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A clerodane diterpene from Polyalthia longifolia as a modifying agent of the resistance of methicillin resistant Staphylococcus aureus.

ABSTRACT

Background: Staphylococcus aureus infections are raising serious concern across the world. The effectiveness of conventional drugs is continuously decreasing due to global emergence of multidrug resistance (MDR) and therefore, new resistance-modifying agents (RMAs) are highly needed.

Hypothesis: Clerodane diterpene 16[alpha]-hydroxycleroda-3,13(14)-Z-dien-15,16-olide (CD) from leaves of Polyalthia longifolia (Sonn.) Thwaites (Annonaceae) as RAM will be useful in improving the current treatment strategies for staphylococcal infections.

Study design: In the present study, we determine the resistance-modifying activity of CD using clinical isolates of MRSA. Further, the influence of CD on innate immune response was also evaluated in vitro and in vivo. The nature of potential interactions was determined by fractional inhibitory concentration indices (FICIs) calculated from microdilution assays and time-kill curves.

Results: The result of in vitro combination study showed that CD significantly reduced MIC of fluoroquinolones up to 16-folds (FICI 0.315-0.500), while in S. aureus infected Swiss albino mice model, combination of CD with norfloxacin, significantly (p < 0.01, p < 0.001) lowered the systemic microbial burden in blood, liver, kidney, lung and spleen tissues in comparison to CD, norfloxacin alone as well as untreated control. Flow cytometry analysis clearly showed that CD significantly inhibited EtBr efflux and extended post-antibiotic effect. In qRT-PCR analysis, CD alone as well as in combination, significantly modulated the expression of various efflux pump genes including norA up to 2-fold in clinical isolate MRSA-ST2071. Further, the in vitro combination study of the CD (10, 5, 2.5 [micro]g/ml) along with the norfloxacin (10 [micro]/ml) depicted a significant decline in the pro-inflammatory cytokines, 1L6 and TNF-[alpha]. In septic shock mice model, CD did not exhibit any significant changes in the level of pro-inflammatory cytokines.

Conclusion: This is the first report on drug resistance-modifying potential of CD through inhibition of MDR efflux pump.

Keywords:

Clerodane diterpene

MRSA

Fluoroquinolones

Efflux pump inhibitor

Resistance- modifying agents

Introduction

Efflux mediated multidrug resistance (MDR) has become serious issue in the management of bacterial infections (Costa et al. 2013). MDR is associated with the over-expression of transporter proteins that efficiently expel a broad range of structurally different compounds resulting in reduced intracellular concentration to an ineffective level (Webber and Piddock 2003; Rouveix 2007). A number of clinically relevant bacteria, most remarkably Staphylococcus aureus utilize these transporter proteins as part of their resistance strategy. NorA, a multidrug transporter protein of S. aureus confers resistance to a broad spectrum of compounds including fluoroquinolones, reserpine, and ethidium bromide (Poole 2000; DeMarco et al. 2007). Apart from NorA, other efflux pumps, NorB and NorC, also contribute to quinolone/fluoroquinolone resistance in S. aureus (Truong-Bolduc et al. 2005, 2006). Emergence of MDR pathogenic bacterial strains has increased attention in pharmaceutical research for identifying novel efflux pump inhibitors (EPIs). Several plant-derived compounds, such as reserpine, baicalein, the diterpenoids totarol and carnosic acid are known to inhibit NorA-induced ethidium bromide efflux (Stavri et al. 2007; Abreu et al. 2012). Previously, we reported potent anti-Methicillin resistant S. aureus (MRSA) activity of the clerodane diterpene 16[alpha]-hydroxycleroda-3,13(14)-Z-dien-15,16-olide (CD) Fig. 1 isolated from leaves of Polyalthia longifolia (Sonn.) Thwaites (Annonaceae) by disrupting the bacterial membrane (Gupta et al. 2013). In the present study, we further investigated synergistic interactions and resistance-modifying potential of CD against clinical isolates of MRSA through in vitro and in vivo assays.

Material and methods

Bacterial strains and growth media

Clinical isolates of MRSA (n = 15) were procured from Clinical Microbiology Laboratory of Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPG1MS) Lucknow, India. Clinical isolates were characterized and maintained as reported earlier (Gupta et al. 2012). The strains were grown in Muelier-Hinton broth (MHB; Hi-media, India), which was equilibrated with Tris buffered saline (Sigma-Aldrich, St Louis, MO, USA).

Antimicrobial agents

CD was isolated and characterized as described earlier (Gupta et al. 2013). The experimental optical rotation of CD was [[alpha]].sub.D] = -66.1[degrees](c = 0.7 in CHC13), which was in complete agreement to that reported in the literature (Misra et al. 2010). The purity of CD was determined by HPLC (Shimadzu LC-20A) equipped with a PDA detector. HPLC analysis was carried out on a reversed-phase water 30 mm x 4.6 mm, 5 [micro]m C18 column maintained at room temperature with a flow rate of 0.6ml/min. 20 [micro]l aliquots of CD were injected and UV detection was made at 220 nm. The mobile phase was composed of MeCN/H.sub.2]O (92:08, v/v). The HPLC analysis showed that the purity of CD was > 99% (Fig. S1). Fluoroquinolone antibiotics, norfloxacin, ciprofloxacin and ofloxacin were purchased from Sigma-Aldrich, St Louis, MO, USA.

In vitro combination study

The fractional inhibitory concentrations (FICs) were evaluated by the checkerboard assays (Novy et al. 2011). Serial dilutions (2-fold) of fluoroquinolone antibiotics such as norfloxacin, ciprofloxacin and ofloxacin (1000-1.953 [micro]g/ml) prepared in horizontal rows of microtiter plates were subsequently diluted vertically by 2-fold serial dilutions of CD (100-0.78 [micro]g/ml). The synergy between CD and the antibiotics was evaluated with the fractional inhibitory concentration index (FICI). F1C1 is a simple mathematical approach to describe interactions quantitatively described previously by Elion et al (1954).

FIC (antibiotics) = MIC of antibiotics in combination / MIC of antibiotics alone

FIC (CD) = MIC of CD in combination / MIC of CD alone

FICI = FIC (antibiotics) + FIC (CD)

FICI < 0.5 = synergy, FICI > 0.5-4 = 0.5-4 = additivity/ indifference, FICI > 4.0 = antagonism (Bonapace et al. 2000).

Time-kill studies

Time-kill study of the combination of norfloxacin and CD was evaluated using a method as described previously (Eliopoulus and Moellering 1996). Each analysis was done in triplicate with a control without test sample. Time-kill curves were derived by plotting logio cfu/ml against time (h). Synergy was defined as bactericidal activity ([greater than or equal to] 2 log10 difference in CFU/ml) of the combination compared to the single agent after 24 h incubation (Hu et al. 2002).

Ethidium bromide accumulation and efflux assay

Ethidium bromide (EtBr) accumulation assay was carried out using flow cytometer (LSR II, BD Bioscience, USA) following the method described earlier by Kalle and Rizvi (2011). Efflux inhibition assay was done spectrofluorometrically (FluoStar Omega, BMG, Germany) by measuring increase in fluorescence intensity of EtBr over the time using the clinical isolate MRSA-ST2071 as reported earlier by Kalia et al. 2012.

Post-antibiotic effect (PAE)

PAE was determined as described previously (Kalia et al. 2012). An inoculum (final 1.5 x [10.sup.6] CFU/ml) of clinical isolate MRSAST2071 was prepared by adding 0.05 ml of an inoculum having turbidity equivalent to 0.5 McFarland standard (1.5 x [10.sup.8] CFU/ml) into each tube containing 5 ml fresh broth with norfloxacin at 1/2 MIC concentration alone as well as in combination with CD. Samples (0.005 ml) from each tube were diluted (1:1000) with fresh broth (5 ml) and incubated at 37[degrees]C with shaking for 2h. A sample from each tube was subjected to viability counts on MHA before exposure and immediately after dilution (0 h), and thereafter every 1 h until visual turbidity was observed in the control tube. The PAE was calculated by the viable count method wherein the difference in the time for growth in the exposed culture (treated) and the corresponding unexposed control (control) was recorded and represented by the formula.

PAE = Treated - Control

Real time PCR (qRT-PCR) analysis

The expression of different efflux pump genes (norA, norB, norC, mepA, mdeA) was analyzed in the presence of CD alone (0.25 MIC), norfloxacin alone (0.25 MIC) as well as in combination (0.25 MIC CD+ 0.25 MIC norfloxacin) in the treated and non-treated bacterial cells of MRSA-ST2071. Cells were grown to mid-log phase in the presence of sub-lethal concentrations of CD and norfloxacin alone as well as in combination. The real-time quantification of RNA template was analyzed by SYBR GreenER qPCR supermix (Invitrogen, Carlsbad, CA, USA) using 7900HT fast real time PCR system (Applied Biosystems, USA). Observations were recorded in terms of LogRQ after normalization with 16S rRNA which was used as an endogenous control (Couto et al. 2008).

Effect of CD alone and in combination on innate immune mechanism

The effect of the CD alone and its combination with norfloxacin were studied for its effect on the innate immune system through their exposure to peritoneal macrophages isolated from healthy mice under aseptic conditions (Agrawal et al. 2013).

The stock solutions of CD, norfloxacin and their combinations were maintained in DMSO. A volume of 10 [micro]l of these solutions was instilled in the macrophages to achieve a final concentration of CD at 10, 5 and 2.5 [micro]g/ml. Further, the cells were stimulated by lipopolysaccharides, LPS (E. coli 050: B5; Sigma Chemical Co.) at 1 [micro]g/ml and allowed to incubate for 16-18 h. The remaining medium was collected for further cytokine quantification, while the plate with adherent cells was subjected to MTT assay. TNF-[alpha] and IL-6 in culture supernatants were quantified through ELISA using BD OptEIA kit as per manufacturer's instructions (Becton Dickinson, USA).

Effect of treatments on septic shock in vivo

CD was orally administered at three dosages 5, 50 and 100mg/kg in Swiss mice (20 [+ or -] 2g). After 90 min, the animals were intraperitoneally injected with lipopolysaccharide, LPS (200 micro]g/kg) and observed for any acute reactions. Blood was collected after 90 min to obtain the serum for cytokine analysis.

In vivo anti-staphylococcal activity of combination of CD with norfloxacin

The therapeutic efficacy of the combinations (CD and norfloxacin) was evaluated through intraperitoneal (i.p.) route using Swiss albino mice model. Five groups, each with six female mice (5-6 weeks old, weighing 18-22 g), were infected by intravenous injection of 0.2 ml ([10.sup.6] CFU/ml) of S. aureus (SA-96). Combinations at different doses (interaperitonial route) Nor at 0.3125 +CD at 12.5, Nor at 0.3125 + CD at 25mg/kg, along with norfloxacin alone (1.25, 0.625 and 0.312 mg/kg) and CD alone (25 and 12.5mg/kg body weight) comprised the treatment groups. The vehicle control group was administered also through intraperitoneal (i.p.) route with an equivalent volume of 0.1 % cremophor (Fluka, VA, USA). The treatment commenced 2h after the infection and continued till day 7 post-infection once daily. Blood was collected from the retro-orbital plexus 24 h after the last dose for bacterial load which was estimated through plate counting on brain heart infusion agar. All the animals were then sacrificed for the collection of lung, liver, kidney and spleen tissues. The tissue homogenates were prepared in 2 ml of chilled, normal saline solution with a glass tissue homogenizer under aseptic conditions. Homogenates were suitably diluted and plated on agar plates to enumerate the bacterial load per gram of tissue (Gupta et al. 2012; Patel et al. 2004). Bacterial elimination was assessed by comparing the reduction of bacterial load of each organ in the infected groups and vehicle control.

Statistical analysis

One-way analysis of variance was used to analyze 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 [less than or equal to] 0.01, p [less than or equal to] 0.001.

Ethical clearance

The study was approved (Protocol number AH-2012-07) 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

In vitro combination study

The combination of CD with norfloxacin, showed strong synergistic interaction with 4- to 16- folds reduction against 9 of the 15 clinical isolates tested with FICI 0.315-0.500 while the remaining 6 combinations exhibited additive interaction (FICI 0.624-0.750). The CD/ciprofloxacin combination showed synergistic interaction with 4- to 8-folds reduction against 6 clinical isolates (FICI 0.324). In combination with ofloxacin, CD showed synergistic interaction with 4- to 8-folds reduction against 8 clinical isolates of MRSA with FICI 0.324 (Table 1). Since CD exhibited up to 16-fold reduction in MIC of norfloxacin against MRSA-ST2071, it was used in further experiments. MRSA-ST2071 also exhibited resistance toward most of the clinically used antibiotics (data not shown).

Combination effect of CD with norfloxacin on bacterial killing kinetics

Time-kill kinetics study revealed that CD and norfloxacin alone, each one at 1/2MIC concentration diminished the viability of bacterial cells (MRSA-ST2071) by 2-fold after 24 h of incubation. The combination of 15.62 [micro]g of CD/ml and 250 [micro]g of norfloxacin/ml (concentrations that are one-half of their MICs), diminished the viability of bacterial cells up to 4-folds resulting after 24 h of incubation indicates synergistic bactericidal activity (Fig. 2a). Norfloxacin, ciprofloxacin and ofloxacin alone, that are used as positive controls diminished the viability of bacterial cells (MRSA-ST2071) by up to 8-fold after 24 h of incubation at 4MIC concentration (Fig. 2b, c and d).

EtBr accumulation and efflux assay

EtBr accumulation assay using flow cytometer clearly indicates that CD blocks the MDR efflux pump of MRSA-ST2071 as shown by increase in EtBr fluorescence in the cells. Significant shift (1.671-fold) in the intensity of red fluorescence was observed inside the cells in the presence of CD as compared to untreated control (Fig. 3a), Similarly, spectrofluorometric assay revealed the decrease in EtBr efflux over the time period of 30 min in case of CD treated cells as compared to untreated bacterial cells (Fig. 3b).

Results of PAE

PAE of CD and norfloxacin alone as well as in combination were determined at sub minimum inhibitory concentrations (0.5MIC). PAE value obtained was 1.1 h for norfloxacin and 1.4 h for CD against MRSA-ST2071 alone, while in combination, it was 2.33 h (Table 2).

qRT-PCR analysis

qRT-PCR analysis of different efflux pump genes (norA, norB, norC, mepA, and mdeA) revealed that in the presence of norfloxacin, all genes were found to be over-expressed, while in combination (norfloxacin and CD), down regulation of these genes was observed as compared to norfloxacin alone by 1.98 (norA), 1.72 (norB), 1.74 (norC), 1.97, (mepA), and 1.91 (mdeA) folds (Fig. 4).

Effect of the treatments on innate immune mechanism

The in vitro combination study of CD (10, 5 and 2.5 [micro]g/ml) along with norfloxacin (10 [micro]g/ml) depicted a significant decline in pro-inflammatory cytokines, IL-6 and TNF-[alpha] (Table 3). Norfloxacin treated macrophages showed a concentration-dependent lowering in the level of cytokines, while CD also depleted the stimulated re sponse against the LPS treatment (p < 0.001). CD in combination with norfloxacin was able to reduce the level of cytokines significantly. The highest concentration in combination i.e., norfloxacin at 10 [micro]g/ml along with CD at 10 [micro]g/ml showed a 4-fold depletion in IL-6 titer (571.199 [+ or -] 4.094) and 6-fold decrease in TNF-[alpha] level (56.735 [+ or -] 1.495) when compared to the LPS stimulated macrophages. A similar significant decrease was also observed at lower concentrations of 5 and 2.5 [micro]g/ml. Since norfloxacin at 10|xg/ml was found to deplete the cytokines most effectively, an analysis of variance in comparison to the combination groups was also made. However, norfloxacin at 10 [micro]g/ml in combination with CD at 10 and 5 [micro]g/ml significantly (p< 0.001) decreased the level of both cytokines.

Effect of CD treatments in septic shock mice model

In the animals induced with septic shock through LPS, there was an increased production of cytokines along with the appearance of diarrhea. The treatment of CD at three doses (5, 50 and 100mg/kg) in LPS stimulated septic shock mice did not show any significant changes in the level of pro-inflammatory cytokines (Table 4).

In vivo anti-staphylococcal activity of combination

The staphylococcal burden on various tissues and blood upon treatment with combination of CD and norfloxacin at various doses are shown in Fig. 5a and 5b, respectively. In kidney, liver, spleen and lung tissues, a significant reduction in staphylococcal burden was observed (p < 0.001) in a dose-dependent manner. Similar observations were recorded in case of blood (p < 0.01) as well (Fig. S2). Norfloxacin alone was used as positive control against SA-MTCC96.

Discussion

Efflux pumps have been implicated in MDR generation toward numerous classes of antibiotics in both Gram-positive as well as Gram-negative bacteria (Mandal et al. 2014). Combination of efflux pump inhibitor (EPI) with antibiotics is expected to increase the intracellular concentration of antibiotics that are expelled by efflux pumps, decrease the intrinsic bacterial resistance, reverse the acquired resistance associated with efflux pumps over-expression, and reduce the frequency of the emergence of multidrug resistant strains (Zechini and Versace 2009; Holler et al. 2012). In continuation of our previous report describing anti-MRSA activity of CD (Gupta et al. 2013), we further investigated the synergy/ resistance-modifying potential (RAMs) of CD with fluoroquinolone antibiotics and its efflux pump inhibitory potential. It was observed that CD could significantly reduce the MICs of norfloxacin, ciprofloxacin and ofloxacin with FICI [less than or equal to] 0.50 (Table 1). Since CD exhibited up to 16-fold reduction in MIC of norfloxacin against the clinical isolate MRSA-ST2071 was used in further experiments. Time-kill curve represents a useful method for evaluating the kinetic interactions between bacterial cells and antimicrobial agents. Results of time-kill kinetics revealed that CD in combination with norfloxacin at 1/2AMIC concentrations of each one significantly reduced the viability of bacterial cells in comparison to CD and norfloxacin alone (Fig. 2a).

Flow cytometric analysis clearly showed that CD inhibits an MDR efflux pump which was evident from the significant shift in the intensity of red fluorescence indicating the accumulation of EtBr inside the bacterial cells. Similar observations were also reported earlier by Kalle and Rizvi (2011), wherein celecoxib was found to inhibit bacterial MDR efflux pump. This observation was further validated via spectrofluorometric assay and the results showed a decrease in EtBr efflux over the time period of 30 min in case of CD treated cells as compared to untreated bacterial cells. This observation indicates a strong interference of CD in the efflux of EtBr (Fig. 3a, 3b).

CD prolonged the post-antibiotic effect of norfloxacin which is the phenomenon of continued suppression of growth after a short exposure of bacterial cells to the antimicrobial agents (Kalia et al. 2012) as shown in Table 2. The result of both the efflux pump inhibition and the PAE were found to be comparable with the known efflux pump inhibitor reserpine (Kumar et al. 2008).

Most of the antibiotics during the treatment of infections are transported outside from bacterial cells by over expression of efflux pump gene products (Mullin et al. 2004; Piddock 2006). In S. aureus, the major facilitator super family (MFS) proteins are predominant, followed by multidrug and toxin extrusion (MATE) family (Kaatz et al. 2005). Over-expression of genes of these families like norA, norB, norC, mdeA and mepA confer MDR in S. aureus (Li and Nikaido 2009).

The over-expression of MFS and MATE family efflux genes such as norA, norB, norC, mdeA and mepA were observed upon the treatment of bacterial cells with norfloxacin while in presence of CD alone as well as in combination (norfloxacin and CD), down-regulation of these genes was observed as compared to norfloxacin alone. The result of expression study was found to be comparable with positive control reserpine (Fig. 4). A number of known compounds (synthetic and natural molecules) such as berberine, 5-methoxyhydnocarpin, reserpine (Wright and Sutherlan 2013), palmatine (Kumar et al. 2013) and capsaicin (Kalia et al. 2012) have been reported as inhibitors of major facilitator efflux pumps. The in vitro combination study of the CD (10, 5 and 2.5 [micro]g/ml) along with norfloxacin (10 [micro]g/ml) depicted a significant decline in the pro-inflammatory cytokines, 1L-6 and TNF-[alpha] in comparison to the CD and norfloxacin alone indicating toward the potent role of the CD in developing a better enhanced response (Table 3). In in vivo septic shock model, we observed that the diarrheal condition in the CD-treated mice were of much less acute nature than the LPS induced untreated counterparts. However, CD did not exhibit any significant changes in the level of pro-inflammatory cytokines (Table 4). This might be due to the fact that the septic shock model through LPS induction is an acute shock model (90 min) and the effects of test compounds might not have been metabolized to generate an expected response.

In vitro combination data was further validated through in vivo experiments using Swiss albino mice and during the experimental period no mortality was recorded in any group. Combination of CD with norfloxacin exhibited significant reduction (p< 0.001; p < 0.01) in systemic staphylococcal burden of lung, liver, kidney and spleen tissues along with blood. Combination of norfloxacin with CD (0.3125 +12.5/25 mg/kg) exhibited up to 1.8-folds staphylococcal load reduction in different tissues and blood in comparison to untreated controls (Fig. 5).

Conclusion

This is the first report on drug resistance-modifying potential of CD through inhibition of MDR efflux pump. CD can enhance the efficacy of fluoroquinolones antibiotics and showed the potential in combination therapy for the treatment of MRSA infections.

http://dx.doi.org/10.1016/J.phymed.2016.03.001

ARTICLE INFO

Article history:

Received 5 January 2015

Revised 27 February 2016

Accepted 2 March 2016

Abbreviations: MIC, minimum inhibitory concentrations; CD, clerodane diterpene; CFU, colony-forming unit; CLSI, Clinical and Laboratory Standards Institute; FICI, fractional inhibitory concentration index; MTCC, microbial type culture collection; MFS, major facilitator superfamily; MRSA, methicillin-resistant Staphylococcus aureus: NOR, norfloxacin; CIP, ciprofloxacin; OFX, ofloxacin; RES, reserpine; EPI, efflux pump inhibitor; MDR, multi drug resistant; LPS, lipopolysaccharide; MATE, multidrug and toxin extrusion; RAMs, resistance- modifying agents; PAE, post antibiotic effect; EtBr, ethidium bromide; IL, interleukin; TNF, tumor necrosis factor; ST, sputum; P, pus; B, blood.

Conflict of interest

The authors declare that they have no conflict of interest Acknowledgment

We are grateful to Prof. K.N. Prasad, Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGIMS) Lucknow, India for providing clinical isolates of MRSA. VKG is thankful to Indian Council of Medical Research, New Delhi for Senior Research Fellowship. This work was a part of in-house project MLP-02 with funding from CSIR Network Project BSC0203 CSIR-CIMAP, Lucknow, India.

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2016.03.001.

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Vivek Kumar Gupta (a), Nimisha Tiwari (a), Priyanka Gupta (a), Surjeet Verma (b,1), Anirban Pal (a), Santosh Kumar Srivastava (b,1), Mahendra Pandurang Darokar (a), *

(a) Molecular Bioprospection Department, CSIR-Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow- 226015, India

(b) Medicinal Chemistry Division, CSIR-Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow- 226015, India

* Corresponding author. Tel.: +91 522 2718532; fax: +91 522 2342666.

E-mail address: mpdarokar@yahoo.com, mp.darokar@cimap.res.in (M.P. Darokar).

(1) www.cimap.res.in

Table 1
In vitro antibacterial activity of CD alone as well as in
combination with fluoroquinolone antibiotics against clinical
isolates of S. aureus.

Strains        Origin   CD               MIC of antibiotics
                        ([micro]g/ml)   alone (([micro]g/ml))

                                        NOR   CIP     OFX

MRSA-ST1745    Sputum   31.25           500   62.50   62.50
MRSA-ST2071    Sputum   31,25           500   62.50   62.50
MRSA-P4620     Pus      31.25           250   62.50   62.50
MRSA-P4627     Pus      15.62           500   62.50   62.50
MRSA-P4423     Pus      15.62           250   62.50   31.25
MRSA-B10760    Blood    15.62           500   62.50   62.50
MRSA-ST3151    Sputum   15.62           250   62.50   62.50
MRSA-ST5457    Sputum   15.62           125   62.50   31.25
MRSA-B6264     Blood    31.25           250   62.50   31.25
MRSA-P6642     Pus      15.62           125   31.25   31.25
MRSA-ST2438    Sputum   15.62           250   62.50   62.50
MRSA-B5944     Blood    15.62           500   62.50   31.25
MRSA-87700     Blood    15.62           250   62.50   31.25
MRSA-B10732    Blood    31.25           250   62.50   62.50
MRSA-ST10342   Sputum   31.25           250   62.50   62.50

Strains        MIC of NOR in presence      MIC of CIP in presence
               of CD (([micro]g/ml))       of CD (([micro]g/ml))

               MIC of                      MIC of
               NOR      FICI          FR   CIP      FICI          FR

MRSA-ST1745    62.50    0.375 (S)      8    7.81    0.324 (S)     8
MRSA-ST2071    31.25    0.315 (S)     16    7.81    0.324 (S)     8
MRSA-P4620     62.50    0.750 (A/I)    4    7.81    0.324 (S)     8
MRSA-P4627     62.50    0.375 (S)      8   15.62    0.750 (A/I)   4
MRSA-P4423     62.50    0.750 (A/I)    4    7.81    0.624 (A/I)   8
MRSA-B10760    62.50    0.375 (S)      8    7.81    0.624 (A/I)   8
MRSA-ST3151    31.25    0.375 (S)      8    7.81    0.624 (A/I)   8
MRSA-ST5457    31.25    0.500 (S)      4    7.81    0.624 (A/I)   8
MRSA-B6264     31.25    0.375 (S)      8    7.81    0.324 (S)     8
MRSA-P6642     31.25    0.750 (A/I)    4    7.81    0.749 (A/I)   4
MRSA-ST2438    62.50    0.750 (A/I)    4    7.81    0.624 (A/I)   8
MRSA-B5944     31.25    0.315 (S)     16    7.81    0.749 (A/I)   8
MRSA-87700     62.50    0.750 (A/I)    4   15.62    0.749 (A/I)   4
MRSA-B10732    31.25    0.500 (S)      8    7.81    0.324 (S)     8
MRSA-ST10342   62.50    0.750 (A/I)    4    7.81    0.324 (S)     8

Strains        MIC of OFX in presence
               of CD (([micro]g/ml))

               MIC of
               OFX      FICI          FR

MRSA-ST1745    7.81     0.324 (S)     8
MRSA-ST2071    7.81     0.324 (S)     8
MRSA-P4620     7.81     0.324 (S)     8
MRSA-P4627     7.81     0.324 (S)     8
MRSA-P4423     3.90     0.324 (S)     8
MRSA-B10760    7.81     0.624 (A/I)   8
MRSA-ST3151    7.81     0.624 (A/I)   8
MRSA-ST5457    7.81     0.749 (A/I)   4
MRSA-B6264     7.81     0.749 (A/I)   4
MRSA-P6642     7.81     0.749 (A/I)   4
MRSA-ST2438    7.81     0.324 (S)     8
MRSA-B5944     7.81     0.749 (A/I)   4
MRSA-87700     7.81     0.749 (A/I)   4
MRSA-B10732    7.81     0324 (S)      8
MRSA-ST10342   7.81     0.324 (S)     8

FICI: [less than or equal to] 0.5 = synergy; 0.5-4 =
additivity/indifference; >4 antagonism.

Abbreviations for interpretations: S, synergy; A/I,
additivity/indifference; ANT, antagonism; FIG, fractional
inhibitory concentrations index; FR, fold reduction; MIC,
minimum inhibitory concentration; CD, clerodane diterpene;
NOR, norfloxacin; CIP, ciprofloxacin; OFX, ofloxacin; MRSA,
methicillin resistant S, aureus; ST, sputum; P, pus; B, blood.

Table 2
Post-antibiotic effect (PAE) of norfloxacin alone
and in combination with CD against MRSA-ST2071.

Compounds   Mean PAE (h) + SD

            CD                   Norfloxacin

0.5MIC      1.40 [+ or -] 0.02   1.10 [+ or -] 0.02

Compounds   CD +                 Reserpine +
            norfloxacin          norfloxacin

0.5MIC      2.33 [+ or -] 0.27   2.15 [+ or -] 0.13

* Each value is the mean [+ or -] SD.

Abbreviations for interpretations: PAE, post-antibiotic effect;
CD, derodane diterpene; MIC, minimum inhibitory concentrations;

Table 3
Quantification of pro-inflammatory cytokines IL-6 and TNF-[alpha] upon
stimulation in macrophages with LPS in presence of CD and norfloxacin
alone as well as in combination.

      In vitro
(Dose in [micro]g/ml)     IL-6 (pg/ml) Mean [+ or -] SE

Norfloxacin (10)         877.163 [+ or -] 16.346 ***
Norfloxacin (5)          998.296 [+ or -] 4.861 ***
Norfloxacin (2.5)       1053.234 [+ or -] 41.742 ***
CD (10)                 1219.399 [+ or -] 14.457 ***
CD (5)                  1500.825 [+ or -] 13.745 ***
CD (2.5)                1603.087 [+ or -] 51.826 ***
Norfloxacin +CD (10)     571.199 [+ or -] 4.094 ***, (a)
Norfloxacin +CD (5)      737.409 [+ or -] 14.626 ***, (a)
Norfloxacin +CD (2.5)    825.734 [+ or -] 33.398 ***, ns
Cells+LPS+Media          2665.71 [+ or -] 226.82
Cells+Media                57.07 [+ or -] 16.207
Media                                   0

      In vitro
(Dose in [micro]g/ml)    TNF (pg/ml) Mean [+ or -] SE

Norfloxacin (10)        105.506 [+ or -] 2.476 ***
Norfloxacin (5)         122.210 [+ or -] 9.450 ***
Norfloxacin (2.5)       126.303 [+ or -] 2.525 ***
CD (10)                 146.619 [+ or -] 3.685 ***
CD (5)                  141.457 [+ or -] 1.081 ***
CD (2.5)                179.373 [+ or -] 4.609 ***
Norfloxacin +CD (10)     56.735 [+ or -] 1.495 ***, (a)
Norfloxacin +CD (5)      82.974 [+ or -] 3.662 ***, (a)
Norfloxacin +CD (2.5)    96.841 [+ or -] 2.565 ***, ns
Cells+LPS+Media         301.557 [+ or -] 7.45
Cells+Media             -66.788 [+ or -] 2.653
Media                                  0

Each value is the mean [+ or -] SE.

*** p < 0.001, Groups versus LPS.

(a) p < 0.001, Combinations groups (Nor + CD 10, 5 and 2.5
[micro]g/ml) versus norfloxacin (10 [micro]g/ml) ns, non significant.

Abbreviations for interpretations: LPS, lipopolysaccharide;
IL, interleukin; TNF, tumor necrosis factor; CD, derodane
diterpene; NOR, norfloxacin.

Table 4
Level of pro-inflammatory cytokines (IL6 and TNF-[alpha])
in Swiss albino mice treated with different doses of CD.

                      IL-6 (pg/ml)                  TNF (pg/ml)
In vivo             Mean [+ or -] SE             Mean [+ or -] SE

CD@5 mg/kg       6.403 [+ or -] 1.685       502.482 [+ or -] 262.834
CD@50 mg/kg     11.015 [+ or -] 2.870       276.905 [+ or -] 49.227
CD@100 mg/kg     9.690 [+ or -] 2.549       153.916 [+ or -] 93.706
LPS@200 ug/kg   15.705 [+ or -] 4.132 ***   230.641 [+ or -] 65.136 ***
Normal                      0                            0

Each value is the mean [+ or -] SE.

*** - p < 0.001, Normal versus LPS.

Abbreviations for interpretations: LPS, lipopolysaccharide;
IL, interleukin; TNF, tumor necrosis factor; CD, derodane diterpene.
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Author:Gupta, Vivek Kumar; Tiwari, Nimisha; Gupta, Priyanka; Verma, Surjeet; Pal, Anirban; Srivastava, Sant
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9INDI
Date:Jun 1, 2016
Words:5871
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