Approaches have been made to find new targets for antimicrobial activity, use of combination agents that are effective against more than one target in the cell, or new delivery mechanisms to maximize the concentration of antimicrobial agents at the site of infection, but relevant clinical evidence with respect to combining agents, has not been well elucidated for treatment against MDRSP strains. Given the astronomical costs involved in the research and development of a new drug and also the time required to take it from ‘the bench to the bedside’, utilization of combination therapy using known antibiotics should be a preferred as a cost-effective choice for therapy. In the current study we have used a murine pneumococcal pneumonia model to compare the efficacy of monotherapy with combination therapy by administering a single intravenous dose of AMP and AZM. From the bacterial growth and magnitude of inflammation (leukocyte infiltration into the lungs, lung cox-2 and high pulmonary vascular permeability) observed in our case supported the mouse model of pneumococcal pneumonia.
Use of β-lactam agents such as AMP, may increase and complicate the problem because these agents lyse the bacterial cell wall leading to release of proinflammatory substances such as cell wall components and cytotoxins which are recognized by the innate immune system and which trigger the inflammatory response [42, 43]. It was observed that macrolides (erythromycin) and macrolide-like agents (AZM, clindamycin, telithromycin), at sub-MIC concentrations, were potent inhibitors of pneumolysin production by both susceptible and resistant strains of Streptococcus pneumoniae, with doxycycline being somewhat less effective, while amoxicillin, ceftriaxone, and tobramycin were ineffective. AZM alone is unlikely to be preferred as resistance rates of community isolates of S. pneumoniae are high . But owing to its anti-inflammatory effects and broader spectrum of activity it might be a realistic candidate [45–48]. In addition AZM retained its anti-inflammatory activity against a resistant strain when used in combination therapy. This finding suggests that there might be clinical benefit independent of antibiotic susceptibility pattern .
Azithromycin (AZM) and ampicillin (AMP) in combination against an azithromycin resistant strain was reported to cure secondary pneumonia in mice. Thus we choose AZM and AMP as combinatorial antibiotic therapy even though we found the S. pneumoniae (AMRI-SP-1) was resistant to AMP or AZM applied in single doses. Furthermore, in a murine model of secondary, influenza-associated pneumococcal pneumonia, the lowest survival rate in antibiotic-treated animals was observed in those treated with AMP only, while the highest rates were noted in those treated with inhibitors of protein synthesis (AZM or clindamycin) only, or in combination with AMP . Improved survival with AZM was associated with an attenuated inflammatory response, manifested as lower numbers of inflammatory cells and pro-inflammatory cytokines in the lungs, and less severe histopathological changes. Therefore, antibiotic selection based solely on the grounds of antimicrobial potency may be inappropriate in some clinical settings, particularly serious infections caused by toxin-producing pathogens with high bacterial loads . In this situation, circumstances permitting, administration of an inhibitor of bacterial protein synthesis, either prior to, or together with a compatible bactericidal agent may be justified to reduce the potential risk of an antibiotic-associated inflammatory reaction. Based on laboratory, experimental animal, and limited clinical data, potential strategies to address this complex clinical problem include combining an inhibitor of bacterial protein synthesis (preferably one with secondary anti-inflammatory properties, i.e. a macrolide), with a cell-wall active agent. Thus, our choice of AMP along with AZM as combinatorial therapy against the multi-drug resistant S. pneumoniae (AMRI-SP-1) in this mouse model of pulmonary infection was hypothesized to be an effective combination therapy. AZM exhibits anti-inflammatory activities independent of its antimicrobial properties . This antibiotic resulted in clinical cure in S. pneumonia infected mice, although it is unclear whether the improved outcomes are solely the result of the mechanism of action or whether they are the result of this factor in addition to the anti-inflammatory properties of the drug . The exact mechanisms of action for the macrolides like azithromycin that have this anti-inflammatory action are still not completely defined, although it is known that they act by various molecular, cellular, and bacterial mechanisms. It might be due to decreased chemotaxis, migration, and cellular activity in neutrophils and macrophages and concomitant decrease in IL-6, TNF-α, IFN-γ and PGE2 in the air way passages after azithromycin administration.
Determining the drug levels in serum as a function of time is essential for estimating the concentration of the antibiotic that are necessary to inhibit (MIC) or to be bactericidal (MBC) to microorganisms. Drug concentration in the blood (plasma, serum) has been correlated to in vivo bacterial eradication. β- lactam antibiotics such as AMP are unevenly distributed in tissue, with a tissue: serum ratio < 1:1 for most sites. They are distributed mostly in the blood and extracellular fluid that represents about 20% of the total body mass. Conversely macrolides have high tissue: serum ratios (> 2:1) and are found predominantly inside cells. Concentrations of these drugs are therefore lower extracellularly while concentrations of β-lactams are higher . AMP has been known to exhibit-time dependent killing which means a long time above MIC (T > MIC) or a large ratio of area under the curve (AUC) to MIC (AUC/MIC) is predictive of a successful treatment outcome . Concentration dependent drugs like AZM are characterized by a steeper pharmacodynamic (PD) function; the steeper the PD function, the more efficient is the bacterial killing which increases commensurately with antibiotic concentration.
The PK and PD parameters suggest that ampicillin was widely distributed in the extracellular fluid and into tissues. A rapid distribution of the drug between blood and the extravascular tissue compartment was achieved which was consistent with that found in the literature. Azithromycin remained in circulation for a longer duration and was available in the tissue bed or at the site of infection thus exerting its bactericidal and anti-inflammatory effect there. It was reported that amoxicillin (AMX), a β-lactam antibiotic, was able to clear the infection of two resistant pneumococci (MICs 1 and 2 μg/ml) if the dose was increased . However, in a mouse pneumonia model, significant bactericidal effect was not achieved on penicillin resistant pneumococci strains for which the MIC was ≥ 2 mg/L, even with a dose/MIC ratio of 200 . In another study with penicillin resistant pneumococci strain (MIC 4 mg/L), a killing of 2 to 3 log10 within the first 6 h was observed, independent of Cmax ranging from 2 to 20 times the MIC. Regrowth occurred after 12 h in a majority of the experiments . Thus an increased Cmax and larger AUC were not sufficient to achieve a predictable killing for that strain. The findings from our present study also supports this observation that AMP though administered at a 4 times greater dose compared to AZM, achieved a greater Cmax and AUC but was not effective in clearing the bacterial load from the lungs in group of mice treated with AMP alone. So the need for studying highly resistant pneumococci is paramount to seek an explanation for this observation and determine its prevalence.
Macrolides induce a biphasic effect on the host. First, they have direct antimicrobial activity by stimulating the host defense against bacteria via stimulation of leukocyte degranulation, phagocytosis and oxidative burst. Secondly, after the acute infection, neutrophils that are primed by cytokines or pneumolysin are inhibited by macrolides, that leads to amelioration of the inflammatory response. Another potential explanation for the beneficial effects of macrolides is reduction in bacterial load with less cell wall lysis than beta-lactam antibiotics; this results in a more gradual reduction in bacterial load and, therefore, a more gradual release of immunologically reactive components, which may prevent an extended systemic inflammatory response . In our study, changes in bacterial density in lung for AMP or AZM monotherapy were similar against S. pneumoniae isolate. For the AMP + AZM combination regimens, we found changes in lung bacterial density to be greater compared with AMP or AZM monotherapy for AMRI-SP-1. Clearance of bacteria from blood also increased after combined antibiotic therapy than the AMP or AZM alone at 3 h after initiation of antibiotic therapy. Therefore, it may be suggested that the outcome in pneumococcal pneumonia was improved when combination antibiotic therapy was initiated at early stages of S. pneumoniae infection even when the bacteria was resistant to that antibiotic [16, 17].
Subsequently, we determined whether neutrophils were critical for combined antibiotic-mediated protection in the pneumococcal infection. Animals infected with S. pneumoniae and 24 h later that were remained untreated showed heavy infiltration of PMN affecting the lung inflammation. However, co-administration of AMP and AZM after the pneumococcal challenge led to reduced PMN infiltration in lungs.
The timing of the observed decrease in neutrophil numbers and inflammatory mediators argues against a causal link between decreased inflammation and host protection. Specifically, combined antibiotic treatment reduced the pulmonary bacterial burden as early as at 2 h after initiation of antibiotic treatment, whereas the decreased inflammatory response was also apparent until 6 h post antibiotic treatment. In addition to this discrepancy in timing, there is substantial evidence that the recruitment of neutrophils and regulatory release of proinflammatory mediators are protective against S. pneumoniae-induced mortality.
High IL-6 concentrations were found in the lungs of mice infected with SP. In addition relatively higher serum IL-6 levels has been reported after intravenous injection of wild type SP than after administration of pneumolysin (PLY) negative mutant SP suggesting the induction of an inflammatory response in the pulmonary compartment in the early phase of pneumococcal pneumonia . During lung inflammation in acute phase, damaged alveolar capillary and epithelial membranes by PMN leading to leakage of protein rich edema fluid into the alveolar space, and formation of hyaline membranes which impaired gas exchange have been reported . At early stages of infection the permeability of lung vasculature is increased due to enhanced release of proinflammatory cytokines (TNF-α, IFN-γ and IL-6) [58–60]. Hence, decrease in extravasations after initiation of combined antibiotic therapy after 3 h of post antibiotic treatment may be due to reduced lung TNF-α, IFN-γ and IL-6 level and increased anti-inflammatory cytokine (IL-10), which is sustained until 6 hours post antibiotic treatment.
The inflammatory cytokine response in the lung is characterized by intense elevation IL-6, TNF-α and IFN-γ which was decreased after combined treatment. A subsequent increase in IL-10 after combinatorial treatment, which is an anti-inflammatory cytokine that inhibits macrophage and neutrophil production, is the beginning of the anti-inflammatory response that prevents an uncontrolled inflammatory response. IL-6 has been considered as a marker for the severity of bacterial challenge represents a relevant marker for the evolution of a host response and high IL-6 concentrations have been found in the lungs of mice infected with SP . Therefore, reduced IL-6 in combined antibiotic treated mice might be responsible for decreased inflammation in mouse lungs along with reduced lung TNF-α and IFN-γ after antibiotic treatment.
We observed that IFN-γ, TNF-α, IL-6 but not IL-10 production was increased initially 18 hours post-infection and decreased gradually thereafter following treatments with AMP and AZM. Therefore, it is likely that increased TNF-α and IFN-γ released into the circulation after infection by the administration of S. pneumonia cells or their exotoxins demonstrated a detrimental effect on the host. We found that severity of pneumonia is associated with altered balance of inflammatory cytokines, and conversely, altering the balance of inflammatory cytokines has a significant impact on the severity of pneumococcal pneumonia. It was reported that azithromycin at concentrations of 1, 5 and 10 μg/ml have been demonstrated to affect in various degree of production of IL-1, IL-6 and IL-10, GMCSF and TNF-α by human monocytes. Most remarkably, azithromycin resulted in a significant decrease of TNF-α in 100% of individuals and treatment with clarithromycin resulted in a significant decrease in IL-6 and TNF-α in 86% of individuals respectively [62, 63].
Of several pneumococcal pneumonia-related molecular pathways with anti-inflammatory actions, we chose to focus on IL-10 as a representative of cytokine in this class. IL-10 appears to be valuable for attenuating inflammatory damage to human lung . Since serum cytokines were considered as a reflection of inflammation induced by pathogens anti-inflammatory cytokines like IL- 10 continues to increase even at 6 hours after treatment of mice with AMP and AZM. This IL-10 level increment dictates the resolution of inflammation and may be a positive prognostic indicator for recovery of pneumonia due to the combined therapy. IL-10 inhibits the production of reactive oxygen and reactive nitrogen intermediates when monocyte and macrophages are activated by IFN-γ and therefore may be important in determining the outcome of pneumonia. As lack of IL-10 causes impaired clearance of bacteria leading to a more destructive cause of pneumonia, therefore, this elevated IL-10 in the combined antibiotic treated mice might be essential for efficient elimination of bacteria and therapy for protection against pneumococcal pneumonia. IL-10 is often considered as the master regulator in immunity from infection . IL-10 reduces both the extent and the duration of inflammation, the outgrowth of pneumococci, and mortality .
Therefore, the present finding indicated that in the presence of concurrent treatment with AMP + AZM may lead to elevated circulating IL-10 that might influence bacterial outgrowth, suggesting that only in the latter phases of pneumococcal pneumonia is IL-10 essential for host defense. It was reported that IL-10 given at latter stages of infection prevented severe inflammation and lung edema and facilitated bacterial clearance in mice treated with ceftriaxone . However, whether elevated systemic IL-10 during combined therapy could modulate the blood and lung levels of antibiotics , either AMP or AZM have not been tested in our case. Previous data also indicated a beneficial role for IL-10 as an adjunctive therapy to antibiotics against pneumococcal pneumonia in mouse model . These protective effects might have resulted from decreased pulmonary inflammation and better availability of the drug to the infected sites. Better bacterial clearance was also reported in other in vivo studies with IL-10 .
Immunoblot analysis of lung tissue homogenate showed that COX-2 level was significantly increased at 18 h post-infection in case of the S. pneumonia (AMRI-SP1), which was gradually decreased at 1, 2, 3 and 4 h post antibiotic treatment. After treatment with AMP along with azithromycin, COX-2 level was significantly decreased on 4 h post therapy. Similar reduction in prostaglandin, nitric oxide, TNF-α, and IL-6 levels has been previously reported in murine macrophages treated with 5 to 80 μM of azithromycin . Given its constitutively expressed nature and predominant role in prostaglandin synthesis during bacterial infection, potential strategies for drug resistant bacteria based on COX pathways or inhibiting COX-2 . These data collectively support that combinatorial antibiotic treatment mediated COX-2 inhibition or strategies that disrupt prostaglandin signaling pathways as useful adjunctive therapies in treating persistent and multi-drug resistant infection.
The combined antibiotic therapy promoted the infiltration of peripherally circulating neutrophils into the lungs, leading to bacterial clearance, COX-2 pathway in lungs and the lung cytokines might determine the outcome of interactions with microbes in the lungs. Insights into the functional roles of cytokines, and regulatory factors in mediating pulmonary immune responses may contribute to rationally designing and appropriately using therapeutic and prophylactic agents. This basic study may be considered for strategies aimed at altering leukocyte recruitment, bacterial clearance and pulmonary inflammation in order to improve host defense (e.g. against antibiotic-resistant organisms).