Transference of bioactive compounds from support ...

Science of the Total Environment 639 (2018) 921–928

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Transference of bioactive compounds from support plants to the termites Constrictotermes cyphergaster (Isoptera) Iamara Silva Policarpo a, Alexandre Vasconcellos a, Thiago Pereira Chaves b, Joanda Paolla Raimundo c, Ana Cláudia D. Medeiros d, Henrique D.M. Coutinho e,⁎, Rômulo Romeu Nóbrega Alves f a

Departamento de Sistemática e Ecologia, CCEN, Universidade Federal da Paraíba, Laboratório de Termitologia, 58051-900 João Pessoa, PB, Brazil Universidade Federal do Piauí, Campus Professora Cinobelina Elvas, Bom Jesus, PI 64900-000, Brazil Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Estadual da Paraíba, Campina Grande, Paraíba 58.429-500, Brazil d Laboratório de Desenvolvimento e Ensaios de Medicamentos, Centro de Ciências Biológicas e da Saúde, Universidade Estadual da Paraíba, Campina Grande, Paraíba 58.429-500, Brazil e Laboratório de Microbiologia e Biologia Molecular, Universidade Regional do Cariri, Crato, CE 63105-000, Brazil f Departamento de Biologia, Universidade Estadual da Paraíba, Laboratório de Termitologia, 58051-900 João Pessoa, PB, Brazil b c

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• The use of animals for the treatment of different human diseases is common in traditional medicine. • Termites are among the species most commonly used in folk medicine. • Potential microbiological activities of termites may be associated with their relationships with plants. • The antimicrobial potential of ethanol extracts of the bark of supporting plants is higher than the antimicrobial potential of the termite C. cyphergaster extracts; • The combination of the extracts of C. cyphergaster and its nests with antibiotics produces a strong synergistic activity.

a r t i c l e

i n f o

Article history: Received 3 February 2018 Received in revised form 14 May 2018 Accepted 14 May 2018 Available online xxxx Keywords: Traditional medicine Termites Medicinal plants Bioprospecting Semiarid

a b s t r a c t This study aims to investigate the microbiological potential of the termite species Constrictotermes cyphergaster (Silvestri, 1901) and its support plants. We collected five C. cyphergaster nests from three different support plant species. Microbiological assays were performed on these extracts using the serial microdilution method in triplicate to measure the minimum inhibitory concentration (MIC) of each microorganism for the analysed extract. The ethanol extracts of the termite C. cyphergaster showed no significant activity against strains of Staphylococcus aureus and Escherichia coli, with an MIC N1000 μg mL-1. Only the extracts of the nests and termites with the nest had the same MICs. These results were in contrast to the extracts of Spondias tuberosa (Umbuzeiro), Poincianella pyramidalis (Catingueira), and Amburana cearensis (Cumaru), which demonstrated significant activity against S. aureus with MICs b1000 μg mL-1. The modulating activity of the extracts tested in the present study demonstrated potentiation of most antibiotics across the bacterial strains tested when combined with the extracts for both S. aureus and E. coli. These results indicate that the extracts tested in the present study may be composed of animal and vegetable origins with the potential to modify the activity of antibiotics and thus may aid in antimicrobial therapy. © 2018 Elsevier B.V. All rights reserved.

⁎ Corresponding author at: Universidade Regional do Cariri, Urca, Rua Cel. Antonio Luis 1161, Pimenta, 63105-000, Brazil. E-mail address: [email protected] (H.D.M. Coutinho). https://doi.org/10.1016/j.scitotenv.2018.05.173 0048-9697/© 2018 Elsevier B.V. All rights reserved.

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I.S. Policarpo et al. / Science of the Total Environment 639 (2018) 921–928

1. Introduction Biodiversity is an invaluable source of information and bioactive chemicals that support human health (Chivian, 2002). Plants and animals have been documented in several geographical regions (WHO, 2002; Ferreira et al., 2012; Alves and Rosa, 2013; Van Vliet et al., 2017; Hajdari et al., 2018) as sources of remedies in traditional medicine worldwide. Although plants and their derivatives constitute most of the products used in traditional medicine, whole animals or their parts and animal sub-products are also important constituents of materia medica in different human societies (Marques, 1995; Alves and Rosa, 2013). Among the invertebrates, insects have played an important role as sources of therapeutic products in traditional medicine (Costa Neto, 2005; Costa Neto et al., 2006; Dossey, 2010). Insects and the products derived from them have been used by human cultures for medicinal purposes in different geographical regions (Figueirêdo et al., 2015; Kritsky, 1987; Morris, 2004; Costa Neto, 2005; Dossey, 2010). For an array of reasons, insects and their biological defence systems offer a important source of chemicals with great potential for use as novel medicinal compounds (Dossey, 2010; Dettner, 2011; Alves and Albuquerque, 2013). Termites (Isoptera) are an insect group that is commonly used in traditional folk medicine (Wilsanand, 2005; Coutinho et al., 2009; Figueirêdo et al., 2015). In Brazil, several termite species are commonly used for the treatment of human diseases, including bronchitis, influenza, whooping cough, asthma, sinusitis, hoarseness and tonsillitis (Alves et al., 2013). Studies with animal extracts have demonstrated the efficiency of some species against bacterial strains (Coutinho et al., 2009, 2010; Chaves et al., 2014). Termites feed on living and dead plant material. Some termite species use trees as support for nest construction, and some of these support trees are used in folk medicine (Wilsanand, 2005; Albuquerque et al., 2007). This scenario provides an good research opportunity in the field of bioprospection because the potential microbiological activity of animals may be associated with their relationships with plants. Possible correlations between medicinal flora and fauna need to be evaluated in pharmacological studies (World Resources Institute, 2000), and the use of animals and plants in folk medicine may help identify and further characterize useful species. Therefore, termites provide an excellent opportunity to evaluate the medicinal properties of biological resources and the importance of the interactions between the animals and plants used in traditional medicine. Additionally, the possible pharmacological activity of termites will contribute to a greater appreciation for these animals, which are usually known for their negative aspects associated with economic losses. Bacterial infections are currently the focus of public health, mainly due to the significant growth of bacterial resistance. Infections caused by Staphylococcus aureus are the most common, showing a greater difficulty in treatment due to its resistance to various antibiotics (Tortora et al., 2008). The species Pseudomonas aeruginosa is the leading cause of nosocomial infections, attacking the skin, urinary tract, ear, and eye (Murray et al., 2004). Escherichia coli are the most common species of the genus Escherichia, associated with severe urinary tract infections, meningitis and gastroenteritis (Murray et al., 2004; Tortora et al., 2008). In the present study, we investigated Constrictotermes cyphergaster, which is one of the most important termite species that build conspicuous nests (with visible structures) in ecosystems with sparse vegetation in South America (Melo and Bandeira, 2004). This species is considered dominant among other termite species in the caatinga (Neotropical dry forest). Additionally, C. cyphergaster actively participates in the decomposition of plant organic matter and nutrient cycling (Vasconcellos et al., 2007). Bezerra-Gusmão et al. (2013) observed that the supporting plants used by this termite species were shrubs that occurred at a high density, including Poincianella pyramidalis (Tul). LP Queiroz (Catingueira), which affected the distribution of termite mounds in

arid environments. We investigated the plant species P. pyramidalis (Catingueira) of the Fabaceae family, Spondias tuberosa Arruda (Umbuzeiro) of the Anacardiaceae family, and Amburana cearensis (Allemo) AC Smith (Cumaru) of the Fabaceae family, which are used by C. cyphergaster as support. These plants are widely used in folk medicine for treating diseases (Silveira and Pessoa, 2005; Lins-neto et al., 2010; Almeida et al., 2010; Medeiros et al., 2012). Therefore, this study aimed to assess whether (i) the antimicrobial potential of termites depended on the supporting plants, (ii) the supporting plants had higher or lower antimicrobial potential than the termites and (iii) variation was present in the antimicrobial potential of the extracts of the supporting plants, nests and termites. In this context, this study investigated the microbiological potential of the termite species Constrictotermes cyphergaster and its supporting plants.

2. Materials and methods 2.1. Study site and sampling The termite material was collected in the Private Reserve of Natural Heritage (Reserva Particular do Patrimônio Natural - RPPN) Fazenda Almas (7°28′S and 36°53′W), in São José dos Cordeiros, state of Paraíba, Brazil (Barbosa et al., 2007) (Fig. 1). During the study period, 15 samples were collected from nests that used Poincianella pyramidalis (Tul). LP Queiroz (Catingueira) (5 samples) and Amburana cearensis (Allemao) AC Smith (Cumaru) (5 samples), both of the Fabaceae family, and Spondias tuberosa Arruda (Umbuzeiro) (5 samples) of the Anacardiaceae family as supporting plants. The samples were collected from different specimens of each plant species. The specimens were randomly selected in the collection area, and the collected specimens were distanced at least 50 m from one another. We collected termites, the inner wall of the nest, and the stem bark from these specimens. The samples were collected using a hatchet. The nests and termites were transferred to sterilized glass containers, and samples of the stem bark of the supporting plants were stored in Kraft paper bags. The termite species was identified by Prof. Alexandre Vasconcellos from the Systematics and Ecology Department (Federal University of Paraíba - UFPB). Two samples were deposited in the Isoptera Collection of the Exact Sciences of Nature Center UFPB under the numbers 2047 and 2048. The botanical identification was carried out in the “Prof. Lauro Pires Xavier Herbarium” (JPB), Systematics and Ecology Department, Federal University of Paraíba, where vouchers specimens were deposited with following reference numbers: JPB 30.589 for Amburana cearensis, JPB 34.322 for Spondias tuberosa and JPB 41.167 for Poincianella pyramidalis.

2.2. Preparation of extracts The collected animals were manually separated from the nest and divided into three samples as follows: i) termites only, ii) nests only, and iii) termites and nests at a 1:1 ratio. The collected stem bark was broken down into small fragments, dried in a forced air oven at 40 °C until the complete stabilization of moisture, ground in a knife mill, and sieved through a 10-mesh sieve to obtain bark powder, which was used to prepare the extracts. Twenty grams was extracted from each sample by cold soaking using ethanol as the solvent for 5 days at room temperature. Subsequently, after filtration, the extracts were concentrated on a rotary evaporator at 40 °C until complete evaporation of the solvent was achieved. The 60 obtained samples included 15 samples of termites alone, 15 samples of nests alone, 15 samples of termites and nests, and 15 samples of stem bark, being isolated from each support plant in which they were collected.


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Fig. 1. Location of the municipality of São José dos Cordeiros, Paraíba, and the RPPN Fazenda Almas. (Source: Santos, 2012)

2.3. Strains Standard strains of Staphylococcus aureus and Escherichia coli were used; their resistance profiles are shown in Table 1. The strains were maintained in Mueller-Hinton agar slants and were cultured before the assay at 37 °C for 24 h on culture plates containing the same culture medium. All strains were obtained from the Laboratory of Microbiology of Universidade Estadual da Paraíba – UEPB. 2.4. Active pharmaceutical ingredient (API) All tested APIs were obtained from Sigma Chemical Corp. (St. Louis, MO, USA) and dissolved in sterile water prior to use.

The MIC of the antimicrobials was determined in the presence and absence of sub-inhibitory concentrations (125 μg mL-1). The culture plates were incubated as described above, and the assays were conducted in triplicate. 2.6. Chemical assays 2.6.1. Determination of total polyphenols, total flavonoids and condensed tannins The total polyphenol content of plant extracts was measured using spectrophotometry in the visible region by the method of Folin– Ciocalteu described by Chandra and Mejia (2004) with minor modifications. The total flavonoids were determined by the method described by Meda et al. (2005). The content of condensed tannins was verified through the method described by Makkar and Becker (1993).

2.5. Determination of the minimum inhibitory concentrations (MICs) and modulatory activity

2.7. Statistical analysis

The minimum inhibitory concentrations (MICs) were determined by microdilution in 96-well plates (CLSI, 2012) using Mueller-Hinton broth. The modulatory activity of the extracts on bacterial resistance to antimicrobials was evaluated as detailed by Coutinho et al. (2010a).

The results are expressed as the geometric means obtained using two-way ANOVA, followed by Bonferroni's post hoc test. The data were analysed using GraphPad Prism version 5.0, with p N 0.001 (Matias et al., 2013)

Table 1 Bacterial resistance profiles.

3. Results and discussion

Bacteria

Source

Resistance profile

Staphylococcus aureus (ATCC 25923) Escherichia coli (ATCC 25922) Staphylococcus aureus 345

ATCC

–

ATCC

–

Catheter tip Catheter tip

OXA, PEN, AZI, SFM, CFO, NOR, AMP, GENT

Escherichia coli 534

AMP, CRO, NOR, CAZ, ATM, TET, COM, GEN, CFL, CLI, SFM

OXA = oxacillin; PEN = penicillin; AZI = azithromycin; SFM = sulfamethoxazole + trimethoprim; CFO = cefoxitin; NOR = norfloxacin; AMP = ampicillin; GENT = gentamicin; CRO = ceftriaxone; CAZ = ceftazidime; ATM = aztreonam; TET = tetracycline; CPM = cefepime; CFL = cephalothin CLI = clindamycin.

The ethanol extract of Constrictotermes cyphergaster demonstrated no clinically significant activity against the S. aureus and E. coli strains, with MICs N1000 μg mL-1. Similarly, the ethanol extracts of the nests alone and termites combined with nests showed MICs N1000 μg mL-1. These results indicate the lack of variation of the antimicrobial potential of the extracts of C. cyphergaster, the nests, and their combinations. Conversely, this variation was evident in the comparison of the MICs of the extracts of the supporting plants because some specimens had MICs ≤1000 μg mL-1 against S. aureus (Tables 2, 3, and 4). The bark extracts of S. tuberosa and P. pyramidalis (Catingueira) showed moderate activity against standard and resistant strains of S. aureus, with MIC values of 500 μg mL-1 (Tables 2 and 3). The bark extract

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of A. cearensis did not show significant antimicrobial activity, with MICs of 1000 μg mL-1 for most specimens; however, variation was observed in the antimicrobial potential of the specimens of this species (Table 4). None of the tested concentrations of the plant extracts inhibited the growth of the standard and resistant E. coli strains. The bark extract of P. pyramidalis presented a MIC of 500 μg mL-1 against the standard and resistant S. aureus strains, indicating moderate antimicrobial activity; this activity was higher than the activity of the bark extracts of the other investigated supporting plants (Table 3). However, variation in the MIC of the bark extract of S. tuberosa was observed against the S. aureus strains; for instance, specimen 4 (St 4) had a MIC of 1000 μg mL-1, whereas the other specimens presented MIC values of 500 μg mL-1 (Table 2). We also observed variation in the MIC of the bark extracts of A. cearensis, with specimen 2 (Ac 2) not showing strong antimicrobial activity (MIC N1000 μg mL-1), whereas the other specimens presented MIC values of 1000 μg mL-1 (Table 4). In a complementary manner, we also evaluated whether the extracts combined with antibiotics exerted antibacterial activity against the multidrug resistant strains of E. coli and S. aureus. For this purpose, we selected the ethanol extracts of C. cyphergaster together with the nest and bark extracts of S. tuberosa, A. pyramidalis, and P. cearensis from the first collected specimens of the samples used in the antimicrobial activity analysis because no significant differences in the MICs of the extracts were found for C. cyphergaster, the nests, their combinations, and the supporting plants of the other specimens against the bacterial strains. The results of the assays to evaluate the modulatory activity of the antibiotics indicated that the combination of the extracts with a MIC of 125 μg mL-1 significantly reduced the MICs of most of the tested antibiotics, with the exception of clindamycin against S. aureus (Fig. 2) and ciprofloxacin and ceftazidime against E. coli (Fig. 3). The results indicated the synergistic effect of the extracts of C. cyphergaster and its nests and the antibiotics against S. aureus, particularly the combination with chloramphenicol, which resulted in a marked reduction of the MIC from 500 to 250 μg mL-1. A reduction in the MIC was also observed for ceftriaxone (250–62.5 μg mL-1) when combined with the extract of C. cyphergaster and the nest of a specimen of S. tuberosa (CcSt) and P. pyramidalis (CcPp) (Fig. 2). Moreover, the synergistic effect of the S. tuberosa, P. pyramidalis, and A. cearensis extracts with chloramphenicol and levofloxacin allowed the reduction of the MIC from 500 to 250 μg mL-1. In contrast to the extracts of C.

cyphergaster and its nests, the S. tuberosa and P. pyramidalis extracts showed no synergistic effect with ceftriaxone. Similarly, the extract of C. cyphergaster combined with the extract of the nests of a specimen of S. tuberosa (CcSt) did not show a synergistic effect when combined with levofloxacin. In contrast, a reduction in the MIC was observed when levofloxacin was combined with the other extracts (Fig. 2). The results of the assays with E. coli 534 indicated synergism of all extracts with levofloxacin, including reduction of the MIC from 62.5 to 7.8 μg mL-1 with the combination of the extract of C. cyphergaster with its nests made with the supporting plants. Similarly, the Pp and Ac extracts decreased the MIC of levofloxacin from 62.5 to 15.62 μg mL-1. The MIC of cefazolin against E. coli 534 was 49.60 μg mL-1. However, the combination of cefazolin with CcSt, St, and Ac reduced the MIC to 31.25 μg mL-1. Moreover, the CcPp and Pp extracts did not

Table 2 Minimum inhibitory concentrations (MICs) of the ethanol extracts of Constrictotermes cyphergaster, the termite nests, and the bark of Spondias tuberosa (μg/mL); Cc: Extract of C. cyphergaster; Nc: Extract of the C. cyphergaster nest; Cc+Nc: Extract of termites together with the nest; St: Extract of the S. tuberosa bark.

Table 4 Minimum inhibitory concentrations (MICs) of the ethanol extracts of Constrictotermes cyphergaster, the nests, and the bark of Amburana cearensis (μg mL−1); Cc: Extract of C. cyphergaster; Nc: Extract of the C. cyphergaster nest; Cc + Nc: Extract of termites together with the nest; Ac: Extract of the A. cearensis bark.

Extratos

Cc 1 Nc1 Cc + Nc1 St 1 Cc 2 Nc 2 Cc + Nc 2 St 2 Cc 3 Nc 3 Cc + Nc 3 St 3 Cc 4 Nc 4 Cc + Nc 4 St 4 Cc 5 Nc 5 Cc + Nc 5 St 5

Microorganismos

Table 3 Minimum inhibitory concentrations (MICs) of the ethanol extracts of Constrictotermes cyphergaster, the nests, and the bark of Poincianella pyramidalis (μg/mL); Cc: Extract of C. cyphergaster; Nc: Extract of the C. cyphergaster nest; Cc+Nc: Extract of termites together with the nest; Pp: Extract of the P. pyramidalis bark. Extracts

Cc 1 Nc 1 Cc + Nc 1 Pp 1 Cc 2 Nc 2 Cc + Nc 2 Pp 2 Cc 3 Nc3 Cc + Nc 3 Pp 3 Cc 4 Nc4 Cc + Nc 4 Pp 4 Cc 5 Nc 5 Cc + Nc 5 Pp 5

Extracts

SA (ATCC 25923)

SA 345

EC (ATCC 25922)

EC 534

N1000 N1000 N1000 500 N1000 N1000 N1000 500 N1000 N1000 N1000 500 N1000 N1000 N1000 1000 N1000 N1000 N1000 500

– – N1000 1000 – – – – – – – – – – – – – – – –

N1000 N1000 N1000 N1000 N1000 N1000 N1000 N1000 N1000 N1000 N1000 N1000 N1000 N1000 N1000 N1000 N1000 N1000 N1000 N1000

– – N1000 N1000 – – – – – – – – – – – – – – – –

Cc 1 Nc 1 Cc + Nc 1 Ac 1 Cc 2 Nc2 Cc + Nc 2 Ac 2 Cc 3 Nc 3 Cc + Nc 3 Ac 3 Cc 4 Nc4 Cc + Nc 4 Ac 4 Cc 5 Nc 5 Cc + Nc 5 Ac 5

Microorganisms SA (ATCC 25923)

SA 345

EC (ATCC 25922)

EC 534

N1000 N1000 N1000 500 N1000 N1000 N1000 500 N1000 N1000 N1000 500 N1000 N1000 N1000 500 N1000 N1000 N1000 500

– – N1000 500 – – – – – – – – – – – – – – – –


– – N1000 N1000 – – – – – – – – – – – – – – – –

Microorganisms SA (ATCC 25923)

SA 345

EC (ATCC 25922)

EC 534

N1000 N1000 N1000 1000 N1000 N1000 N1000 N1000 N1000 N1000 N1000 1000 N1000 N1000 N1000 1000 N1000 N1000 N1000 1000

– – N1000 1000 – – – – – – – – – – – – – – – –


– – N1000 N1000 – – – – – – – – – – – – –

–


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Fig. 2. Modulatory activity of the ethanol extracts of Constrictotermes cyphergaster and supporting plants on the resistance of S. aureus to antibiotics. ***: Significant value with p b 0.001; ns: Non-significant value with p N 0.05. LEV: levofloxacin; CLO: chloramphenicol; CLI: clindamycin; CFO: Ceftriaxone; CcSt: Extract of C. cyphergaster and the nest of a specimen of S. tuberosa; CcPp: Extract of C. cyphergaster and the nest of a specimen of P. pyramidalis; CcAc: Extract of C. cyphergaster and the nest of a specimen of A. cearensis; St: Extract of the S. tuberosa bark; Pp: Extract of the P. pyramidalis bark; Ac: Extract of the A. cearensis bark.

exhibit significant activity when combined with cefazolin (Fig. 3). Our results indicated no synergism between the extracts and ciprofloxacin and ceftazidime. Through chemical tests, it was possible to appoint the presence and concentration of substances in the extracts that confirmed significant modulating activity. The concentration of these compounds is shown in Table 5 and was expressed in milligrams equivalent to the standards used. Regarding the antimicrobial activity, a MIC greater than 1000 μg mL1 was found for the ethanol extracts of C. cyphergaster and its nests against the E. coli and S. aureus,strains whereas the S. tuberosa, P. pyramidalis, and A. cearensis extracts did not exhibit clinically significant activity against the E. coli strains. This MIC was considered high because Rios and Recio (2005) reported in a study of medicinal plants that the MICs of extracts with antimicrobial activity should be less than 1000 μg mL-1. Similarly, other authors reported that plant extracts with MICs less than 100 μg mL-1 had high inhibitory activity, extracts with MICs ranging from 100–500 μg mL-1 had moderate activity, extracts with MICs ranging from 500–1000 μg mL-1 had weak activity, and extracts with MICs larger than 1000 μg mL-1 lacked antimicrobial activity (Fabry et al., 1998; Holetz et al., 2002; Dall Angol et al., 2003; Tanaka et al., 2005). Considering these ranges, the ethanol extracts of S. tuberosa

and P. pyramidalis revealed moderate activity (500 μg mL-1) and the extract of A. cearensis revealed weak activity (1000 μg mL-1) against the tested S. aureus strains. The correlation between the MICs of the ethanol extracts of C. cyphergaster and its nests indicated that these extracts were inactive against the S. aureus and E. coli strains and that all of the plant extracts were inactive against the E. coli strains. The absence of inhibitory activity of these ethanol extracts against the Gram-negative bacterium (E. coli) may be correlated with structural differences in the outer membrane between this bacterial species and the Gram-positive S. aureus because this membrane acts as a barrier to the entry of the active substances present in the extracts (Urzua et al., 1998). The increased bacterial resistance may also be due to the presence of the potentially active compounds at very low concentrations or the lack of metabolites active against this bacterial species in the extracts (Koneman et al., 2001). Trabulsi and Alterthum (2005) reported that the higher rate of inhibition of Gram-positive bacteria occurred because 90% of the cell walls of these bacteria were made of peptidoglycans. In contrast, the cell walls of Gram-negative bacteria were more complex, which increased the resistance of these bacterial species against antimicrobials. The moderate activity of the supporting plants against the S. aureus strains indicated that the antimicrobial potential of the bark ethanol extracts of S. tuberosa, P. pyramidalis, and A. cearensis was higher than the

Fig. 3. Modulatory activity of the ethanol extracts, Constrictotermes cyphergaster, and supporting plants on the resistance of E. coli to antibiotics. ***: Significant value with p b 0.001; ns: non-significant value with p N 0.05. LEV: levofloxacin; CIP: ciprofloxacin; CFZ: cefazolin; CFT: Ceftazidime; CcSt: Extract of C. cyphergaster and the nest of a specimen of S. tuberosa; CcPp: Extract of C. cyphergaster and the nest of a specimen of P. pyramidalis; CcAc: Extract of C. cyphergaster and the nest of a specimen of A. cearensis; St: Extract of the S. tuberosa bark; Pp: Extract of the P. pyramidalis bark; Ac: Extract of the A. cearensis bark.

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Table 5 Concentration of secondary metabolites (mg g-1) determined for the extract of C. cyphergaster and the nest of a specimen of S. tuberosa (CcSt); Extract of C. cyphergaster and the nest of a specimen of P. pyramidalis (CcPp); Extract of C. cyphergaster and the nest of a specimen of A. cearensis (CcAc); Extract of the S. tuberosa bark (St); Extract of the P. pyramidalis bark (Pp) and the extract of the A. cearensis bark (Ac). Extract

Total polyphenols

Flavonoids

Tannins

Pp CcPp St CcSt Ac CcAc

36.94 ± 0.451 74.51 ± 0.151 17.47 ± 0.201 70.20 ± 0.231 81.05 ± 0.241 79.02 ± 0.191

19.09 ± 0.782 23,09 ± 0.372 8.51 ± 0.312 21.98 ± 0.382 25.36 ± 0.272 24.58 ± 0.202

59.08 ± 0.693 171.22 ± 0.773 47.17 ± 0.723 170.69 ± 0.833 190.69 ± 0.813 186.96 ± 0.803

1 2 3

Gallic acid equivalent (GAE). Quercetin equivalent (QE). Catechin equivalent (CE).

activity of C. cyphergaster and its nests. The antimicrobial activity of the analysed supporting plants was clearly demonstrated. The activity of the S. tuberosa extract against S. aureus reinforced the findings of Rocha et al. (2013), who used the same microdilution method and found a similar activity of the S. tuberosa extract against S. aureus. Similarly, other studies have confirmed the antimicrobial activity of S. tuberosa. Costa et al. (2013) used a different methodology (disc agar diffusion) and found that the S. tuberosa extract was effective against S. aureus and Enterococcus faecalis. Carvalho (2012) found that that Streptococcus and Candida were sensitive to this plant extract. Similarly, the high antimicrobial activity of P. pyramidalis demonstrated in our study supported the results of Lima et al. (2006), who found that ethanol extracts of the bark and leaves of this plant species were active against S. aureus. Moreover, the aqueous extract of P. pyramidalis showed strong activity against oral microorganisms, indicating its potential for the treatment of oral diseases (Alviano et al., 2008). A phytochemical research of P. pyramidalis extracts revealed the presence of several compounds with recognized antimicrobial activity, including ursolic acid, quercetin, catechin, sitosterol, flavonoids, and gallic acid (Saraiva et al., 2012). Therefore, the biological activity presented in our study and the above-mentioned studies might be a result of the activity of these constituents. Our results indicated that the combination of the tested extracts with antibiotics potentiated the activity of most antibiotics against the E. coli and S. aureus strains. The use of natural products in combination with antibiotics has been well investigated, and the products of medicinal fauna have shown the potential to enhance the action of antibiotics against multidrug-resistant bacteria (Coutinho et al., 2009, 2010, 2014; Ferreira et al., 2011; Santos et al., 2012; Oliveira et al., 2014). Although the antimicrobial activity of the extracts of C. cyphergaster and its nests was not significant, the modulatory activity of these extracts was considered significant because the combination of the extracts with most tested antibiotics reduced the MIC against resistant strains of S. aureus and E. coli and potentiated the activity of levofloxacin and cefazolin against strains of EC 534 and the activity of levofloxacin, chloramphenicol, and ceftriaxone against strains of SA 345 (Figs. 2 and 3). Similarly, Coutinho et al. (2009) observed a reduction in the MICs of gentamicin and neomycin, indicating modulatory activity of the Nasutitermes corniger extract. The study by Chaves et al. (2014) also reported a synergism of the N. corniger extract and antibiotics against E. coli and S. aureus strains, indicating that compounds from N. corniger and possibly other termite species could change the modulatory activity of antibiotics against multidrug-resistant bacteria. The chemical exam performed in this work indicated the presence of flavonoids, polyphenols and tannins in the extracts tested in the modulatory activity (Table 5). It is possible that these metabolites affect the lipid bilayer of the bacteria, disrupting the cell membrane and enhancing the influx of antibiotics and their effect (Chaves et al., 2014). The content of the total number of tannins was high compared to total flavonoids and polyphenols, this compound can act on the metabolism in

microorganisms through inhibition of enzymes, the electron transport system, oxidative phosphorylation and inactivation of microbial adhesins and proteins of the cell envelope (Scalbert, 1991; Cowan, 1999). The synergism between the extract of C. cyphergaster and its nests and the antibiotics may be due to the secondary metabolites existent in the extracts that are synthesized by plants because C. cyphergaster is xylophagous, and the secondary metabolites of the supporting plants used as food source are found in the digestive system of the animal and in the faeces used to build the nests (Medeiros, 2004; Lima and CostaLeonardo, 2007). Therefore, the possible antimicrobial potential of C. cyphergaster is associated with the plant used as support. Dixon et al. (2001) reported that the synergistic activity of natural products and antibiotics might be due to secondary metabolites, such as flavonoids and tannins, which are produced by plants in response to microbial infections. No significant differences in the modulatory activity of the antimicrobials from C. cyphergaster and its nests and the plant antimicrobials were observed, and this activity was not increased when the extracts were combined with the tested antibiotics. Previous studies have confirmed the activity of supporting plants. For instance, the study of Figueredo et al. (2013) found a reduction in the MIC and consequently the potentiation of the activity of gentamicin and amikacin combined with an A. cearensis extract against E. coli strains. Similarly, Alencar et al. (2015) found modulatory activity of antibiotics from the extracts of species of the genus Spondias, suggesting that these species were sources of natural products with the potential to modulate the activity of antibiotics against multidrug-resistant bacterial strains. In a broader perspective, the antimicrobial activity of products isolated from termites has been evidenced, including the peptides spinigerin and termicin isolated from Pseudocanthotermes spiniger, which show antibacterial and antifungal activity. These peptides are found in the salivary glands and granules of red blood cells of this termite species, respectively (Lamberty et al., 2001). Similarly, studies on the molecular biology of species of genus Nasutitermes demonstrated their potential to produce antimicrobial peptides (Bulmer and Crozier, 2004, 2006). Termites and other social insects build nests that contain an associated microbiota, which includes microorganisms that live in symbiosis with these insects and provide protection against bacterial and fungal infections (Chaves et al., 2014). This microbiota includes actinomycetes, some of which have been isolated and had their antimicrobial activity demonstrated. Visser et al. (2012) isolated Actinobacteria from the nests of Microtermes sp., Macrotermes natalensis and Odontotermes spp. and demonstrated their antimicrobial activity. Moreover, Bonfim (2012) confirmed the activity of Actinobacteria and Bacillus isolated from the nests of Nasutitermes against Gram-positive bacteria. The ethanol extracts of C. cyphergaster and its nests and the bark extracts of the analysed supporting plants strongly modulated the activity of most of the tested antibiotics. The synergistic activity of these extracts indicates the high potential for the use of natural products in combination with synthetic drugs in the Brazilian pharmaceutical market. Among the products of plant and animal origin, extracts are considered complex mixtures, and this complexity limits microbial adaptability. Therefore, microorganisms have a low likelihood of acquiring resistance against these products (Coutinho et al., 2009b). Furthermore, when combined with antibiotics, extracts can act directly against bacterial species by either modulating or increasing the activity of specific antibiotics, thereby reversing the natural resistance of bacteria to specific antibiotics (Souza et al., 2014). 4. Conclusion Our results indicate that the antimicrobial potential of ethanol extracts of the bark of supporting plants is higher than the antimicrobial potential of the C. cyphergaster extracts. Moreover, we found variation


in the antimicrobial potential of the extracts of C. cyphergaster and its nests. Conversely, the combination of the extracts of C. cyphergaster and its nests with antibiotics produces a strong synergistic activity because C. cyphergaster is a xylophagous species. These results indicate that the antimicrobial potential of this termite species depends on the plants used as support.

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