We appreciate the comments of the reviewers related to our manuscript. All the queries
have been addressed as follows:
As per the comments of reviewer 1:
Comments
Answers
Whether the inoculum doses were verified after Each time, after administration of bacteria
i.n or i.p inoculation of the bacteria
intranasally or intraperitoneally in mice, the
same volume of inoculum was plated on
nutrient agar plates and bacterial number
was estimated so as to confirm whether the
administered dose is correct or not.
Method 2.9.2, Enzyme activity following A schematic representation of the
incubation with antisera: in this experiment, the experiment is as follows:
authors tried to show that depolymerase activity Depolymerase was pre-incubated with
didn’t change following antisera treatment by antiserum raised against it/naïve serum (heat
phagocytic killing of bacterial cell test. Yet the inactivated at 560C for 30 min) for 60 min at
experiment had some problems. Firstly, the 370C
complements in normal mouse serum used for
opsonisation may kill the bacteria directly. Bacteria (108 cfu/ml) treated with the preTherefore the final killing of Klebsiella was incubated depolymerase for 60 min at 370C.
combination of both complements and
macrophages.
Bacteria washed twice with HBSS and
Secondly, the mouse sera used for preincubation bacterial number was estimated.
with depolymerase also contained functional
complement system, which may opsonize
bacteria
if
not
inactivated
before Bacteria opsonized with normal mouse
enzyme/bacteria co-incubation. Also, the serum for 20 min at 37oC and bacterial
concentration of the sera was not clear in this number was estimated thereafter.
experiment.
Opsonized bacteria (108 cfu/ml) incubated
with macrophages (106/ml) and samples
collected after various time intervals (0min,
30 min, 60 min, 90 min, 120 min, 180 min)
and count estimated.
Thus, as represented above: Before
preincubation of depolymerase with
antiserum containing antibodies against it,
the antisera was heated at 560C for 30 min to
inactivate the complement. This prevented
complement mediated lysis of bacteria,
when depolymerase preincubated with
antisera was used for treatment of bacteria.
After enzyme treatment, bacteria were
washed twice with HBSS and bacterial
number was estimated. It was found to be
108cfu/ml indicating no bacterial killing.
Then bacteria were opsonized with normal
serum. After opsonization bacterial number
was determined and it was found to be
108cfu/ml same as initial count, indicating
no cell death due to complement mediated
lysis rather it led to deposition of
complement components on the bacterial
surface that enhances phagocytic uptake).
After opsonization, bacteria were incubated
with macrophages (106/ml) and bacterial
number was estimated at 0 hr (it was the
same i.e 108cfu/ml) and at various time
intervals as depicted above.
The method followed for performing the
phagocytic killing is the same as done by
Hampton, M.B., Winterbourn, C.C. 1999 J.
Immunol. Met. 232: 15–22. They have also
used normal serum (without complement
inactivation) for opsonizing bacteria before
phagocytosis.
Method 2.9.1 and Figure 6: The data was not
presented clearly. And it is not clear how many
infected untreated mice used in this study. A
straightforward way is to present and do
statistical analysis of the log10CFU of each
group directly.
There are at least 78 distinct capsular serotypes
in K. pneumoniae. It is necessary to understand
the specificity of the depolymerase to different
capsular serotypes. Is there any article discussed
this issue or whether the authors can test the
function of this enzyme on the other serotype,
e.g. K1.
The antisera raised against depolymerase
had a antibody titer of 1000. It has been
mentioned in section 3.5 of results.
Method 2.9.1 and its corresponding Figure 6
have been modified as suggested by the
reviewer. Three mice groups were put up in
the experiment: immunized infected treated,
naïve infected treated and naïve infected
untreated. Each of these had 10 mice.
The data is now presented as Log10CFU/ml
in Figure 6 as suggested by the reviewer.
Depolymerases are highly specific for their
capsular serotypes. This is reported in
literature (Geyer et al. 1983; Pure and Appl
Chem., Vol. 55, No. 4, pp: 637-653). If at
all, one enzyme acts on more than one
capsular serotype, it acts on similar bonds
which might exist in some of the 78 capsular
serotypes of Klebsiella. Although, the
capsular serotypes K1 and K2 are the most
frequently encountered in clinical infections
but they have quite distinct structures
[K1:
→ 4) − α − L − Fucp − (1 → 3) −
β − D − Glcp − (1 → 4) − β − D −
GlcAp − (1 →]n
[K2: →)4-Glc-(1→3)-𝛼-Glc-(1→4)-𝛽-Man-
Fig 1, please specify whether the significant
reduction in bacterial count is for
each day (2, 3, 5, 7)
(3←1)-𝛼-GlcA)-(1→]n
So, an enzyme specific for K2 capsule
cannot act on K1 capsular serotype.
The significant reduction in bacterial count
between different groups is for days 2, 3, 5,
7. This has been mentioned in the Legend
for fig 1
We apologize for the error. It is not 100
CFU/ml. actually the dose administered i.p.
was 100 CFU/0.1ml. i.e. 100 µl containing
100 bacteria was administered i.p in mice.
Line 160, line 254, the authors mentioned
intraperitoneal administration of 100 CFU/ml of
K. pneumoniae, yet 100 CFU/ml is only the
concentration of the bacteria, please specify the
volume of the inoculum to calculate the exact
inoculation dose.
Line 208-209, 296, specify the volume of Intranasally, a dose of 50 µl containing 104
inoculums
CFU was administered to mice. It has been
corrected throughout the manuscript.
Many grammar mistakes such as ling 255, 260: Grammatical errors have been corrected in
liver(s), kidney(s), spleen(s)
lines 255 and 260
As per the comments of reviewer 2:
COMMENTS
ANSWERS
About bacterial physiological stage, the authors
used the overnight culture, it should be either
stationary or death phase. Bacteria in different
growth phases will show different virulence and
permeability, therefore working efficiency for
antibiotic
is
different
(PLoS.Genetics.
2013.9:e1003421). However, Method 2.9.2, the
authors used bacteria in log-phase. It makes me
confused.
During preliminary experiments, log phase
culture as well as overnight grown culture of
K. pneumoniae B5055 was used. It was
centrifuged and 2 washings given with
saline. Then the required count was adjusted
for in vivo administration in mice. Acute
lung infection as well as septicemia, was
established in mice with log phase or
stationary phase cells. Then the animals
were treated with gentamicin and the
bacterial count was determined. The counts
in both cases were same. This indicated that
under in vivo conditions bacteria in either
state responded in a similar manner to
antibiotic
treatment.
Moreover,
the
reference: PLoS.Genetics. 2013.9:e1003421
has reported response of bacteria in different
phases of growth to antibiotic in vitro. In our
Further, there is no exact information about
bacterial dosages in different kinds of injections,
why they used 104 in intranasal instillation
(method 2.4) or 100 CFU/ml in intraperitoneal
injection or 108 in vitro macrophage assay,
if 104 CFU/ml (OD=0.03) in intranasal
instillation, taking 50 μl, then 500 CFU should
be in the intranasal experiment. It is impossible
to inject 1 ml by intranasal method, usually
<0.02 ml, excess volume or rapid injection will
induce suffocation and death.
lab also, a study conducted by Singla et al.
2012 shows how bacteria in different growth
phases respond differently to antibiotic
treatment in vitro
(Susceptibility of
different phases of biofilm of Klebsiella
pneumoniae to three different antibiotics).
Under in vivo conditions (as in our study),
antibiotic action against the bacteria depends
on the dose of bacteria used for infection
rather than the growth phase.
Similarly, in the ex vivo macrophage
experiment also (Method 2.9.2), we tried
using similar counts of overnight culture as
well as log phase culture and incubated them
with macrophages. The results of phagocytic
killing were similar in both the cases,
indicating that it actually depends on the
bacterial number rather than the growth
phase. But, since the whole experiment i.e.
macrophage isolation and its interaction
with bacteria had to be completed in a single
day (as decrease in macrophage viability
takes place with time) therefore, ultimately
log phase cells were chosen for the
experiment.
Different doses 102,103, 104, 105, 106, 107,
108 have been tried in our laboratory for
inducing acute lung infection after intranasal
administration
and
septicemia
after
intraperitoneal administration. The dose
which gave 100% infection without causing
any mortality was chosen for further work.
(i.e. 104cfu in 50µl for i.n infection and
102cfu in 100 µl for systemic infection).
Furthermore, since acute lung infection is
compartmentalized
infection
whereas
septicemia is systemic infection therefore a
higher dose is required for the former while
a higher than 102cfu i.p. dose resulted in
death of mice within 24 hours.
For phagocytosis, the killing efficacy
depends on the MOI i.e ratio of bacteria and
macrophages. In our study, we tried
different MOIs i.e 1, 10, 100,1000. But the
best results were obtained with MOI 100. It
could
be
achieved
using
8
6
10 bacteria:10 macrophages
or
107bacteria:105macrophages
or
6
4
10 bacteria:10 macrophages.
The
phagocytic killing was same with either of
these till the MOI is 100. We selected 1st
out of these.
For intranasal administration, a volume of
50µl containing 104 cfu was used.
Post-injection time: About post intraperitoneal
injection of depolymerase, after 24 h in
intranasal administration, or, after 6 h in
intraperitoneal injection of bacteria, then inject
depolymerase, what is the scientific basis? The
bacterial population should increase after 24 h,
then 50 μg of depolymerase is enough to cover
bacterial effects, not only population, but also
virulent effects in mice?
Post infection administration time
depolymerase was selected as follows:
of
After establishment of acute lung infection
model, it was treated with gentamicin (1.5
mg/kg) at 0hr, 6hr, 12hr, 24hr, 48 hr,
24hr+48hr post infection. In the first 3
groups significant reduction in bacterial
count was observed on the peak day (day 3)
whereas in the other groups, gentamicin
could not control the infection alone. These
results indicated that antibiotic is effective
during the initial time till the bacteria has
not completely established itself or started to
proliferate in the lungs. Once it colonizes,
proliferates in the lung and CPS production
is maximal, gentamicin is no longer
effective. Moreover, in clinical situations
also, it takes some time to initiate antibiotic
treatment. Thus, depolymerase was injected
24 hr post infection, to check whether it can
remove the CPS and render the bacteria
susceptible to gentamicin.
The bacterial load, 24 hr post infection, was
4.2 logs (Figure 1, day1). It was similar to
the intranasal dose administered to mice.
Depolymerase injected at 24 hr was not
directly bactericidal rather it degraded the
CPS matrix encasing the bacteria thus
rendering them susceptible to gentamicin
and components of immune system.
Similarly, during systemic infection, after
administration of 102 CFU i.p. if gentamicin
was administered immediately after
infection it resulted in significant reduction
in bacterial count but if administered 6 hr
post infection, the infection had spread so
much that no significant reduction in
bacterial count was observed. Thus,
depolymerase was given 6 h post infection
and it improved gentamicin susceptibility.
LD50 of Klebsiella pneumonia B5055 will be Yes, the lethal dose of Klebsiella
variable in different administration. (Please see: pneumoniae B5055 used by us is different
Microbes and Infections. 2011. 13: 1045-1051). after
intranasal
or
intraperitoneal
administration. The dose causing 100%
infection and no mortality i.e 104 cfu by
intranasal
route
and
102cfu
by
intraperitoneal route, used in our study is
also different.
The authors used intraperitoneal injection (50 μg We appreciate your suggestion on studying
of depolymerase), and there was no immune
the distribution of enzyme in various organs
responses to this enzyme. It is interesting to
using antibody directed against it and
know the enzyme distribution by systematic
correlating it with bacterial distribution in
circulation in different organs and it can be
various organs. But, we would like to
detected by using antibody of depolymerase.
mention that this would be carried out as a
Since the data showed different bacterial
part of the next student’s project.
distribution among mice organs, both data
combined would give the full spectra of the
enzyme which was working in different places.
What was happened during the 2-5 day, the
After intranasal instillation of bacteria in
authors should give some
mice, bacteria overcome the innate immune
explanation? Why did the bacterial population
response operating in the respiratory tract
reached the peak at the 3rd day
and establishes in the lungs by day1.
Histopathological analysis of the lung tissue
(please see Figures 1, 3)? Why were
showed that on day 1 mice develop mild
depolymerase and gentamicin less
effective in the 3rd day, but had better effective pneumonia (Indian J Med Res 118, July 2003,
pp 47-52). Thereafter, bacteria proliferate in
in the 4th day?
the lungs, express virulence factors resulting
in a corresponding increase in bacterial
number. Lungs of mice sacrificed on day 2
post infection reveal moderate changes. The
bacteria multiply and reach a peak by day 3.
Thus, the animals showed well-developed
pneumonia with abscess formation and
destruction of alveoli (Indian J Med Res 118,
July 2003, pp 47-52). At day 3, tissue injury
characterized by increased levels of nitric
oxide, free radicals and pro-inflammtory
cytokines is also maximum. Beyond Day 3,
bacteria is tackled by the activated host








immune response resulting in decrease in
bacterial number. Histopathology of mice
sacrificed on day 7 PI showed resolving
pneumonia and macrophages dominating in
the affected areas (Indian J Med Res 118, July
2003, pp 47-52).
Depolymerase
and
gentamicin
were
comparatively less effective on the 3rd day
because the bacterial infection and tissue
injury was at its peak and they were unable
to tackle it alone. Since, the infection is
compartmentalized in the lung therefore as
the bacterial number decreases by day 4/5,
the agents become more effective.
Introduction: CPS synthesis can be
 Reference has been added
blocked by deleting and down-regulating
 Spellings have been corrected
the genes for capsule biosynthesis
 References have been written in a
(please add the reference)
uniform pattern
Enterobactericiae --? Enterobacteriaceae
 Standard deviation has been added to
Please write the references in uniform
the day 7 results in figure 1
pattern including title, pages and
 The arrow in Figure 5c represents
volumes.
peribronchial inflammation. It has
Figure 1: the 7th day, no standard
been added in the legend for figure
derivation??
5c
Please indicate what mean of arrow in
 The word ‘reclamation’ has been
Figure 5c.
removed from the title. The title has
been modified.
Please consider to alter the title, because
“Reclamation” is not the right
 In Figure 6 legend, 104 CFU/ml has
presentation in this work.
been replaced with 104 CFU/50 µl.
Figure 6 in legend:is it wrong, not 10000,
 Yes CPS reacts with macrophage
but should be 500.
mannose receptor (as reported in ref
27 in the manuscript). This has been
About discussion: lines 342-344, K2 CPS
should react with some cellular receptors,
mentioned in the discussion
eg. mannose binding receptor, etc,
therefore, please making sure this
viewpoint.