Purification and Physicochemical Characterization of Anti-Gardnerella vaginalis Bacteriocin HV6b Produced by Lactobacillus fermentum Isolate from Human Vaginal Ecosystem
Anti-Gardnerella vaginalis, Lactobacillus fermentum bacteriocin HV6b
producing strain was isolated from vaginal swabs of healthy, fecund females.
It was identified, purified and further characterized physicochemically. The
relative molecular mass of bacteriocin was found to be less than 7 kDa. Maximum
bacteriocin production was observed at 37°C, pH 6.5 after 16-18 h of incubation
Proteinase K and pepsin strongly inhibited bacteriocin production. The bacteriocin
has purified by absorption desorption method and characterized by reverse phase
liquid chromatography. It exhibited high thermal stability and showed inhibitory
activity over wide range of pH. The study revealed the possibility of using
bacteriocin for bio-medical use and the L. fermentum strain as probiotic.
to cite this article:
Baljinder Kaur, Praveen Balgir, Bharti Mittu, Ashish Chauhan and Balvir Kumar, 2013. Purification and Physicochemical Characterization of Anti-Gardnerella vaginalis Bacteriocin HV6b Produced by Lactobacillus fermentum Isolate from Human Vaginal Ecosystem. American Journal of Biochemistry and Molecular Biology, 3: 91-100.
Received: May 08, 2012;
Accepted: June 25, 2012;
Published: October 04, 2012
Many scientist in the field of biology and chemistry have given significant
contribution to science and technology by utilizing the natural resources (Chauhan
and Kaith, 2012; Aan et al., 2011; Abd
El-Hady, 2011; Abd-El-Hady and Abd El-Baky, 2011;
Abdi et al., 2010; Raja and
Thilagavathi, 2011; Issaoui et al., 2011;
Das et al., 2011; Rocco,
2011; Adedayo, 2012). Bacteriocins are biologically
active peptides produced by several bacterial species against related species
(Tagg et al., 1976). Bacteriocins have been classified
into Class I, Class II, Class III, IV and Class V (Klaenhammer
et al., 1993; Maqueda et al., 2004;
Kawai et al., 2004).
Bacterial Vaginosis (BV) is an inflammation most common infection in the reproductive
age. Bacterial vaginosis is an extremely prevalent vaginal condition and the
cause of vaginitis among both pregnant and non-pregnant women. BV is a polymicrobial,
superficial vaginal infection that occurs in the vagina and includes several
strains of germs that cause bacterial vaginosis. It occurs when the natural
balance of bacteria in the vagina is disturbed. Typically vagina contains a
balance of healthy bacteria, known as lactobacilli, and dangerous bacteria,
known as anaerobes. Lactobacillus microflora, the intrinsic stability of the
resident micro flora is paramount to women's health. The lactobacilli species
inhabit the vaginal tract and plays an important role in the maintenance of
health and prevention of infection (Amsel et al.,
1983; Ronnqvist et al., 2007; Tomas
et al., 2003).
G. vaginalis recent studies have suggested that BV is a significant
risk factor for upper genital tract infections in pregnant women that can result
in adverse outcomes of pregnancy, Abnormal Vaginal Flora (AVF) before 14 gestational
weeks is a risk factor for Preterm Birth (PTB) including preterm delivery bacterial
colonization of the genital tract, subsequent preterm delivery, late miscarriage,
low birth weight of infants, premature rupture of membranes, premature labor
and impaired fetal development. BV may increase susceptibility to infection
by human immune deficiency virus. However, with the exception of evidence for
a relationship between G. vaginalis and Prevotella bivia, very
little is known about the interactions between the microorganisms associated
with BV. G. vaginalis is the predominant microorganism associated with
Bacterial Vaginosis (BV) (Amsel et al., 1983).
A survey of BV has been conducted among women in Delhi, India according to which
a high percentage of woman was found to have BV. Increased rate of BV 43.5%
was observed in 2008 (Madhivanan et al., 2009).
Initially G. vaginalis was sensitive to antibiotics but now recurrent
infections might occur due to the survival of metronidazole or clindamycin resistant
bacteria in the vagina (Nagaraja, 2008). An overall
68% resistance of G. vaginalis to metronidazole at very high rate in
the population was observed. Recurrence rates of up to 30% within three months
after treatment have been reported in literature (Hay, 2000).
Bacteriocin provides an alternative and attractive proposition for control of
BV. The study reports the production, purification and physicochemical characterization
of a new novel protein of Lactobacillus fermentum isolated from healthy
human vagina and its anti Gardnerella vaginalis activity has been discussed
that remained unexplored.
MATERIALS AND METHODS
Isolation and identification: One hundred vaginal swabs of healthy ladies
of reproductive age group were collected from gynecologist (GMCH, Sector-32,
Chandigarh) in the year 2009. They were aseptically sterilized in saline (NaCl)
solution and transferred in MRS broth (pH 6.5) at 37°C for 18-20 h. Supernatants
of overnight grown cultures were isolated and analyzed for detection of bacteriocin
activity. The culture was purified by repeated streaking and purity was checked
by gram staining.
Preparation of culture supernatant: The bacteriocin producing lactic
acid bacteria were grown in MRS broth at 37°C for 18-20 h. The lactobacilli
culture was centrifuged at 13,000 rpm for 10 min and boiled for 20 min then
the supernatant was collected.
Bacteriocin assay: Bacteriocin activity was detected by agar well diffusion
method. Twenty microliter culture supernatant was transferred to the wells in
Casman media plates supplemented by selective media and 5% defibrinated blood.
The plates kept for 2 h at 4°C and then incubated at 37°C for overnight
and examined for the presence of inhibition zones around the wells and the antimicrobial
activity expressed in millimeter (mm). Gardnerella vaginalis ATCC14018
was procured from American Type Culture Collection for demonstrating the anti-
Gardnerella activity. Culture was revived and maintained in the Casman medium
supplemented by Gardnerella supplement and 5% w/v defibrinated human
blood. Bacteriocin activity assay was performed using spot-on-lawn method (Pucci
et al., 1988), agar well diffusion method (Toba
et al., 1991).
Production by adsorption and desorption: Bacteriocin was obtained and
purified by pH dependent adsorption and desorption method on to producer cells.
The protocol relies on the property of several bacteriocins to adsorb to the
producer cells at neutral pH and their release after being treated with a low
pH. Culture of Lactobacillus fermentum inoculated initially in MRS broth
was grown to late log phase (18 h, incubated at 37°C). The culture broth
was then heated in boiling bath and allowed to cool. The pH was adjusted to
6.5 and kept for overnight stirring. Cells were harvested by centrifugation
for 20 min at 9000 rpm and culture pellet were washed twice in 5 mM sodium phosphate
buffer (pH 6.5) and re-suspend the pellet in 10 mL of 100 mM NaCl solution that
had adjusted pH 1.5 with 5% (v/v) phosphoric acid. The suspension was stirred
overnight and cells were harvested by centrifugation at 9000 rpm for 30 min.
They should be stored at -20°C. Activity of bacteriocin was confirmed by
well-diffusion assay showing inhibition of 22 mm against the G. vaginalis
ATCC14018 (Yang et al., 1992; Ronnqvist
et al., 2007; Tomas et al., 2003).
Purification reverse-phase (RP) HPLC: This analytical technique has
been shown to be extremely valuable for the analysis of these antimicrobial
peptides, since bacteriocins are generally resistant to different organic solvents
used as mobile phases and the high pressures employed trough the chromatographic
technique. The bacteriocin extract is loaded onto the HPLC column. The mobile
phase consisted of acetonitrile and HPLC-grade water containing 0.1% Trifluoroacetic
Acid (TFA). The sample was loaded on the C-18 column and separated by a linear
biphasic gradient of 20 to 80% acetonitrile over 30 min at a flow rate of 0.5
Protein identification: peptide mass finger printing to identify protein:
Proteins were identified by mass spectrometry, the most powerful techniques
in protein chemistry, giving a 100 fold increase in sensitivity and 10 fold
increase in speed. Accurate measuring of the mass-to-charge ratio of the resulting
peptides gives enough information to identify the proteins in the sample. This
can easily be done using the Matrix-Assisted Laser Desorption Ionization Mass
Spectrometry (MALDI-MS) procedure. This technique offers a fingerprint unique
for the particular protein or protein mixtures. The experimental obtained mass-to-charge
values can be matched against theoretical obtained mass data from already identified
protein sequences and a score depending on the correlation can be given. This
way of identifying proteins is called peptide mass searching or fingerprint
MALDI-TOF MS and Liquid chromatography-Mass spectrometry.
MALDI-TOF analysis of pure protein: MALDI-TOF mass spectrometry was
performed on Brukers Daltonics. Mass spectra were obtained in the positive ion
mode, with an accelerating voltage of 25 kV. One microliter of the trypsin digested
protein was mixed with 1 μL of matrix (10 mg of sinapinic acid and 0.1%
4-hydroxy-α-cyanocinnamic acid) in 1 mL of distilled water and 0.3 μL
of this mixture was applied to the Teflon-coated plate. BSA was used for calibration.
Liquid chromatography-mass spectrometry: The chromatographic system
consisted of a Agilent MS-6460 triple quadra pole, equipped with an auto sampler.
Analysis was done using an Agilent eclipse C-18 5 μm column 150x4.6 mm
internal diameter with a flow of 0.5 mL min-1. The mobile phase was
0.1% formic acid 70% in purified water and 30% ACN. The injection volume 5 μL.
The system was coupled online to HPLC-MS-MS-triple quadruple. Mass spectrometer
equipped with a pneumatically assisted electrospray ionization source (Barber
et al., 1981).
Anti-Gardnerella activity: The overnight grown Gardnerella
vaginalis culture (cells-1x105-7) 1 mL culture was transferred
to six different Effendorfs with different concentrations of bacteriocin. Spreading
was done on Casman media plates and kept overnight at 37°C for colony counting.
RESULTS AND DISCUSSION
Isolation and purification: Lactobacillus fermentum was isolated
from healthy vaginal ecosystem of 11 bacteriocin producing lab strains. Only
8 isolates namely HV6A, HV6B, HV54A, HV59A, HV59C, HV59D, HV69, HV75 were found
to inhibit G. vaginalis. But, only HV6b was showing maximum inhibition
against G. vaginalis that was selected for further study and identified
as Lactobacillus fermentum and its culture supernatant showed a vast
Production by adsorption and desorption: The crude and purified bacteriocin
was purified and then subjected to bio-assay; the results obtained are given
in Table 1. The result clearly shows that the purified bacteriocin
has significantly increased activity and a high recovery was observed (Kawai
et al., 1997; Miteva et al., 1998;
Boris et al., 2001). The crude protein was showing
6.5x105 activity units per mL and pure protein was showing 4.4x106
activity units per mL. The specific activity have been increased 20.84
folds while the total recovery of pure protein is approximately 32%.
Evaluation of physicochemical characterization and stability of bacteriocin
Effect of temperature on bacteriocin assay: Temperature stability
was investigated by heating 50 μL of each culture supernatant at 40, 60,
80, 100°C and 121°C for 30 min. The samples were then assayed for antimicrobial
activity using the well diffusion method (Fig. 1). It is active
even at 90°C for 20 min. The results clearly shows that it has its maximum
activity at 37°C and up to 90°C it indicates that the temperature does
not affect the activity of bacteriocin. It remains stable even at 90 for 20
Effect of pH on bacteriocin assay: To determine the effect of pH, purified
bacteriocin was adjusted at 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0
and incubated for 2 h at 37°C. Each pH treated bacteriocin was assayed by
well diffusion method (Table 2, Fig. 1).
The result obtained clearly show the higher growth at pH 6-6.5 and 9.5 due to
maximum production of bacteriocin at 6.5, 9.5.
Effect of time on bacteriocin assay: To determine the effect of time
of incubation, the bacteriocin production was observed by taking supernatant
after 4, 8, 12,16, 20, 22, 26 to 48 h. Each supernatant was assayed by well
diffusion method. It indicates maximum production of bacteriocin in between
16-20 h that has optimized our method of bacteriocin production (Table
3, Fig. 1).
Effect of various enzymes and surfactants: Effect of enzymes on activity
have been observed by adding different enzymes (proteolytic enzymes, proteinase
k or α-chymotrypsin) to bacteriocin, the results clearly indicate that
the bacteriocin activity was lost after enzyme treatment.
|| Purification data after absorption-desorption method
|Purification folds represents increase in specific activity,
Recovery % is remaining protein concentration
|| Inhibition zone at different pH
|-: No inhibition
|| Anti microbial activity depicting effect of zones at different
(a) Dilutions, (b) pH and (c) Temperature
Stability of bacteriocin during storage: It retains its activity for
3 to 5 months at refrigerator (0-4°C) and for one year at deep freezer (-20°C)
in liquid and lyophilized form. It showed its stability even after prolonged
storage. To determine the stability of bacteriocin, it was stored in an incubator
(37°C), the refrigerator (4°C) and freezer (-20°C). At different
time interval (every 30 days) the bacteriocin were taken from the stored bacteriocin
for detection of antimicrobial activity using well diffusion assay and degradation
pattern were observed on RP-HPLC.
|| Inhibition zone at different time intervals
|-: No inhibition
|| HPLC pattern of the bacteriocin
A single peak of bacteriocin remained constantly same throughout (Fig.
2). The reverse phase HPLC of the bacteriocin gave a single peak at 2 minute
time interval of 2.6 intensity and it gave the same results after preservation
till 8 months there after it started forming the degradation product. Reverse
phase HPLC has confirmed its stability. It was performed at regular intervals
of time at absorbance of 280 nm. There was a decrease in absorbance at 280 with
time and the single peak starts splitting after a prolonged storage which indicates
degradation after 1 year of storage at -20°C. The X axis refers to retention
time and Y axis refers to Absorbance Unit Full Scale (AUFS). The absorbance
at 280 nm is 2.7632 AU, and retention time is 2.214 min.
MALDI-TOF: After trypsin digestion the bacteriocin was fragmented that
give the fragmentation pattern (after deleting the peaks of contamination and
trypsinzation: 842.51, 1045.56, 2211.10, 2225.12. Protein identification was
performed by sending trypsin digested peptide masses to bacterial databases
of National Centre for Biotechnology Information (NCBI) and Swiss port using
the MASCOT (Matrix Science) Peptide Mass Finger printing programme.
|| The MALDI TOFF of the bacteriocin
The mono-isotopic masses were used and the mass tolerance was set to 0.5 kDa.
Almost no match to the protein data was available in various protein search
engines that indicates it may be an unidentified noble protein. The following
peak list in the m/z ratio was available: 644.013, 650.010, 656.015, 665.986,
672.000, 681.970, 768.537, 842.501, 855.043, 871.026, 877.021, 882.577, 892.99,
1051.731, 1060.053, 1082.034, 2211.040, 2225.067, 2233.051, 2248.050, 3323.831,
3337.828 (Fig. 3) (Yang et al.,
LCMS: The resulting MS/MS spectra of trypsin digested protein (Fig.
4) were submitted to MASCOT for searching. The Y-axis is peak intensity
and the X-axis refers to m/z. Searching was against gram positive bacteria NCBI
and Swiss port databases and was performed but the but their ion score of individual
peptides failed to reach a significant score level that indicates that the data
of this particular protein has yet not been notices or explored (Wu
et al., 2009).
Evaluation of the anti-Gardnerella vaginalis activity: The bacteriocin
was evaluated for anti-Gardnerella vaginalis activity. The results obtained
(Table 4) were significant as seen in Fig. 1,
a/b/c and table shows that number of colonies have been decreased drastically
with increase in the concentration of protein and 500 μg of bacteriocin
completely inhibit the growth of-Gardnerella vaginalis which has given
us a excellent treatment of bacterial vaginosis. 100 μg of protein sample
kills 50% of bacteria, while 500 μg of protein completely kills G.
|| LCMS mass fragmentation patter of the bacteriocin
|| Effect of bacteriocin on viability of G. vaginalis
Based on the presented results, it may be concluded that the antimicrobial
entity procured from the culture supernatant of L. fermentum is a peptide
with a diverse spectrum of antimicrobial activity. It shows stability in acidic,
neutral and basic pH range (up to pH 11), it is sensitive to proteolytic cleavage.
Heat stability at various temperatures allows bacteriocin HV6b competitive advantage
to use L. fermentum as probiotic preparations and bacteriocin for the
treatment of bacterial vaginosis and other infections. Maldi TOF- TOF peptide
mass fingerprinting and LCMS-MS mascot ion search results indicates that it
is a novel protein. It was effective against Gardnerella vaginalis.
Our special thank, to Prof. Raj Bahadur, Director of Govt. Medical College
and Hospital, Sector 32 Chandigarh, Prof. Anju Huria Head of Department Obstt.
and Gynae, GMCH Chandigarh and nurse Uma and Dr. Darshan Jot, Gynecologist,
Phase 10, Mohali for their support in collecting the samples.
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