K. pneumoniae accounts for 25-43% of nosocomial pneumonias caused by gram negative bacteria. It has a rapid progressive clinical course often complicated by multilobular involvement and lung abscesses  which leaves little time to institute effective antimicrobial treatment. As a result, the mortality rates may reach or exceed 50% even in treated cases . The voluminous capsular layer is involved in adhesion, maintenance, proliferation and development of infections by this pathogen . Owing to an increase in the MDR and NDM-1 strains, the WHO recommended ampicillin and gentamicin as first line of treatment have proved to be ineffective . Thus a demand for newer antimicrobials, not affected by resistance mechanisms has gained momentum in the last decade .
Promising results obtained after using A. punctata derived depolymerase that lyses K2 capsule of K. pneumoniae, thereby improving gentamicin efficacy against planktonic and biofilm cells of K. pneumoniae (data not presented) during in vitro studies, prompted us to evaluate its in vivo therapeutic efficacy. Post infection administration of single bolus of bacterial depolymerase, resulted in significant drop (~2 log) in bacterial titers during pulmonary infection. Kabha et al.,
 have reported that Klebsiella with certain CPS types like K21a, containing mannose-a-2-mannose and rhamnose-a-2/3-rhamnose sequences are readily recognized by the macrophage mannose receptor followed by their ingestion and killing. In contrast, strains with K2 CPS lacking these sequences are not recognized, thus allowing expression of virulence factors and bacterial proliferation in various organs. Denudation of K2 capsule by Aeromonas derived enzyme possibly led to improved uptake and killing of Klebsiella by alveolar macrophages in lungs.
Studies by Hoffman et al.  and Durante-Mangoni et al.  have reported wide use of aminoglycosides to treat bacterial infections of heart, lung and urinary tract. Thus, gentamicin was chosen for treating K. pneumoniae induced acute lung infection. When used at 2.5 mg/kg or 5 mg/kg post infection, highly significant reduction (P < 0.001) in bacterial load was observed (data not presented) indicating its high efficacy at these concentrations. When 1.5 mg/kg gentamicin was administered at 0 h, 6 h, 12 h, 24 h, 48 h, 24 h + 48 h post infection, significant reduction in bacterial load was observed in the first 3 groups on peak day (day 3) (data not presented). But in the other groups, gentamicin (1.5 mg/kg) alone could not control the infection. Even when injected daily till 7th day post infection, no significant protection was observed. These results indicated that gentamicin was effective during the initial time when bacteria have not completely established themselves or started to proliferate in the lung tissue. Once bacteria colonized, proliferated in the lung and CPS production was maximal, gentamicin was no longer effective. Moreover, in clinical situations also, it takes some time to initiate antibiotic treatment. Furthermore, Lavender et al. , have reported that early stages of K. pneumoniae airway infections might involve biofilm formation. As suggested by Kristian et al. , biofilm formation inhibits effectiveness of antibiotic treatment, prevents deposition of host defense components including C3b and IgG and facilitates bacterial communication leading to expression of virulence determinants. Therefore, A. punctata derived depolymerase was co-injected 24 h post infection with gentamicin to check whether it could remove CPS and render the bacteria susceptible to gentamicin. A reduction of ≥99% in bacterial titers was seen. This can be attributed to the enzyme mediated dispersal of CPS matrix leading to improved susceptibility of bacteria towards an otherwise ineffective gentamicin concentration. Depolymerase and gentamicin were comparatively less effective on the 3rd day because bacterial infection and tissue injury were at its peak and they were unable to tackle it on their own. Since the infection was confined to lung only, hence, as the bacterial number decreased by day 4/5, the agents became more effective. Thus enzyme based therapy helped to overcome the limitations offered by CPS including slow penetration of aminoglycosides due to electrostatic interactions with mucus and biofilm matrix. Disruption of capsular layer also possibly led to improved opsonization and increase in effector function of leukocytes leading to significant reduction in bacterial count. Reports by Brown, , using antibiotics and mucolytics in cystic fibrosis patients have also suggested that the latter provided symptomatic relief by decreasing mucus viscosity, thereby facilitating bacterial clearance.
Spread of bacteria on intraperitoneal administration led to bacteremia and colonization of liver, kidney, spleen and lungs. After intraperitoneal administration, the enzyme gained quick access to these organs via systemic circulation and through capsule removal facilitated bacterial clearance by mononuclear phagocytic system operating in these organs. Merino et al.  suggested that during planktonic growth, K2 capsule of Klebsiella causes complement activation, but activated complement components bind far from cell membrane hence, no cell lysis occurs. Highly significant reduction of bacterial count in blood on treatment of mice with enzyme can be attributed to capsule removal which facilitated deposition of complement components resulting in cell death. Decapsulating enzymes like endosialidase against E. coli K1 and poly-glutamic acid depolymerase against B. anthracis have been shown to prevent the spread of infections by these bacteria in experimental animals by enhancing their killing by complement, neutrophils and macrophages [35–39]. Moreover, reports showing synergy between enzyme and antibiotic as observed in this study, have also appeared previously in Gram positive infections caused by Staphylococcus and Pneumococcus
During acute lung infection, bacterial components (CPS and LPS), macrophage and neutrophil mediators (oxygen radicals, proteolytic enzymes) and complement components induce an inflammatory response. Thus, in the present study significant increase was observed in levels of pro-inflammatory as well as anti-inflammatory cytokines during compartmentalized pneumonia. Witzenrath et al.  and Zelmer et al.  have reported reduction in cytokine expression after treatment of Streptococcus pneumoniae infection with lytic enzyme (Cpl-1) and E. coli K1 infection with endosialidase (endoE). Similarly, in our study significant reduction in cytokine expression was observed in treated animals. It protected mice from pathogen induced damage and helped in clearance of invading bacteria. In enzyme treated animals, during systemic infection, reduction in cytokine expression was highly significant, in comparison to that observed during compartmentalized pneumonia. This indicates that during respiratory infection, enzyme although interrupts the course of infection, but does not completely diminish, local tissue response to bacterial invasion. This might be due to extensive proliferation of the pathogen on mucosal layer of respiratory tract in biofilm mode contrary to the presence of planktonic bacteria during systemic spread. Histopathological examination of lung tissue of infected, untreated animals showed well-developed pneumonia with neutrophil infiltration, abscess formation and destruction of alveoli as previously described in our laboratory . In contrast, lung tissue of mice treated with enzyme alone showed signs of peribronchial inflammation but lung alveoli were completely devoid of neutrophils. On the other hand, no signs of inflammation and neutrophil extravasation was observed in mice treated with enzyme and gentamicin. Thus it could be concluded that, enzyme mediated capsule removal did not allow high bacterial density to be reached, reduced the residence time of bacteria in mice and sensitized decapsulated bacteria to gentamicin and components of the immune system.
Since, proteins are immunogenic when delivered systemically, therefore the issue of neutralizing antibodies interfering with activity of depolymerase after its in vivo administration was addressed. Inspite of the presence of antibodies, the ‘enzybiotic’ was equally active in immunized and naïve mice. Moreover, pre-incubation of enzyme with its antisera did not hinder the overall bacterial killing by immune cells. This might be due to presence of antibodies directed against epitopes that do not contribute to therapeutic potential of protein or due to a higher binding affinity of the enzyme for its substrate in comparison to the antibody’s affinity for enzyme .