Figure
4: Comparison of zone of inhibition against
antibiotics on depolymerase treated and untreated sample of K. pneumoniae (Ampicillin-A,
Ampicillin/sublactam-As, Piperacillin/tazobactum-Pt, Cefoparazone-Cfs,
Cefotaxime-Ce, Amikacin-Ak, Ceftadizime-Ca, Ceftriaxzone-Ci, Netillin-Nt,
Tobramicin-Tb and Gentamicin-G). Bars represent standard deviation.

4 Discussion:

K. pneumoniae
is a major cause of nosocomial infections including respiratory, urinary tract,
wound infections, septicemia, bacteremia, pyogenic liver abscess etc (Wu et al.,
2011). CPS of K. pneumoniae is
antiphagocytic, mediates complement resistance, induces cytotoxicity during
infection of lung epithelial cells and helps in the establishment of intracellular
biofilm communities (Cano et al.,
2009; Rosen et al., 2008). Capsular
material forms bulky bundles of ?brillous structures that covers the bacterial
surface in immense layers and comprises 90% of the biofilm (Podschun and
Ullmann, 1998). Hence, CPS acts as an excellent target for compounds aimed at replacing or
supplementing antibiotic treatment for microbial infections.CPS
synthesis can be blocked by down regulating the genes for capsule biosynthesis
(Cress et al., 2014). Attempts have
also been made to target the phosphoregulatory
proteins involved in synthesis of capsular polysaccharides in Streptococcus pneumoniae, Staphylococcus
aureus and Escherichia coli (Ericsson
et al., 2012). The therapeutic attempts include
‘capsule stripping’ by using naturally occurring compounds or engineered
bacteria or phages that secrete pathogen specific CPS depolymerizing enzymes
(Cress et al., 2014). The capsule
depolymerization, using enzymes of bacterial origin was first employed during
the preantibiotic era by administrating a partially purified capsule
depolymerase to control pneumococcal infection in mice, rabbits and monkeys
(Goodner et al., 1932; Francis et al., 1934). Hoogerheide (1940) worked
on the degradation of K. pneumoniae
capsule using an enzyme from Bacillus
spp. and an enzyme from genus Aeromonas
having depolymerase activity against K.
pneumoniae capsule has been reported (Bansal et al., 2015). Other than this, there are no reports available in
literature describing the use of enzymes from unrelated bacterial genera for
disrupting the capsule of K. pneumoniae.

Enrichment of media with alginate has
been used for the isolation of alginate lyases, capable of degrading alginate
containing capsular polysaccharide of Pseudomonas
spp. from various environmental sources (Nakagawa et al.,
1998). In pursuance of this observation, enrichment of soil/sewage samples in
minimal media containing capsular polysaccharide extracted from different
strains of K. pneumoniae was
attempted. This technique led to successful isolation of thirty two
depolymerase producing bacteria. The isolate number ’30’ was able to produce
maximal amount of depolymerase extracellulary and it was found to be having
broad spectrum activity, as it showed depolymerase activity against seven out
of the ten clinical strains selected for this study. On the basis of
biochemical test and 16 s rRNA sequencing, the isolate was identified as B. siamensis (99.93% sequence similarity) and sequence has been
submitted to the NCBI/Nucleotide databases under accession no. MG018338. This is the first
report wherein we demonstrate the production of depolymerase enzyme by B. siamensis as
till today, no report on the  production
and optimization of broad spectrum capsular depolymerase is available in literature.

  Statistical
optimization has been a subject of central importance in a range of industrial
production processes. Hence, to standardize the optimum culture conditions,
physicochemical parameters were optimized in order to augment the production of
capsular depolymerase. In literature, medium has been standardized for the
optimal production of capsular depolymerase from a gram negative bacterium
(Bansal et al., 2013). However, no
such medium has been identified for any gram positive bacteria for the
production of capsular depolymerase. So using one variable at time (OVAT)
method, the capsular depolymerase production was increased from 0.9 to 1.15 IU/ml. In
our study maximum enzyme production was found to be at 96 hour which depicts
that  depolymerase production is
inducible in nature and dependent on initial depletion of simple sugars
initially but later on utilization of CPS leads to enhanced production of
depolymerase enzyme has been reported by Bansal (2013).  Significant variables necessary for enhancing
capsular depolymerase production were selected using Plackett Burman design. A
positive role of magnesium sulphate, pH and peptone screened during Plackett
Burman design was observed on enzyme yield. Magnesium sulphate showed a
positive response on depolymerase activity as it is suggested that the
inorganic salts may act as cofactor in capsular depolymerase production
(Castillo et al., 1976,  Nagai et
al., 2009 and Bansal et al., 2013). pH plays a significant role in
application of therapeutic enzymes, in present study capsular depolymerase
activity was enhanced by elevated pH in production media and may be used in
bacterial infections as pathogenic bacteria are capable of surviving and
growing at alkaline pH (Padan et al., 2005). Peptone as a major nitrogen
source showed a positive effect on the production of depolymerase.

RSM applied for optimization of
depolymerase production showed the significance of variety of three selected
factors from PB analysis at different levels. A high
level of similarity was observed between the experimental (1.92 IU/ml) and predicted
values (1.89 IU/ml)
that showed the accurateness and applicability of RSM to optimize the capsular
depolymerase production. Central composite design (CCD) enabled us to study and
explore the physicochemical conditions that led to a 115.5 percent increase
in enzyme production than unoptimized conditions. Stripping of capsule was seen
after treatment of K. pneumoniae with depolymerase as compared to
untreated culture which was also supported by the differential antibiotic
susceptibility pattern of depolymerase treated and untreated cells. Antibiotic
susceptibility remarkably increased for the antibiotics tested against
depolymerase treated cells than untreated cells. Bansal (2015) has
also been reported that depolymerase improves susceptibility of K.
pneumoniae to gentamicin also supports present investigation. Present
study showed that a broad spectrum depolymerase from B. siamensis SCVJ30 can be used to
produce capsular depolymerase and studied further for its prophylactic and therapeutic applications.

5 Conclusion:

Recent study demonstrates that the broad
spectrum capsular depolymerase can be obtained from natural sources. This was its first kind of study where broad
spectrum capsular depolymerase producing bacterium was isolated and
characterized although earlier capsular depolymerase from bacteria and phage
were isolated against a single standard strain. Capsular depolymerase
production could be improved by controlling a range of physicochemical
variables. Other than OVAT method, a statistical approach such as RSM has
proved to be a valuable, powerful model for rapid identification of positive
parameters and development of optimal culture conditions with a limited number
of experimental runs. Increased antibiotic susceptibility to K. Pneumoniae
by depolymerase treatment lay emphasis on the probable therapeutic role of
broad spectrum depolymerase in developing alternative strategies against drug
resistant K. pneumoniae infections.

Acknowledgments:
The above work was supported by UGC, New Delhi (India)

Conflict of
interest: The author(s) declare(s) that there is no conflict
of interest.

Availability of
data: 16S rRNA sequence of isolate is available at
Genbank NCBI database.