Effects of associations of tannins from Anacardium occidentale and Anadenanthera colubrina with cephalosporin against bovine Staphylococcus aureus isolates Efeitos da associação de taninos de Anacardium occidentale e Anadenanthera colubrina à cefalosporina sobre Staphylococcus aureus isolados de bov

Arq. Inst. Biol., v.85, 1-8, e0582016, 2018 RESUMO: A associação de compostos naturais, isolados de plantas medicinais, com antibióticos convencionais, com mecanismos de ação semelhantes, torna‐se uma estratégia alternativa e viável para superar o problema da resistência. Assim, nosso objetivo foi avaliar a atividade antimicrobiana in vitro de substâncias tânicas presentes na casca de Anacardium occidentale e Anadenanthera colubrina associa‐ das à cefalexina, sobre amostras de Staphylococcus aureus. Avaliamos essa associação por meio da determinação da concentração mínima inibitória. Dessa forma, taninos e a cefalexina foram dissolvidos de forma seriada em água destilada em concentrações variando de 0,976 mg/mL a 500 mg/mL e 2 μg/mL a 512 μg/mL, respectiva‐ mente. Quando associados, inibiram o crescimento de S. aureus formando halos que variaram de 0,9 a 46 mm com concentração mínima inibitória de 7,8 mg/mL (taninos)/ 4 μg/mL (cefalexina). O efeito resultante da associação de substâncias, natural e sinté‐ tica, com mecanismos de ação semelhantes, apresentou resultados superiores aos observados quando testados isoladamente. Podemos concluir que os taninos e a cefalexina tiveram sua ação antimicro‐ biana potencializada quando utilizados em associação, permitindo o uso de uma menor concentração, mantendo seu efeito antibac‐ teriano sobre cepas de S. aureus.


ABSTRACT:
The association of natural compounds isolated from medicinal plants with conventional antibiotics, both with similar mechanisms of action, have become a viable alternative strategy to overcome the problem of drug resistance.This study aimed to evaluate the in vitro antimicrobial activity of tannic substances present in the bark of Anacardium occidentale and Anadenanthera colubrina against samples of Staphylococcus aureus when in combination with cephalexin.These combinations were evaluated by determining the minimum inhibitory concentration (MIC).For this purpose, tannins and cephalexin were serially dissolved in distilled water at concentrations ranging from 0.976 mg/mL to 500 mg/mL and 2 mg/mL to 512 mg/mL, respectively.When combined, the compounds inhibited S. aureus growth forming halos ranging from 0.9 to 46 mm with an MIC of 7.8 mg/mL (tannins) and 4 µg/mL (cephalexin).The resulting effect of the combination of natural and synthetic substances with similar mechanisms of action presented better results than when tested alone.Thus, the conclusion is that both the tannins and cephalexin had their antimicrobial action enhanced when used in combination, enabling the use of lower concentrations while maintaining their antibacterial effect against strains of S. aureus.

INTRODUCTION
The emergence of antibiotic resistant Staphylococcus aureus strains occurs due to improper and abusive use of antimicrobial agents, leading to their resistance to drugs in current use.This has become a public health problem (MASURANI;TAVARES, 2007), and the treatment of bacterial infection cases related to this strain is becoming increasingly difficult.This has promoted a growing interest in scientific circles for compounds obtained from medicinal plants, which are effective against microorganisms resistant to conventional medicines.They can minimize side effects and are more affordable.Among the tannins, there is a pool of components, and their synergisms are often preponderant (ALMEIDA et al., 2012;MENEZES et al., 2014;HIGINO et al., 2015;PEREIRA et al., 2015;CAVALCANTI-DANTAS et al., 2016).
Tannins act directly against cellular organelles and within the cell membranes of various microorganisms, inhibiting their growth (MIN et al., 2008;RODRIGUES et al., 2014;AGOSTINI-COSTA et al., 2015).Recent studies indicate that tannins present great potential in binding proteins and adhesins, interfering directly in the substrate availability necessary for bacterial metabolism and growth (JONES et al., 1994;HASLAM, 1996;GUIMARÃES-BEELEN et al., 2006;RODRIGUES et al., 2014).
Among the antibiotics most prescribed to treat infections caused by S. aureus, 1st generation cephalosporin (Cephalexin) presents action against many bacteria including: S. aureus, Staphylococcus epidermides, Streptococcu spneumoniae, Streptococcus pyogenes, Eschericha coli, Haemophilus influenzae, Proteus mirabilis, Klebsiella pneumoniae and Moraxella catarrhalis (PRESCOTT et al., 2002;RODRIGUES;BERTOLDI, 2010;SAEKI et al., 2011), and constitutes a first option for skin infections, in which Staphylococcus aureus is the most probable causative agent (WHITE, 1996;PRESCOTT et al., 2002).Considering the increasing prevalence of multidrug-resistant S. aureus, which causes many diseases in humans and in animals (FREITAS et al., 2005;RUEGG, 2007), the use of tannins, associated with conventional antibiotics, can reduce morbidities and combat disease.In this context, tannins isolated from Anadenanthera colubrina (Vell) Brenan (Red Angico), and Anacardium occidentale Linn.(Cashew) may be good therapeutic candidates.They can be used alone (MENEZES et al., 2014;GONÇALVES et al., 2015;HIGINO et al., 2015;PEREIRA et al., 2015), or as mentioned above, in association with antibiotics.However, studies focusing on their solitary (or associated) biological activities have not been found in the literature.This study aimed to evaluate the in vitro antimicrobial activity of tannin substances present in the bark of A. occidentale and A. colubrina when associated with cephalexin against multidrug-resistant S. aureus samples of bovine origin.

Anadenanthera colubrina (Vell) Brenan and Anacardium occidentale L.
Stem husks of "Red Angico" (A. colubrina) and "Cashew" (A.occidentale) were collected and prepared in NUPEÁRIDO, the Semi-arid Research Nucleus of Universidade Federal de Campina Grande (UFCG).The plants were collected (four samples), and deposited at the Prof. Lauro Pires Xavier Herbarium and the Pharmaceutical Technology Laboratory reference collection, both at Universidade Federal da Paraíba (UFPB).The identification of the samples was carried out in the Virtual Herbarium of Flora and Fungi (INCT) registered with the code CEN 50716 and CEN 6191 respectively.

Extraction of tannin substances for antimicrobial activity evaluation
The husks were packed in plastic bags so as to not lose moisture.In the laboratory, two samples were used; they were cut into smaller fragments, homogenized, weighed and dried in an oven at 103±2°C for 48 h for determination of moisture content (dry basis).The husks were then air dried and milled in a forage harvester.The material was classified, and what passed through a 2 x 2 cm mesh sieve was used.After this process, four samples were collected; two of these were ground in a Willey-type mill to obtain smaller and more homogeneous particle sizes aimed to quantify the tannins present in A. colubrina and A. occidentale in accordance with PAES et al. (2006).The extraction of tannins was performed in water, using a rotary digester, at a temperature of 70±5ºC for 2 h.For each 2 kg of husks, 10 L of water were respectively added (1:5).Each sample was submitted to two extractions (GUANGCHENG et al., 1991).The solution obtained was homogenized, strained, and placed in aluminum trays, and then placed in an oven with forced ventilation and kept at 70±3ºC until complete moisture evaporation.The dry material, final product of the evaporation process, was crushed in a household multiprocessor and sifted in a 60 mesh sieve; corresponding to pure tannin.

Acquisition of Staphylococcus aureus strains
Collection of milk samples and nasal swabs were performed in NUPEÁRIDO, the Semi-arid Research Nucleus of Universidade Federal de Campina Grande (UFCG), in the municipality of Patos, State of Paraíba, Brazil.
Samples were obtained from 30 naturally infected lactating cattle.After washing the udders with soap and water, drying with a paper towel, and disinfecting the ostium of the udders with ethyl alcohol at 70° GL, approximately 5 mL of milk was collected from each mammary quarter for the California Mastitis Test reagent (CMT), aseptically, with an inclined pipe positioned horizontally.
The nasal swab was collected, and all samples were stored in sterile threaded pipes; identified and shipped under refrigeration in boxes made of insulated material.All materials were sent for processing, microbiological examination and S. aureus identification in the Genetics and Microbiology Laboratory of UFPB/CCEN.
In order to identify multidrug-resistant strains, the sensitivity to antibiotics was previously evaluated in three different categories using disk diffusion tests in Petri plates containing Muller Hinton agar (MHA), in accordance with YOUN et al. (2011).In this study, 11 strains of S. aureus were chosen: one strain of S. aureus (ATCC 25925) and ten of strains of S. aureus from bovine origin obtained from the udders (104U, 120U, 125U, 787U, 250U, 275U, 282U, 301U, 305U and 335U).The strains were tested against β-lactams (ampicillin and penicillin), as well as aminoglycosides (neomycin, streptomycin, and tetracycline).

Antimicrobial activity
Tannins were dissolved in sterile distilled water and filtered in a 0.22 µm membrane filter (TPP ® ) at 25 mg of tannin for each mL of distilled water.The initial solution was diluted serially, obtaining concentrations ranging from 500 mg/mL to 0.976 mg/mL; these were added individually to the S. aureus bacterial cultures.
The bacteria were previously grown in BHI medium (Brain Hearth Infusion, Sigma™) at 37°C for 24 h, and the suspensions were adjusted to the Nephelometric 0.5 McFarland standard scale turbidity standard.This corresponded to approximately 1.5x10 8 colony-forming units (CFU)/mL, which were diluted in BHI broth for a concentration of 1.5x10 5 CFU/mL.
Aliquots of 50 µL were removed from these tubes and disseminated in Petri plates containing Muller Hinton agar (MHA).Holes were made in the culture mediums with a 6 mm diameter glass tube and filled with 25 µL of tannin and cephalexin diluted in distilled water.The initial concentration of tannin for serial dilution was 500 mg/mL, and cephalexin at 512 µg/mL.This concentration was selected in accordance with the results obtained by SULEIMAN et al. (2013).The plates were kept in the oven at 37°C for 24 h.Minimum inhibitory concentration (MIC) was considered as the lowest concentration of antibiotic that completely inhibited bacterial growth (CLSI, 2010) for the diameter of the bacterial growth inhibition halos (BARRY, 1991;JORGENSEN et al., 1999) formed around each hole.The halos were measured and the results displayed in millimeters (mm).
A qualitative method was used, which allowed classifying the bacterial samples as susceptible or resistant to the association used (CLSI, 2010).Strains were considered sensitive when presenting halos greater than 10 mm, according to the preliminary studies (PEREIRA et al., 2015;MENEZES et al., 2014;CAVALCANTI-DANTAS et al., 2016).

STATISTICAL ANALYSIS
Sensitivity testing was carried out in triplicate and evaluated using analysis of variance by the Kruskal-Wallis test, followed by Dunn's test, comparing tendencies of bacterial growth inhibition (CALEGARI-JACQUES, 2003).Tests were considered significant when the p-value was less than 5%.The data were also recorded in the form of a database in the computer program SPSS (Statistical Package for Social Sciences) for Windows ® , version 15.0; and analyzed by means of descriptive and inferential statistics.

RESULTS
Initially, the resistance profiles of S. aureus strains against β-lactams (ampicillin and penicillin) and aminoglycosides (neomycin, streptomycin, and tetracycline) in this way were verified and considered as S. aureus strains susceptible or resistant to the association used, thus corroborating analyses conducted by BARRETO et al. (2014).The protocol used in this study allowed the determination of the antimicrobial activity (MIC) of stem bark tannins from A. occidentale and A. colubrina when associated with cephalosporin (Cephalexin) against multidrug-resistant S. aureus samples of bovine origin.
The inhibition halos for A. occidentale and A. colubrina associations together with cephalexin had diameters ranging from 0.9 to 46 mm at all dilutions, thus presenting 100% effectiveness at the 1:64 dilution (Table 1).Inhibition of growth occurred homogeneously and according to the concentration of the tannic substance in associations with cephalexin.
Another six strains tested showed resistance to the Cephalexin -A.colubrina association; corresponding to 54.5% of the samples (halos with less than 9 mm; Table 2).In view of the inhibition halos when Cephalexin and the tannins were used separately, the A. occidentale tannin showed an MIC of 62.5 mg/mL, with halos of 11 mm, the A. colubrina tannin obtained an MIC of 31.25 mg/mL, with halos that ranged from 10 to 13 mm, and Cephalexin obtained an MIC of 32 µg/mL with halo dimensions that ranged from 29 to 33 mm.

DISCUSSION
From the results obtained, it can be observed that the tannins presented significant pharmacological effect.In the in vitro assay, both tannins, when associated with cephalexin, presented potentiated antimicrobial action against multidrug-resistant S. aureus.S. aureus presents variability in developing resistance to multiple antimicrobial agents, causing physiological disorders and disease processes in individuals (MONROE; LEWIS, 2000).This variability contributes to its survival and dissemination in hospital environments (MUSUMECI et al., 2003;MAIA et al., 2009).
Currently, there are many limitations for medicines when treating diseases caused by multiresistant microorganisms (ENTENÇA; MOREILLON, 2008).Compounds obtained from medicinal plants present various biological activities against pathogenic Gram-positive and Gram-negative species (ABUBAKAR, 2009;AHMED et al., 2010), which allows their use.Compounds such as tannins can be used either pure or in association with conventional antimicrobial agents, representing a greater strategy to overcome multidrug-resistant S. aureus, among others.
In the present study, the samples studied were sensitive to low concentrations of tannins when associated with cephalexin.According to LOGUERCIO et al. (2005).The antimicrobial activity of tannins occurs through interaction between tannin and the bacterial cell wall.SCALBERT (1991) states that this interaction interferes directly in transport as well, the availability of essential ions and nutrients to maintain the microbial metabolism.MENEZES et al. (2014) andPEREIRA et al. (2015) investigated the antimicrobial activity of tannins isolated from the stem bark of A. luxembourg against dental biofilm formations of S. aureus, S. mutans, S. mitis, S. sanguis, Streptococcus oralis, Streptococcus salivarius, and Lactobacillus casei, which all showed promise with MICs that ranged from 0.062 mg/mL to 31.25 µg/mL (MENEZES et al., 2014;PEREIRA et al., 2015).Tannins of other native species from Brazil's caatinga biome (exclusively) were tested against S. aureus, Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis, and presented excellent antimicrobial activities (GONÇALVES et al., 2015;HIGINO et al., 2015;PEREIRA et al., 2015).PEREIRA et al. (2015) evaluated the activity of tannin isolated from the bark of Mimosa tenuiflora and demonstrated an MIC of 31.2 µg/mL against S. aureus.The antimicrobial activity of tannins isolated from the bark of Dalbergia stipulacea was verified by PEREIRA et al. (2015) against strains of S. aureus of animal origin, and obtaining an MIC of 62.5 µg/mL.More recent data regarding the antimicrobial activity of condensed P. stipulacea tannin was observed by CAVALCANTI-DANTAS et al. (2016), having found an MIC of 31.25 mg/mL against strains of S. aureus.In the present study, the methodology was based on studies by these authors.
The antimicrobial activity of the tannins obtained from the bark of Mimosa arenosa was observed in both Gram-positive and Gram-negative bacteria such as: S. aureus, E. coli, P. aeruginosa, and B. subtilis with MICs ranging from 250 to 1000 µg/mL (GONÇALVES et al., 2015).The authors observed that the antibacterial activity exerted by tannins from M. arenosa was bacteriostatic against all strains tested.
The results in this study demonstrated synergism between cephalexin and the tested tannins against multidrug-resistant S. aureus samples.The tannins of A. occidentale and A. colubrina presented respective MICs of 62.5 mg/mL and 31.2 mg/mL when used pure.Cephalexin, when used alone, presented an MIC of 32 µg/mL.The associations of tannins with the antibiotic were more expressive.We saw antimicrobial action even at dilutions of 1:64 considering the MICs for tannin contents at 7.8 mg/mL, and at 8 µg/mL for Cephalexin.This demonstrates that the effect of the antibiotic was potentiated by the tannin addition, which, even in low concentrations, promoted better antimicrobial action.This likely occurred due to their interactions, and to synergism in their mechanisms of action.
Cephalexin interrupts the synthesis of peptidoglycan and inhibits the action of the enzyme involved in cell membrane trans-peptidation, preventing connections between tetra-peptide chains of peptidoglycan, and promoting lysis of bacterium cell wall (SPINOSA, 2011).Tannins are oligomers and polymers formed by poly-condensation of two or more flavanols and flavandiol, which in turn act similarly by forming tannin-protein complexes and/or polysaccharides at the bacterial cell membrane.The formation of these complexes leads to protein solubility reductions, promoting aggregation and precipitation of proteins present in the membrane, and inhibiting the action of microbial enzymes, thus depriving the organism of substrates essential to growth.This causes lysis of the plasma membrane (GUIMARÃES-BEELEN et al., 2006;RODRIGUES et al., 2014;CAVALCANTI-DANTAS et al., 2016).In accordance with SPINOSA (2011), substances that act against the plasma membrane of the bacterial cell present mechanisms of action that promote modification of its structure, and interfere in the transport systems of nutrients, ions, and other molecules.Synergism of these two substances promotes an increase of physiological response (SPINOSA, 2011;RANG et al., 2012), and may be responsible for the results obtained in the present study.
In the present study, a potentiating effect was observed in the association of two substances obtained from medicinal plants and a conventional medicine.Both present a similar mechanism of action.The results demonstrated that the combined action between them was significantly greater than the sum of the effects achieved by each one alone.Through this association, it can be observed that the tannins present a significant pharmacological effect when associated with cephalexin, and that both the tannins and cephalexin had their in vitro antimicrobial action maximized against the tested multiresistant microorganisms.In vivo studies need to be performed to test dosage safety and to reduce the overuse of the antibiotic, in order to maintain its antibacterial effect against multidrug-resistant S. aureus strains.According to SAR et al. (2012), the main advantage in using associated antibiotics that have the same mechanism of action is that the association allows potentiation of their effects, with consequent reductions in the drug dose, while maintaining therapeutic efficacy and promoting its effects.
Currently, there is a growing number of publications concerning the use of medicinal plants and their metabolites in in vitro experimental models against multidrug-resistant S. aureus (MANSOURI, 1999;HEYMAN et al., 2009;MENEZES et al., 2014;PEREIRA et al., 2015;CAVALCANTI-DANTAS et al., 2016).However, more systematized information is needed concerning possible clinical and pathological aspects resulting from such associations of conventional medicines with tannins.

CONCLUSION
When used in association, the tannins and cephalexin presented augmented antimicrobial action.The results of this study suggest that the antibacterial activity demonstrated against multiresistant strains of S. aureus was achieved due to a potentiating effect introduced by the tannins.Thus, tannins may be used in association with cephalexin against diseases caused by multidrug-resistant S. aureus.These findings, in favor of such economically viable compounds, add to an important list of new alternatives to help in infection control.

Table 1 .
Mean±standard deviation of the minimum inhibitory concentration (MIC); with diameters of inhibition halos in millimeters (mm), on solid medium; association of cephalexin with Anacardium occidentale tannin; against Staphylococcus aureus.

Table 2 .
Mean ± standard deviation of the minimum inhibitory concentration (MIC); with diameters of inhibition halos in millimeters (mm), on solid medium; association of Cephalexin with Anadenanthera colubrina extract against Staphylococcus aureus.