%) and minimum with LBS 3 (67.42%, 57.61%,
50.67%) respectively at 0.15%, 0.3% and 0.45% (w/v) of bile salt
Effect of different concentrations of bile salt on morphology (Fig. 1).
(roughness) of bacteria was analyzed using SEM as described by The survival of LAB in low pH of stomach is important for
Soo-Hwan et al. [25] with some modifications. Bacterial cells after bearing the initial acid stress. The effect of acidity on the viability of
treatment with bile salt (015e0.45 % w/v) were fixed in 3% (v/v) all isolates was assessed by their growth in different incubation pH
glutaraldehyde buffered with 0.1 M sodium phosphate buffer (pH in MRS broth. Out of twelve isolates, five were resistant to acid pH
7.2) for 3 h and then washed three times in the same buffer. Further value 3 (with resistance  50%). Highest resistance was reported
cells were dehydrated in a graded alcohol series (30, 50, 70, 90, with LBS 2 (60.52%) and lowest with LBS 11 (52.62%) at pH 3 (Fig. 2).
100%). The specimens were dried, coated with thin layer of gold and However, no isolate was found resistant at pH 2 (with percentage
mounted onto stubs using double-sided carbon tape and examined resistance <50%).
for change in surface roughness using SEM.
3.1.2. Antimicrobial activity
2.6. Statistical analysis One of the major properties for an isolate to be used as probiotic
is its ability to inhibit microbial pathogens. In the present study,
All characterization experiments were done in triplicates and antimicrobial effects of all isolates against selected pathogenic
standard deviation was calculated. Statistical evaluation was also bacteria were studied. All isolates inhibited the growth of S. aureus
carried out using GraphPad Prism 6.0. A p-value below 0.05 was whereas only one isolate (LBS 2) inhibited the growth of E. coli, S.
considered to be statistically significant. typhimurium with inhibition zone diameter of 10.66 ± 0.57 mm and
10.33 ± 0.57 mm respectively in addition to S. aureus (11.33 ±
3. Results 0.57 mm). However, highest zone of inhibition (14 ± 1 mm) against
S. aureus was observed with the isolate LBS 8 (Table 1).
Thirty bacterial isolates were initially obtained from thirty dairy
samples collected from Solan district in Himachal Pradesh, India. 3.1.3. Antibiotic susceptibility test
Out of thirty, 14 isolates were from milk sample and 16 isolates were The susceptibility to antibiotics is a common feature of probiotic
from curd samples. Characterization of isolates was performed on bacteria. The antibiotic susceptibility of all isolates was assessed by
the basis of their morphological characteristics viz., type of colony, disc diffusion method using MHA medium and results are shown in
color, margin, elevation, opacity and presence of pigment and the Table 2. The isolate LBS 5 was found resistant to six antibiotics
Lactobacillus specific biochemical tests. Based on morphological, and the isolate LBS 2 was resistant to five antibiotics. The antibiotic

Fig. 1. Effect of different concentration of bile salt 0.15e0.45% (w/v) on survival of Lactobacillus isolates (LBS1-12) after 24 h incubation at 37  C in MRS broth.
120 A. Kumar, D. Kumar / Anaerobe 33 (2015) 117e123

Fig. 2. Effect of different acid pH values on survival of Lactobacillus isolates (LBS 1e12) after 24 h incubation at 37  C in MRS broth.

resistance traits among probiotic microorganisms are advanta- arginine dihydrolysis, lysine decarboxylase and indole test were
geous to survive the gastrointestinal tract during antibiotic treat- found negative for this isolate. This isolate showed positive growth
ment. Some resistance genes might be present in resistant at 25e42  C, pH 5e9 and 2.5e5% NaCl (w/v), however, no growth
microorganisms and mechanism associated with this resistance is was observed with increase in temperature beyond 55  C, pH 9 and
still unknown. 6% NaCl respectively. The isolate LBS 2 showed positive results for
fermentation with dextrose, fructose, galactose, lactose, maltose,
3.2. Identification and characterization of selected isolate LBS 2 mannitol, rhamnose, sorbitol, sucrose, trehalose and mannose and
no fermentation was observed with xylose, adonitol, inulin, cello-
The isolate LBS 2 showed maximum resistance with bile salt biose, dulsitol, inositol, melibiose, raffinose and salicin. Based on
(72.36%) at 0.30% (w/v), acid pH value 3 (60.52%), inhibited growth morphological, biochemical and physiological characteristics the
of all tested microbial pathogens and was resistant to five antibiotics isolate LBS 2 showed similarity in phenotypic properties with L.
and was selected for further identification and characterization. rhamnosus.
The PCR amplification of 16S rDNA of LBS 2 resulted in a single
band of about 1500 bp product which corresponds to the expected
3.3. Phenotypic characterization and genotypic characterization of
size of the 16S rDNA gene. Both forward and reverse sequence was
LBS 2 obtained after sequencing and assembly sequence was generated
but in the present study only the forward sequence was used.
The selected Lactobacillus (LBS 2) with maximum probiotic po- BLAST analysis of the obtained sequence with the available nucle-
tential properties was further characterized at IMTECH, Chandi-
otide database in the GenBank, showed 99% similarity of the LBS 2
garh, India for its phenotypic characteristics viz. morphological, isolate with the L. rhamnosus (Fig. 3). The sequence was deposited
biochemical and physiological properties. Morphological investi-
in GenBank with accession no. KJ562858. The comparative analysis
gation of LBS 2 showed that colonies were round, smooth, raised, of LBS 2 sequences with the published sequences of different
entire margins, Gram positive rods arranged in clusters, non-spore
Lactobacillus species, using the SeaView Version 4 program
forming and non-motile. Various biochemical tests viz. methyl red, confirmed its similarity with the L. rhamnosus KF806539.1 and
citrate utilization, Voges Proskaeur, casein, starch and urea hydro-
demonstrated the phylogenetic distances in a generated Neighbor-
lysis, nitrate reduction, H2S production, cytochrome oxidase, joining rooted tree (Fig. 3).

Table 1 3.4. In vitro epithelial cell adherence assay and bile effect analysis
In vitro antimicrobial activity of Lactobacillus isolates against some selected patho- using SEM
genic bacteria in MHA after 24 h incubation at 37  C.

Isolate Test organism with IZD mean (mm) ± S.D. The ability of microbe to adhere to intestinal mucosa is an
important selection criterion for its probiotic use. The Lactobacillus
Staphylococcus aureus E. coli Salmonella typhimurium
isolates with more than 15 adhered cells per epithelial cell were
LBS 1 13.33 ± 0.57 e e considered positive and the results of adherence of LBS 2 to in vitro
LBS 2 11.33 ± 0.57 10.66 ± 0.57 10.33 ± 0.57
LBS 3 10.66 ± 0.57 e e
epithelial cells is shown in Fig. 4.
LBS 4 10.33 ± 0.57 e e The effect of different concentration of bile salt after incubation
LBS 5 12 ± 1 e e of Lactobacillus rhamnosus LBS 2 at 37  C for 24 h on its cell
LBS 6 11 ± 1 e e morphology (roughness) was studied with SEM. The results
LBS 7 11.66 ± 0.57 e e
showed that with an increase in amount of bile salt from 0.15% to
LBS 8 14 ± 1 e e
LBS 9 10.66 ± 0.57 e e 0.45% the roughness of bacterial cell surface also increased (Fig. 5).
LBS 10 11.33 ± 0.57 e e
LBS 11 10.66 ± 0.57 e e 4. Discussion
LBS 12 11 ± 1 e e

IZD ¼ Inhibition Zone Diameter, e ¼ no activity. In the present study twelve Lactobacillus spp. isolated from dairy
A. Kumar, D. Kumar / Anaerobe 33 (2015) 117e123 121

Table 2
Antibiotic susceptibility of Lactobacillus isolates using disc diffusion method with MHA after 24 h incubation at 37  C.

Antibiotic Lactobacillus isolate (LBS 1e12)

1 2 3 4 5 6 7 8 9 10 11 12

Co-trimoxazole (25 mcg) R R R R R R R þþ þþþ þ R R

Amikacin (30 mcg) þþþ R þþ þþ þþ þ þ þþ þþ þþ þþ þ
Amoxycillin (10 mcg) R R R R R R þþ þþ þþ R R R
Penicillin-G (10 mcg) þ þ þþ þþ þ þþ þþþ þþ þþ þþ þþ þþþ
Ciprofloxacin (10 mcg) þþ þ þþþ þ R þþ þþ þþþ þþ þþþ þþ þþ
Vancomycin (10 mcg) R R þþ R R R R þþ R þþ R þþ
Tetracycline (10 mcg) þþþ þ þþþ þþ R R þþþ þþþ þ þþþ R þþþ
Gentamycin (10 mcg) R R þþ R R þþ þþþ þþ þþ þþ þþ þþ

R ¼ resistant, þ ¼ less sensitive, þþ ¼ moderately sensitive, þþþ ¼ highly sensitive.

Fig. 3. Phylogenetic tree (neighbor-joining) of isolate LBS 2 based on the 16S rDNA sequence. The scale bar represents relative sequence similarities.

Fig. 4. In vitro epithelial cell adherence of Lactobacillus rhamnosus LBS 2. A) Control (only crop epithelium), B) crop epithelium with positive adhered cells.
122 A. Kumar, D. Kumar / Anaerobe 33 (2015) 117e123

Fig. 5. Effect of different bile salt (0.15e0.45% w/v) incubation of Lactobacillus rhamnosus LBS 2 for 24 h at 37  C on cell morphology (roughness) as studied under scanning Electron
Microscope (20,000). A) Control (Without bile salt), B) 0.15%, C) 0.30% and D) 0.45% bile salt (w/v).

samples were characterized for probiotic properties and results should be resistant to certain antibiotics to survive in the gastro-
were compared with the existing literature. In screening study intestinal tract [37,38]. The presence of antibiotic-resistance genes
eight, out of twelve isolates were bile resistant and Lactobacillus in many LAB and transfer of plasmids and conjugative transposons
isolates showing resistance 50% at 0.3% (w/v) bile salt were to and from LAB, have been reported in Lactobacillus species [39]. In
considered as bile resistance strains [16]. Highest resistance general, glycopeptide, aminoglycoside (amikacin, kanamycin,
(72.36% and 68.62%) was observed with isolate LBS 2 at 0.3% and streptomycin and gentamycin) and sulfamethoxazole resistance
0.45% of bile salt respectively. Gilliland [26] reported that lactoba- has been described in LAB, and in most cases it is associated with
cilli isolated from animal intestines showed high tolerance to bile their natural and intrinsic resistance due to membrane imperme-
salt than isolates from milk products. Similar results were reported ability, probably complemented by potential efflux mechanism
by Patel et al. [27]. Gilliland et al. [28] observed high variability resistance [40e42]. Intrinsic resistance is not horizontally trans-
among Lactobacillus acidophilus strains isolated from calf intestinal ferable and poses no risk in non-pathogenic bacteria [43]. Hoque
contents to grow in vitro in the presence of bile salts. Garriga et al. et al. [44] found that the Lactobacillus spp. were resistant to b-
[29] reported the selected LAB strains with resistant to 4% bile salts. lactams due to presence of b-lactamase in the isolates. Zhou et al.
The resistance variability among lactobacilli is due to the presence [45] reported that new probiotic strains; L. rhamnosus HN001 and
of bile salt hydrolase (BSH), an enzyme that reduces toxic effects by HN067 were resistant to fusidic acid, kanamycin, nalidixic acid,
conjugating bile [30]. neomycin, polymyxin B and vancomycin due to their intrinsic
The acid pH stability results of tested isolates in the present resistance and without transmissible antibiotic resistance genes. In
study are in accordance with the reported literature. Isolates another report L. rhamnosus BFE 7442 was resistant against cipro-
showing 50% resistance at acid pH value 3 were considered acid floxacin, gentamycin and streptomycin and demonstrated that the
tolerant [17]. In present study five isolates were acid resistant and resistant genes might be present in probiotic strains but are silent.
highest resistance was observed with LBS 2 (60.52%) at acid pH Genetic basis and associated resistance mechanisms towards some
value 3. Idoui et al. [31] also showed resistance of Lactobacillus antibiotics are still unknown and need to be explored [46,47].
plantarum BJ0021 at pH 3. Idoui [32], showed that in comparison to The isolate LBS 2 was selected for further study as it showed
the Lactobacillus isolated from the gastrointestinal tracts of human, highest resistance against bile salt and acid pH value in in vitro
strains of Lactobacillus fermentum, Lactobacillus gasseri and Lacto- experiments. It also showed broad spectrum microbial pathogen
bacillus delbrueckii subsp. bulgaricus were having better acid inhibition, resistance to five antibiotics and was further investi-
tolerance. gated for phenotypic and genotypic characterization and was
In present study, antimicrobial activity of twelve selected iso- identified as L. rhamnosus LBS 2 by 16S rDNA sequencing.
lates was tested against selected clinical isolates. Isolate LBS 2 In vitro epithelial cell adherence and bile salt effect was studied
inhibited the growth of E. coli, S. typhimurium and S. aureus and with SEM analysis. L. rhamnosus LBS 2 showed positive epithelial
showed broad spectrum antimicrobial activity. Garriga et al. [29] cell adherence test. The results of the epithelial cell adherence were
reported inhibition of one or more enteric indicator strains compared with the existing literature and isolates with an adhesion
(E. coli, Salmonella enteritidis) by Lactobacillus paracasei subsp. efficacy of 15 bacteria per epithelial cells are considered positive
Paracasei. L. acidophilus strains isolated from infant faeces had weak [48]. Idoui [32], reported adherence specificity of L. fermentum HG3
antibacterial activity on E. coli and Yersinia enterocolitica [33]. and L. gasseri HG8, to chicken intestinal epithelium. Jamaly et al.
Daeschel [34] reported that the antimicrobial effect of LAB is due to [48] reported that all the tested strains were able to adhere to rat
the production of lactic acid, reduction of pH, acetic acid, diacetyl, ileum epithelial cells. Effect of bile salt on surface morphology of L.
fatty acids, aldehydes and other compounds. Whereas, as per rhamnosus showed that with an increase in concentration of bile
another report the antimicrobial action is due to the potential of salt the smoothness of bacterial surface changed.
LAB bacteriocin and peptides having inhibitory properties [35].
In antibiotic test all isolates showed varying antibiotic suscep- 5. Conclusion
tibility pattern. Isolate LBS 5 was resistant to six antibiotics and LBS
2 was resistant to five antibiotics. As reported in literature, specific In conclusion one potential probiotic isolate LBS 2 was obtained
antibiotic resistance traits among probiotic strains may be desirable after screening of twelve Lactobacillus isolates for the probiotic
[36]. In case of co-administration of probiotics with antibiotics they properties. This isolate LBS 2 from the household curd sample of
A. Kumar, D. Kumar / Anaerobe 33 (2015) 117e123 123

Solan, district of Himachal Pradesh was identified as L. rhamnosus Press, San Dieago, California, 1998.
[22] J. Sambrook, D.W. Russel, Molecular Cloning: a Laboratory Manual, third ed.,
LBS 2 after phenotypic and genotypic characterization. High resis-
Cold Spring Harbor Laboratory, New York, 2001, pp. 1.72e5.77.
tance to bile salt, tolerance to acid pH values, broad spectrum mi- [23] Chemical Laboratory Standards Institute (CLSI, Methods for Dilution Antimi-
crobial pathogen inhibition and adherence to epithelial cells seems crobial Susceptibility Tests for Bacteria that Grow Aerobically, 2006. Approved
to be a potential advantage of this culture for use as probiotic standard e seventh addition, CLSI document M7- A7 (ISBNI -56238 e 587-9).
CLSI, Wayne, Pennsylvania 19087-1898 USA.
culture. Isolate LBS 2 was resistant to five antibiotics, out of tested [24] M.M. Annika, M. Manninan, H. Gylienberg, The adherence of lactic acid bac-
eight. Antibiotic resistant traits among probiotic strains may be teria to the columnar epithelial cells of pigs and calves, J. Appl. Bacteriol. 55
desirable if administered alongwith antibiotics. However, detailed (1983) 241e245.
[25] K. Soo-Hwan, L. Hyeong-Seon, R. Deok-Seon, C. Soo-Jae, L. Dong-Seok,
molecular investigation has to be conducted to establish the J. Korean, Antibacterial activity of silver-nanoparticles against Staphylococcus
mechanism of the antibiotic resistance genes in this isolate. aureus and Escherichia coli, Microbiol. Biotechnol. 39 (2011) 77e85.
[26] S.E. Gilliland, Beneficial interrelationships between certain microorganisms
and human: candidate microorganisms for use as dietary adjuncts, J. Food
Acknowledgments Prot. 42 (1979) 164e167.
[27] H.M. Patel, S.S. Pandiella, R.H. Wang, C. Webb, Influence of malt, wheat and
The authors are thankful to Dr. Rohit Goyal, Faculty of Phar- barley extracts on the bile tolerance of selected strains of Lactobacilli, Food
Microbiol. 21 (2004) 83e89.
maceutical Sciences, Shoolini University, Solan, India for his help in [28] S.E. Gilliland, T.E. Staley, L.J. Bush, Importance of bile tolerance of Lactobacillus
epithelial cells adherence assay and Er. Sandeep, Indian Institute of acidophilus used as dietary adjunct, J. Dairy Sci. 67 (1984) 3045e3051.
Technology, Mandi, India for SEM analysis in the present study. [29] M. Garriga, M. Pascual, J.M. Monfort, M. Hugas, Selection of lactobacilli for
chicken probiotic adjuncts, J. Appl. Microbiol. 84 (1998) 125e132.
[30] M. Du Toit, C.M. Franz, L.M. Dicks, U. Schillinger, P. Haberer, B. Warlies,
References F. Ahrens, W.H. Holzapfel, Characterisation and selection of probiotic lacto-
bacilli for a preliminary mini pig feeding trial and their effect on serum
[1] FAO/WHO, Evaluation of Health and Nutritional Properties of Probiotics in cholesterol levels, faeces pH and faeces moisture content, Int. J. Food Micro-
Food Including Powdered Milk and Live Lactic Acid Bacteria, 2001. Food and biol. 40 (1998) 93e104.
Agriculture Organization of the United Nations and World Health Organiza- [31] T. Idoui, E. Leghouchi, N.E. Karam, Selection of Lactobacillus plantarum BJ0021
tion Report, for rabbit probiotic adjuncts, Int. J. Prebiotics Probiotics 2 (2007) 188e193.
[2] G. Klein, A. Pack, C. Bonaparte, G. Reuter, Taxonomy and physiology of pro- [32] T. Idoui, probiotic potential of lactic acid bacteria isolated from human gut,
biotic lactic acid bacteria, Int. J. Food Microbiol. 41 (1998) 103e125. TOJSAT 2 (2012) 47e51.
[3] E.G. Zoetendal, E.E. Vaughan, W.M. deVos, A microbial world within us, Mol. [33] V. Xanthopoulous, E. Litopoulou-Tzanetaki, N. Tzanetaki, Characterization of
Microbiol. 59 (2006) 1639e1650. Lactobacillus isolates from infant faeces as dietary adjuncts, Food Microbiol. 17
[4] N.A. Hasan, J.F. Frank, Starter cultures and their use, in: E.H. Marth, J.L. Steele (2000) 205e215.
(Eds.), Applied Dairy Microbiology, second ed., Marcel Dekker, New York, [34] M.A. Daeschel, Antimicrobial substances from lactic acid bacteria for use as
2001, pp. 152e155. food preservatives, Food Technol. 43 (1989) 164e167.
[5] J.P. Vernoux, V. Coeuret, S. Dubernet, M. Bernardeau, M. Gueguen, Isolation, [35] M.K. Strus, H. Pakosz, A. Gociniak, E. PrzondoMordarska, H. Roynek, F. Pituch,
Characterisation and Identification of Lactobacilli Focusing Mainly on Cheeses A. Meisel-Miko, P.B. Heczko, Antagonistic activity of Lactobacillus strains
and Other Dairy Products, vol. 83, INRA, EDP Sciences, 2003, pp. 269e306. against anaerobic gastrointestinal tract pathogens (Helicobacter pylori,
[6] A.M. Azcarate-Peril, R.R. Raya, Methods for plasmid and genomic DNA isola- Campylobacter coli, Campylobacter jejuni, Clostridium difficile), Med. Dosw.
tion from lactobacilli, in: J.F.T. Spencer, A.L.R.D. Spencer (Eds.), Food Micro- Mikrobiol. 53 (2001) 133e142.
biology Protocols. Totowa, Humana Press Inc;, New Jersey, 2001, p. 135. [36] W.P. Charteris, P.M. Kelly, L. Morelli, J.K. Collins, Antibiotic susceptibility of
[7] R. Fuller, Probiotics in man and animals: a review, J. Appl. Bacteriol. 66 (1989) potentially probiotic Lactobacillus species, J. Food Prot. 61 (12) (1998)
365e378. 1636e1643.
[8] F.J. Carr, D. Chill, N. Maida, The lactic acid bacteria, a literature survey, Crit. [37] P.L. Shivram, P.P. Vishwanath, Assessment of probiotic potential of Lactoba-
Rev. Microbiol. 28 (2002) 281e370. cillus sp. isolated from cheese and preparation of probiotic ice cream, Int. J.
[9] A.M.P. Gomes, F.X. Malcata, Bifidobacterium spp. and Lactobacillus acidophilus: Res. Ayurveda Pharm. 3 (4) (2012) 532e536.
biological, biochemical, technological and therapeutical properties relevant [38] G. Dixit, D. Samarth, V. Tale, R. Bhadekar, Comparative studies on potential
for use as probiotics, Trends Food Sci. Technol. 10 (1999) 139e157. probiotic characteristics of Lactobacillus acidophilus strains, Eurasian J. Biosci.
[10] L. Ebringer, M. Ferencik, N. Lahitova, L. Kacani, D. Michalkova, Antimutagenic 7 (2013) 1e9.
and immunostimulatory properties of lactic acid bacteria, World J. Epidemiol. [39] I. Yoshiyuki, K. Yasushi, A. Kensuke, H. Yoshiko, S. Takashi, S. Tadao, Con-
Commun. Health 43 (1995) 356e361. jugative plasmid from Lactobacillus gasseri LA39 that carries genes for pro-
[11] A.C. Ouwehand, S. Salminen, E. Isolauri, Probiotics: an overview of beneficial duction of and immunity to the circular bacteriocin Gassericin A, Appl.
effects, Antonie van Leeuwenhoek 82 (2002) 279e289. Environ. Microbiol. 75 (19) (2009) 6340e6351.
[12] M.T. Liong, N.P. Shah, Bile salt deconjugation ability, bile salt hydrolase ac- [40] C.A. Elkins, L.B. Mullis, Bile-mediated aminoglycoside sensibility in Lactoba-
tivity and cholestrol co-precipitation ability of Lactobacilli strains, Int. Dairy J. cillus species likely results from increased membrane permeability attribut-
15 (2005) 391e398. able to cholic acid, Appl. Environ. Microbiol. 70 (2004) 7200e7209.
[13] L. Ong, A. Henriksson, N.P. Shah, Development of probiotic cheddar cheese [41] P.R. Rauta, M. Dhupal, B. Nayak, Screening and characterization of potential
containing L. acidophilus, L. paracasei, L. casei and Bifidobacterium spp. and the probiotic lactic acid bacteria isolated from vegetable waste and fish intestine,
influence of these bacteria on proteolytic patterns and production of organic Int. J. Curr. Microbiol. Appl. Sci. 2 (8) (2013) 234e244.
acid, Int. Dairy J. 16 (2006) 446e456. [42] S.A. Liasi, T.I. Azmi, M.D. Hassan, M. Shuhaimi, M. Rosfarizan, A.B. Ariff,
[14] A. Kumar, D. Kumar, Isolation and characterization of bacteria from dairy Antimicrobial activity and antibiotic sensitivity of three isolates of lactic acid
samples of Solan in Himachal Pradesh for identification of Lactobacillus spp, bacteria from fermented fish product, Budu, Malays. J. Microbiol. 5 (1) (2009)
Int. J. Pharm. Sci. Rev. Res. 25 (2014) 110e114. 33e37.
[15] M. Gouy, S. Guindon, O. Gascuel, SeaView version 4: a multiplatform graphical [43] S. Mathur, R. Singh, Antibiotic resistance in food lactic acid bacteria-A review,
user interface for sequence alignment and phylogenetic tree building, Mol. Int. J. Food Microbiol. 105 (2005) 281e295.
Biol. Evol. 27 (2010) 221e224. [44] M.Z. Hoque, F. Akter, K.M. Hossain, M.S. Rahman, M.M. Billah, K.M.D. Islam,
[16] C.G. Vinderola, J.A. Reinheimer, Lactic acid starter and probiotic bacteria, a Isolation, identification and analysis of probiotic properties of Lactobacillus
comparative “in vitro” study of probiotic characteristics and biological barrier Spp. from selective regional yoghurts, World J. Dairy Food Sci. 5 (2010) 39e46.
resistance, Food Res. Int. 36 (2003) 895e904. [45] J.S. Zhou, C.J. Pillidge, P.K. Gopal, H.S. Gill, Antibiotic susceptibility profiles of
[17] D.V. Sieladie, N.F. Zambou, P.M. Kaktcham, A. Cresci, F. Fonteh, Probiotic new probiotic Lactobacillus and Bifidobacterium strains, Int. J. Food Microbiol.
properties of Lactobacilli strains isolated from raw cow milk in the western 98 (2) (2005) 211e217.
highlands of Cameroon, Innov. Rom. Food Biotechnol. 9 (2011) 12e28. [46] A.S. Hummel, C. Hertel, W.H. Holzapfel, C. Franz, Antibiotic resistances of
[18] National Committee for Clinical Laboratory Standards (NCCLS, Performance starter and probiotic strains of lactic acid bacteria, Appl. Environ. Microbiol. 73
Standard for Antimicrobial Disc Susceptibility Test, Approved Standard NCCLS (3) (2007) 730e739.
Publication, Villanova. PA, USA, 1993, pp. M2eA5. [47] L.I.I. Ouoba, V. Lei, L.B. Jensen, Resistance of potential probiotic lactic acid
[19] A.W. Bauer, W.M.N. Kirby, J.C. Sherris, M. Turck, Antibiotic susceptibility bacteria and bifidobacteria of African and European origin to antimicrobials:
testing by a standardized single disk method, Am. J. Clin. Pathol. 45 (1966) determination and transferability of the resistance genes to other bacteria, Int.
493e496. J. Food Microbiol. 121 (2) (2008) 217e224.
[20] J.G. Holt, N.R. Krieg, J.T.S. Sneath, S.T. Williams, Bergey's Manual of Determi- [48] N. Jamaly, A. Benjouad, M. Bouksaim, Probiotic potential of Lactobacillus
native Bacteriology, ninth ed., Williams and Wllkins, London, UK, 1994. strains isolated from known popular traditional Moroccan dairy products, Br.
[21] W.F. Harrigan, Laboratory Methods in Food Microbiology, third ed., Academic Microbiol. Res. J. 1 (2011) 79e94.