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Journal of Meningitis
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Resveratrol Susceptibility of Streptococcus pneumoniae and Neisseria meningitidis Strains Isolated in the State of Minas Gerais, Brazil, from 2007 to 2013

Dhian Renato Almeida Camargo1,2, Policarpo Ademar Sales Junior3, Marluce Aparecida Assunção Oliveira2 and Roney Santos Coimbra1*

1Neurogenomics, Centro de Pesquisas René Rachou, FIOCRUZ. Av. Augusto de Lima, 1715, Barro Preto, Belo Horizonte, MG, Brazil, 30190-002

2Service of Bacterial and Fungal Diseases, Fundação Ezequiel Dias (FUNED). Rua Conde Pereira Carneiro, 80, Gameleira, Belo Horizonte, MG, Brazil, 30510-010

3Functional Genomics and Proteomics of Leishmania ssp and Trypanosoma cruzi, Centro de Pesquisas René Rachou, FIOCRUZ. Av. Augusto de Lima, 1715, Barro Preto, Belo Horizonte, MG, Brazil, 30190-002

*Corresponding Author:
Roney Santos Coimbra
Department of Neurogenomics
Research Centre René Rachou
FIOCRUZ. Av. Augusto de Lima, 1715
Barro Preto, Belo Horizonte
MG, Brazil, CEP 30190-002
Tel: +55-31-3349-7871
Fax: +55-31-3295-3115
E-mail: [email protected]

Received Date: October 29, 2015 Accepted Date: November 19, 2015 Published Date: November 28, 2015

Citation: Camargo DRA, Junior PAS, Oliveira MAA, Coimbra RS (2015) Resveratrol Susceptibility of Streptococcus pneumoniae and Neisseria meningitidis Strains Isolated in the State of Minas Gerais, Brazil, from 2007 to 2013. J Meningitis 1:101. doi: 10.4172/2572-2050.1000101

Copyright: © 2015 Camargo DRA, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Objective: Evaluation of the in vitro susceptibility to Resveratrol of a bacterial collection representing the S. pneumoniae and N. meningitidis strains prevalent in the Brazilian state of Minas Gerais from 2007 to 2013. Methods: One reference strain of S. pneumoniae (ATCC 49619), and sixty-three strains (31 S. pneumoniae, and 32 N. meningitidis) isolated from patients with meningitis and available at the certified strains collection of Ezequiel Dias Foundation were tested. The susceptibility to Resveratrol was tested on blood agar containing this drug at eight concentrations ranging from 25 mg/L to 200 mg/L diluted in 0.5% ethanol, and control plates with blood agar with 0.5% ethanol. Pneumococci were also tested for susceptibility to currently available antimicrobials used to treat meningitis using E-test and disc diffusion methods. The association between pneumococcal susceptibility to Resveratrol and to any other antibiotic tested was assessed with chi-square test, and the toxic doses of Resveratrol were determined upon L929 mammalian cells. Results: The MIC100 for Resveratrol was 75 mg/L for meningococci (range: 50-75 mg/L), and 200 mg/L for pneumococci (range: 125-200 mg/L). There was no association between pneumococcal susceptibility to Resveratrol and to any currently available antimicrobials tested suggesting different modes of action. However, low selectivity indices (SI) calculated as the ratio between the IC100 in L929 cells and the MIC values were found for meningococci (0.332) and pneumococci (0.125). Conclusion: Resveratrol inhibited the growth of all N. meningitidis and S. pneumoniae strains causing meningitis in the state of Minas Gerais, Brazil, from 2007 to 2013. Our results, despite the low selectivity indices observed, may warrant further studies to assess the potential of Resveratrol derivates as antimicrobial alternatives to treat meningococcal and pneumococcal infections.

Keywords

Streptococcus pneumonia; Neisseria meningitides; Resveratrol; 3,5,4’-trihydroxylstilbene; Bacterial meningitis; Antibacterials; Natural Compounds

Introduction

Neisseria meningitidis and Streptococcus pneumoniae are the most prevalent etiological agents of bacterial meningitis in Brazil [1]. S. pneumoniae resistance to penicillin has become a major global concern, and resistance has been linked to worse clinical outcomes in patients with pneumococcal meningitis [2]. In a recently published report, the World Health Organization warned that its six surveillance regions had national reports of 25% resistance or more for pneumococci, in some cases exceeding 50% resistance or nonsusceptibility to penicillin [3]. Despite the urgent need for new antimicrobials, the high cost and complexity of the process of new antibiotics discovery has hindered the release of new drugs. Therefore the repurposing of existing drugs with known pharmacokinetics and safety profiles turns out to be an attractive approach to fight against multi resistant bacteria. Aligned with this purpose, Docherty and collaborators [4] reported that Resveratrol (3,5,4’-trihydroxylstilbene) selectively inhibited one reference strain of N. meningitidis (ATCC 13090). Resveratrol is a natural compound produced by grapevines, peanuts and other plants in response to interactions with pathogens. Antifungal, antibacterial, antiviral, and antiparasitic activities of Resveratrol have been reported [5-8].

In the present study we investigated the in vitro susceptibility to Resveratrol of a bacterial collection representing the S. pneumoniae and N. meningitidis strains prevalent in the Brazilian state of Minas Gerais from 2007 to 2013.

Materials and Methods

The bacterial collection tested in this study comprised sixty-three strains (31 S. pneumoniae, and 32 N. meningitidis) isolated from patients with meningitis, which have been identified using standard methods and maintained in the certified strains collection of Ezequiel Dias Foundation. In addition, one reference strain of S. pneumoniae (ATCC 49619) was also included in this study. The susceptibility to Resveratrol (Sigma-Aldrich, St Louis, MO), expressed as the minimum inhibitory concentration (MIC), was determined on blood agar containing this drug at eight concentrations ranging from 25 to 200 mg/L diluted in 0.5% ethanol, and control plates with blood agar with 0.5% ethanol [4]. All strains were tested in duplicates and incubated at 37°C, for 24 h, under ambient air or atmosphere with 5% CO2. MIC100 was defined as the lowest concentration of Resveratrol that completely inhibited any visible growth.

Pneumococci were also tested for susceptibility to ceftriaxone and penicillin using E-test method, and to chloramphenicol, clindamycin, erythromycin, ofloxacin, oxacylin, rifampicin, tetracycline, trimethoprim-sulfametoxazol, and vancomycin using the disc diffusion method (Probac, São Paulo, Brazil). Experimental procedures and interpretation of results were performed according to clinical and laboratory standards institute (CLSI) clinical breakpoints [9]. In order to shed light on the mode of action of Resveratrol, we tested the association between the susceptibility to this drug and to the antibiotics mentioned above. Briefly, pneumococci were divided into two groups according to their MIC100 to Resveratrol: a) 200 mg/L; b) ≤ 175 mg/L. Then, a confusion matrix was built for each antibiotic tested distributing the strains into the groups “a” and “b” above according to their susceptibility or resistance to the respective antibiotic (Table 1 exemplifies a confusion matrix). Associations were assessed with chi-square test using GraphPad 5.0 (GraphPad Software, San Diego, CA).

    RSV MIC100=200 mg/L RSV MIC100 ≤ 175 mg/L
Susceptible Strains 731/09; 148/10; 080/11; 84/11; 143/11; 149/11; 511/11; 43/12 317/08; 144/09; 435/09; 779/09; 345/11
  N 8 5
Resistant or Intermediate Strains 883/07; 1070/07; 1180/07; 176/08; 305/08; 262/08; 619/08; 159/08; 620/09; 127/10; 421/10; 029/11; 120/11; 197/11; 380/12 295/08; 585/10; 124/11
  N 15 3

Table 1: Confusion matrix of trimethoprim-sulfametoxazol resistance versus Resveratrol MIC100 of Streptococcus pneumoniae strains. MIC: minimum inhibitory concentration.

Aiming to determine the toxic doses of Resveratrol upon L929 mammalian cells (mouse C3H/An connective tissue-ATCC CCL-1; cultured in Roswell Park Memorial Institute medium (RPMI)+2 mM Glutamine+10% foetal bovine serum (FBS)), after 4 days of compound exposure, 10% Alamarblue dye (Invitrogen, San Diego, CA) was added and the absorbance at 570 and 600 nm was measured after 4-6 h. The cell viability was expressed as the percentage of difference in the reduction between treated and untreated cells [10].

Results and Discussion

Resveratrol inhibited all S. pneumoniae and all N. meningitidis strains tested (Tables 2 and 3). Some strains showed higher MICs when incubated under atmosphere with 5% CO2 than in ambient air. The MIC100 was 75 mg/L for N. meningitidis (MIC range: 50 mg/ml-75 mg/L), and 200 mg/L for S. pneumoniae (MIC range: 125 mg/L-200 mg/L). It is worth noting that the reference strain of N. meningitidis tested by Docherty and collaborators [4] had MIC of 125 mg/L.

Strain ID Year of isolation Age (years) Gender Serotype CEF CLI CO ERI OF OXA PEN RIF ST TT VAN MIC100 RSV (mg/L)-ambient air MIC100 RSV (mg/L) - atmosphere with 5% CO2
883/07 2007 41 F 11A S S S S S S S S R S S 200 200
1070/07 2007 57 M 11F S S S S S S S S R S S 200 200
1180/07 2007 29 M 6A/C S S S S S S S S S S S 200 200
176/08 2008 33 M 7C S S S S S S S S R S S 200 200
295/08 2008 2 F 1 S S S S S S S S R S S 150 150
305/08 2008 9 F 6A/C S S S S S S S S R S S 200 200
317/08 2008 57 F 34 S S S S S S S S S S S 125 125
262/08 2008 9 F 6A/C S S S S S S S S R S S 200 200
619/08 2008 46 M 6A/C S S S S S S S S R S S 200 200
159/09 2009 77 M 23B S S S S S R R S R S S 200 200
144/09 2009 77 M 35B S S S S S S S S S S S 150 175
435/09 2009 71 M 28A S S S S S S S S S R S 125 125
620/09 2009 22 F 9N S S S S S S S S I S S 200 200
731/09 2009 1 M 7F S S S S S S S S S S S 125 200
779/09 2009 58 M 15C S S S S S S S S S S S 150 175
127/10 2010 41 M 6C S S S S S R R S R S S 200 200
148/10 2010 12 M 13 S S S S S S S S S S S 150 200
421/10 2010 75 F 10A S S S S S S S S R S S 125 200
585/10 2010 50 F 9V S R S R S S S S R S S 175 175
029/11 2011 55 M 23F S S R S S R R S R R S 175 200
080/11 2011 12 M 24F S S S S S S S S S S S 200 200
84/11 2011 3 F 12F S S S S S S S S S S S 200 200
120/11 2011 4 F 18C S S S S S S S S I S S 200 200
124/11 2011 13 M 19A S S S S S S R S R S S 150 175
143/11 2011 3 M 6B S S S S S S S S S S S 200 200
149/11 2011 <1 F 9N S S S S S S S S S S S 200 200
345/11 2011 <1 M 4 S S S S S S S S S S S 175 175
197/11 2011 4 F 3 S S S S S R R S R S S 175 200
511/11 2011 45 M 9V S S S S S S S S S S S 200 200
380/12 2012 32 M 14 S S S R S R R S R S S 200 200
43/12 2012 48 M 3 S S S S S S S S S S S 150 200

Table 2: Epidemiological data and antimicrobial susceptibility of Streptococcus pneumoniae strains. Epidemiological data and results of susceptibility tests to antibiotics and Resveratrol of pneumococci strains causing meningitis in Minas Gerais, Brazil, from 2007 to 2013. M: male; F: female; S: susceptible; I: intermediate; R: resistant; MIC: minimum inhibitory concentration; CEF: ceftriaxone; CLI: clindamycin; CO: chloramphenicol; ERI: erythromycin; OF: ofloxacin; OXA: oxacylin; PEN: penicillin; RIF: rifampicin; TT: tetracycline; ST: trimethoprimsulfametoxazol; VAN: vancomicin; RSV: Resveratrol

Strain ID Year of isolation Age (years) Gender Sero group MIC100 RSV (mg/L)-ambient air MIC100 RSV (mg/L) - atmosphere with 5% CO2
78/08 2008 7 F Y 50 50
92/08 2008 73 M W135 75 75
264/08 2008 14 F B 75 75
563/08 2008 58 F W135 75 75
100/09 2009 NA NA W135 75 75
119/09 2009 NA NA W135 75 50
281/09 2009 15 M Y 75 75
24/10 2010 12 F C 50 75
35/10 2010 50 M C 50 75
57/10 2010 57 F C 50 50
56/10 2010 11 M C 50 50
108/10 2010 44 F C 75 75
131/10 2010 5 F B 75 75
132/10 2010 1 F Y 50 50
138/10 2010 64 M C 50 50
177/10 2010 5 F C 75 75
201/10 2010 6 F B 50 75
215/10 2010 10 M C 50 50
42167 2012 21 F C 50 75
42320 2012 6 F C 75 75
15/12 2012 4 M B 75 75
78/12 2012 30 M C 50 75
86/12 2012 15 F C 75 75
96/12 2012 22 M C 50 75
101/12 2012 40 M C 50 75
170/12 2012 NA NA NA 50 50
175/12 2012 <1 M B 50 50
326/12 2012 73 F W135 75 75
345/12 2012 18 M B 75 75
616/12 2012 <1 F B 50 75
843/12 2012 18 F B 75 75
17/13 2013 39 M C 75 75

Table 3: Epidemiological data and Resveratrol susceptibility of Neisseria meningitidis strains. Epidemiological data and results of susceptibility test to Resveratrol of meningococci strains causing meningitis in Minas Gerais, Brazil, from 2007 to 2013. M: male; F: female; NA: non available; MIC: minimum inhibitory concentration; RSV: Resveratrol

Although the antimicrobial activity of Resveratrol and some of its derivatives and natural oligomers has been demonstrated against various pathogenic bacteria, the mode of action of this drug has not been elucidated yet [11,12]. Preliminary studies suggest that the antibacterial activity of Resveratrol involves "quorum sensing" proteins, hyperpolarization of the bacterial membrane potential, inhibition of macromolecules biosynthesis, and inhibition of the virulence factor type III secretion system (T3SS) [13-15]. For the 32 pneumococci tested, there was no association between high (200 mg/L) or low (≤ 175 mg/L) levels of Resveratrol susceptibility and the susceptibility to any of the antibiotics tested. These findings suggest that Resveratrol and the antibiotics often used to treat pneumococcal infections do not share the same mode of action.

Furthermore, 58.1% of pneumococci clinical isolates, representing the strains occurring in Minas Gerais from 2007 to 2013, were nonsusceptible (resistant or intermediate) to trimethoprimsulfamethoxazole, while only a low percentage were resistant to erythromycin (6.4%), or clindamycin (3.2%). All isolates tested in the present study were susceptible to ceftriaxone. Similar results had been previously reported by Mantese and collaborators [16] who found 79.5% nonsusceptibility to trimethoprim-sulfamethoxazole, 11.3% resistance to erythromycin, 11.3% to clindamycin, and 5.6% to ceftriaxone (5.6%) among Brazilian pneumococci isolates. In Germany, the overall pneumococci non-susceptibility rates were 11.0% for trimethoprim-sulfamethoxazole, 5.5% for clindamycin, 0.7% for levofloxacin, and 8.5% for tetracycline [17].

Resveratrol at 25 mg/L induced 100% cellular death (IC100) in L929. The Selectivity Index (SI), a parameter used to estimate the therapeutic dose window of a candidate drug, was calculated as the ratio between the IC100 in L929 mammalian cell line and the MIC values. The SIs were 0.332 for meningococci and 0.125 for pneumococci, indicating low therapeutic selectivity.

In conclusion, Resveratrol inhibited the growth of all N. meningitidis and S. pneumoniae strains causing meningitis in the state of Minas Gerais, Brazil, from 2007 to 2013. To our knowledge, this is the first report of pneumococcal susceptibility to Resveratrol, and the first study to assess the antibacterial effect of this drug against a strain collection with epidemiological relevance for meningitis.

The lack of association between pneumococcal susceptibility to Resveratrol and the antibiotics tested herein suggests that the former does not share the same mode of action with the currently available antimicrobials used to treat meningitis. Our results, despite the low selectivity indices observed, may warrant further studies to assess the potential of Resveratrol derivates as antimicrobial alternatives to treat meningococcal and pneumococcal infections.

Acknowledgement

The authors thank the program for technological development of tools for health-PDTIS-Fiocruz for use of its facilities. RSC and MAAO conceived this study, and participated in its design and coordination and helped draft the manuscript. DRAC and PASJ carried out the microbiological and statistical analysis and helped drafted the manuscript. All authors read and approved the final manuscript.

Conflict of interest

The authors have no conflict of interest to declare.

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