Mechanisms of action and in vivo antibacterial efficacy assessment of five novel hybrid peptides derived from Indolicidin and Ranalexin against Streptococcus pneumoniae

Bacterial culture and assay medium

S. pneumoniae clinical strains used in this study were obtained from University of Malaya Medical Centre (UMMC). Columbia agar with 5% sheep blood was used to culture the bacteria. Mueller-Hinton broth (MHB) was used for synergism assay and cationally adjusted as described in the guidelines of Clinical and Laboratory Standards Institute (2012).

Transmission electron microscopy (TEM)

Bacteria were prepared for TEM according to the guidelines of the Electron Microscopy Unit at the Faculty of Medicine, University of Malaya. S. pneumoniae cultures were grown overnight on Columbia agar with 5% sheep blood and suspended in cationally adjusted Mueller-Hinton broth (CAMHB) at 10⁸ CFU/ml. Pneumococcal suspensions were incubated with hybrid peptides RN7-IN10, RN7-IN9, RN7-IN8 and RN7-IN6 at 125 µg/ml and with RN7-IN7 at 500 µg/ml (8 × of their respective MIC) in a 1.5 ml eppendorf tube for 1 h at 37 °C under 5% CO₂. Cells in cationally adjusted Mueller-Hinton broth (CAMHB) were used as an untreated control. After an hour of incubation, the pneumococcal cells were centrifuged to discard the medium, washed thrice with 10 mM phosphate buffer saline at pH 7.3 and fixed overnight in 4% (v/v) glutaraldehyde. All samples were washed twice with cacodylate buffer, incubated for 2 h in osmium tetroxide buffer (OsO₄ 1: 1 cacodylate), washed twice with cacodylate buffer and then incubated overnight in cacodylate buffer. All the samples were washed with distilled water twice and incubated for 10 min with uranyl acetate. After this, all samples were then washed twice with distilled water and dehydrated in an ascending series of ethanol: 35% (10 min), 50% (10 min), 70% (10 min), 95% (15 min), and thrice in 100% ethanol (15 min). After dehydration, samples were incubated with propylene oxide (15 min), propylene oxide 1:1 Epon (1 h), propylene oxide 1:3 Epon (2 h) and finally incubated overnight with Epon. All the samples were embedded in agar 100 resin at 37 °C for 5 hr and maintained at 60 °C until viewing. Reichert Ultramicrotome copper grids 3.05 mm (300 square mesh) (Agar Scientific, Essex, United Kingdom) were used to prepare Ultrathin sections. Ethanol-based uranyl acetate and lead citrate were used to stain the samples for 5 min. Transmission electron microscope (Leo Libra 120) was used to capture the images.

Scanning electron microscopy (SEM)

S. pneumoniae with a starting inoculum of 1 × 10⁸ CFU/ml in CAMHB medium, was treated with hybrid peptides RN7-IN10, RN7-IN9, RN7-IN8 and RN7-IN6 at 125 µg/ml and with RN7-IN7 at 500 µg/ml of respective peptides and incubated for 1 h at 37 °C for under 5% CO₂. After incubation, 20 µl of the untreated and treated cell suspensions were transferred onto membrane filters and processed as described by the standard guidelines provided by Electron Microscopy Unit, Faculty of Medicine, University of Malaya. Briefly, the bacterial samples were fixed overnight with 4% glutaraldehyde at 4 °C and then washed twice with sodium cacodylate buffer for 10 min each. In the second fixation, 1% osmium tetroxide was used to fix the samples for 1 h at 4 °C and then washed twice with distilled water for 10 min each. All the samples were then dehydrated through a serially graded ethanol (30%, 50%, 70%, 80%, 90%, 95% and twice in 100%) for 15 min each, followed by dehydration in ethanol:acetone mixtures (3:1, 1:1 and 1:3) for 15 min each and three rounds of pure acetone for 20 min each. The samples were then dried for 1 h and kept in a desiccator before the examination. The samples were then mounted on stubs, coated with gold in sputter coater and viewed under the FEI-Quanta 650 Scanning Electron Microscope.

ATP efflux assay

The amount of ATP released from pneumococcal cells incubated with hybrid peptides was measured as described previously, with a slight modification (Tanida et al., 2006). The ATP determination kit (Molecular Probes, Eugene, OR, USA) was used to measure the amount of ATP released based on the luciferin/luciferase method according to the manufacturer’s instructions. Briefly, S. pneumoniae were grown overnight on Columbia agar with 5% sheep blood. Bacterial suspensions were spectrophotometrically adjusted to (1 × 10⁷ CFU/ml) and incubated with hybrid peptides RN7-IN10, RN7-IN9, RN7-IN8 and RN7-IN6 at 125 µg/ml and with RN7-IN7 at 500 µg/ml. The amount of ATP released form the pneumococcal cells were measured at three time points 1, 2 and 3 h. The samples were then centrifuged at 5,000 rpm for 5 min, and ATP efflux was subsequently estimated using an ATP standard curve. Values were obtained from three independent experiments. Ceftriaxone and erythromycin were used as positive controls.

Gel retardation assay

This assay was carried out as described previously, with minor modifications (Li et al., 2013). S. pneumoniae were grown overnight on Columbia agar with 5% sheep blood. A few bacterial colonies were transferred into a 1.5 ml eppendorf tube containing phosphate buffered saline (PBS). The bacterial cells were centrifuged, PBS was discarded and 50 µl of TE buffer containing 0.08 g/ml of lysozyme and 150 U/ml of mutanolysin was added to the cells. Genomic DNA was isolated from pneumococcal cells using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany), following the manufacturer’s giudlines. The optical density ratio of 260 nm and 280 nm (OD₂₆₀∕OD₂₈₀ = 1.83) was used to measure the purity of the DNA. Genomic DNA (250 ng) was incubated with hybrid peptides at various concentrations (0.24–500 µg/ml) in 12 μl at room temperature for 10 min. 2 μl of loading buffer were added to the mixture and the migration of DNA through 1% agarose gel was evaluated by electrophoresis in 1 × Tris borate–EDTA buffer (45 mM Tris–borate and 1 mM EDTA at pH 8.0) and spotted by the fluorescence of gel stain (Gel Red, BIOTIUM, Fremont, CA, USA). The natural peptide indolicidin was used as positive controls, ceftriaxone and erythromycin were used as negative controls.

Synergistic effect

Pneumococcal cells were cultured overnight using Columbia agar with 5% sheep blood at 37 °C under 5% CO₂, resuspended in cation-supplemented Mueller-Hinton broth and adjusted to 5 ×10⁵ CFU/ml, following CLSI guidelines. Combinations of hybrid AMPs with each other and with standard drugs (ceftriaxone and erythromycin) were assessed for their synergistic effects by the checkerboard titration method described previously, with minor modification (Bajaksouzian et al., 1996). Briefly, 50 µl of eight serial two-fold dilutions of drug B starting at 4 × MIC were added to each column of the plate followed by 50 µl of a fixed 0.25 × MIC of drug A, this yielded eight peptide-peptide combinations at different ratios. 100 µl of bacterial suspension (5 ×10⁵ CFU/ml) were then added to each well and the plates were incubated for 24 h at 37 °C under 5% CO₂. The fractional inhibitory concentration (FIC) index of each combination was calculated according to the following formula: ${\displaystyle \text{FICI}=\frac{\text{MIC of drug A in combination}}{\text{MIC of drug A alone}}+\frac{\text{MIC of drug B in combination}}{\text{MIC of drug B alone}}.}$

MIC A in combination and MIC B in combination represent the MICs of drug A and B tested in combination. MIC A alone and MIC B alone represent the MICs of drug A and B in standalone. FIC index values were interpreted as follows: two drugs have synergy if FIC ≤ 0.5, additive or indifference if 0.5 < FIC ≤ 4.0 and antagonism if FIC > 4.0. The experiment was done in triplicate.

In vivo assessment

In vivo toxicity

In order to evaluate the possible toxic effects correlated to peptides administered in mice, four hybrid peptides RN7-IN10, RN7-IN9, RN7-IN8 and RN7-IN6 were chosen for in vivo toxicity assessment, due to their promising in vitro antibacterial activity (Jindal et al., 2015). Mice were separated into four groups (each with four mice) and were injected with respective peptides at 1 h, 12 h, and 24 h (three-dose regimen) via IP, SC, and IN administration routes. The hybrid peptides were first administered at high doses (100 mg/kg for IP route, 100 mg/kg for SC routes and 20 mg/kg for IN route). Any abnormal behavior was recorded and survival of mice was noted as well. In case adverse effects such as high physical stress, severe lethargy, physical inactiveness, and/or death were detected, lower graded doses were given. All the administered mice were monitored for seven days or until death occurred. At day seven post administration, all animals were sacrificed and blood and organs were collected. Untreated mice were used as a control group.

In vivo antipneumococcal activity

Two pneumococcal infection models developed previously in our lab (Le et al., 2015) were used to assess the therapeutic efficacy of peptides in vivo. The systemic infection model was used to mimic pneumococcal bacteremia in humans and the pneumococcal pneumonia model was used to mimic pneumococcal pneumonia in humans. A highly virulent strain was used in both the models. The bacterial isolate was grown overnight on Columbia agar with 5% sheep blood at 37 °C under 5% CO₂. The bacterial suspension was adjusted to OD₆₂₅ 0.08-0.1 (1 ∼ 2 × 10⁸ CFU/ml). Mice tested for lethal systemic infection were inoculated with 1.5 × 10² CFU/mouse (100 μl) via IP route. Mice used to assess the Pneumococcal pneumonia model were inoculated with pneumococcal cells of 5 × 10³ CFU/mouse (50 μl) via the intrathoracic route. Both the infection models caused 100% death within 2 to 4 days post-infection.

After 1 hr of inoculation, Mice receiving treatment were randomized and divided into six groups. RN7-IN10 and RN7-IN8 were tested at three different doses for each (5 mg/kg, 10 mg/kg and 20 mg/kg) using a group of 10 mice. Graded doses of ceftriaxone (5 mg/kg, 10 mg/kg, 20 gm/kg, 40 mg/kg and 80 mg/kg) were also tested to assess the in vivo antibacterial activity of this antibiotic. Only mice injected with PBS were served as uninfected control. Mice injected with bacterial inoculum were used as untreated control group and given sterile distilled water only. Survival of mice was documented for seven days or until death. After seven days, the experiment was ended, the blood and homogenates of the five major organs (kidney, brain, spleen, liver and lung) of the surviving mice were plated on Columbia agar with 5% sheep blood, to detect the presence of pneumococcal cells.

In vivo synergy assessment of peptide/peptide and peptide/ceftriaxone

After evaluating the in vivo antibacterial activity of the hybrids in the standalone mode, the in vivo efficiency of the hybrid peptides in combination with each other and with the standard antibiotic ceftriaxone was carried out. Graded doses of peptide and ceftriaxone were chosen and prepared at 2 × the desired concentration separately in 1 ml tubes, and the volume was 0.1 ml. Just before injection, both the drugs were combined, giving the final desired concentration at a volume of 0.2 ml. The synergetic effect was then performed in infection models (n = 10).

Anesthesia and necropsy

Mice used to evaluate the in vivo toxicity and antibacterial activity of the hybrid peptides using the subcutaneous (SC) and intranasal (IN) administration routes, were anesthetized using a combination of a standard dose of xylazine (ilium xylazil-20, 10 mg/kg) and ketamine (Narketan^®-10, 100 mg/kg) through intraperitoneal (IP) injection. After seven days of treatment, the in vivo toxicity and antibacterial efficacy experiments were ended and the surviving mice were anesthetized. Blood samples for Hematological and biochemical analysis were collected via cardiac puncture using a 25G syringe (BD bioscience, San Jose, CA, USA). Whole blood for heamatological analysis was collected in 500 µl dipotassium EDTA microtainer tubes (BD Bioscience, USA). About 500 µl of blood collected an eppedorff tube and centrifuged at 8000 rpm for 5 min and then the serum was transferred into a new 1.5 ml tube for biochemistry analysis. The mice were then euthanized by cervical dislocation, dissected and the following organs were collected for histopathology evaluation: lung, kidney, brain, liver and spleen.

Hematological and biochemical analysis

For whole blood analysis, the parameters were number of red cells (RBC), number of white cells (WBC), lymphocytes, monocytes, eosinophil, granulocytes, haemoglobin (Hgb), mean corpuscular volume (MCV), hematocrit (HCT), platelet Counts (PLT), Mean corpuscular haemoglobin (MCH) and corpuscular haemoglobin concentration (MCHC). For biochemistry analysis, the parameters were alanine transaminase (ALT), creatinine, alkaline phosphatase (ALP), aspartate aminotransferase (AST), total bilirubin and urea.

Histopathological examination

The following organs were collected from all the dissected mice: lung, kidney, brain, spleen, and liver. All tissues were fixed in 10% (v/v) buffered formalin and processed for paraffin embedding. Hematoxylin–eosin (HE) was used to stain the histological sections at the histopathology laboratory, Veterinary Laboratory Service Unit, University Putra Malaysia (UPM).

Statistical analysis

GraphPad Prism 5 was used to perform the Statistical analysis. The results were expressed as mean ± standard deviation. Two-way ANOVA with Bonferroni post-test was used to analyze the significance of the difference between the treated groups and control in ATP assay. One-way ANOVA with post-hoc Dunnett-t test was used to assess the statistical difference between the blood haematogram and blood serum biochemistry parameters of the treated and the untreated control groups in the in vivo toxicity assay. Kaplan–Meier analysis with log-rank test (Mantel-Cox) was used to generate the survival curve for each treated group versus untreated control, for both in vivo antibacterial activity and in vivo synergy assays.

Effects of hybrid peptides on cell morphology and membrane permeability

TEM and SEM studies were performed to observe the damaging effect of the hybrid peptides on the pneumococcal cell wall/membrane. The images obtained clearly indicated that all the hybrid peptides were capable of disrupting the integrity of bacterial membranes. As shown in Fig. 1A, the untreated cells appeared with complete cell wall and plasma membrane and therefore preserved the normal integral shape of S. pneumoniae. The pneumococcal capsular polysaccharide appeared as a thin layer sheltering the whole cell and the cytoplasm of the cell was compactly packed and occupied the entire space (Fig. 1A, arrow 1). Incubation of pneumococcal cells with hybrid peptides had led to a dramatic effect on the morphology of bacterial surface. After 1 h of incubation, the hybrid peptides were able to breach the intactness of the cell wall and/or plasma membrane, causing membrane breakage and loss of fragments (Figs. 1B–1F, arrow 2). Additionally, our TEM results have revealed that treatment with hybrid peptides has led to the leakage of the cytoplasmic components to the outer environment through the disruption of the cell wall. As a result, huge halos were detected in the inner space of all these treated cells, leading to cell collapse and death (Figs. 1B–1F, arrow 3). Moreover, the TEM results also revealed partial disconnection of the cell wall from the cell membrane in pneumococci treated with hybrid peptides, especially those treated with RN7-IN9, RN7-IN8, RN7-IN7 and RN7-IN6 (Figs. 1C–1F, arrow 4).

Scanning electron microscopy (SEM) was employed to understand the impact of the hybrid peptides on the morphology of S. pneumoniae. As presented in Fig. 2, the hybrid peptides were able to induce significant morphological alterations to pneumococcal cells. The untreated S. pneumoniae displayed normal and smooth surface (Fig. 2A, arrow 1), whereas S. pneumoniae treated with hybrid peptides at 8 × MIC appeared with a rough and injured surface (Figs. 2B–2F, arrow 2). The numerous fragments observed on the bacterial surface are an indication of cell wall breakage and fragments loss upon treatment with hybrid peptides. This result indicates that hybrid peptides could disrupt and damage the integrity of cell wall/membrane or breach the membrane, which was in agreement with the result of TEM.

In order to evaluate the permeability of the membrane and leakage of intracellular components upon treatment with hybrid peptides, the level of ATP in the supernatant following contact of the pneumococcal cells with hybrid peptides was determined using the ATP release assay. After 1 h of treatment with hybrid peptide, the levels of ATP released after 1 h of treatment with RN7-IN10 and RN7-IN9 were the highest among the five hybrid peptides tested (54.5 ± 4.7 and 42.27 ± 92 pM respectively) (Fig. 3). The ATP efflux steadily decreased, and the ATP release reached 25.42 ± 3.51 pM and 22.9 ± 3.22 pM after 3 h of treatment with RN7-IN10 and RN7-IN9 (Fig. 3). On the other hand, the quantities of ATP released from pneumococcal cells upon incubation with RN7-IN8, RN7-IN7 and RN7-IN6 after 1 h were 39.03 ± 0.2 pM, 22.35 ± 0.9 pM and 14.8 ± 0.35 pM. The levels of ATP release from pneumococcal cells treated with RN7-IN8, RN7-IN7 and RN7-IN6 were 20.54 ± 1.03 pM, 13.02 ± 2.26 pM and 11.47 ± 0.32 pM respectively after 3 h of treatment (Fig. 3). However, all the hybrid peptides showed better capacity in efflux ATP from pneumococcal cells in comparison with standard antibacterial drugs ceftriaxone and erythromycin. The efflux levels of ATP by ceftriaxone and erythromycin treated cells after 1 h of incubation were 5.24 ± 1.43 pM and 0.49 ± 0.004 pM, respectively (Fig. 3).

DNA retardation activity

To clarify the influence of the hybrid peptides on pneumococcal genomic DNA, the retardation of DNA by the hybrid peptides at various concentrations was assessed by analyzing electrophoretic movement of pneumococci DNA bands through the agarose gel (1%, w/v). Our results clearly indicated that like their parent peptide indolicidin, all the five hybrid peptides were capable of inhibiting DNA migration through the gel at a concentration of 62.5 µg/ml (Figs. 4A–4F). On the other hand, the standard drugs ceftriaxone and erythromycin could not prevent the migration of DNA band through the agarose gel up to a concentration of 500 µg/ml (Figs. 4G and 4H).

In vitro synergistic effects of peptide/peptide and peptide/antibiotic combinations

The in vitro antibacterial activity of peptide/peptide and peptide/antibiotic combinations was evaluated using the chequerboard dilution assay. Our results reveal that combinations of hybrid peptides (RN7-IN10, RN7-IN9, RN7-IN8, RN7-IN7, and RN7-IN6) with each other showed synergistic effects, with FICI of less than 0.5 (Table 2), regardless of the susceptibility of pneumococcal isolates towards standard drugs. Likewise, combinations of standard drugs ceftriaxone and erythromycin with all five hybrid peptides presented synergistic effects with fractional inhibitory concentration (FIC) index of ≤ 0.5 against both isolates of S. pneumoniae, regardless of their susceptibility to antibiotics (Table 2). These results indicate that all the hybrid peptides were able to enhance the antibacterial activity of both the standard drugs ceftriaxone and erythromycin.

In vivo toxicity of hybrid peptides

The in vivo toxicity of four hybrid peptides namely RN7-IN10, RN7-IN9, RN7-IN8 and RN7-IN6 was evaluated following a three dose regimen with the mice at 1 h, 12 h and 24 h using three different administration routes. The results revealed that in the case of mice treated with all hybrid peptides via subcutaneous (SC) injection at the maximum dose (100 mg/kg), no animal death or hypersensitivity reactions were observed up to seven days post-treatment. Minor differences were noted for mice given RN7-IN10 via SC, which displayed significantly lower granulocytes (p = 0.0172) and ALP (p = 0.0037) (Table S1, highlighted in yellow), while Mice treated with RN7-IN6 displayed significantly higher platelet counts (p = 0.0487) in comparison with the control group (Table S1, highlighted in blue). Histopathological studies were performed with the lung, brain, liver, spleen and kidney of control and treated animals. No histological abnormalities were detected in the organs of any group, as all the tissue sections were normal and did not display differences with the control group (Fig. S1). Similarly, no abnormal physical behavior was noted upon giving the mice hybrid peptides via the intranasal (IN) route. However, treatment with RN7-IN9 displayed significantly lower MCV (p = 0.001) (Table S2, highlighted in yellow) than the control group. Mice treated with RN7-IN6 displayed significantly lower percentage of granulocytes (p = 0.0482) (Table S2, highlighted in blue), as compared to the control group. Histological examination of the organs collected from all the treated and control groups did not expose any histopathological changes (Fig. S2).

In terms of the intraperitoneal (IP) administration route, all four hybrid peptides caused death and/or high physical stress when injected at a concentration of 100 mg/kg. Therefore, low graded doses were attempted until we reached the maximum dose at which no signs of stress or abnormal behavior were evident. Hybrid peptides RN7-IN10 and RN7-IN8 did not display any sign of toxicity when injected at 20 mg/kg; no death occurred in any of the treated mice up to seven days post-treatment. RN7-IN9 and RN7-IN6 were non-toxic when injected at 10 mg/kg. None of the five major organs of the treated mice revealed any significant histological abnormality, as compared to the untreated control group (Fig. S3). However, mice treated with RN7-IN9 (10 mg/kg) via IP route showed significantly lower lymphocytes (p = 0.0445) and lower ALP (p = 0.0187) (Table S3, highlighted in yellow), while RN7-IN6 treated mice had lower ALT (p = 0.0425) when compared to the control group (Table S3, highlighted in blue).

In vivo antibacterial efficacy of hybrid peptides

Two peptides, RN7-IN10 and RN7-IN8, which showed the fastest killing kinetics (Jindal et al., 2015) and exhibited less toxic effects in vivo were selected to evaluate their in vivo antibacterial efficacy via IP route. Both hybrid peptides were tested at three different doses (5 mg/kg, 10 mg/kg and 20 mg/kg) in three treatment regimens (1 h, 12 h and 24 h post-infection). In the pneumococcal bacteremia model, both RN7-IN10 and RN7-IN8 failed to treat any of the infected mice at 5 mg/kg. At a dose of 10 mg/kg, 10% of the infected mice survived after treatment with RN7-IN10 (p = 0.0018), whereas 30% of the mice was able to survive after treatment with RN7-IN8 (p = 0.0002). However, at a dose of 20 mg/kg, 30% of the mice treated with RN7-IN10 survived (p < 0.0001), while 50% of the mice survived when treated with hybrid peptide RN7-IN8 (p = 0.0002) (Fig. 5). No pneumococci were detected from the blood of the mice that survived and none of the mice showed presentation of illness, as compared to the untreated group which was severely ill and inactive. Treatment via SC and IN routes had no impact on infected mice up to seven days post-infection and none of the mice survived.

In addition to our designed peptides, ceftriaxone, as a standard drug, was used to treat infected mice via IP route at 5 mg/kg, 10 mg/kg, 20 mg/kg, 40 mg/kg, and 80 mg/kg and the survival was 10%, 30%, 40%, 70% and 90% up to seven days post-infection (p < 0.0001) (Fig. 6). In the pneumococcal pneumonia model, none of the mice treated with both peptides via IP, SC and IN routes survived up to seven days post-infection and therefore, this model was excluded from further studies.

Combinations of peptide-peptide were also assessed for their ability to treat infected mice with pneumococcal bacteremia. Two combinations were used, 5 mg/kg + 5 mg/kg and 10 mg/kg + 10 mg/kg to treat mice via IP route at three regimens 1 h, 12 h and 24 h. The results indicate that the combination of 5 mg/kg + 5 mg/kg was able to treat 40% of the mice and protected them from death up to seven days post-infection (p = 0.0003), while increasing the dose to 10 mg/kg of each peptide resulted in 60% of the infected mice surviving the pneumococcal infection (p < 0.001) (Fig. 7). These findings are in agreement with our in vitro synergism results which showed that hybrid peptides are able to enhance the biological activity of each other.

In vivo synergy assessment of hybrid peptide RN7-IN8 in combination with ceftriaxone

Among the hybrid peptides RN7-IN10 and RN7-IN8, standalone treatment with RN7-IN8 at 20 mg/kg was found to confer significant survivability on mice infected by a highly virulent pneumococcal clinical isolate via IP route. To assess the synergistic effect of RN7-IN8 in combination with the standard drug ceftriaxone (CTX), three different doses of RN7-IN8 (5 mg/kg, 10 mg/kg and 20 mg/kg) and ceftriaxone (5 mg/kg, 10 mg/kg and 20 mg/kg) were tested, using the same bacteremia infection model in three treatment formulations: RN7-IN8₅–CTX₅(5 mg/kg of RN7-IN8 and 5 mg/kg of CTX), RN7-IN8₁₀–CTX₁₀(10 mg/kg of RN7-IN8 and 10 mg/kg of CTX) and RN7-IN8₂₀–CTX₂₀(20 mg/kg of RN7-IN8 and 20 mg/kg of CTX). Using groups of 10 mice, the combinations of RN7-IN8 and ceftriaxone RN7-IN8₅–CTX₅(5 mg/kg of RN7-IN8 and 5 mg/kg of CTX), RN7-IN8₁₀–CTX₁₀(10 mg/kg of RN7-IN8 and 10 mg/kg of CTX) and RN7-IN8₂₀–CTX₂₀(20 mg/kg of RN7-IN8 and 20 mg/kg of CTX) led to survival rates of 60%, 80% and 100% in mice infected with highly virulent pneumococcal strain up to seven days post-infection (p < 0.0001) (Fig. 8). Our results displayed that treatment using combinations of peptide-antibiotics conferred higher survival rate than peptide and antibiotic in their stand-alone form. In addition, all treated mice which survived from the infection appeared physically active and none of them showed signs of abnormal behavior.

Histopathological evaluation

All the histopathological examinations of the mice infected with bacteremia model with and without treatment are presented in Figs. 9 and 10. Out of the five major organs examined, the lung and spleen of the infected animals were the most severely affected. A number of histopathological changes were observed in the lung of the infected mice. As compared to the uninfected control group, the lung of the infected and untreated group exhibited extensive vascular congestion with foci consolidation. Heavy permeation of the red blood cells into the alveolar spaces strongly denoted pulmonary hemorrhage (Fig. 9A, arrow a). The greatly congested lung appeared with little alveolar spaces (Fig. 9A, arrow b). This is in contrast to the uninfected group, where the normal lung displayed greatly aerated alveolar spaces with a thin layer of the alveolar wall (Fig. 9B). Severe tissue injuries was also noticed in the spleen of the infected group (Fig. 10A). Unlike the normal spleen which showed normal red and white pulps (Fig. 10B, arrow b), the infected spleen demonstrated depleted splenocytes with no white matter/germinal center (Fig. 10A, arrow a). No significant histopathological lesions were observed in other organs, such as the brain, liver, kidney and heart.

For the respective treatments of infected mice including hybrid peptide RN7-IN8 at 20 mg/kg, combination of hybrid peptides RN7-IN10 and RN7-IN8 (10 mg/kg + 10 mg/kg) and combination of RN7-IN8 and ceftriaxone (5 mg/kg + 5 mg/kg, 10 mg/kg + 10 mg/kg and 20 mg/kg + 20 mg/kg), it was noticed that although lesions, inflammatory events and the degree of tissues damage were found in the organs, the degree and severity of the damage were significantly less than the infected control group. Unlike the lung of the untreated mice which exhibited severe inflammation and the alveolar spaces were about 90% congested (Fig. 9A), all the lungs harvested from the treated mice revealed only low level of congestion and minor thickening of the alveolar wall, even though these histological changes were still noticeable in the mice (Figs. 9C–9G). Treatment of infected mice with a combination of hybrid peptide RN7-IN8 and ceftriaxone at three different dosages (5 mg/kg – 5 mg/kg, 10 mg/kg – 10 mg/kg and 20 mg/kg – 20 mg/kg) showed gradual decrease in the degree of congestion and damage (Figs. 9E–9G). Lungs harvested from mice treated with a combination of RN7-IN8 and ceftriaxone at 20 mg/kg + 20 mg/kg (Fig. 9G) which presented 100% mice survival, were similar to those harvested from the uninfected control mice (Fig. 9B) in degree of normality. Likewise, all the spleens of treated mice displayed no or minimum damage, with the white and red pulps being clearly observed (Figs. 10C–10G), as compared to the infected one (Fig. 10A). No significant tissue damage was observed in the brain, kidney and liver in both treated and untreated mice.