Antibacterial activity of Lamiaceae plant extracts in clinical isolates of multidrug-resistant bacteria

FELIPE V. DE ASSIS FLÁVIA L. SIQUEIRA ISABELA E. GONÇALVES RAFAEL P. LACERDA RAFAELA A. NASCIMENTO STHÉFANE G. ARAÚJO JÉSSICA T. ANDRADE KARINA M.S. HERRERA LUCIANA A.R.S. LIMA JAQUELINE M.S. FERREIRA About the authors

ABSTRACT

The antibacterial activity of plant extracts of the Lamiaceae family was evaluated against clinical isolates of multi-resistant Gram-negative bacteria by broth microdilution technique. Promising results were obtained considering that all extracts were active for at least two bacterial species with MIC ranging from 0.5 to 2.0 mg/mL.

Key words:
antibacterial; clinical isolates; Lamiaceae extracts; multidrug-resistant

INTRODUCTION

Antibiotic resistance is spreading faster than the introduction of new compounds into clinical practice, causing a public health crisis (Ling et al. 2015LING LL ET AL. 2015. A new antibiotic kills pathogens without detectable resistance. Nature 517: 455-462.). Nosocomial bacterial infections have increased significantly, resulting in prolonged hospitalization, morbidity and treatment costs and hospital stay (Vincent et al. 2009VINCENT JL ET AL. 2009. International study of the prevalence and outcomes of infection in Intensive Care Units. JAMA 302(21): 2323-2329.). The increase of bacterial resistance to the main antibiotics used in therapy emphasize the need to develop new and more effective antibacterial drugs (Cataneo 2010CATANEO C. 2010. Sensibilidade dos critérios para isolamento de pacientes admitidos num hospital especializado em oncologia. Tese de Mestrado. Ribeirão Preto: Universidade de São Paulo, 84 p. (Unpublished)., Kumarasamy et al. 2010KUMARASAMY KK ET AL. 2010. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet Infect Dis 10(9): 597-602.).

The plant extracts are presented as a promising source for the search of new substances, because they have a higher molecular diversity when compared to products synthetic chemically (Novais et al. 2003NOVAIS T, COSTA JFO, DAVID JPL, DAVID JM, QUEIROZ LP, FRANÇA F, GIULIETTI AM, SOARES MBP AND SANTOS RR. 2003. Atividade antibacteriana em alguns extratos de vegetais do semi-árido brasileiro. Rev Bras Farmacogn 13(2): 8-11., Veiga Junior 2008, Surendra et al. 2016aSURENDRA TV AND ROOPAN SM. 2016a. Photocatalytic and antibacterial properties of phytosynthesized CeO2 NPs using Moringa oleifera peel extract. J Photochem Photobiol B 161: 122-128.). To be invaded by bacteria, fungi, parasites, viruses or other agents, plants synthesize molecules defense with antimicrobial activity (Haida et al. 2007HAIDA K, PARZIANELLO L, WERNER S, GARCIA DR AND INÁCIO CV. 2007. Avaliação in vitro da atividade antimicrobiana de oito espécies de plantas medicinais. Arq Ciênc Saúde Unipar 11(3): 185-192.). Plants contribute to a variety of chemical compounds with antimicrobial properties and scientific research to determine the therapeutic potential of these substances is relevant for possible antibiotic properties against resistant pathogens (Duarte et al. 2005DUARTE MCT, FIGUEIRA GM, SARTORATTO A, REHDER VL AND DELARMELINA C. 2005. Anti-Candida activity of Brazilian medicinal plants. J Ethnopharmacol 97: 305-311., Surendra et al. 2016b, Dinesh et al. 2017DINESH M, ROOPAN SM, SELVARAJ CI AND ARUNACHALAM P. 2017. Phyllanthus emblica seed extract mediated synthesis of PdNPs against antibacterial, heamolytic and cytotoxic studies. J Photochem Photobiol B 167: 64-71.). Thus, this study aims to evaluate the antimicrobial activity of ethanol extracts in prevalent multidrug-resistant Gram negative bacteria in nosocomial infections.

MATERIALS AND METHODS

The Lamiaceae species were collected in Carmópolis de Minas, Minas Gerais, Brazil, in April 2011. The plants materials were identified by Dr. Alexandre Salino and the voucher specimens were deposited at the Instituto de Ciências Biológicas Herbarium, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil (Mentha sp. BHCB 147244, Ocimum basilicum BHCB 147240, Plectranthus barbatus BHCB 147241, and Rosmarinus officinalis BHCB 147245).

The fresh plant material was extracted by cold maceration in ethanol P.A (Vetec, Brazil) for a period of 10 days at room temperature. After it was filtrate and concentrated in a rotary evaporator (IKA RV10, Germany) at 40 °C under reduced pressure and lyophilized (Líotop K105, Biobrás, Brasil) to yield ethanol extract (Mentha sp. 10.37 g, Ocimum basilicum 2.78 g, Plectranthus barbatus 3.17 g, and Rosmarinus officinalis 6.30 g) (Araújo et al. 2014ARAÚJO SG, ALVES LF, PINTO MEA, OLIVEIRA GT, SIQUEIRA EP, RIBEIRO RIMA, FERREIRA JMS AND LIMA LARS. 2014. Volatile compounds of Lamiaceae exhibit a synergistic antibacterial activity with streptomycin. Braz J Microbiol 45(4): 1341-1347.).

Twelve clinical isolates of multidrug-resistant Gram-negative bacteria were selected for this study, three of each species: Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa. Isolates from clinical samples including urine, tracheal aspirate and broncoalveolar lavage, were provided by Laboratório de Microbiologia do Hospital São João de Deus, Divinópolis, MG, Brazil. Underwent identification and antimicrobial susceptibility testing using the VITEK-2 (Bio-Merieux, Marcy l’Etoile, France) automated system.

The minimum inhibitory concentration (MIC) was determined by the microdilution broth method performed in accordance with the guidelines of the Clinical and Laboratory Standards Institute, with modifications (CLSI 2012). The extracts were diluted in dimethylsulfoxide (DMSO) (Sigma-Aldrich, USA) 20% in at concentrations 2, 1, 0.5 and 0.25mg/mL. Mueller Hinton broth (MH) medium (Himedia, India) without samples or solvents was used as a control for sterility. The antibiotics streptomycin 1.0 mg/mL plus penicillin 0.6 mg/mL (Sigma-Aldrich, USA) and DMSO (Sigma-Aldrich, USA) used in the dilution of the compounds were included as positive and negative controls, respectively. The MIC was assessed based on the lowest concentration of sample required to complete inhibit microbial growth detected as the lack of visible turbidity plus spectrophotometer (625 nm). The experiments were performed in triplicate and repeated three times in independent experiments.

RESULTS

The he profile resistance of clinical isolates performed by VITEK-2 (Bio-Merieux, Marcy l’Etoile, France) automated system were showed in Table I. The antimicrobial activity of ethanol extracts in prevalent multidrug-resistant Gram negative bacteria in nosocomial infections was evaluated. The values of MIC are shown in Table II.

TABLE I
Resistance profile of clinical isolates Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa against three antibiotic classes used in medical clinic.
TABLE II
Minimal inhibitory concentration (MIC) from crude extracts of Lamiaceae family against Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa multidrug-resistant bacteria strains.

The P. barbatus extract showed activity against strains of all species, with best results, with MIC ranging from 0.5 to 2.0 mg/mL. Mean while, O. basilicum extract was active against the A2 (A. baumannii) and E2 strains (E. coli), with both showing a MIC of 2.0mg/mL. The R. officinalis extract inhibited the growth of two A. baumannii strains, A1 and A2, with MIC of 1.0 and 2.0 mg/mL, respectively, and two K. pneumoniae strains, K2 and K3, with MIC of 2.0 and 0.5 mg/mL, respectively. Mentha sp. extract inhibited all P. aeruginosa strains and the A. baumannii A2 strain, showing MICs of 2.0 mg/mL. Negative control with DMSO 20% showed no activity.

DISCUSSION

In this work, have been used clinical samples that show resistance to major classes of antibiotics used in medicine, and the results show promising activity of the studied extracts. The activities of Lamiaceae species were superior than that of synthetic compounds Isoquinolin-1-yl-2- (cycloalk2-enylidene) hydrazines derivatives evaluated by Manivel et al. (2009MANIVEL P, ROOPAN SM, KUMAR RS AND KHAN FN. 2009. Synthesis of 3 substituted isoquinolin-1-yl-2-(cycloalk-2-enylidene) hydrazines and their antimicrobial properties. J Chil Chem Soc 54(2): 183-185.), which were inactive against E. coli, Proteus mirabilis, Salmonella typhi and S. aureus. In the present study, the best result of inhibition of antibacterial activity was obtained by extract of P. barbatus, which showed activity against all tested samples of species, presenting MIC of 0.5 mg/mL against a strain of P. aeruginosa. The extract of R. officinalis was active against two samples of A. baumannii and two samples of K. pneumoniae, with MICs ranging from 0.5 to 2.0 mg/mL, the results are also considered promising, however, the antimicrobial activity of the natural compounds of the Lamiaceae family could be potentiated through the use of silver nanoparticles, as described by Madhumitha et al. (2015MADHUMITHA G, ELANGO G AND ROOPAN SM. 2015. Bio-functionalized doped silver nanoparticles and its antimicrobial studies. J Sol-Gel Sci Technol 73: 476-483.).

Many studies have demonstrated antibacterial activity of plants commonly used by traditional medicine (Rakholiya and Chanda 2012RAKHOLIYA K AND CHANDA S. 2012. In vitro interaction of certain antimicrobial agents in combination with plant extracts against some pathogenic bacterial strains. Asian Pac J Trop Biomed, p. S876-S880., Tekwu et al. 2012TEKWU EM, PIEME AC AND BENG VP. 2012. Investigations of antimicrobial activity of some Cameroonian medicinal plant extracts against bacteria and yeast with gastrointestinal relevance. J Ethnopharmacol 142: 265-273.). Ríos and Recio (2005RÍOS JL AND RECIO MC. 2005. Medicinal plants and antimicrobial activity. J Ethnopharmacol 100: 80-84.) suggested that the presence of activity is very interesting in the case of concentrations below 0.1 mg/mL for extracts. On the other hand, Fabry et al. (1998FABRY W, OKEMO PO AND ANSORG R. 1998. Antibacterial activity of East African medicinal plants. J Ethnopharmacol 60(1): 79-84.) defined as active the crude extract with MIC < 8 mg/mL. In a study carried out by Holetz et al. (2002HOLETZ FB, PESSINI GL, SANCHES NR, CORTEZ DA, NAKAMURA CV AND FILHO BP. 2002. Screening of Some Plants Used in the Brazilian Folk Medicine for the Treatment of Infectious Diseases. Mem Inst Oswaldo Cruz 97(7): 1027-1031.) was considered that if the extracts displayed an MIC less than 0.1 mg/mL, the antimicrobial activity was good; from 0.1 to 0.5 mg/mL the antimicrobial activity was moderate; from 0.5 to 1.0 mg/mL the antimicrobial activity was weak; over 1.0 mg/mL the extract was considered inactive. In this study, however, MIC lower than 1.0 mg/mL were considered as promising.

Several studies have demonstrated antibacterial activity of plant extracts. Gemechu et al. (2013GEMECHU A, GIDAY M, WORKU A AND AMENIET G. 2013. In vitro anti-mycobacterial activity of selected medicinal plants against Mycobacterium tuberculosis and Mycobacterium bovis strains. BMC Complement Altern Med 13: 291.) tested the methanol extract of Ocimum basilicum against Mycobacterium tuberculosis and M. bovis. The MIC ranged from 0.025 to 0.1 mg/mL, and from 0.025 to 0.050 mg/mL, respectively. Extract of P. betle was evaluated against multidrug-resistant bacteria, P. aeruginosa, A. baumanni, E. coli and K. pneumoniae, and exhibited MIC ranging from 0.312 to 0.625 mg/mL (Valle et al. 2015VALLE DL, ANDRADE JI, PUZON JJM, CABRERA EC AND RIVERA WL. 2015. Antibacterial activities of ethanol extracts of Philippine medicinal plants against multidrug-resistant bacteria against clinical isolates of P. aeruginosa, A. baumannii, E. coli and K. pneumoniae. Asian Pac J Trop Biomed 5(7): 532-540.).

In a study conducted Kowero et al. (2016KOWERO EE, LEWERI C AND CHACHA M. 2016. Evaluation of antibacterial activity of five selected medicinal plants in Tanzania against Gram negative bacteria. Eur J Med Plant 12(2): 1-7.) P. barbatus displayed a relatively wide MIC range of 3.12 to 12.5 mg/mL. The P. barbatus extracts demonstrated MIC values of 3.12 mg/mL against S. typhi and K. oxytoca. Additionally, P. barbatus exhibited MIC value of 3.12 mg/mL against P. aeruginosa. Likewise, essential oils of P. barbatus showed antibacterial activity against E. coli, Proteus vulgaris, Bacillus cereus, P. aeruginosa, Staphylococcus aureus, and S. aureus (multidrug-resistant) (Galvão et al. 2013GALVÃO RFF, COSTA JGM, RODRIGUES FFG AND CAMPOS AR. 2013. Study of the interference between Plectranthus species essential oils from Brazil and aminoglycosides. Evid-Based Compl Alt 2013: 1-7.). These findings corroborate with the present findings, where promising antibacterial activity was found for P. barbatus extract against P. aeruginosa with MIC (0.5 to 1.0 mg/mL) lower than that reported by Kowero et al. (2016).

Additionally, Araújo et al. (2014ARAÚJO SG, ALVES LF, PINTO MEA, OLIVEIRA GT, SIQUEIRA EP, RIBEIRO RIMA, FERREIRA JMS AND LIMA LARS. 2014. Volatile compounds of Lamiaceae exhibit a synergistic antibacterial activity with streptomycin. Braz J Microbiol 45(4): 1341-1347.) observed that ethanol extracts exhibited a synergetic effect with streptomycin. Data showed synergistic effects of P. barbatus extract and streptomycin against P. aeruginosa ATCC 27853 and E. coli ATCC 43895 serotype O157:H7, as well as, synergistic effect of R. officinalis extract and streptomycin against E. coli ATCC 43895 serotype O157:H7.

In order to identify the compounds present in ethanol extracts of Lamiaceae family, Araújo et al. (2014ARAÚJO SG, ALVES LF, PINTO MEA, OLIVEIRA GT, SIQUEIRA EP, RIBEIRO RIMA, FERREIRA JMS AND LIMA LARS. 2014. Volatile compounds of Lamiaceae exhibit a synergistic antibacterial activity with streptomycin. Braz J Microbiol 45(4): 1341-1347.) investigated these extracts by gas chromatography/mass spectrometry (GC/MS). Phytol was identified in all ethanol extracts, camphor and verbenone compound are present in P. barbatus and R. officinalis, cadinene was identified in Mentha sp. and O. basilicum (Araújo et al. 2014). Santoyo et al. (2005SANTOYO S, CAVERO S, JAIME L, IBAÑES E, SENÕRÁNS FJ AND REGLERO G. 2005. Chemical composition and antimicrobial activity of Rosmarinus officinalis L. essential oil obtained via supercritical fluid extraction. J Food Prot 68: 790-795.) observed that camphor and verbenone showed activity against S. aureus and Escherichia coli. Vukovic et al. (2007VUKOVIC N, MILOSEVIC T, SUKDOLAK S AND SOLUJIC S. 2007. Antimicrobial activities of essential oil and methanol extract of Teucrium montanum. Evid Based Complement Alternat Med 4: 17-20.) demonstrated activity of cadinene against several microorganisms including P. aeruginosa and K. pneumoniae (Vukovic et al. 2007). These findings explain the activity of extracts against Gram negatives bacteria.

In view of the clinical importance of the bacteria studied in this work, the results obtained with the four extracts of Lamiaceae encourage new studies to isolate compounds and studying them to better elucidate its antimicrobial effect. This report probably enables the discovery of new antimicrobial compounds of those used in medical clinical.

ACKNOWLEDGMENTS

The authors are grateful to Prof. Dr. Alexandre Salino for his assistance in plant identification and to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and Universidade Federal de São João del-Rei (UFSJ) for the financial support.

REFERENCES

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Publication Dates

  • Publication in this collection
    16 Apr 2018
  • Date of issue
    Apr-June 2018

History

  • Received
    13 Dec 2016
  • Accepted
    20 Dec 2017
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