Anaerobe 33 (2015) 117e123

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Anaerobe
journal homepage: www.elsevier.com/locate/anaerobe

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
Keywords:
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

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

http://dx.doi.org/10.1016/j.anaerobe.2015.03.004
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
and sugar (glucose) fermentation test) as reported in our earlier
publication [14] and twelve selected Lactobacillus isolates were
given code as LBS 1-LBS 12 [8 from household curd (LBS 1,2,4,5,7,
9,10,12) and 4 from household milk (LBS 3,6,8,11)] and used in the
present study.
For the antimicrobial activity clinical isolates of Staphylococcus
aureus, Escherichia coli and Salmonella typhimurium were obtained
from the Post Graduate Institute of Medical Education and Research
(PGIMER), Chandigarh, India and maintained on Nutrient agar
medium (HiMedia, India) at 4
C after 24 h growth at 37
C.
2.2. Characterization of isolates for probiotic properties
All twelve Lactobacillus isolates were screened for their resis-
tance against bile salt, acid pH values, antibiotics and ability to
inhibit selected microbial pathogens. Inoculums of each isolates
was prepared using standard protocol M7-A7- CLSI (Chemical
Laboratory Standard Institute) [15] in test tubes containing 5 ml of
broth medium and incubated at 37
C for 24 h. The turbidity of
inoculum was maintained with 0.5 McFarland standards, contain-
ing 107e108 CFU/ml.
2.2.1. Bile and acid tolerance
All isolates were investigated for bile salt tolerance following the
method of Vinderola and Reinheimer [16]. Briefly, 0.2 ml of each
isolate inoculum suspension (107e108 CFU/ml) was added to 10 ml
of MRS broth containing different concentrations of bile salt
(0.15e0.45% w/v) and MRS broth without bile salt as control and
incubated at 37
C for 24 h. Optical density (OD), was recorded at
560 nm after incubation and compared with the control. Lactoba-
cillus isolates showing resistance more than 50% at 0.3% (w/v) bile
salt were considered as bile resistant as this is the minimum range
for being a probiotic culture.
The ability of isolates to tolerate the acid pH values was deter-
mined as described by Sieladie et al. [17]. Briefly, 1 ml of inoculum
(107e108 CFU/ml) of the each isolate was transferred into 10 ml of
MRS broth at pH 2, 3 and 5 respectively and OD was taken after 24 h
incubation at 37
C against the control (pH 7.0). Isolates showing
resistance more than 50% at pH 3 were considered acid tolerant
strains.
The percentage resistance in both cases (bile/acid pH values)
was calculated as:
Increment of OD in MRS broth with bile salt=pH 2; 3; 5
% Resistance ¼
Increment of OD in MRS broth without bile salt =at pH 7
2.2.2. Antimicrobial activity
Antimicrobial activity of the isolates against pathogenic bacteria
viz. E. coli, S. aureus and S. typhimurium was determined by standard
well diffusion method using Mueller Hinton Agar (MHA) plates
[18]. The 100 ml inoculum (107e108 CFU/ml) of each bacterium was
spread on MHA plates. Wells were made in each inoculated plate
and filled with 30 ml of cell free supernatant of Lactobacillus isolates
obtained after 24 h growth in MRS broth and centrifugation of the
inoculums at 4000 rpm for 10 min. Ciprofloxacin (30 mg/ml) was
used as positive control and MRS broth was used as negative con-
trol. All supernatant broths were neutralized to pH 6.5 to maintain
that the inhibition is not due to lactic acid but by antimicrobial
substances. All MHA plates were incubated at 37
C for 24 h and the
zone of inhibition was measured using Hi antibiotic zone scale. The
inhibition zone diameter of 10 mm and above was considered as
positive antimicrobial effect.
2.2.3. Antibiotic susceptibility test
Antibiotic susceptibility of all isolates was determined by the
standard disc diffusion method on MHA plates [19]. After adjusting
the turbidity standard of bacterial inoculum (107e108 CFU/ml) a
sterile cotton swab was dipped into each inoculum and swab
streaked over the entire surface of respective MHA plates. Now
different antibiotic discs (vancomycin, amoxycillin, amikacin, gen-
tamycin, tetracycline, chloramphenicol, co-trimoxazole and cipro-
floxacin) were placed on these test plates with the help of sterile
forceps and incubated at 37
C for 24 h and the inhibition zone
diameter was measured with Hi antibiotic zone scale.
The isolate LBS 2 was used for the further investigation viz.
phenotypic and genotypic characterization and scanning electron
microscopy (SEM) study to see in vitro cell adherence assay and
effect of bile salt on its morphology by surface roughness study.
2.3. Phenotypic and genotypic characterization of LBS 2
The phenotypic characterization of isolate LBS 2 was performed
on the basis of its morphological (colony morphology, Gram's re-
action, spore formation and motility), biochemical (methyl red,
citrate utilization, Voges Proskaeur, casein, starch and urea hydro-
lysis, nitrate reduction, H2S production, cytochrome oxidase, argi-
nine dihydrolysis, lysine decarboxylase and indole test) and
physiological characteristics (growth at varying temperature
25e42
C, pH 5e9 and 2.5e5% (w/v) NaCl) at Institute of Microbial
Technology (IMTECH), Chandigarh, India [20,21]. The isolate LBS 2
was further identified by 16S rDNA gene sequencing. Briefly,
genomic DNA was extracted by the standard chloroform-isoamyl
alcohol method as described by Sambrook et al. [22]. PCR amplifi-
cation of the 16S rDNA was performed using the following forward
and reverse primers: 8F: 50 AGA GTT TGA TCC TGG CTC AG 30 and
1492R: 50 ACG GCT ACC TTG TTA CGA CTT 30 . The polymerase chain
reaction (PCR) mixture consisted of 7.50 mL of DNase-RNase free
water, 12.50 mL (1) of 2 PCR master mix, 1.0 mL (10 pmole) of each
primer and 30 ng of DNA template. The PCR reaction was performed
in 25 mL volumes for 30 cycles at 95
C, 30 s at 52
C, and 1 min at
72
C with additional extension for 10 min at 72
C. Amplified DNA
 100
was examined by electrophoresis in 1% agarose with 5 mL aliquots of
PCR product using non-amplified DNA as a negative control. The
16S rDNA PCR product was purified by QIAquick Gel Extraction Kit
(Qiagen, India). The purified PCR product was sequenced (forward
and reverse sequence) by Xcelris lab, Ahmadabad, India. In the
present study only the forward sequence was used. The sequence
was compared with the nucleotide database from the NCBI Gen-
Bank using the BLAST program. A similarity of >98% to the 16S
rDNA sequence of the reference Lactobacillus rhamnosus was used
as a criterion for the identification. The obtained 16S rDNA
sequence was deposited in the NCBI GenBank database. A phylo-
genetic tree was generated from the alignment of the deposited
sequence by the Neighbor-joining method using Sea-View version
4 [23].
 A. Kumar, D. Kumar / Anaerobe 33 (2015) 117e123
2.4. In vitro epithelial cells adherence assay
The in vitro adherence of LBS 2 cells to epithelial cells was
studied using rat ileum as a model. Epithelial cells were prepared
according to the method described by Annika et al. [24]. Small
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
in PBS and incubated at 4
C for 30 min to remove the surface
mucus and then washed thrice with PBS. Epithelial cells were
scrapped into sterilized PBS. One ml of bacterial inoculum
(107e108 CFU/ml) was mixed with 1 ml of the cell suspension of
epithelial cells. The mixture was incubated at 37
C for 30 min. The
in vitro adhesion of LBS 2 to epithelial cells was observed using FEI
Nova Nanosem 450, Scanning Electron Microscope.
2.5. Effect of bile salt on morphology of LBS 2
Effect of different concentrations of bile salt on morphology
(roughness) of bacteria was analyzed using SEM as described by
Soo-Hwan et al. [25] with some modifications. Bacterial cells after
treatment with bile salt (015e0.45 % w/v) were fixed in 3% (v/v)
glutaraldehyde buffered with 0.1 M sodium phosphate buffer (pH
7.2) for 3 h and then washed three times in the same buffer. Further
cells were dehydrated in a graded alcohol series (30, 50, 70, 90,
100%). The specimens were dried, coated with thin layer of gold and
mounted onto stubs using double-sided carbon tape and examined
for change in surface roughness using SEM.
2.6. Statistical analysis
All characterization experiments were done in triplicates and
standard deviation was calculated. Statistical evaluation was also
carried out using GraphPad Prism 6.0. A p-value below 0.05 was
considered to be statistically significant.
3. Results
Thirty bacterial isolates were initially obtained from thirty dairy
samples collected from Solan district in Himachal Pradesh, India.
Out of thirty, 14 isolates were from milk sample and 16 isolates were
from curd samples. Characterization of isolates was performed on
the basis of their morphological characteristics viz., type of colony,
color, margin, elevation, opacity and presence of pigment and
Lactobacillus specific biochemical tests. Based on morphological,
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.
119
cultural and biochemical characteristics, 12 bacterial isolates (8
from curd and 4 from milk) were considered to belong to the genus
Lactobacillus as reported in our previous publication [14] and these
were further characterized for the probiotic potential and the re-
sults are discussed in the following sections.
3.1. Characterization of isolates for probiotic properties
3.1.1. Bile and acid tolerance
Bile salt resistance is one of the criteria for any microbial strain
to be used as a probiotic culture. The presence of bile in the in-
testine affects the viability of LAB. All twelve isolates were grown in
different concentrations of bile salt and results showed that only
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
(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
(Fig. 1).
The survival of LAB in low pH of stomach is important for
bearing the initial acid stress. The effect of acidity on the viability of
all isolates was assessed by their growth in different incubation pH
in MRS broth. Out of twelve isolates, five were resistant to acid pH
value 3 (with resistance  50%). Highest resistance was reported
with LBS 2 (60.52%) and lowest with LBS 11 (52.62%) at pH 3 (Fig. 2).
However, no isolate was found resistant at pH 2 (with percentage
resistance <50%).
3.1.2. Antimicrobial activity
One of the major properties for an isolate to be used as probiotic
is its ability to inhibit microbial pathogens. In the present study,
antimicrobial effects of all isolates against selected pathogenic
bacteria were studied. All isolates inhibited the growth of S. aureus
whereas only one isolate (LBS 2) inhibited the growth of E. coli, S.
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 ±
0.57 mm). However, highest zone of inhibition (14 ± 1 mm) against
S. aureus was observed with the isolate LBS 8 (Table 1).
3.1.3. Antibiotic susceptibility test
The susceptibility to antibiotics is a common feature of probiotic
bacteria. The antibiotic susceptibility of all isolates was assessed by
disc diffusion method using MHA medium and results are shown in
the Table 2. The isolate LBS 5 was found resistant to six antibiotics
and the isolate LBS 2 was resistant to five antibiotics. The antibiotic
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-
geous to survive the gastrointestinal tract during antibiotic treat-
ment. Some resistance genes might be present in resistant
microorganisms and mechanism associated with this resistance is
still unknown.
3.2. Identification and characterization of selected isolate LBS 2
The isolate LBS 2 showed maximum resistance with bile salt
(72.36%) at 0.30% (w/v), acid pH value 3 (60.52%), inhibited growth
of all tested microbial pathogens and was resistant to five antibiotics
and was selected for further identification and characterization.
3.3. Phenotypic characterization and genotypic characterization of
LBS 2
The selected Lactobacillus (LBS 2) with maximum probiotic po-
tential properties was further characterized at IMTECH, Chandi-
garh, India for its phenotypic characteristics viz. morphological,
biochemical and physiological properties. Morphological investi-
gation of LBS 2 showed that colonies were round, smooth, raised,
entire margins, Gram positive rods arranged in clusters, non-spore
forming and non-motile. Various biochemical tests viz. methyl red,
citrate utilization, Voges Proskaeur, casein, starch and urea hydro-
lysis, nitrate reduction, H2S production, cytochrome oxidase,
arginine dihydrolysis, lysine decarboxylase and indole test were
found negative for this isolate. This isolate showed positive growth
at 25e42
C, pH 5e9 and 2.5e5% NaCl (w/v), however, no growth
was observed with increase in temperature beyond 55
C, pH 9 and
6% NaCl respectively. The isolate LBS 2 showed positive results for
fermentation with dextrose, fructose, galactose, lactose, maltose,
mannitol, rhamnose, sorbitol, sucrose, trehalose and mannose and
no fermentation was observed with xylose, adonitol, inulin, cello-
biose, dulsitol, inositol, melibiose, raffinose and salicin. Based on
morphological, biochemical and physiological characteristics the
isolate LBS 2 showed similarity in phenotypic properties with L.
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
size of the 16S rDNA gene. Both forward and reverse sequence was
obtained after sequencing and assembly sequence was generated
but in the present study only the forward sequence was used.
BLAST analysis of the obtained sequence with the available nucle-
otide database in the GenBank, showed 99% similarity of the LBS 2
isolate with the L. rhamnosus (Fig. 3). The sequence was deposited
in GenBank with accession no. KJ562858. The comparative analysis
of LBS 2 sequences with the published sequences of different
Lactobacillus species, using the SeaView Version 4 program
confirmed its similarity with the L. rhamnosus KF806539.1 and
demonstrated the phylogenetic distances in a generated Neighbor-
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
were compared with the existing literature. In screening study
eight, out of twelve isolates were bile resistant and Lactobacillus
isolates showing resistance 50% at 0.3% (w/v) bile salt were
considered as bile resistance strains [16]. Highest resistance
(72.36% and 68.62%) was observed with isolate LBS 2 at 0.3% and
0.45% of bile salt respectively. Gilliland [26] reported that lactoba-
cilli isolated from animal intestines showed high tolerance to bile
salt than isolates from milk products. Similar results were reported
by Patel et al. [27]. Gilliland et al. [28] observed high variability
among Lactobacillus acidophilus strains isolated from calf intestinal
contents to grow in vitro in the presence of bile salts. Garriga et al.
[29] reported the selected LAB strains with resistant to 4% bile salts.
The resistance variability among lactobacilli is due to the presence
of bile salt hydrolase (BSH), an enzyme that reduces toxic effects by
conjugating bile [30].
The acid pH stability results of tested isolates in the present
study are in accordance with the reported literature. Isolates
showing 50% resistance at acid pH value 3 were considered acid
tolerant [17]. In present study five isolates were acid resistant and
highest resistance was observed with LBS 2 (60.52%) at acid pH
value 3. Idoui et al. [31] also showed resistance of Lactobacillus
plantarum BJ0021 at pH 3. Idoui [32], showed that in comparison to
the Lactobacillus isolated from the gastrointestinal tracts of human,
strains of Lactobacillus fermentum, Lactobacillus gasseri and Lacto-
bacillus delbrueckii subsp. bulgaricus were having better acid
tolerance.
In present study, antimicrobial activity of twelve selected iso-
lates was tested against selected clinical isolates. Isolate LBS 2
inhibited the growth of E. coli, S. typhimurium and S. aureus and
showed broad spectrum antimicrobial activity. Garriga et al. [29]
reported inhibition of one or more enteric indicator strains
(E. coli, Salmonella enteritidis) by Lactobacillus paracasei subsp.
Paracasei. L. acidophilus strains isolated from infant faeces had weak
antibacterial activity on E. coli and Yersinia enterocolitica [33].
Daeschel [34] reported that the antimicrobial effect of LAB is due to
the production of lactic acid, reduction of pH, acetic acid, diacetyl,
fatty acids, aldehydes and other compounds. Whereas, as per
another report the antimicrobial action is due to the potential of
LAB bacteriocin and peptides having inhibitory properties [35].
In antibiotic test all isolates showed varying antibiotic suscep-
tibility pattern. Isolate LBS 5 was resistant to six antibiotics and LBS
2 was resistant to five antibiotics. As reported in literature, specific
antibiotic resistance traits among probiotic strains may be desirable
[36]. In case of co-administration of probiotics with antibiotics they
should be resistant to certain antibiotics to survive in the gastro-
intestinal tract [37,38]. The presence of antibiotic-resistance genes
in many LAB and transfer of plasmids and conjugative transposons
to and from LAB, have been reported in Lactobacillus species [39]. In
general, glycopeptide, aminoglycoside (amikacin, kanamycin,
streptomycin and gentamycin) and sulfamethoxazole resistance
has been described in LAB, and in most cases it is associated with
their natural and intrinsic resistance due to membrane imperme-
ability, probably complemented by potential efflux mechanism
resistance [40e42]. Intrinsic resistance is not horizontally trans-
ferable and poses no risk in non-pathogenic bacteria [43]. Hoque
et al. [44] found that the Lactobacillus spp. were resistant to b-
lactams due to presence of b-lactamase in the isolates. Zhou et al.
[45] reported that new probiotic strains; L. rhamnosus HN001 and
HN067 were resistant to fusidic acid, kanamycin, nalidixic acid,
neomycin, polymyxin B and vancomycin due to their intrinsic
resistance and without transmissible antibiotic resistance genes. In
another report L. rhamnosus BFE 7442 was resistant against cipro-
floxacin, gentamycin and streptomycin and demonstrated that the
resistant genes might be present in probiotic strains but are silent.
Genetic basis and associated resistance mechanisms towards some
antibiotics are still unknown and need to be explored [46,47].
The isolate LBS 2 was selected for further study as it showed
highest resistance against bile salt and acid pH value in in vitro
experiments. It also showed broad spectrum microbial pathogen
inhibition, resistance to five antibiotics and was further investi-
gated for phenotypic and genotypic characterization and was
identified as L. rhamnosus LBS 2 by 16S rDNA sequencing.
In vitro epithelial cell adherence and bile salt effect was studied
with SEM analysis. L. rhamnosus LBS 2 showed positive epithelial
cell adherence test. The results of the epithelial cell adherence were
compared with the existing literature and isolates with an adhesion
efficacy of 15 bacteria per epithelial cells are considered positive
[48]. Idoui [32], reported adherence specificity of L. fermentum HG3
and L. gasseri HG8, to chicken intestinal epithelium. Jamaly et al.
[48] reported that all the tested strains were able to adhere to rat
ileum epithelial cells. Effect of bile salt on surface morphology of L.
rhamnosus showed that with an increase in concentration of bile
salt the smoothness of bacterial surface changed.
5. Conclusion
In conclusion one potential probiotic isolate LBS 2 was obtained
after screening of twelve Lactobacillus isolates for the probiotic
properties. This isolate LBS 2 from the household curd sample of
 A. Kumar, D. Kumar / Anaerobe 33 (2015) 117e123
Solan, district of Himachal Pradesh was identified as L. rhamnosus
LBS 2 after phenotypic and genotypic characterization. High resis-
tance to bile salt, tolerance to acid pH values, broad spectrum mi-
crobial pathogen inhibition and adherence to epithelial cells seems
to be a potential advantage of this culture for use as probiotic
culture. Isolate LBS 2 was resistant to five antibiotics, out of tested
eight. Antibiotic resistant traits among probiotic strains may be
desirable if administered alongwith antibiotics. However, detailed
molecular investigation has to be conducted to establish the
mechanism of the antibiotic resistance genes in this isolate.
Acknowledgments
The authors are thankful to Dr. Rohit Goyal, Faculty of Phar-
maceutical Sciences, Shoolini University, Solan, India for his help in
epithelial cells adherence assay and Er. Sandeep, Indian Institute of
Technology, Mandi, India for SEM analysis in the present study.
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