Bacteri
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  • 1. This article was downloaded by: [SENESCYT ]On: 12 December 2012, At: 15:26Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20 Anti-Staphylococcus aureus action of three Caatinga fruits evaluated by electron microscopy a b Luís Cláudio Nascimento da Silva , Jana Messias Sandes , c d Maurício Magalhães de Paiva , Janete Magali de Araújo , Regina b e Célia Bressan Queiroz de Figueiredo , Márcia Vanusa da Silva & a Maria Tereza dos Santos Correia a Laboratório de Glicoproteínas, Departamento de Bioquímica, Universidade Federal de Pernambuco, Pernambuco, Brazil b Laboratório de Biologia Celular, Departamento de Microbiologia, Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz – Pernambuco, Brazil c Laboratório de Microscopia Eletrônica e Microanálise, Centro de Tecnologias Estratégicas do Nordeste, Pernambuco, Brazil d Laboratório de Genética de Microrganismos, Departamento de Antibióticos, Universidade Federal de Pernambuco, Pernambuco, Brazil e Laboratório de Produtos Naturais, Departamento de Bioquímica, Universidade Federal de Pernambuco, Pernambuco, Brazil Version of record first published: 14 Sep 2012.To cite this article: Luís Cláudio Nascimento da Silva, Jana Messias Sandes, Maurício Magalhãesde Paiva, Janete Magali de Araújo, Regina Célia Bressan Queiroz de Figueiredo, Márcia Vanusa daSilva & Maria Tereza dos Santos Correia (2012): Anti-Staphylococcus aureus action of three Caatingafruits evaluated by electron microscopy, Natural Product Research: Formerly Natural ProductLetters, DOI:10.1080/14786419.2012.722090To link to this article: http://dx.doi.org/10.1080/14786419.2012.722090PLEASE SCROLL DOWN FOR ARTICLE
  • 2. Full terms and conditions of use: http://www.tandfonline.com/page/terms-and- conditions This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.Downloaded by [SENESCYT ] at 15:26 12 December 2012
  • 3. Natural Product Research 2012, 1–5, iFirst SHORT COMMUNICATION Anti-Staphylococcus aureus action of three Caatinga fruits evaluated by electron microscopy Luı´ s Claudio Nascimento da Silvaa, Jana Messias Sandesb, Maurı´ cio Magalhaes de ´ ˜ Paivac, Janete Magali de Araujod, Regina Celia Bressan Queiroz de Figueiredob, ´ ´ Marcia Vanusa da Silvae and Maria Tereza dos Santos Correiaa* ´ a Laborato´rio de Glicoproteı´nas, Departamento de Bioquı´mica, Universidade Federal de Pernambuco, Pernambuco, Brazil; bLaborato´rio de Biologia Celular, Departamento de Microbiologia, Centro de Pesquisas Aggeu Magalha˜es, Fundaca˜o Oswaldo Cruz – Pernambuco, ¸ Brazil; cLaborato´rio de Microscopia Eletroˆnica e Microana´lise, Centro de Tecnologias Estrate´gicasDownloaded by [SENESCYT ] at 15:26 12 December 2012 do Nordeste, Pernambuco, Brazil; dLaborato´rio de Gene´tica de Microrganismos, Departamento de Antibio´ticos, Universidade Federal de Pernambuco, Pernambuco, Brazil; eLaborato´rio de Produtos Naturais, Departamento de Bioquı´mica, Universidade Federal de Pernambuco, Pernambuco, Brazil (Received 26 March 2012; final version received 11 July 2012) This study evaluated the antibacterial activity of Anadenanthera colubrina, Libidibia ferrea and Pityrocarpa moniliformis fruit extracts against clinical strains of Staphylococcus aureus. The samples were active for all S. aureus strains (minimum inhibitory concentration: 0.38–3.13 mg mLÀ1), including the multi- resistant strain. The morphological changes suggested the cell wall as the main action target. The treated-cells also lose their ability to form aggregates. The analysis suggests cell wall impairment, which causes the loss of viability and death. This study showed for the first time the morphologic alterations involved in the anti-S. aureus action of fruits of A. colubrina, L. ferrea and P. moniliformis. These findings indicated that these fruit extracts are sources of bioactive compounds that can be used as antibacterial agents. Keywords: Anadenanthera colubrina; Libidibia ferrea; Pityrocarpa moniliformis; Staphylococcus aureus; bactericidal action mechanism; Caatinga biome 1. Introduction Staphylococcus aureus has been considered the most important human pathogen in the twenty-first century, due to its exceptional capacity to acquire resistance to antibiotics. This bacterium usually causes superficial skin infections and, occasionally, deep-seated infections that can spread through the blood stream and ultimately lead to sepsis. Staphylococcus aureus infections are also associated with intravascular devices and foreign bodies (Foster, 2005; Tang & Stratton, 2010). Taken together, these factors point to the urgent need for new antimicrobial agents against S. aureus. The Brazilian Caatinga is marked by an accentuated dryness (rainfall is usually less than 600 mm yearÀ1; Sampaio, Giuiietti, Virginio, & Gamarra-Rojas, 2002), its plants have developed unique chemical features that have made them an excellent source of bioactive antibacterial compounds. Indeed, many Caatinga plants are used in traditional medicine *Corresponding author. Email: mtscorreia@gmail.com ISSN 1478–6419 print/ISSN 1478–6427 online ß 2012 Taylor & Francis http://dx.doi.org/10.1080/14786419.2012.722090 http://www.tandfonline.com
  • 4. 2 L.C.N. da Silva et al. (Cartaxo, Souza, & Albuquerque, 2010) and various research works have confirmed their uses (da Silva et al., 2011). In this study we present the inhibitory activity of Anadenanthera colubrina (ACHE), Libidibia ferrea (LFHE) and Pityrocarpa moniliformis (PMHE) fruit extracts against different clinical strains of S. aureus and the ultra-structural alterations involved. 2. Results and discussion The extracts effectively inhibited the growth of S. aureus strains, including those with drug resistance (Table 1). The LFHE presented a higher inhibitory action (p 5 0.05) with minimum inhibitory concentration (MICs) ranging from 0.39 to 0.781 mg mLÀ1 and minimum bactericidal concentrations (MBCs) from 3.12 to 6.25 mg mLÀ1. ACHE and PMHE had the same variation of MIC (1.56–3.13 mg mLÀ1) and MBC (6.25 mg mLÀ1) (Table 1). DMSO did not affect the bacterial growth. Transmission electronic microscopy (TEM) of untreated S. aureus showed homoge- neous cytoplasm electron density, intact cell wall and a characteristic splittingDownloaded by [SENESCYT ] at 15:26 12 December 2012 system (Figure 1.1A–C). The extracts induced drastic morphological changes in S. aureus (Figure 1.1D–L). Treated bacteria presented a corrugated and thicker cell wall. Some cells showed cell wall detachment (black arrow in Figure 1.1E) and discontinuity of membranous profiles (white arrow in Figure 1.1F). The appearance of electron-lucent region in the cell periphery, the increase of cell volume and the presence of perforations in the bacterial surface (asterisk in Figure 1.1J) are indicative of disturbance in these structures (Dı´ az-Visurraga, Garcia, & Cardenas, 2010). We also observed abnormal division processes with alteration in the splitting complex and in some case the complete absence of a septum structure. The abnormally-shaped daughter cells might result from interference with the growth of cross-wall or from the displacement of the cross-wall formation site. In scanning electron microscopy (SEM), the control were heavily colonised with the formation of grape-like structures of dividing and normally-shaped bacteria (Figure 1.2A). Bacterial clusters were rarely observed in treated cultures (Figure 1.2B–I). The presence of a bubble protruding from the cell wall surface (arrow in Figure 1.2C and D) and holes in their cell walls were also observed (arrow in Figure 1.2E). A considerable increase in the cell volume and the appearance of perforation of the cell wall was commonly found in treated cells, mainly at MBCs (Figure 1.2F–I). The biofilm formation was also inhibited. The cells showed deep roughening, peeling of cell wall and small holes in surface (arrow in Figure 1.2H and I). In the phytochemical screening, flavonoids were found in all the extracts as well as carbohydrates such as maltose (ACHE and PMHE), glucose (ACHE) and sucrose (LFHE). The presence of gallic tannins was observed in LFHE (gallic acid and ellagic acid) and PMHE (gallic acid), while saponins were detected only in PMHE. 3. Experimental 3.1. Preparation of the hydroalcoholic extracts The samples were collected in the Parque Nacional do Catimbau (Pernambuco, Brazil), a preservation area of Caatinga biome. The voucher numbers and the extract preparation were described in da Silva et al. (2011). 3.2. Antimicrobial assays Staphylococcus aureus strains were isolated from medical material and provided by the ´ Departamento de Antibioticos, Universidade Federal de Pernambuco (Suppl. 1).
  • 5. Downloaded by [SENESCYT ] at 15:26 12 December 2012Table 1. Anti-S. aureus activity of plant extracts. Libidibia ferrea Anadenanthera colubrina Pityrocarpa moniliformis Inhibition Inhibition InhibitionStrain (mm) MICa MBCa MBC/MIC (mm) MICa MCBa MBC/MIC (mm) MICa MCBa MBC/MIC 02 15.3 Æ 3.8 0.39 6.25 16 14.7 Æ 0.6 1.56 6.25 4 15.0 Æ 1.0 1.56 6.25 4660 16.0 Æ 1.0 0.78 3.13 4 12.0 Æ 2.7 3.13 6.25 2 19.7 Æ 2.3 3.13 6.25 2663 17.0 Æ 1.0 0.78 3.13 4 13.5 Æ 0.5 3.13 6.25 2 11.3 Æ 2.3 3.13 6.25 2670 15.3 Æ 3.8 0.39 6.25 16 15.3 Æ 1.5 1.56 6.25 4 16.7 Æ 0.6 3.13 6.25 2672 17.3 Æ 2.1 0.39 3.13 8 15.3 Æ 1.5 1.56 6.25 4 19.5 Æ 0.5 3.13 6.25 2676 14.7 Æ 0.6 0.39 6.25 16 13.7 Æ 0.6 1.56 6.25 4 15.0 Æ 1.7 1.56 6.25 4677 15.0 Æ 1.0 0.39 3.13 8 12.7 Æ 0.6 3.13 6.25 2 10.7 Æ 1.3 3.13 6.25 2679 14.7 Æ 6.6 0.39 3.13 8 13.7 Æ 0.6 3.13 6.25 2 10.0 Æ 2.0 3.13 6.25 2687 13.3 Æ 0.6 0.39 6.25 16 13.0 Æ 1.0 1.56 6.25 4 16.0 Æ 1.0 3.13 6.25 2689 15.3 Æ 1.2 0.39 6.25 16 13.3 Æ 0.6 3.13 6.25 2 16.3 Æ 0.6 3.13 6.25 2Note: aThe MIC and MBC values are expressed in mg mLÀ1. Natural Product Research 3
  • 6. 4 L.C.N. da Silva et al. Figure 1. Effects of extracts on the ultrastructure of S. aureus as observed by TEM (1.1) and SEM (1.2). 1.1A–C: untreated bacteria showing; 1.1D–F: LMHE-treated bacteria; 1.1G–I: ACHE-treatedDownloaded by [SENESCYT ] at 15:26 12 December 2012 cells; 1.1J–L: PMHE-treated cells (bars: A ¼ 2 mm; B, F, I ¼ 0.2 mm; C–E, H, J–L ¼ 0.5 mm and G ¼ 1 mm). 1.2A: treated cells; 1.2B and C: ACHE-treated cells; 1.2D–F: LMHE-treated bacteria; 1.2G–I: PMHE-treated cells (bars: A, C, I ¼ 4 mm; B, D, F, G, H ¼ 3 mm and E ¼ 5 mm). The antibacterial activity were evaluated by the disc diffusion method and the determination of MIC and MBC through microdilution assay, using a Resazurin solution (0.01%) as a growth indicator. 3.3. Electron microscopy After incubation (6 h at 37 C) the cells were Exed (2.5% glutaraldehyde/4% paraformal- dehyde in 0.1 mol LÀ1 phosphate buffer, pH 7.2), pos-fixed (1% osmium tetroxide/0.8% potassium ferricyanide/5 mmol LÀ1 CaCl2 in 0.1 mmol LÀ1 cacodylate buffer), dehydrated (graded acetone series) and embedded using an epoxy-embedding medium kit (Fluka, Switzerland). Ultrathin sections were stained with lead citrate and uranyl acetate and observed by TEM. For SEM, bacterial cells were processed as describe above and allowed to adhere onto a poly-L-lysine-coated coverslip for 5 min at room temperature. The cells were dehydrated (graded ethanol), critical-point dried, attached to aluminium mounting stubs, sputter coated with 20 nmol LÀ1 gold and imaged with a Quanta 200 F (FEI company). 3.4. Phytochemical analysis The phytochemical tests to detect the presence of tannins, flavonoids, anthocyanins, saponins, coumarins, quinones, anthraquinones, tannin, reducers compounds and alkaloids were performed according to the method described by Harborne (1998). 4. Conclusion Our results demonstrated that A. colubrina, L. ferrea and P. moniliformis are promising sources of bioactive compounds against S. aureus and, probably, the cell wall and/or plasma membrane is the main site of extract action. The observed activities confirm the traditional use of A. colubrina and L. ferrea, and contributes to the recent indications of biotechnological potential of P. moniliformis indicated by our group. Our research findings indicate the possibility for sustainable utilisation of natural resources from the Caatinga.
  • 7. Natural Product Research 5 The isolation and chemical characterisation of these extracts are being performed by our group. Acknowledgements The authors wish to thank the Brazilian agencies (Conselho Nacional de Desenvolvimento Cientı´ fico ´ e Tecnologico, Coordenacao de Aperfeicoamento de Pessoal de Nı´ vel Superior and Fundacao de ¸ ˜ ¸ ¸ ˜ ` Amparo a Ciencia e Tecnologia do Estado de Pernambuco) for the grant. ˆ References Cartaxo, S.L., Souza, M.M.A., & Albuquerque, U.L. (2010). Medicinal plants with bioprospecting potential used in semi-arid northeastern Brazil. Journal of Ethnopharmacology, 131, 326–342. da Silva, L.C.N., Silva-Junior, C.A., Souza, R.M., Macedo, A.J., Silva, M.V., & Correia, M.T.S. (2011). ´ Comparative analysis of the antioxidant and DNA protection capacities of Anadenanthera colubrina, Libidibia ferrea and Pityrocarpa moniliformis fruits. Food and Chemical Toxicology, 49, 2222–2228. Dı´ az-Visurraga, J., Garcı´ a, A., & Cardenas, G. (2010). Lethal effect of chitosan-Ag (I) Elms on Staphylococcus ´Downloaded by [SENESCYT ] at 15:26 12 December 2012 aureus as evaluated by electron microscopy. Journal of Applied Microbiology, 108, 633–646. Foster, T.J. (2005). Immune evasion by staphylococci. Nature Reviews Microbiology, 3, 948–958. Harborne, J.B. (1998). Phytochemical methods. London: Chapman & Hall. Sampaio, E.V.S.B., Giuiietti, A.M., Vı´ rginio, J., & Gamarra-Rojas, C.F.L. (2002). Vegetaca e flora da caatinga ¸ ˜o (1st ed.). Recife: Associacao Plantas do Nordeste. ¸ ˜ Tang, Y.W., & Stratton, C.W. (2010). Staphylococcus aureus: an old pathogen with new weapons. Clinics in Laboratory Medicine, 30, 179–208.