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Molecular Microbiology Research (Online) 2013, Vol.3 No.1 1-8
ISSN 1027-5595
http://mmr.sophiapublisher.com
3
3 Biofilm and its Determination
Biofilm is a structural complex of bacteria in which
they are enclosed and composed of self-made polymeric
matrix. These make connections to inert and free
living surfaces which enhance the secure growth in the
environment (Prakash et al., 2003). Biofilms are
causing more and more problems in daily life like
production of different diseases due to contamination
of medical equipment, food industry and environ-
mental sets because of biofilm’s particular charac-
teristics like resistance to UV light, antibiotics,
biocide chemicals, amplification of genomic change,
changed biodegradability and increased secondary
metabolite production (Costerton et al., 1987). As
aggregation of cells leads to clump formation due to
production of biofilm, bacteria are not susceptible to
macrophage facilitated engulfment and become
resistant to some antibiotics (Monzon et al., 2002).
Poly-N-acetyl β 1, 6 glucosamine (PNAG), a surface
polysaccharide is a key component of the
staphylococcus biofilm matrix which is synthesized
by proteins encoded by the intercellular adhesion
ica
operon and it can elicit a protective immune response
(Maira-Litran et al., 2004). Another biofilm consti-
tuent among mastitis isolates is the Bap protein but it
is not so much common (Cucarella et al., 2001).
Dhanawade et al. (2010) investigated the role of
biofilm production by
S. aureus
as vital virulence
factor by determining the biofilm contents adopting
different phenotypic and genotypic methods. They
collected 102
S. aureus
samples from subclinical
bovine mastitic cases. Frequency of biofilm producers
was 48.03% when identified by Congo red agar (CRA)
method while it was 36.27% using tube method. In
tissue culture plate method (TCP) without and with
de-staining, the frequency of this trait was 19.60% and
29.41%, respectively. By using standardized
polymerase chain reaction (PCR), 102
S. aureus
isolates were investigated for the detection of
intracellular adhesion genes,
ica
A and
ica
D
responsible for biofilm formation. Out of the pool,
both genes were present in 36 (35.29%) strains.
Considering PCR as a standard test, CRA and TCP
without de-staining were the most sensitive and
specific tests, respectively. PCR technique was
standardized to detect the
ica
A and
ica
D genes is
trustworthy for identifying biofilm forming potential
of
S. aureus
leading to quick determination of biofilm
producing staphylococci.
Mathur et al. (2006) compared the three methods of
biofilm detection. To detect the biofilm trait, 152
clinical isolates of Staphalococci were curtained by
tissue culture plate (TCP), tube method (TM) and
Congo red agar (CRA) method. Of the 152
staphylococcal isolates, 57.8% (88) exhibited biofilm
production when TCP method was used and strains
were further categorized as high (n= 22; 14.47 %) and
intermediate (n= 60; 39.4 %) biofilm producer
organisms while 46.0 % (70) isolates were weak or
non-biofilm producers. While using TM, it was
difficult to discriminate between weak biofilm
producers from biofilm negative isolates. The study
showed that CRA method does not show a
relationship with either of the other two methods for
detecting biofilm formation. The results showed that
the TCP method was the most profound, precise and
reproducible method to detect the biofilm production
by staphylococci.
Vasudevan et al. (2003) conducted a study to
evaluate
in vitro
slime production, biofilm formation
and presence of genes associated with biofilm
production
i.e.
ica
A and
ica
D in
S. aureus
isolated
from bovine mastitic samples. They used CRA method
and 32 out of 35 tested isolates produced slime while
only 24 of the microbes were biofilm producer
in vitro
.
However, all of the 35 sequesters contained the
ica
locus
i.e. ica
A and
ica
D genes. The experiment
showed the
ica
genes among
S. aureus
mastitis
isolates were highly prevalent and their presence was
not always accompanied with
in vitro
formation of
slime or biofilm. They suggested that phenotypic and
genotypic tests may be used in combination for
determination of biofilm formation in
S. aureus.
Ammendolia et al.
(1999) reported that slime-
producing strains were amongst coagulase-negative
staphylococci in equal proportion as reported in other
studies. Unexpectedly, a great proportion of
S.
aureus
strains were able to produce this extracellular