Isolation, Characterization, Statistical Optimization ...

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Original Article

Isolation, Characterization, Statistical Optimization, and Application of a Novel Broad‑Spectrum Capsular Depolymerase against Klebsiella pneumoniae from Bacillus siamensis SCVJ30 Sanjay Chhibber, Vijay Singh Gondil, Jasreen Kaur Department of Microbiology, Basic Medical Sciences, Panjab University, Chandigarh, India

Abstract Background: Bacterial resistance is one of the most challenging and emerging public healthcare crisis in the modern era. Along with antibiotic degrading strategies, bacteria also evolved to produce extracellular polymers such as capsular polysaccharide (CPS) which not only provides immune protection but also act as a permeability barrier to antibiotics. The use of therapeutic enzymes alone and in combination with antibiotics has opened a new window for clinicians and researchers. Methods: In the present study, isolation of broad-spectrum capsular depolymerase bacterium was attempted from a number of environmental samples followed by 16SrRNA characterization. Optimization of capsular depolymerase production was performed by the one variable at time (OVAT) method and response surface methodology (RSM). Capsular depolymerase was partially purified using ammonium sulfate saturation method. Capsule stripping effects of depolymerase were analyzed using microscopic visualization of the capsule and antibiotic susceptibility test. Results: Thirty-two capsular depolymerase producing bacteria were isolated in this study and broad-spectrum depolymerase producing Isolate-30 was characterized as Bacillus siamensis SCVJ30 according to the 16srRNA sequencing. Depolymerase production was optimized using OVAT method and RSM. Relatively high yields (1.92 IU/ml) of capsular depolymerase were obtained in a medium containing 1 mg magnesium sulfate, 7 mg peptone and at 9 pH. A 115% increase in capsular depolymerase production was observed under optimal conditions than unoptimized conditions. Microscopic visualization of the capsule and antibiotic susceptibility testing postulates the positive effect of depolymerase on antibiotic effi cacy against Klebsiella pneumoniae. Conclusion: Further characterization of the enzyme will help in developing broad-spectrum depolymerase as a potent therapeutic agent against drug-resistant strains. Keywords: Antibiotic resistance, capsular depolymerase, Klebsiella pneumoniae, placket Burman design, response surface methodology

Introduction Capsular polysaccharide (CPS) is an outermost layer of the bacterial cell wall which is acidic in nature and composed of repeats of basic units of four to six sugars. CPS contains glucuronic acid in combination with hexose and deoxyhexose sugars and plays an important role in adherence, protects bacteria against desiccation, bacteriophages, and immune response.[1] Strains possessing capsules can evade immune surveillance by mechanisms such restricted phagocytosis due to their hydrophilic nature of capsule and net negative charge, which repels the negatively charged phagocytes.[2] CPS may prevent activation of immune response by masking the pathogen‑associated molecular patterns that are recognized by Toll‑like receptors.[3,4] Moreover, the presence of capsule on the cell may lead to the scarce binding, degradation, or masking of Access this article online Quick Response Code:

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DOI: 10.4103/bbrj.bbrj_40_18

complement complexes.[5] Besides immune averting, CPS also provides a permeability barrier to antibiotics due to which they are unable to counter the Klebsiella pneumoniae infections. Traditional antibiotic therapy for countering Gram‑negative infections generally includes treatment with broad‑spectrum antibiotics,[6] but emergence of drug‑resistant strains and side effects of antibiotics limit their in vivo applications. CPS is an excellent target for alternative compounds which can be used for supplementing conventional antibiotic therapy for treating Address for correspondence: Prof. Sanjay Chhibber, Department of Microbiology, Basic Medical Sciences, Panjab University, Chandigarh, India. E‑mail: [email protected] This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution‑NonCommercial‑ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non‑commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms. For reprints contact: [email protected]

How to cite this article: Chhibber S, Gondil VS, Kaur J. Isolation, characterization, statistical optimization, and application of a novel broad-spectrum capsular depolymerase against Klebsiella pneumoniae from Bacillus siamensis SCVJ30. Biomed Biotechnol Res J 2018;2:125-31.

© 2018 Biomedical and Biotechnology Research Journal (BBRJ) | Published by Wolters Kluwer - Medknow

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bacterial infections. Recently, enzymes have been identified as a useful adjunct to antibiotics for acute as well as chronic infections as they enhance the efficacy of antimicrobials, which may lead to better management of infections[7] and improve the bactericidal effect of antibiotics.[8] Therapeutic application of enzymes includes removal or disruption of the capsule using bacteria or bacteriophage that can produce pathogen‑specific CPS depolymerizing enzymes. [1,9,10] Capsular depolymerase‑derived from Aeromonas punctata has been successfully used to alter the infection progression during K. pneumoniae‑mediated pneumonia and septicemia.[8] In the present study, an attempt on isolation, characterization, and optimization by the one variable at time (OVAT) and response surface methodology (RSM) method of a broad spectrum depolymerase from bacteria was made, which can degrade the CPS of different clinical strains of K. pneumoniae, thereby resulting in the development of a potential therapeutic agent against different K. pneumoniae infections.

Methods Bacterial strains and growth conditions

K. pneumoniae B5055, used in the present study was procured from Dr. Mathia Trautmann, University of Ulm, Germany and other clinical strains of K. pneumoniae were procured from Post Graduate Institute of Medical Education and Research, Chandigarh. All bacterial isolates were grown in nutrient broth at 37°C and subcultured periodically.

Preparation and estimation of substrate

CPS from different strains of K. pneumoniae was extracted by the method given by Hanlon et al., (2001) and used as substrate for isolation of depolymerase‑producing bacteria. Estimation of CPS was done according to the method of Dubois.[10]

Isolation of appropriate depolymerase‑producing bacteria Soil and sewage samples were screened for the isolation of depolymerase‑producing bacteria specific for CPS of K. pneumoniae by the method of Hoogerheide.[11]

Assay for depolymerase activity

Depolymerase activity was determined in supernatant on the basis of the release of reducing sugars using DNSA method. [12] One unit of enzyme activity was defined as micromoles of reducing sugars released/ml/min. The bacterial isolate giving the maximum yield and activity was selected for further study.

Identification of the selected isolate

The identification of the selected bacterial isolate was done according to the Bergey’s Manual of Systematic Bacteriology[13] and 16S rRNA gene amplification using universal primers. The bacterial isolate was identified using Ez‑Taxon and NCBI‑BLAST. The phylogenetic analysis of bacterial isolate was performed with all closely related taxas using MEGA6 by Kimura 2‑parameter method. 126

Optimization of various physiochemical parameters by one variable at a time method

Different culture conditions for capsular depolymerase production were optimized. Optimization of various nutritional and environmental parameters including different substrate concentrations (0–1 mg/ml), temperature (4°C–45°C), pH,[4‑9] incubation time (24–120 h), different nitrogen sources, and production volumes (25–100 ml) was carried out by classical OVAT method.

Plackett‑Burman design for the selection of significant variables

A total of 11 factors based on OVAT and literature search were selected, and a total of 12 experiments were designed using Design Expert10 software[14] as shown in Table 1. The effect of individual factors on enzyme activity was calculated by: Ei =

(∑ Pi + − ∑ Pi − ) N

where E i is the effect of i parameter, P i+ and P i‑ are responses (depolymerase activity) of runs at which the parameter was at its high level and low level, respectively, and N is the total number of runs.

Central composite design of response surface methodology

The most influencing factors, magnesium sulfate, peptone, and pH were selected and tested at their low and high values, 20 runs were generated as shown in Table 2. The RSM model was validated for further analysis. Three‑dimensional response surface plots were also constructed to find interaction among significant factors on depolymerase production/yield.

Partial purification of the depolymerase

The cell‑free supernatant after centrifugation (200 ml) was precipitated using fractional (50–80%) ammonium sulfate saturation. The precipitate‑containing the concentrated enzyme was dialyzed and the protein thus obtained was treated as partially purified depolymerase enzyme and used for further experiments. Table 1: Experimental variables for production of depolymerase Variables

Symbol code

Inoculum size A CPS B Casein C Peptone D Yeast extract E Meat extract F Ammonium chloride G Incubation period H pH I Production volume J Magnesium sulfate K CPS: Capsular polysaccharide

Units

Minimum value

Maximum value

% mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml Days ‑ ml mg/ml

0.5 0.2 0 0 0 0 0 2 6 50 0

1.5 0.3 0.2 5 1.5 1.5 1 4 8 100 0.5

Biomedical and Biotechnology Research Journal ¦ Volume 2 ¦ Issue 2 ¦ April-June 2018


Visualization of bacterial capsule after depolymerase treatment

K. pneumoniae B5055 was treated with depolymerase (2 IU/ml) and incubated overnight at 37°C, whereas in control tube, only buffer was added. Next day, bacterial depolymerase treated and untreated cultures were taken on a clean glass slide. A drop of Indian ink dye was mixed with culture and using another clean glass slide, the suspension was spread uniformly on the slide. Slides were then observed under a light microscope (×100), clear hallow depicts the presence of capsule around bacterial cells against a dark background.

Antibiotic susceptibility testing

Lawn of K. pneumoniae B5055 was made by spreading 100 µl of culture from the treated and untreated cultures on nutrient agar plates as described in above section. The hexa‑disks (Himedia) of different antibiotics including ampicillin (10 µg), ampicillin/ sublactam (10/10 µg), amoxicillin/clavulanic acid (20/10 µg), piperacillin/tazobactum (100/10 µg), ticarcillin/clavulanic acid (75/10 µg), cefoparazone (175/10 µg), cefuroxime (30 µg), cefotaxime (30 µg), amikacin (30 µg), carbenicillin (100 µg), ceftadizime (30 µg), ceftriaxzone (30 µg), netillin (30 µg), piperacillin (100 µg), tobramicin (10 µg), and gentamicin (10 µg) were treated with the plate. After overnight incubation, the zone of inhibition around each antibiotic disc was measured and compared.

Results and Discussion Isolation and selection of depolymerase‑producing bacteria specific for different strains of Klebsiella pneumoniae

Thirty‑two capsular depolymerase‑producing bacteria were isolated from sewage and soil samples. The zone of clearance was observed on plates‑containing CPS extracted from different strains of K. pneumoniae [Supplementary Data S1]. The activity of depolymerase from environmental isolates was checked on the CPS extracted from 10 different clinical isolates of K. pneumoniae [data not shown here, Supplementary Data S2]. Depolymerase activity of the Isolate No. 30 was observed on CPS of 7 out of the 10 strains tested (70%).

in Figure 1. This sequence was also validated using Ez‑Taxon and showed the similarity of 99.93% with B. siamensis KCTC 13613. The sequence has been submitted to the NCBI/ Nucleotide databases under accession no MG018338 and named as B. siamensis SCVJ30.

Production optimization of depolymerase using physiochemical parameters Optimization of various physiochemical parameters by one variable at a time method

To increase the yield of depolymerase, its production was optimized by classical one variable at a time method. An increase in the enzyme activity, i.e., from 0.9 to 1.15 IU/ml, was achieved under following optimized conditions, namely, 0.2 mg/ml substrate concentration, 96‑h incubation time, 37°C temperature, pH 8.0, 1% peptone, and 75 ml production volume as given in Table 3.

Screening the significant parameters for depolymerase production by PB analysis

As shown in pareto chart [Figure 2], out of 11 different factors, six factors, namely, magnesium sulfate, pH, peptone, ammonium chloride, incubation time, and yeast extract showed positive effects on the production. Out of these, magnesium sulfate, pH and peptone had showed the highest influence on the production. For the enhancement of depolymerase yield central composite design (CCD) was formulated using magnesium sulfate, pH, and peptone for further optimization.

Optimization of depolymerase production through response surface methodology

A CCD was formulated to examine the optimum levels of magnesium sulfate, pH, and peptone showing the highest influence in PB analysis [Table 2]. By applying multiple regression analysis on the experimental data, a predictive

Selection of depolymerase producing bacterium

The Isolate 30 that showed maximum extracellular enzyme activity in nutrient broth supplemented with CPS at 72 h (0.9 IU/ml) and was selected for further study.

Identification of the Bacillus siamensis SCVJ30

The morphological and biochemical characteristics of the selected Isolate SC 30 (SCVJ30) were studied and according to the Bergey’s Manual of Systematic Bacteriology, on the basis of biochemical characteristics the isolate could be placed in the genus “Bacillus.” The complete 16S rRNA gene was sequenced, and the (1492 bp) analysis clearly confirmed that strain was a member of the genus Bacillus and exhibited maximum similarity with the 16S rRNA sequence Bacillus siamensis KCTC 13613 (99.93% sequence similarity) as shown

Figure 1: Phylogenetic tree based on 16S rRNA gene sequences showing the evolutionary relationship among bacterial isolates drawn in MEGA6 using Kimura 2‑parameter method. 16S rRNA gene sequence accession numbers are given within parentheses. Bootstra P values are given at nodes. Scale bar 0.02 substitutions per nucleotide position


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Table 2: Optimization of enzyme production through response surface methodology Run order

Magnesium sulfate (mg)

Peptone (mg)

pH

Actual value (IU/ml)

Predicted value (IU/ml)

Residual value (IU/ml)

1 1.5 1.8409 1.5 1 0.159104 1 1 0.5 1.5 1 1 0.5 1 1 1.5 1 0.5 1 0.5

7 5 7 9 7 7 7 7 5 5 7 10.3636 5 3.63641 7 9 7 9 7 9

9 10 9 10 9 9 9 9 8 8 10.6818 9 10 9 9 8 7.31821 8 9 10

1.92±0.041 0.60±0.017 0.40±0.016 1.60±0.037 1.84±0.045 0.81±0.023 1.90±0.053 2.20±0.064 0.87±0.032 0.75±0.053 0.59±0.047 0.60±0.044 0.090±0.062 0.99±0.073 1.79±0.049 0.65±0.028 1.16±0.069 0.30±0.028 1.69±0.075 0.90±0.051

1.89 0.43 0.71 1.29 1.89 0.53 1.89 1.89 1.16 0.86 0.85 0.87 0.24 0.75 1.89 0.47 0.93 0.44 1.89 0.77

0.031 0.17 −0.31 0.31 −0.049 0.28 0.011 0.31 −0.29 −0.11 −0.26 −0.27 −0.15 0.24 −0.099 0.18 0.23 −0.14 −0.20 0.13

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Table 3: Optimization of capsular depolymerase by one variable at time method

Substrate concentration Temperature pH Incubation time Production volume Nitrogen source

Range

Optimum range

Optimum activity (IU/ml)

0‑1 mg/ml 4°C‑45°C 4‑9 24‑120 h 25‑100 ml ‑

0.2 mg/ml 37°C 8 96 h 75 ml Peptone

0.90 0.90 0.97 1.05 1.08 1.15

quadratic polynomial equation was constructed to illustrate a correlation between significant factors and depolymerase production. R= +1.89 + 0.055 × A + 0.035 × B − 0.025 × C + 0.083 × AB + 0.12 × AC + 0.31 × BC − 0.45 × A2 − 0.38 × B2 − 0.35 × C2 where R is the depolymerase activity; A, B, and C were magnesium sulfate, peptone, and pH, respectively. The analysis of variance for the model is summarized in Table 4. The P F

0.74 0.041 0.017 8.396E‑003 0.054 0.12 0.77 2.89 2.09 1.79 0.089 0.15 0.030

8.36 0.46 0.19 0.095 0.61 1.35 8.66 32.57 23.53 20.17

0.0013 0.5110 0.6695 0.7648 0.4517 0.2719 0.0147 0.0002 0.0007 0.0012

Significant

4.93

0.0523

Not significant

Model 6.68 9 A‑magnesium sulfate 0.041 1 B‑peptone 0.017 1 C‑pH 8.396E‑003 1 AB 0.054 1 AC 0.12 1 BC 0.77 1 A2 2.89 1 B2 2.09 1 C2 1.79 1 Residual 0.89 10 Lack of fit 0.74 5 Pure error 0.15 5 Cor total 7.57 19 C.V %=27.25, R2=0.8827, R2 (pred)=0.2283, R2(adj)=0.7771

0.09 X1 = A: magnesium sulphate X2 = B: peptone Actual Factor C: pH = 9

Untreated cells were surrounded by a capsule which was seen as clear arena around the bacteria against a dark background, whereas in treated samples, less number of encapsulated bacteria were found [Supplementary Data S3].

2.5

Enzyme Activity (u/ml)

Design-Expert® Software Factor Coding: Actual Original Scale Enzyme Activity (u/ml) Design points above predicted value Design points below predicted value 2.2

2 1.5 1

Antibiotic susceptibility testing

0.5 0

9

1.5 1.3

8

1.1

7

0.09 X1 = B: peptone X2 = C: pH Actual Factor A: magnesium sulphate = 1


B: peptone


0.9

6

0.7 5

0.5

A: magnesium sulphate (mg)

2.5 2 1.5 1 0.5 0

10

9 9.5

8 9

X1 = A: magnesium sulphate X2 = C: pH Actual Factor B: peptone = 7


0.09

7 8.5

C: pH


For antibiotic susceptibility testing 18 antibiotics were used. Out of these, five antibiotics did not show any zone of inhibition in both depolymerase treated and untreated samples, indicating that K. pneumoniae B5055 was resistant to these antibiotics. Ceftadizime showed zone of inhibition in the treated sample but not in untreated sample, indicating that K. pneumoniae B5055 became susceptible to antibiotic after the capsule was removed. In case of other antibiotics, the zone of inhibition considerably increased depicting the increase in susceptibility of K. pneumoniae B5055 to these antibiotics after the removal of the capsule [Figure 4].

6 8

Discussion

B: peptone

5

2.5 2 1.5 1 0.5 0

10

1.5 1.3

9.5

1.1

9

C: pH

0.9

8.5

0.7 8

0.5

A: magnesium sulphate (mg)

Figure 3: (a‑c) Three‑dimensional response surface plots presents the interaction of (a) magnesium sulfate and peptone (b) pH and peptone (c) pH and magnesium sulfate on capsular depolymerase production from Bacillus siamensis

Visualization of bacterial capsule

To validate the capsule degrading the ability of depolymerase, treated bacterium and untreated bacterium were visualized under light microscope followed by negative staining.

K. pneumoniae is a leading cause of nosocomial infections including respiratory, urinary tract, wound infections, septicemia, bacteremia, and pyogenic liver abscess.[15] CPS of K. pneumoniae is antiphagocytic in nature, mediates complement resistance, induces cytotoxicity to lung epithelial cells during infection and acts as extracellular support for the establishment of biofilms.[16,17] Capsular material forms bulky extracellular structures that covers the bacterial surface in layers and comprises 90% of the biofilm.[18] Hence, CPS can be targeted to counter drug resistance microbial pathogens. CPS synthesis can be blocked by downregulating the capsule biosynthesis genes and targeting the phosphoregulatory proteins involved in the synthesis of CPSs.[1,19] Capsule depolymerization, an another approach by which capsule can be removed using enzymes was first employed during the pre‑antibiotic era by administrating a partially purified capsule depolymerase to control pneumococcal infections.[20,21] Hoogerheide[11] in their study reported the degradation of K. pneumoniae capsule


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Treated sample

Zone of inhibition (mm)

Untreated sample

10

5

G

Tb

N t

C i

C a

Ak

C e

C fs

Pt

As

A

0 Antibiotic

Figure 4: Comparison of zone of inhibition against antibiotics on depolymerase treated and untreated sample of Klebsiella 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

using an enzyme from Bacillus spp. and an enzyme from genus Aeromonas having depolymerase activity against K. pneumoniae capsule has been also reported.[8] Other than this, there are no reports available in the literature describing the use of enzymes from unrelated bacterial genera for disrupting the capsule of K. pneumoniae. Alginate has been used for enrichment of media for isolation of alginate lyases, capable of degrading alginate containing CPS of Pseudomonas spp. from various environmental sources.[22] In pursuance of this observation, enrichment of soil and sewage samples in media containing CPS extracted from different strains of K. pneumoniae was attempted which led to the successful isolation of 32 depolymerase producing bacteria. The isolate number ‘30’ was able to produce maximal amount of depolymerase extracellulary and having broad‑spectrum capsule degrading 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 reveal the production of depolymerase enzyme by B. siamensis as till date, no report on the production and optimization of broad‑spectrum capsular depolymerase is available in the literature. Statistical optimization has been an important parameter in designing a production process to achieve maximum yield of product. Hence, to standardize the optimum culture conditions, physicochemical parameters were optimized to augment the production of capsular depolymerase. In literature, the medium has been standardized for the optimal production of capsular depolymerase from a Gram‑negative 130

bacterium.[23] However, no such medium has been identified for any Gram‑positive bacteria for the production of capsular depolymerase. By employing one variable at time (OVAT) method, depolymerase production was optimized and increased from 0.9 to 1.15 IU/ml. In our study, maximum enzyme production was found to be at 96 h which depicts that depolymerase production is inducible in nature and dependent on initial depletion of simple sugars initially, but later on the utilization of CPS leads to enhanced production of depolymerase enzyme has been reported by Bansal et al.[23] Significant variables necessary for enhancing depolymerase production were screened using Plackett–Burman design and showed a positive role of magnesium sulfate, pH, and peptone on enzyme yield. Magnesium sulfate showed a positive response to depolymerase activity as it is suggested that the inorganic salts may act as cofactor in capsular depolymerase production.[23‑25] pH plays a significant role in the application of therapeutic enzymes, in the present study capsular depolymerase production 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.[26] Peptone as a major nitrogen source showed a positive effect on the production of depolymerase. RSM based optimization of depolymerase production showed the significance of variety of three selected factors from PB analysis. The adequate similarity was observed between the experimental values (1.92 IU/ml) and predicted values (1.89 IU/ml) which can be correlated with the accurateness and applicability of RSM in the optimization process. CCD explored the physicochemical conditions that led to a 115.5% increase in depolymerase production than unoptimized conditions. Stripping of the capsule was seen after treatment of K. pneumoniae with depolymerase as compared to an 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 et al.[8] have also been reported that depolymerase improves susceptibility of K. pneumoniae to gentamicin also supports the present investigation. The 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.

Conclusion A recent study reveals that the broad spectrum capsular depolymerase can be obtained from natural sources. This is its first kind of study where broad‑spectrum capsular depolymerase‑producing bacterium has been isolated and characterized. 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 following depolymerase treatment emphasized on the probability of its therapeutic role in treating multidrug‑resistant strains of K. pneumoniae.

Availability of data

16S rRNA sequence of isolate is available at Genbank NCBI database [Supplementary Data S4].

Financial support and sponsorship

The above work was funded by UGC‑MANF, New Delhi (India) to Mr. Vijay Singh Gondil.

Conflicts of interest

There are no conflicts of interest.

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