ECOBIOS, Vol. 6.

(1&2), 2013 ISSN: 0972-6446

(International Journal for Biology, Ecology and Allied Sciences)

Antibacterial activity of Staphylococcus hominis against

Gram negative organism
S. D. Purkhayastha1,2, M. K. Bhattacharya1*, H. K. Prasad2, H.Upadhyaya1,
K. Pal3 , G. D. Sharma4 , S Das Lala5
1
Dept. of Botany & Biotechnology, Karimganj College, Karimganj-788710 Assam
2
Dept. of Life Science and Bioinformatics Assam University, Silchar-788011, Assam
3
Dept. of Biotechnology & Medical Engineering, NIT Rourkela, Rourkela-769008, Orissa
4
Bilaspur University, Bilaspur-495001, Chhattisgarh
5
Department of Gynaecology, Hospital of Redcross Society, Karimganj-788710, Assam, India

ABSTRACT
Staphylococcus infections are one of the most common problems in clinical microbiology
worldwide. It is a gram positive bacteria and an opportunistic pathogen. Due to use and
misuse of antibiotics over the world, increased risks of multidrug resistance have become a
threat and is a big challenge to find an alternatives of antibiotics in medical microbiology.
Recently a number of LAB species Bacillus subtilis, Streptococcus thermophiles etc. have
been using as probiotics. Staphylococcus hominis reported in this study showed
antagonistic activity against both gram positive and gram negative bacteria. The isolate may
be explored as probiotic candidate after further experiments to assess its probiotic nature.

Keywords: Antimicrobial activity, Staphylococcus hominis, antagonistic.

*E mail. mrinalkxj@yahoo.co.in
Published by
The Society for Biometry, Ecology & Econometrics (BEES), Karimganj, Assam, India 21
ECOBIOS, Vol. 6. (1&2), 2013 ISSN: 0972-6446

INTRODUCTION MATERIALS AND METHODS

Probiotic potential of Staphylococcus Sample collection
hominis has been studied as an anti-
Vaginal samples were collected after
staphylococcus aureus agent isolated from
obtaining permission of the ethical
microbiota of a healthy women1. The
committee. The samples were obtained by
antagonist isolated by them exhibited
a gynecologist with informed consent of
desirable probiotic trade such as acid
the volunteers. Vaginal sample were
tolerance, bile resistance and adherent to
collected from the Red Cross Hospital,
epithelial cell line. The bacterium also
Karimganj, Assam after obtaining
produce a bacteriocin with unique
informed consent. The women aged 18-30
molecular weight and high antimicrobial
years. They were all pre-menopausal,
activity like traditional antibiotics against
non-menstrual and either pregnant or non-
both Gram positive and gram negative
pregnant but generally disease free. The
organisms. In recent years different
swabs were then used for streaking sterile
species of Lactobacillus, Bacillus subtilis,
nutrient agar and de Manns Rogosa
Streptococcus thermophiles etc. are
(MRS) Agar2.
reported as probiotic microorganism.
There is only one report which suggested Isolation of vaginal microbiota
use of Staphylococcus hominis as The inoculated MRS plates were
probiotic agent 1. In view of the above incubated at 37C under aerobic condition
Staphylococcus hominis isolated from for 48hrs. The colonies obtained were
human vagina was used for study of examined through Gram staining
antibacterial activity against Gram technique and catalase test (Table1).
negative organisms.
Table 1-Morphological and biochemical characterization
Sample Name Gram Staining Catalase Test Test for spore formation
Lactobacillus fermentum +ve +ve Non spore forming

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ECOBIOS, Vol. 6. (1&2), 2013 ISSN: 0972-6446

Antibacterial assay and Staphylococcus aureus (MTCC 3160)

separately using McFarland standard 0.5.
Antibacterial assay was done using well
Following this 5mm well were prepared
diffusion method. Mueller-Hinton agar
in each plate and 10l of cell free culture
plates were prepared and the plates were
filtrate of the isolate was loaded in each
seeded with Escherichia coli (MTCC
well3. The plates were incubated for 24
1060), Bacillus subtilis (MTCC3053),
hours at 352C (Table2).
Pseudomonas aeruginosa (MTCC741)
Table 2- Antimicrobial activity of the bacteria
Sample Name Zone size against test organisms (mm)
Escherichia Staphylococcus Bacillus subtilis Pseudomonas
coli aureus aeruginosa
Staphylococcus 10 - 10 -
hominis

Molecular characterization identification of the isolate, the 16S gene

was amplified by using 27s Forward
DNA isolation has been done, using the
primer and 1100 Reverse primer (Table 3)
method described by Vural and Ozgun4
and sequenced.
(2011) with little modifications. For

Table 3-Primer sequence for PCR reaction

Name Sequence Reference

1100R AGGGTTGCGCTCGTTG Turner et al. 1999 [6]
27 F AGAGTTTGATCMTGGCTCAG Lane et al. 1991 [7]

Preparation of PCR reaction mixture- Reaction condition-Reactions were run

Each single reaction mixture (10 l)
contained 1 l of template DNA, 1 l of
each primer (20 picomole), 5 l of
Master Mix 2X (HiMedia) and 2l of
nuclease free water.
under the following conditions: initial
denaturation 94C for 2 min, 35 cycles of
94 C for 30 sec, 49.3C for 30 sec, 72C
for 1min and final extension at 72C for 3
min.

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ECOBIOS, Vol. 6. (1&2), 2013
Gel electrophoresis and analysis-5 l of
each PCR product was run in 1% agarose
gel for two hours. The gel was visualized
in GelDoc EZ imager (Bio-Rad).
16S rDNA sequencing-The 1.5 Kb
amplicons were sequenced by using the
facilities provided by the MERK
Bangalore. After obtaining the sequencing
data, these were analyzed and edited by
using Sequence scanner software (v1.0).
Alignment was done by ClustalW and
DNA contigs were prepared. After that
similarity has been matched using
BLASTn of NCBI database to identify
their closest relatives. The sequences were
submitted in the NCBI data base using the
submission tool BankIt and accession
numbers obtained.
RESULT AND DISCUSSION
Staphylococcus hominis is a gram
positive, catalase positive coccus. The
16S rRNA gene sequencing and
subsequent analysis with bioinformatics
tool showed that the bacteria show 100%
similarity with Staphylococcus hominis,
the sequence has been submitted in by
BanqIt submission tool of NCBI and
accession number obtained (KR264989).
In the present experiment antimicrobial
activity of Staphylococcus hominis was
ECOBIOS, Vol. VI (I&II) 2013
ISSN: 0972-6446
studied using cellfree supernatant against
some potentially pathogenic micro-
organisms viz Escherichia coli (MTCC
1060), Bacillus subtilis (MTCC3053),
Pseudomonas aeruginosa (MTCC741)
and Staphy-lococcus aureus (MTCC
3160) it has been found that, it was active
against Pseudomonas auroginosa (10mm)
and E coli (12mm). Against Bacillus
subtilis and Staphylococcus aureus there
has not been any zone.
Staphylococcus hominis is known as an
opportunistic micro-organism; however,
other scientists have reported that
different micro-organisms implicated in
some opportunistic infections, including
E. coli, Ent. faecium and Ent. faecalis,
also exhibit great potential as
probioticmicro-organisms (Kropec et al.
2005; Willenbrock et al.2007; Henker et
al. 2008; Laukova et al. 2008). Therefore,
the assessment of the safety potential for
using a probiotic candidate from an
atypical species is of special concern and
should be fully investigated1. However, as
per our knowledge this is the second
report of antimicrobial activity of
Staphylococcus hominis isolated from
vaginal microbiota. Sung et al1, first
reported the antimicrobial activity of
Staphylococcus hominis against Gram
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ECOBIOS, Vol. 6. (1&2), 2013
positive organisms but in our study we
reported antagonistic activity against
Gram negative bacteria only.
ACKNOWLEDGEMENT
Authors acknowledge with thanks
financial assistance received under DBT
under NER TWINNING PROJECT vide
DBT Sanction Order No. BT/220/
NE/TBP/2011; 30 May, 2012. We also
acknowledge DBT sponsored Institutional
Biotech Hub, Karimganj College (BCIL/
NER-BPMC/2010 dated 30th November,
2010) for providing infrastructural facility.
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