Bacterial strains, antibiotic
Klebsiella pneumoniae B5055 (O1:K2), obtained from Dr Mathia Trautmann, Department of Medical Microbiology and Hygiene, University of Ulm, Germany, stored in our laboratory in 60% glycerol at -80°C and maintained on nutrient agar slants at 4°C, was used in the study. Gentamicin (Himedia, India) stock solution (2 mg/ml) was prepared in sterile distilled water according to the method of Andrews . It was administered to mice at a final concentration of 1.5 mg/kg body wt. throughout the study.
Bacterial depolymerase isolated from A. punctata (GenBank: KF158411), capable of acting on the K2 capsular polysaccharide of K. pneumoniae B5055 (composed of glucose, mannose and glucuronic acid) was used. Production of bacterial depolymerase was carried out by cultivating A. punctata in a statistically optimized media as standardized in our laboratory . Cell free supernatant containing enzyme was obtained and purified by anion exchange (DEAE) followed by gel filtration chromatography (Sephadex G100). The enzyme was purified to homogeneity and used at a concentration of 50 μg throughout the study. [Decapsulation of K. pneumoniae B5055 after treatment with A. punctata derived depolymerase is depicted in Additional file 1].
Pathogen-free BALB/c mice of either sex, 6–8 weeks old, weighing 20–25 g were procured from the central animal house of Panjab University, Chandigarh, India. Animals were kept in clear polypropylene cages and fed on a standard antibiotic-free diet (Hindustan Lever Products, Kolkata, India) and water ad libitum. The temperature ranged between 18 and 22°C and relative humidity was between 55 and 65%.
Ethics statement: The study was conducted after obtaining approval from the Institutional Animal ethics committee of Panjab University [Approval ID: IAEC/346-356]. All experiment protocols were performed in accordance with the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. All efforts were made to minimize the suffering of animals.
[Note: Bacterial doses corresponding to 102-108 CFU/ml were tried for inducing acute lung infection after intranasal administration or septicemia after intraperitoneal administration. The dose which gave 100% infection without causing any mortality was chosen for this work. (i.e. 104 CFU/50 μl for i.n infection and 102 CFU/100 μl for systemic infection by i.p route)].
Treatment efficacy in acute lung infection model
K. pneumoniae B5055 was cultivated for 24 h at 37°C in nutrient broth. Next day, cells were pelleted and washed twice with normal saline (0.85% NaCl). Bacterial suspension prepared in saline was adjusted to achieve a cell density corresponding to 2 × 105 CFU/ml (O.D = 0.03). Acute lung infection was induced in mice following the method of Held et al.  modified by Yadav et al. . Intranasal instillation of 104 CFU of K. pneumoniae B5055 in a volume of 50 μl was performed by holding the mice in an upright position without any anaesthesia. Infected mice were then randomly divided into four groups with each group comprising 20 animals. Different groups were given one of the following treatments:
Group 1 (control): Mice infected with K. pneumoniae B5055, given normal saline intraperitoneally.
Group 2: Mice infected with K. pneumoniae B5055 followed by intraperitoneal administration of gentamicin (1.5 mg/kg/daily), initiated 24 h post infection.
Group 3: Mice infected with K. pneumoniae B5055 followed by intraperitoneal administration of the A. punctata derived depolymerase (50 μg), 24 h post infection.
Group 4: Mice infected with K. pneumoniae B5055 followed by intraperitoneal administration of depolymerase (50 μg) as well as gentamicin (1.5 mg/kg, daily), 24 h post infection.
[Note: Optimal dose of A. punctata derived depolymerase to be used was chosen based on the results of preliminary experiments carried out using different doses, 25 μg, 50 μg and 75 μg. Dose giving maximum log reduction after in vivo administration was thus selected].
Animals were sacrificed on different days [1–3, 5, 7] post-infection by cervical dislocation and lungs were removed aseptically. Bacterial load, pro-inflammatory and anti-inflammatory cytokine levels were estimated in lung tissue homogenates while histopathological examination was carried out for the intact lung tissue.
Effect on systemic infection
Systemic infection was induced in mice by intraperitoneal administration of 102 CFU of overnight grown and washed K. pneumoniae B5055 in a volume of 0.1 ml. Thereafter, mice were divided into the following 4 groups with each group comprising of 10 animals:
Group 1 (control): Mice infected with K. pneumoniae B5055 and given normal saline intraperitoneally, 6 h post infection.
Group 2: Mice infected with K. pneumoniae B5055, administered gentamicin (1.5 mg/kg) intraperitoneally, 6 h post infection.
Group 3: Mice infected with K. pneumoniae B5055, administered bacterial depolymerase (50 μg), intraperitoneally, 6 h post infection.
Group 4: Mice infected with K. pneumoniae B5055, administered bacterial depolymerase (50 μg) and gentamicin (1.5 mg/kg), intraperitoneally, 6 h post infection.
In each group, blood was taken from mice by retro-orbital puncture and lungs, liver, kidney, spleen were removed aseptically, 24 h post infection. Estimation of bacterial load and pro and anti-inflammatory cytokine was carried out in tissue homogenates.
Quantification of bacteria
Mice were sacrificed on different days post-infection by cervical dislocation. Different organs were removed aseptically and homogenized in 1 ml normal saline. Serial dilutions of the homogenized tissues were made and plated on nutrient agar plates. Plates were incubated at 37°C for 24 h and bacterial counts determined.
Estimation of cytokine levels
Assay for Tumor necrosis factor a (TNF-a), Interleukin-1 (IL-1), Interleukin-10 (IL-10) was performed by ELISA using commercially available cytokine kits (Peprotech). Lungs, liver, kidney, spleen were homogenized in 1 ml lysis buffer containing 0.5% Triton X 100, 150 mM NaCl, 15 mM Tris, 1 mM CaCl2 and 1 mM MgCl2 (pH 7.4). Homogenates were centrifuged at 400 g for 10 min and supernatants were used for estimation of cytokine levels. Appropriate antigen-affinity purified anti-mouse antibody pairs, detection reagents (TMB, BD biosciences) and mouse recombinant cytokines obtained from Peprotech were used as standards (capture antibodies: rabbit anti-mouse IL-1ß/IL-10/TNFa; detection antibodies: biotinylated rabbit anti-mouse IL-1ß/IL-10/TNFa). Absorbance was measured at 450 nm and results were expressed as pg/ml of cytokines released. All assays were performed in triplicates and performed thrice.
Lungs were removed aseptically, immersed in 10% formalin fixative and processed for histological examination. The lung tissue was embedded in paraffin wax and cut into 4–6 μm thick sections using a microtome. The sections were stained with haematoxylin and eosin and assessed for neutrophil infiltration and inflammatory changes.
Generation of antisera
For raising antisera against bacterial depolymerase, 10 Balb/c mice were injected sub-cutaneously on day 0 with 50 μg protein emulsified in CFA (Sigma Aldrich). It was followed by subcutaneous injection with booster doses on days 7, 14 and 21 [50 μg protein emulsified in IFA (Sigma Aldrich)]. Thereafter, mice were bled through retro-orbital puncture on 10th, 17th and 28th day and blood serum was collected. Antibody titer was determined in serum samples using 1:25,000 dilution of goat anti-mouse HRP conjugate (Bangalore genei) as the secondary antibody in an enzyme linked immunosorbent assay. Antibody titer was defined as the reciprocal of dilution that gave an absorbance of 1.0 at 450 nm after a 30-min reaction with the chromogenic substrate (TMB).
Enzyme efficacy in vivo in presence of antisera
Mice were divided into the following 3 groups with each group comprising of 10 animals:
Group 1: Naïve control mice challenged intranasally with 104 CFU of K. pneumoniae B5055 in a volume of 50 μl followed by treatment with normal saline (0.1 ml/i.p), 24 h post infection.
Group 2: Naïve control mice challenged intranasally with 104 CFU of K. pneumoniae B5055 in a volume of 50 μl followed by treatment with bacterial depolymerase (50 μg, i.p), 24 h post infection.
Group 3: Immunized mice (with antibodies against depolymerase) challenged intranasally with 104 CFU of K. pneumoniae B5055 in a volume of 50 μl followed by treatment with bacterial depolymerase (50 μg, i.p), 24 h post infection.
Bacterial count was determined on the peak day (day 3) in lungs homogenates of mice belonging to the three groups and Log10CFU/ml was calculated.
Enzyme activity following incubation with antisera
Bacterial depolymerase was preincubated at 37°C for 60 min with sera obtained from naïve and immunized mice (antibody titer: 1000) (sera was heated at 56°C for 30 min to inactivate complement before use). Following this, log phase K. pneumoniae (108 CFU/ml) were treated with these pre-incubated enzyme samples at 37°C for 60 min. These cells were then washed twice with Hank’s balanced salt solution (HBSS; 10 mM PBS, pH 7.2, containing 1 mM CaCl2, 0.5 mM MgCl2 and 1 mg/ml glucose) and bacterial number was determined. The bacterial cells were then opsonized with 10% normal mouse serum (taken from uninfected mice) for 20 min at 37°C and bacterial number was confirmed. Thereafter, phagocytosis of bacteria by macrophages was performed by the method of Hampton and Winterbourn, .
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. Therefore, it was selected for this study. Briefly, peritoneal macrophages (106/ml) were isolated from the peritoneal lavage collected from pathogen free BALB/c mice. Macrophages were suspended in HBSS containing 10% normal mouse serum. These were incubated with pretreated opsonized bacterial cells (108 CFU/ml) at 37°C in 5% CO2. Samples were withdrawn at appropriate time intervals and an equal volume of ice-cold PBS was added followed by centrifugation at 200 g for 5 min. Supernatant was separated, pellet was washed twice, suspended in PBS with 0.5% triton X solution and incubated at room temperature for 30 min. Intracellular bacteria recovered after 3 h were stained with a LIVE-DEAD assay kit (Molecular Probes) consisting of 30 nM SYTO9 and 15 μM propidium iodide for 15 min in dark. Analysis was done by flow cytometry, using a flow cytometer (BD Biosciences FACS Canto II) and FACSdiva software. A control tube to study phagocytic killing of bacterial cells not treated with depolymerase was also put up simultaneously.
Results were analysed statistically by applying one-way ANOVA (Microsoft Excel 2007) for comparing various parameters in treated and untreated control mice. Differences were considered statistically significant if P-value was less than 0.01.