In vivo efficacy and synergistic interaction of 16α-hydroxycleroda-3, 13 (14) Z-dien-15, 16-olide, a clerodane diterpene from Polyalthia longifolia against methicillin-resistant Staphylococcus aureus

In vivo efficacy and synergistic interaction of 16α-hydroxycleroda-3, 13 (14) Zdien-15, 16-olide, a clerodane diterpene from Polyalthia longifolia against methicillin-resistant Staphylococcus aureus Vivek Kumar Gupta, Surjeet Verma, Anirban Pal, Santosh Kumar Srivastava, et al. Applied Microbiology and Biotechnology ISSN 0175-7598 Volume 97 Number 20 Appl Microbiol Biotechnol (2013) 97:9121-9131 DOI 10.1007/s00253-013-5154-9

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Author's personal copy Appl Microbiol Biotechnol (2013) 97:9121–9131 DOI 10.1007/s00253-013-5154-9

APPLIED MICROBIAL AND CELL PHYSIOLOGY

In vivo efficacy and synergistic interaction of 16α-hydroxycleroda-3, 13 (14) Z-dien-15, 16-olide, a clerodane diterpene from Polyalthia longifolia against methicillin-resistant Staphylococcus aureus Vivek Kumar Gupta & Surjeet Verma & Anirban Pal & Santosh Kumar Srivastava & Pramod Kumar Srivastava & Mahendra P. Darokar Received: 7 March 2013 / Revised: 8 July 2013 / Accepted: 27 July 2013 / Published online: 30 August 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract The Staphylococcus aureus bacterium, a nosocomial pathogen often causing untreatable and lethal infection in patients, mutated to become resistant to all the first-line drugs. The present study details the potential of clerodane diterpene 16α-hydroxycleroda-3, 13 (14) Z-dien-15, 16-olide (CD) isolated from Polyalthia longifolia against methicillin-resistant S. aureus (MRSA) through in vitro and in vivo assays. Minimum inhibitory concentration (MIC) of CD exhibited significant anti-MRSA activity (15.625–31.25 mg/l) against reference strain and seven clinical isolates, while time kill assays at graded MICs indicated 2.78–9.59- and 2.9–6.18-fold reduction in growth of reference strain and clinical isolates of S. aureus, respectively. The combined effect of the CD and 7.5 % NaCl resulted in significant reduction in microbial count within 24 h, indicating the loss of the salt tolerance ability of S. aureus. Further, release of 260-nm absorbing material and flow cytometric analysis revealed an increased uptake of propidium iodide. These assays may indicate the membrane-damaging potential of CD. The molecule CD was found to interact synergistically with clinically used antibiotics (FICI≤0.5) against all clinical isolates. In infected mice, CD significantly (P4.0=antagonism, and FICI>0.5–4=no interaction (Odds 2003).

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FIC A ¼ MIC of A in combination=MIC of A alone FIC B ¼ MIC of B in combination=MIC of B alone FICI ¼ FIC ðAÞ þ FIC ðBÞ :

Toxicity assessment In vivo toxicity in Swiss albino mice was studied in accordance with the ‘Organisation for Economic Co-operation and Development (OECD)’ test guideline No. 423 (Gupta et al. 2012). Briefly, three groups of six female mice were taken: group I served as vehicle control (healthy and uninfected), while groups II and III were administered with 100 mg/kg body weight of CD (i.p.) for 12 days. Group II did not receive any infection, while group III was infected with SA-96. The animals were observed for body weight changes apart from any form of morbidity or mortality, upon which blood was collected for various physiological parameters. The animals were sacrificed for gross organ necropsy and the relative organ weights. Blood was collected for haematological and serum biochemical parameters which were analysed through an autoanalyser (Daytona Randox, USA). Statistical analysis One-way analysis of variance was used to analyse the mean values obtained for the treatment and vehicle groups. Tukey’s test was used to compare the treatment and vehicle groups, and statistical significance was set at p≤0.001.

Fig. 2 Structure of bioactive compound (CD)

m, H-2), 5.19 (1H, m, H-3), 1.70 (1H, m, H-6a), 1.19 (1H, m, H-6b), 1.44 (2H, m, H-7), 1.45 (1H, m, H-8), 1.32 (2H, m, H-10), 1.64 (1H, m, H-11a), 1.50 (1H, m, H-11b), 2.26 (2H, m, H-12), 5.83 (1H, s, H-14), 6.03 (1H, s, H-16), 0.81 (3H, d, J=5.1Hz, H-17), 1.58 (3H, s, H-18), 1.00 (3H, s, H-19), 0.77 (3H, s, H-20); 13C NMR (CDCl3, 75 MHz): δ 18.7 (C-1), 27.2 (C-2), 120.8 (C-3), 144.7 (C-4), 38.6 (C-5), 37.1 (C-6), 27.8 (C-7), 36.7 (C-8), 39.1 (C-9), 46.9 (C-10), 35.2 (C-11), 21.8 (C-12), 171.5 (C-13), 117.2 (C-14), 172.5 (C-15), 99.8 (C-16), 16.4 (C-17), 18.4 (C-18), 20.3 (C-19), 18.6 (C-20).

Ethical clearance Anti-staphylococcal activity The study was approved by the Institutional Bio-safety Committee and Institutional Animal Ethics Committee under the Committee for the Supervision and Experimentation on Animals, Ministry of Environment, Government of India.

Results Isolation and characterization of bioactive constituent CD from P. longifolia The bioactive compound isolated and purified as described earlier was subjected to various spectroscopic analyses. The structure, which was deduced as 16α-hydroxycleroda-3, 13dien-15, 16-olide (1) on the basis of its 1D and 2D NMR, mass and other spectroscopic data (Fig. 2), was further confirmed by comparing all the above spectroscopic data to that reported in literature (Misra et al. 2010): [α]D =−66.1(c=0.7 in CHCl3), ESIMS: m/z 319 [M+H]+, 1 H NMR (CDCl3, 300 MHz): δ 1.52 (2H, m, H-1), 2.03 (2H,

MIC and interaction studies of CD with antibiotics The MIC of CD against seven clinical isolates of MRSA was observed to be in the range of 15.625–31.25 mg/l, while it was 15.625 mg/l against reference strain SA-96 (Table 1). The susceptibility of the clinical isolates of S. aureus (MRSA) to β-lactam and other antibiotics is summarized in Table 1. The combination of CD and oxacillin (β-lactam antibiotic) showed maximum synergy with 10–80-fold reduction in MIC (FICI=0.223 to 0.409) against MRSA isolates. The combination of CD and tetracycline also exhibited strong synergistic interaction, resulting in 4–16-fold reduction (FICI=0.321 to 0.448). The combination of CD with daptomycin showed synergistic effect only against four isolates MRSA-ST 1745, MRSA-ST 2071, MRSA-P 4620 and MRSA-B 10760 with four- to eightfold reduction (FICI=0.325 to 0.450). Similarly, CD in combination with linezolid exhibited synergistic effect only against two isolates MRSA-ST 1745 and MRSA-ST 2071 with fourfold reduction

Author's personal copy 0.699 0.699 0.699 0.781 0.781 0.781 0.650 0.450 0.680 0.3125 0.3125 0.3125 0.448 0.323 0.324

OXA, oxacillin, TET tetracycline, DAP daptomycin, LZD linezolid

FICI: ≤0.5, synergy (bold font indicates synergistically active combinations); 0.5–4, no interaction; 0.4, antagonism

6.25 3.125 6.25 0.409 0.248 0.223 6.25 12.5 25 1.56 1.56 1.56 1.25 1.25 0.65 25 50 25 500 250 500 Pus Blood Sputum MRSA (P 4423) MRSA (B 10760) MRSA (ST 3151)

15.625 15.625 15.625

0.781 0.781 0.781 0.781 0.35 0.325 0.450 0.650 0.625 0.3125 0.3125 0.3125 0.321 0.321 0.350 0.448 6.25 6.25 6.25 6.25 0.25 0.30 0.30 0.409 50 50 50 6.25 3.125 3.125 1.56 1.56 2.5 2.5 1.25 1.25 50 50 25 50 1,000 500 1,000 500 31.25 31.25 31.25 15.625

MIC of LZD FICI MIC of DAP FICI MIC of TET FICI TET

DAP

LZD

MIC of OXA

MIC of TET in the presence of CD

Sputum Sputum Pus Pus

Reduction in the cell viability of S. aureus on agar medium supplemented with 7.5 % NaCl in the presence of CD at 0.5 MIC, MIC and 2MIC concentrations was observed. This treatment reduced the ability of S. aureus to tolerate salt by about threefold at 0.5MIC and MIC concentrations. Parallel to this observation, it was 1.53- and 3.5-fold at 2MIC concentrations in case of clinical isolate MRSA-ST 2071 and reference strain SA-96, respectively, after 24 h of treatment (Fig. 6).

MRSA (ST 1745) MRSA (ST 2071) MRSA (P 4620) MRSA (P 4627)

Loss of salt tolerance

OXA

The treatment of bacterial cells with 4MIC of CD after 30, 60 and 90 min resulted in higher release of cell materials in both strains, which, in turn, produced higher values of OD260nm as compared to that of control. Similarly, at MIC and 2MIC concentrations, an increase in OD260nm was observed. However, at MIC and 2MIC concentrations, no significant difference was observed in OD260nm of both strains (SA-96 and MRSA-ST 2071) over the time period at 30, 60 and 90 min (Fig. 5).

MIC of OXA in the presence of CD

Loss of 260-nm absorbing material (cytoplasmic leakage assay)

MIC of antibiotics alone (mg/l)

The results of the bacteriolysis assay are depicted in Fig. 4, which indicate that there was an initial increase in OD620nm for both strains (SA-96 and MRSA-ST 2071) at MIC, 2MIC and 4MIC till 16 h, after which there was a moderate reduction in OD for 2MIC and 4 MIC at 24 h in the reference strain (SA-96), while in case of the clinical strain (MRSA-ST 2071), there was a moderate reduction of all MIC concentrations at 24 h.

CD (mg/l)

Bacteriolysis assay

Origin

Mode of action studies

Strains

The treatment of clinical isolate MRSA-ST 2071 and a reference strain SA-96 with CD at MIC, 2MIC and 4MIC concentrations reduced the viability significantly. In case of clinical isolate MRSA-ST 2071, viability reduced up to sixfold, while in case of the reference strain, reduction was up to ninefold at 4MIC after 24 h of incubation (Fig. 3). However, at MIC concentration, reduction in viability was only up to threefold in both strains.

Table 1 In vitro inhibitory activity (MIC) of CD alone as well as in combination with β-lactam and other antibiotics against clinical isolates of S. aureus (MRSA)

Bacterial-killing assay

MIC of DAP in the presence of CD

MIC of LZD in the presence of CD

(FICI=0.3499 to 0.4499) (Table 1). Since MRSA-ST 2071 showed higher resistance towards the maximum number of antibiotics (data not shown), it was selected for further study.

0.3499 0.4499 0.600 0.699

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Fig. 3 Time kill curves of SA-96 (a) and MRSA-ST 2071 (b) upon treatment with CD and vancomycin at MIC, 2MIC and 4MIC concentrations. One percent dimethyl sulphoxide was used as control. The data are expressed as mean±SEs

Flow cytometric analysis (propidium iodide uptake assay)

Toxicity studies of CD

Bacterial cells treated with 4MIC of CD for 0, 8 and 24 h resulted in the identification of two sub-populations, PIpositive and PI-negative cells representing dead and viable cells, respectively. The effect of CD was observed to be time dependent, since after 8 h of incubation, the rate of dead cells was 46.17±0.24 %, which was increased to 86.17±0.32 % within 24 h in case of clinical isolate MRSA-ST 2071. Similarly, in case of the reference strain, the rate of dead cells increased from 58.03±0.39 to 88.05±0.47 % within 24 h (Fig. 7).

Both in healthy and septicaemic animals, no significant changes in behavioural, haematological and serum biochemical parameters were observed after the administration of CD (i.p.) at 100 mg/kg body weight over the period of 12 days. Since the concentration of CD used here (100 mg/kg body weight) is eight times higher than the minimum effective dose of CD (12.5 mg/kg body weight), it was considered to be safe. However, a change in body weight was observed in infected mice with and without treatment of CD (11.35 and 5.71 %, respectively) in comparison to control (Table 2).

Anti-staphylococcal efficacy of CD in systemically infected mice

Discussion

The staphylococcal loads on various tissues and blood upon treatment with CD at various doses ranging from 12.5 to 100 mg/kg body weight are shown in Fig. 8a, b, respectively. In kidney, liver, spleen and lung tissues, a significant reduction in bacterial load was observed (P