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Antimicrobial
And Spermicidal
Activity Of Native
And Recombinant
Pediocin CP2:
A Comparative
Evaluation

2012
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Vol. 3 No. 3:2
doi: 10.3823/254

Balvir Kumar*, Praveen P. Balgir, Baljinder Kaur, Bharti
Mittu and Neena Garg
Department of Biotechnology,
Punjabi University, Patiala-147002,
Punjab, India.

Correspondence:

 balvirkumarbt@gmail.com

balvirkumar@yahoomail.co.in

Tel.: +91 175 3046263;
Fax: +91 175 3046262

Abstract
Title: Comparative analysis of antimicrobial and spermicidal activity of native and
rec-pediocin CP2

Background: Bacteriocins produced by lactic acid bacteria (LAB) are extremely
attractive dual-acting candidates for formulating topical personal care products
aimed at contraception and prophylaxis and treatment of bacterial vaginosis (BV).
“Generally recognized as safe” lactic acid bacteria synthesize these antimicrobial
bacteriocins as components of their natural defense system. These are extremely
versatile agents as they could be used as spermicidal agents to not only prevent unwanted pregnancy but also to combat the growing prevalence of sexually
transmitted microbial infections and viral diseases. Probiotic LAB have also been
well documented as being effective in biotherapeutic applications against gastrointestinal pathogens, e.g. Clostridium, Helicobacter pylori, Salmonella, Escherichia
coli, Listeria monocytogenes, and rotaviruses. Therapeutic application of probiotic
strains to protect against gastrointestinal infections may be of great importance
for future medicinal use.

Methods and Findings: Herein, we report studies carried out to comparatively investigate antimicrobial activity of native and recombinant pediocin CP2
against general human pathogens and opportunistic pathogens associated with
BV, gastrointestinal infections and others. Further, we demonstrate the spermicidal
activity of pediocins, on human sperm so that it could be considered as a potent
spermicidal agent.

Conclusions: Pediocins have the ability to target a relatively wide range of pathogenic bacteria which is an important advantage, especially to replace antibiotics
with much safer therapeutics and to combat the ever growing problem of antibiotic resistance, when compared to other antibiotics. They have the potential to
perform a very specific role like treatment of bacterial vaginosis, gut infections
and peptic ulcers. Preliminary experiments with rec-pediocin CP2 have proved its
better effectiveness at fighting Bacteroides, Candida, Escherichia, Enterococcus,
Helicobacter, Gardnerella, Klebsiella, Listeria, Neisseria, Propionibacterium, Staphy­
lococci, Streptococci and Vibrio infections compared to native pediocin CP2. Study
has also established pediocins as potent spermicidal agents with very impressive
market value in the form of personal care products.

This article is available from:
www.acmicrob.com

Keywords: Pediococcus acidilactici, Pediocin CP2, Recombinant pediocin, Antimicrobial peptides, Spermicidal activity

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Introduction
Pediocin CP2 was initially purified and characterized from
Pediococcus acidilactici MTCC 5101 [1-4]. It possesses a
wide spectrum of antimicrobial activity against Aspergillus
flavus, Clostridium sporogenes, Lactobacillus brevis, L. bul­
garicus, Leuconostoc mesenteroides, Listeria monocytoge­
nes, Micrococcus flavus, Neisseria mucosa, Pediococcus aci­
dilactici, P. pento­saceus, Pseudomonas aeruginosa, P. putida,
Staphylococcus albus, S. aureus, Streptococcus mutans and
S. pyogenes, that raised the possibility of its potential as an
ingredient in medical and/or personal care products. Later
on, it was genetically manipulated and its synthetic fusion
gene construct was cloned and expressed in heterologous E.
coli BL21(DE3) using pET32(b) expression vector. It was engineered with the aim to improve its antimicrobial spectrum,
enhance its production and facilitate its purification from
cell lysates[5]. Parental N-terminal sequence of pediocin CP2
(KYYGNGVTCGKHSC) was changed to TKYYGNGVSCTKSGC
as it could improve its biopreservative potential and effectiveness against sour-spoilage of dairy products [6]. Rec-pediocin
expression was induced by adding IPTG to early log phase
cultures of E. coli BL21(DE3) transformed with recombinant
pET32(b)-pedA. Rec-pediocin was purified from cell lysates by
employing two tandem affinity chromatographic steps with
an intermediate enterokinase cleavage step. Highly pure recpediocin was recovered from 2nd affinity column and further
characterized biochemically as well as biologically for its antimicrobial spectrum [5].
BV is a pretty common condition of vagina that occurs when
natural balance of the vaginal microflora gets disturbed due
to infection by bacteria, fungi or Trichomonas. Numerous reports across the world have shown its association with anaerobes like Bacteroides spp. (Prevotella spp.), Escherichia coli,
Gardnerella vaginalis, Mobiluncus spp., Mycoplasma hominis,
Peptostreptococcus spp., Staphylococci, Streptococci, and/or
viruses [7-11]. BV is asymptomatic in approximately one-half
of all cases, but is associated with a wide variety of symptoms
including foul odor, presence of “clue cells” and an increase
in pH of the vagina to >4.5 [12-13]. BV can cause adverse
outcomes of pregnancy, including preterm delivery, premature labor, premature birth, infection of the amniotic fluid,
infection of the uterus after delivery, death of the fetus or
new born and induction of pelvic inflammatory disorder [10,
14-16]. BV enhances susceptibility to infections by HIV [17]
and HSV type 2 [8] as it directly causes an increase in the
rate of viral replication and disease progression [18-19]. Toxins released by BV-associated microorganisms may cross the
placenta and may cause permanent brain damages leading to
development of Parkinson’s disease and schizophrenia in the
affected child [20-22]. BV is also responsible for recurrence of
UTI’s. G. vaginalis may assume a pathogenic role in males too

2

Vol. 3 No. 3:2
doi: 10.3823/254

as infection can extend up to the prostate or bladder especially in immuno-compromised patients who have undergone
a urological procedure [23-25].
Currently, preventive therapies for BV rely almost exclusively
on the use of antibiotics such as clindamycin and metronidazole administered either orally or intravaginally [26] and gives
initial cure rates of approximately 60-90% [27]. However, antibiotic therapy has been reported to impose several side effects such as diarrhea, dizziness, headache, loss of appetite,
nausea or vomiting, stomach pain or cramps [28]. In vitro experiments established the fact that antibiotics clindamycin and
metronidazole could inhibit healthy vaginal Lactobacillus spp.
at concentrations lower than doses topically recommended
for treatment [29-30]. Moreover, emergence of multi-drug
resistant phenotype in BV associated pathogenic bacteria; it
has become imperative to develop alternative therapeutics/
prophylactic measures against these pathogens [31-34].
Till date, nisin is the only bacteriocin that enjoys GRAS (Generally Recognized As Safe) food additive status given by the
Food and Drug Administration (FDA). It was also shown to
have impressive spermicidal activity [35]. An earlier study by
Sutyak et al. [36] that convincingly demonstrated the efficacy of antimicrobial peptide subtilosin to inhibit growth of
G. vaginalis and established its spermicidal activity, is also
of particular importance as this makes it a highly recommendable compound for inclusion in topical BV treatments
and human contraceptive products. They conducted tissue
sensitivity assays using human ectocervical tissue model and
indicated its safety for human use. Subtilosin was shown to
eliminate motility and forward progression of human spermatozoa in a concentration-dependent manner. Results of the
previous studies on other LAB bacteriocins therefore suggest
that antimicrobial peptides (AMPs), including native and recombinant pediocin CP2, may enhance current therapeutics/
prophylactic measures against BV as well as contraception as
they also possess both spermicidal and antimicrobial properties, prompting our investigation.
The human gastrointestinal tract constitute a complex and
diverse ecosystem of microbiota or commensal microflora inhabiting approx. 2,000 different species of aerobic, facultative
and anaerobic bacteria, the majority of which reside in the
intestines [37]. The diversity of gut microbiota is dependent
upon the age, diet and health status of the individual [38].
Intestinal microbiota may also play a critical role in disease
suppression and maintaining gut homeostasis [39-40]. Some
forms of cancers such as gastric cancer [41] and colon cancer [42] are also associated with the bacterial pathogenesis.
Probiotics have been recommended as GRAS biotherapeutic
agents for prevention and cure of human gastrointestinal diseases [37]. Present study is an attempt to unleash the antimi© Under License of Creative Commons Attribution 3.0 License

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crobial properties of a food associated bacteriocin producing
probiotic strain of Pediococcus acidilactici.

Materials and Methods
Procurement and maintenance of cultures
Pediococcus acidilactici MTCC 5101 was revived and maintained in MRS medium; pH6.5 (Lactobacillus Heteroferm
Screen Broth, Himedia) containing 0.1% Tween-80 at 37ºC.
Standard indicator strain of Listeria monocytogenes MTCC
657 was procured from MTCC, Chandigarh, India. It was
maintained as broth and agar cultures in Brain Heart Infusion broth (Himedia) at 37ºC. E. faecalis (NDRI isolate) and P.
acidilactici LB42 lab isolate were grown in MRS medium at
37ºC and maintained as glycerol stocks.

Production and purification of native
pediocin CP2
Overnight grown broth culture of P. acidilactici MTCC 5101
in MRS (supplemented with 0.1% w/v Tween-80; pH 6.5)
was used to prepare pure bacteriocin preparations by conventional adsorption-desorption method as previously described [43]. Its pH was adjusted to 6.5 using 0.1N NaOH.
Cells were then killed by subjecting them to heat treatment
for 20 min in boiling water bath. Broth culture was stirred
overnight at 4°C to facilitate adsorption of the bacteriocin
to their respective producer cells. Dead cells were harvested
by centrifugation for 20 min at 5000 g and 4°C. Pellet was
washed twice with sterile phosphate buffered saline (pH 6.5)
and resuspended in minimum volume of 100mM NaCl (pH
1.5). Suspension was kept stirred overnight at 4°C to facilitate
desorption of the bacteriocin from dead cells. The protein of
interest was finally obtained by centrifuging this suspension
for 30 min at 5000 g and 4°C. Bacteriocin preparation was
then filter sterilized using 0.45 mm filters (Millipore, USA),
as the aim was just to remove dead bacterial cells from cell
free supernatant.

Production and purification of recombinant
pediocin CP2
Expression of recombinant pediocin was obtained in E. coli
BL21(DE3) using IPTG as an inducer. Rec-pediocin was expressed using T7 driven pET32(b)-pedA in periplasm as well
as in the form of inclusion bodies (IBs). IBs were extracted
from the cell lysates by urea lysis and rec-pediocin was renatured using refolding buffer containing 5mM immidazole
and b-mercaptoethanol each. Since rec-pediocin protein
bears two affinity purification tags, thus it was purified from
the crude cell extract by employing Ni-NTA affinity chroma© Under License of Creative Commons Attribution 3.0 License

Vol. 3 No. 3:2
doi: 10.3823/254

tography. Biological activity in the rec-pediocin was induced
after its processing with enterokinase enzyme that digested
its N-terminal fragment bearing N-terminal affinity tags. Final
purification step was based on streptactin affinity chromatography and highly pure rec-pediocin was obtained in the
process. Active fractions were pooled and antimicrobial activity was assayed using standard bacteriocin assay [6].

Bacteriocin activity assay
The antimicrobial activity of all bacteriocin preparations was
confirmed by the well diffusion assay according to the protocol of Cintas et al. [44] and bacteriocin activity was calculated
as arbitrary unit (AU) and expressed as AU/ml as per standard protocol of Pucci et al. [45]. Well diffusion assays were
performed using 50 μl of each dilution against P. acidilactici
LB42 and L. monocytogenes MTCC 657 as reference microorganisms for the determination of a bacteriocin’s biological
activity. Protein estimations were carried out by measuring
OD at 280nm using BSA as a standard.

Determination of antimicrobial activity of
bacteriocins
Antimicrobial spectra of purified bacteriocins, native and recombinant pediocin CP2, were characterized using various
indicator microorganisms including standard lactic acid bacteria, food spoilage organisms and opportunistic pathogens by
well-diffusion assay [44]. Indicator microorganisms were revived and maintained in the recommended media at growth
conditions specified by various culture banks (as indicated in
Table 1).

Semen sample collection and analysis
Pure pediocin preparations were used to study their effect
on the motility of human spermatozoa. Two semen samples
were collected by self-masturbation in a polypropylene tube
on the day of experimentation. Within 1 hour of collection,
the samples were pooled and the total sperm count was calculated using a compound microscope (Olympus; 100X) after
dilution (1:50) of the semen in normal saline. The percent
sperm motility was determined by the progressive (forward)
and non-progressive (vibrating and zig-zag) movements of
sperms observed under a compound microscope. The sperm
count was calculated using Neubauer haemocytometer from
a count of between 100-200 sperms using randomly selected
field views (100X) as prescribed by Sutyak et al. [36].

Treatment of spermatozoa with bacteriocins
A modified Sutyak test was used to determine the effect of
purified bacteriocins on the motility of human spermatozoa

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TABLE 1. Growth conditions of indicator microorganisms used to study antimicrobial spectrum of pediocins.
Microorganism

Gram
Nature

Growth
Medium

Nature

Temp. (oC)
/pH

Inc. Time

S.P.
(Days)

Aspergillus. flavus (lab isolate)

Fungus

PDA

Aerobe

28 / 5.5

3 days

30

A. niger (lab isolate)

Fungus

PDA

Aerobe

28 / 5.5

3 days

30

Bacillus subtilis ATCC 6633a

+ ve

NB

Aerobe

37 / 7.4

24 h

15

Bacteriodes fragilis MTCC 1045

- ve

RCB

Anaerobe

37 / 6.8

5 days

30

B. ovatus MTCC 3298

- ve

RCB

Anaerobe

37 / 6.8

48 h

30

B. vulgatus MTCC 1350

- ve

CMM

Anaerobe

37 / 7.2

72 h

30

Candida albicans ATCC 10231a

Yeast

YEPD

Aerobe

30 / 7.2

48 h

60

C. albicans MTCC 183

Yeast

YEPD

Aerobe

30 / 7.2

48 h

60

Clostridium perfringens MTCC 450

+ ve

RCB

Anaerobe

37 / 6.8

48 h

30

C. sporogenes MTCC 1349

+ ve

RCB

Anaerobe

37 / 6.8

48 h

30

Escherichia coli MTCC 1590

- ve

LB

F. anaerobe

37 / 7.2

24 h

30

E. coli BL21(DE3) MTCC 1679

- ve

LB

F. anaerobe

37 / 7.2

24 h

30

E. coli DH5α MTCC 1652

- ve

LB

F. anaerobe

37 / 7.2

24 h

30

E. coli KL16 MTCC 1650

- ve

LB

F. anaerobe

37 / 7.2

24 h

30

Enterococcus faecalis (Lab isolate)

+ ve

MRS

F. anaerobe

37 / 6.5

24 h

30

E. faecalis (NDRI isolate)b

+ ve

MRS

F. anaerobe

37 / 6.5

24 h

30

E. faecalis ATCC 29212a

+ ve

MRS

F. anaerobe

37 / 6.5

24 h

30

Helicobacter pylori DSMZ 10242

- ve

BHI

Anaerobe

37 / 7.4

48 h

30

Gardnerella vaginalis ATCC 14018

+ ve

CB

Anaerobe

37 / 7.2

48 h

30

Klebsiella pneumoniae NCIM 2883

- ve

NB

F. anaerobe

37 / 7.2

24 h

30

K. pneumoniae MTCC 4030

- ve

NB

F. anaerobe

30 / 7.2

24 h

15

Lactobacillus brevis MTCC 1750

+ ve

MRS

F. anaerobe

30 / 7.4

24 h

30

L. bulgaricus NCDC 253

+ ve

MRS

F. anaerobe

37 / 6.5

24 h

30

L. casei NCIM 2651

+ ve

MRS

F. anaerobe

37 / 6.5

24 h

30

L. helveticus NCIM 2126

+ ve

MRS

F. anaerobe

37 / 6.5

24 h

30

L. leichmanii NCIM 2027

+ ve

MRS

F. anaerobe

37 / 6.5

24 h

30

L. pentosus NCIM 2669

+ ve

MRS

F. anaerobe

37 / 6.5

24 h

30

L. plantarum NCIM 2912

+ ve

MRS

F. anaerobe

37 / 6.5

24 h

30

L. plantarum NCDO 945

+ ve

MRS

F. anaerobe

37 / 6.5

24 h

30

Lactococcus lactis ssp. cremoris MTCC 1484

+ ve

TSY

Aerobe

20 / 6.5

24 h

30

Leuconostoc mesenteroides MTCC 107

+ ve

MRS

Aerobe

25 / 6.5

48 h

30

Listeria monocytogenes MTCC 657

+ ve

BHI

Aerobe

37 / 7.4

24 h

30

Micrococcus flavus ATCC 10240

+ ve

NB

Aerobe

30 / 7.4

48 h

90

Neisseria gonorrhoeae ATCC 19424

- ve

NB

Aerobe

37 / 7.4

24 h

30

N. mucosa MTCC 1772

- ve

NB

Aerobe

37 / 7.4

24 h

30

4

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Pediococcus acidilactici LB 42b

+ ve

MRS

Aerobe

30 / 6.5

24 h

30

P. acidilactici NCIM 2039

+ ve

MRS

Aerobe

37 / 6.5

24 h

30

P. acidilactici NCIM 2292

+ ve

MRS

Aerobe

37 / 6.5

24 h

30

P. pentosaceus NCDC 35

+ ve

MRS

Aerobe

37 / 6.5

24 h

30

Pseudomonas aeruginosa ATCC 10662a

- ve

NB

Aerobe

30 / 7.4

24 h

30

P. aeruginosa MTCC 647

- ve

NB

Aerobe

30 / 7.4

24 h

30

P. putida MTCC 102

- ve

NB

Aerobe

30 / 7.4

24 h

30

Propionibacterium acnes MTCC 1951

+ ve

BHI

Anaerobe

25 / 7.4

5 days

30

P. acnes DSMZ 16379

+ ve

BHI

Anaerobe

25 / 7.4

5 days

30

Proteus mirabilis NCIM 2387

- ve

NB

F. anaerobe

37 / 7.2

24 h

30

Salmonella typhi NCTC 5760a

- ve

BHI

F. anaerobe

37 / 7.4

24 h

15

S. typhimurium MTCC 1251

- ve

BHI

Aerobe

37 / 7.4

24 h

15

Staphylococcus albus ATCC 11631a

+ ve

BHI

Anaerobe

25 / 7.4

5 days

30

S. aureus MTCC 737

+ ve

BHI

Anaerobe

37 / 7.4

24 h

15

S. aureus NCTC 7447a

+ ve

BHI

Anaerobe

37 / 7.4

24 h

30

S. epidermidis MTCC435

+ ve

NB

Anaerobe

37 / 7.4

24 h

30

Streptococcus agalactiae NCIM 2401

+ ve

MRS

Aerobe

37 / 6.5

48 h

30

S. faecalis MTCC 459

+ ve

MRS

Aerobe

37 / 6.5

48 h

30

S. mutans MTCC 890

+ ve

MRS

Aerobe

37 / 6.5

48 h

30

S. pyogenes NCTC 10869a

+ ve

BHI

Aerobe

37/ 7.4

48 h

30

S. thermophilus MTCC 1928

+ ve

MRS

Aerobe

40 / 6.5

24 h

30

Vibrio cholerae ATCC 14104a

- ve

NB

Aerobe

37 / 7.4

24 h

15

Yersinia enterocolitica MTCC 861

- ve

BHI

F. anaerobe

30 / 7.2

12 h

30

F. anaerobe- Facultative anaerobe, Inc. Time- incubation time; S.P. (Days)- subculturing period
a
b

Strains are provided by Orbit Biotech, Punjab, India.
Strains are provided by Prof. R.K. Malik, NDRI, Karnal, India.

[36]. This measured the inhibitory effect of bacteriocins on
sperm mobility after 30 sec exposure time of different concentrations of bacteriocins ranging from 50 to 200 mg/ml, on
diluted semen sample. The motilities of spermatozoa from
random high magnification fields (100X) of the sample were
determined in duplicate using atomic force microscope.

Statistical analysis of data
QI Macros ANOVA was used to calculate all statistical parameters. Statistical tools like ANOVA one factor, regression and
Box Whisker plots were used to determine significance of the
results obtained in duplicate experimental sets.

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Results
In this study the antimicrobial properties and spermicidal
properties of native and rec-pediocin CP2 of P. acidilactici
MTCC 5101 were comparatively evaluated.

Antimicrobial spectrum of bacteriocins
The purpose of this study was to comparatively investigate
the antibacterial activity of native and recombinant pediocin CP2 against some BV associated pathogens including B.
fragilis, B. ovatus, B. vulgatus, yeast C. albicans, E. coli, G.
vaginalis, M. flavus, N. gonorroeae, N. mucosa, P. mirabilis,
Staphylococci, Streptococci and other general human patho-

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gens including C. sporogenes, E. coli, E. faecalis, H. pylori, K.
pneumoniae, L. mesenteroides, L. monocytogenes, P. aeru­
ginosa, P. putida, P. acnes, S. typhi and V. cholerae. Both are
capable of inhibiting a wide spectrum of pathogenic bacteria
and yeast implicated in bacterial vaginosis (BV associated organisms are highlighted in Table 2). Pediocin CP2 native and
recombinant showed growth inhibition of only few strains
of Lactobacilli and Lactococci tested in the study. They did
not show any activity against B. subtilis, C. perfringens, and
Y. enterocoloitica. Antimicrobial activity of rec-pediocin was
significantly improved against tested indicator strains especially against B. vulgatus, C. albicans, E. faecalis, G. vaginalis,
L. monocytogenes, N. gonorroeae, N. mucosa, P. acidilactici,
S. albus, S. aureus and S. mutans than native pediocin CP2.
Results were consistent with the previous report by Tominaga
and Hatakeyama [5]. Incorporation of 5 point mutations in
the rec-pediocin as Thr1, Ser9, Thr11, Ser13 and Gly14 in Nterminal region as compared to native pediocin CP2 produced
by P. acidilactici MTCC5101 significantly enhanced its antimicrobial activity. The differences in the antimicrobial activities
of native and rec-pediocin CP2 were found to be significant
(p<0.05 and Fcrit<F value) according to QI Macros ANOVA
single factor test. Results obtained from regression analysis
indicate coefficient of determination (r2) = 0.9688, adjusted
r2 = 0.9626, multiple R = 0.9843. Data obtained in the experiment was further analyzed with QI Macros Box Whisker plots
where significant improvement in the antimicrobial activity of
rec-pediocin was indicated against strains of Enterococcus,
Gardnerella, Listeria and Neisseria (Figure 1).
TABLE 2. A 
ntimicrobial spectrum of native and recombinant
pediocin CP2.
Indicator strains

6

Diameter of inhibition zone
(mm)
Native
pediocin CP2

Rec-pediocin
CP2

A. flavus (lab isolate)

+

+

A. niger (lab isolate)

-*

-

B. subtilis ATCC 6633

-

-

B. fragilis MTCC 1045

10

12

B. ovatus MTCC 3298

10

12

B. vulgatus MTCC 1350

11

14

C. albicans ATCC 10231

16

19

C. albicans MTCC 183

19

21

C. perfringens MTCC 450

-

-

C. sporogenes MTCC 1349

14

15

E. coli MTCC 1590

-

-

E. coli BL21 DE3 MTCC 1679

-

-

E. coli DH5α MTCC 1652

20

21

E. coli KL16 MTCC 1650

20

21

E. faecalis (Lab isolate)

13

18

Vol. 3 No. 3:2
doi: 10.3823/254

E. faecalis (NDRI isolate)

-

-

E. faecalis ATCC 29212

18

22

H. pylori DSMZ 10242

14

15

G. vaginalis ATCC 14018

15

23

K. pneumoniae NCIM 2883

20

21

K. pneumoniae MTCC 4030

16

18

L. brevis MTCC 1750

18

19

L. bulgaricus NCDC 253

11

13

L. casei NCIM 2651

-

-

L. helveticus NCIM 2126

21

23

L. leichmanii NCIM 2027

-

-

L. pentosus NCIM 2669

-

-

L. plantarum NCIM 2912

-

-

L. plantarum NCDO 945

-

-

L. lactis subsp. cremoris
MTCC1484

16

18

L. mesenteroides MTCC 107

25

27

L. monocytogenes MTCC
657

16

22

M. flavus ATCC 10240

18

20

N. gonorrhoeae ATCC 19424

18

22

N. mucosa MTCC 1772

16

23

P. acidilactici LB 42

14

23

P. acidilactici NCIM 2039

12

13

P. acidilactici NCIM 2292

-

-

P. pentosaceus NCDC 35

-

-

P. aeruginosa MTCC 647

14

15

P. aeruginosa ATCC 10662

13

15

P. putida MTCC 102

12

13

P. acnes MTCC 1951

10

11

P. acnes DSMZ 16379

13

15

P. mirabilis NCIM 2387

21

23

S. typhi NCTC 5760

12

13

S. typhimurium MTCC 1251

-

-

S. albus ATCC 11631

12

16

S. aureus MTCC 737

-

-

S. aureus NCTC 7447

13

16

S. epidermidis MTCC435

-

-

S. agalactiae NCIM 2401

-

-

S. faecalis MTCC 459

-

-

S. mutans MTCC890

13

16

S. pyogenes NCTC 10869

15

17

S. thermophilus MTCC 1928

-

-

V. cholerae ATCC 14104

19

21

Y. enterocolitica MTCC 861

-

-

-* pediocin resistant organism,
+ inhibition at higher concentration.
Data is an average of two independent experiments; P-value
= 1.62E-31, a (level of significance) = 0.05, F crit 1.548 and F
value 39.890.

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FIGURE 1. B 
ox Whisker plot showing
comparative antimicrobial
activity of pediocins.

Inhibition of sperm motility
Pediocins are shown to significantly reduce the motility of
human spermatozoa in a concentration-dependent manner
(Figure 2). The effect of pediocins on the forward progression of human sperms and their aggregation was also observed to be a dose-dependent interaction. Serial dilutions
of bacteriocin preparations showed a steady decrease in
forward progression, with all progression halted at the concentration of 250 mg/ml for rec-pediocin and 300 mg/ml for
native pediocin CP2 as compared to control samples. Tails of
the sperm cells became curved or coiled as result of bacteriocin treatment, indicating their damaged beyond a simple
restriction of movement (Figure 3). Coiling of the sperm tails
is considered to be an abnormality, and may indicate damage to the plasma membrane [36, 46]. The differences in
the proportion of motile spermatozoa in all samples were
found to be significant (p<0.05 and Fcrit<F value) according
to QI Macros ANOVA single factor analysis. So, we can easily
reject the null hypothesis and safely assume that pediocins
retard sperm motility in a dose dependent manner. Results
obtained from regression analysis indicate coefficient of determination (r2) = 0.9631, adjusted r2 = 0.9539, multiple R
= 0.9814 for native pediocin and r2 = 0.9197, adjusted r2 =
0.8996, multiple R = 0.9590 for rec-pediocin. Data obtained

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in the experiment was further analyzed using QI Macros Box
Whisker plots where differences in spermicidal activities of
native and rec-pediocin are indicated (Figure 4).

FIGURE 2. P ediocins retard sperm motility in a concentration
dependent manner.

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FIGURE 3. Images showing effect of pediocins on human spermatozoa a) normal spermatozoa; curved sperm tails; c) coiled sperm tails;
d) and e) sperm aggregation and immobilization.

FIGURE 4. B 
ox Whisker plot showing
differences in spermicidal
activities of native and recpediocin.

Discussion
Probiotic LAB strains are greatly appreciated for fermentation
and preservation of dairy and meat products [47]. Probiotic
LAB synthesize a variety of antagonistic factors such as antimicrobial peptides or bacteriocins, lactic acid, diacetyl, hydrogen peroxide, fungicidal agents etc. that will force the opportunistic pathogens away from the ecological niche conquered
back by the healthy probiotic microflora [48-50]. AMPs or
bacteriocins from GRAS bacterial species have aroused a

8

great deal of industrial interest as they are extremely appealing alternatives to conventional pharmaceutical treatments.
Since these bacteriocins are prone to proteolytic digestion
in the gastrointestinal tract and therefore seem to lack any
potential antigenicity/toxicity in animals [51-52]. AMPs kill
their target cells by inducing drastic changes in membrane
permeability and disrupting proton motive force [53-54].
Many AMPs exhibit structural similarities with members of
eukaryotic channel forming peptides [53]. They may hinder
biosynthesis of DNA, peptidoglycan, proteins and RNA as a
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result of PMF depletion, interfere with the activities of essential enzymes, degrade vital macromolecules such as DNA,
RNA and cause cell lysis [53, 55-56].
Much of the earlier research has been carried out to investigate the biopreservative potential of antimicrobial peptides
synthesized by LAB, but very little has been reported on demonstrating their efficacy in treatment of human diseases such
as BV. In light of above as well as improvement in understanding and knowledge of the complex microbial interactions,
an increase tendency to use live probiotic LAB is urgently
desired for formulating therapeutic health care substances
aimed at prophylactic and control of human gastrointestinal
and urogenital diseases. Colonization of the vaginal ecosystem by health promoting probiotic LAB particularly prevents
infection by competition for available nutrients and mannose
sugar and by interfering binding of pathogens to the cell surface receptors [57-58]. An acidic pH of vaginal secretion aids
in keeping microbial infections away. However, low vaginal
pH alone is not sufficient to inhibit vaginal pathogens and to
prevent BV [48-49]. Thus, bacteriocin based therapeutics are
urgently desired to cure BV as well as to overcome side effects of antibiotic therapy such as diarrhea, poor compliance
and recurrence of vaginal infections.
Both native and rec-pediocin CP2 are also extremely attractive
therapeutic agents aimed at control of gastro-intestinal infections caused by food spoilage bacteria and general opportunistic human pathogens such as Bacteroides, Clostridium,
Coliforms, H. pylori, Listeria, Propionibacterium, Pseudomo­
nas, Salmonella, Staphylococcus and Streptococcus sps. There
is increasing body of evidence that indicate potential of probiotic strains in cure and prevention of gut infections such as rotavirus diarrhea [59], traveller’s diarrhea [60], antibiotic associated diarrhea [61], necrotizing enterocolitis [62-63], inflammatory bowel disease [64-65], H. pylori associated infection
[66-67] and atopic diseases [68]. Growing scientific evidences
have proven efficacy of probiotics in maintaining restoring
and gut homeostasis that have been impressively reviewed
recently by Thirabunyanon in his article on “biotherapy for
and protection against gastrointestinal pathogenic infections
via action of probiotic bacteria” [37]. They are supporting
our investigation as Pediococci possess desirable antimicrobial properties for formulating probiotic dietary supplements,
yogurts, drinks and capsules [1-4].
Most of the published work regarding in vivo efficacy of
bacteriocins has focused on the oral delivery of probiotic
LAB to the gastrointestinal tract, where they will establish
themselves and secrete antagonistic bacteriocins. The use
of live probiotic bacteria may have prophylactic applications,
but the use of purified AMPs appears to be more attractive
for countering an established bacterial infection [69]. Experi© Under License of Creative Commons Attribution 3.0 License

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mental evidence in this regard established potency of an
important class IIa pediocin PA-1 to cure L. monocytogenes
infection in mice [70]. Ideally, an antimicrobial agent should
specifically target disease causing microorganisms with only
minimal impact on the health promoting natural flora. In
fact, the spectrum of activity for these anti-listerial pediocins
may be extremely well suited for targeting specific pathogens in vivo. Inhibitory action of pediocin PA-1 has been
assayed in vitro against screens of common gut bacterial
species including bifidobacteria and at the concentrations
tested, no growth inhibition was observed against any of the
tested reference strain [71-72]. Class IIa pediocins therefore
differ considerably from class I lantibiotic peptides such as
nisin A and nisin Z, as later two inhibited growth of majority
of Gram-positive strains assayed [71-72]. Later on, Bernborn
et al. [73] also provided experimental evidence in support
this and showed that purified pediocin PA-1 fed to rats did
not interfere with normal composition of the mouse intestinal flora. In contrast to it, frequently prescribed antibiotics
strongly inhibit most of the commensal bacteria at much
lower concentrations [72].
Pediocins hold an added advantage over antibiotic therapy as
they are proteinaceous in nature. They therefore are susceptible to proteolytic digestion upon their oral administration.
These peptides can be easily metabolized to simple nontoxic
amino acids and have lower residence time in the gastrointestinal tract as compared to antibiotics. The stability and
residence time of pediocin PA-1 in the gastrointestinal tract
was examined using an artificial system mimicking the human
stomach and small intestine [51]. It retained partial activity
after 90 min in the artificial gastric conditions where a combination of pepsin and highly acidic environment may be responsible for the decrease in pediocin activity observed in the
gastric chamber. Pediocin PA-1 completely lost its activity in
the duodenal compartment, where pancreatin was thought
to be responsible for the ultimate cleavage of bacteriocin.
This is in agreement with in vivo report of Bernborn et al.
[73], as pediocin PA-1 fed to test animals was not detected
in their fecal samples. Encapsulation of antibacterial pediocins
is being suggested here to exploit their full potential in the
form of oral therapeutics as it may preserve their potency as
well as increase their residence time in the gastrointestinal
tract. Although it has not been tested for pediocins as of
yet, partial success reports on the class I bacteriocin nisin are
not lacking in the literature [74-76]. The therapeutic potential
of pediocin PA-1 administered orally to mice infected with
L. monocytogenes has been examined [70]. Treatment with
250 μg of pediocin PA-1 a day for three consecutive days lowered fecal listerial counts by 2-log units. L. monocytogenes
that causes adult septicemia and meningitis, generally crosses
the gut epithelial barrier once it enters the small intestine and
then spreads quickly to the liver, spleen, and central nervous

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system [77]. This bacteriocin treatment was found to decrease
counts of listeriae reaching the liver and spleen [70].
Data from previous studies convincingly demonstrated the
safety of pediocins for human applications in comparison to
other accepted and available products, indicating they could
be safely incorporated into personal care applications aimed
at the treatment of bacterial vaginosis, gastrointestinal disorders, peptic ulcers and UTIs. Results of the present study
established pediocins as a general spermicidal agent. In addition to pediocins, two more bacteriocins namely nisin [35]
and subtilosin [36], were shown to possess impressive spermicidal activity. A possible concern with the therapeutic use
of pediocins is the possibility of an immune response. Bacterial peptides may assume antigenic role, and their introduction could trigger an immune response in humans. To ensure
safety of LAB associated AMPs, pediocin AcH was administered intraperitoneally into mice and rabbits to determine its
antigenic properties. It did not elicit an immune response in
test animals and therefore designated as non-immunogenic
peptide [50]. In fact, antibody responses could be induced
against class IIa bacteriocins but it requires their conjugation
with adjuvant such as polyacrylamide [78] or carrier proteins
such as keyhole limpet hemocyanin [79-80]. A number of previous in vitro studies conducted with the EpiVaginal model on
subtilosin have revealed safety of LAB bacteriocins for topical
application. In vivo testing of the rabbit vaginal irritation (RVI)
system further confirmed the safety of another LAB bacteriocin, Lactocin 160 [81]. Information regarding the cytotoxicity
of class IIa pediocins is relatively limited.

Conclusions
Class IIa pediocins are antagonistic to many important human
pathogens. These bacteriocins have the ability to target a relatively wide range of pathogenic bacteria which is an important
advantage, especially to replace antibiotics with much safer
therapeutics and to combat the ever growing problem of antibiotic resistance, when compared to other antibiotics. They
have the potential to perform a very specific role like treatment of bacterial vaginosis, gut infections and peptic ulcers.
Preliminary experiments with rec-pediocin CP2 have proved it
to be effective at fighting Bacteroides, Candida, Escherichia,
Enterococcus, Helicobacter, Gardnerella, Klebsiella, Listeria,
Neisseria, Propionibacterium, Staphylococci, Streptococci and
Vibrio infections compared to native pediocin CP2. Attempt
at engineering pediocin CP2 of P. acidilactici MTCC 5101 to
improve its potency and stability has been highly encouraging.
Study also established pediocins as potent spermicidal agents
with very impressive market value in the form of personal care
products. The application of class IIa pediocins as therapeutics
is a rapidly developing area, and there is still much to investi-

10

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gate. Continued study of some aspects especially assessment
of toxicity of rec-pediocin on human tissues in vitro/in vivo is
desired in order to exploit their full potential in pharmaceuticals. Further research is desired for having complete insight
into development of triple therapy formulation including a
pH regulator, an antibiotic partner and probiotic LAB/bacteriocin thereof in order to propose effective cure of BV and
gut infections and to prevent their further recurrence upon
completion of antibiotic treatment. In addition, it would be
recommendable to test pediocins against a range of probiotic
Gram-positive bacteria, as they have displayed unexpected
activity. Statistical approaches like response surface methodology in combination with scalable efficient purifications are
now available for the cost effective production of pure bacteriocin. This preliminary study has successfully revealed that
pediocins possess several desirable and useful properties as in
vivo antimicrobial agents. What remains now is to integrate
and exploit current knowledge to fully explore the suitability
of pediocins as in vivo therapeutics.

Acknowledgement
Authors acknowledge the UGC, New Delhi, India, for providing financial assistance in the form of Rajiv Gandhi National
Fellowship to Mr. Balvir Kumar.

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