Bmc Complementary and Alternative Medicine Termite Usage Associated with Antibiotic Therapy: Enhancement of Aminoglycoside Antibiotic Activity by Natural Products of Nasutitermes Corniger (motschulsky 1855)

Background: Several species from Insecta are used as remedies. Among these species, the termite Nasutitermes corniger is commonly used in traditional medicine in Northeast Brazil. The present work tests the modifying antibiotic activity of Nasutitermes corniger, a termite used in folk medicine in Northeastern region of Brazil.


Background
Although medicinal plants are well studied around the world, animals or animal parts have been broadly used in Brazilian traditional medicine and have played a significant role in healing practices [1,2]. Several species from Insecta have been used as remedies [1][2][3][4]. Among these species, the Neotropical termite Nasutitermes corniger is commonly used in traditional medicine in Northeast Brazil. N. corniger is distributed from southern Mexico to northern Argentina and the West Indies and is spread from the semi-arid to tropical rain forest ecosystems [5][6][7][8][9]. In South America, this species is highly adaptable to colo-nizing contrasting habitats in urban, agricultural, and natural environments [10,11]. N. corniger builds arboreal carton nests with a population that can exceed 400,000 individuals/nest, and density that ranges from 22.1 to 47.1 nest/ha in tropical rain forests [10,12,13]. Based on morphologic, genetic and biogeographic evidence, N. costalis has been revised as a synonym of N. corniger, like the congeneric species N. araujoi, N. globiceps and N. tatarendae which are also synonyms of N. corniger [8,9].
With the increase in microbial resistance to antibiotics, the use of natural products represent an interesting alternative for treatment [14,15]. Many products have been evaluated not only for direct antimicrobial activity, but also as resistance modifying agents [16,17]. Several chemical compounds, from synthetic or biological sources, such as phenothiazines and natural products, have direct activity against many bacteria, enhancing the activity of a specific antibiotic, reversing the natural resistance of specific bacteria to several antibiotics, promoting the elimination of plasmids from bacteria such as Escherichia coli, and inhibiting drug-transport functions of the plasma membrane. Inhibition of plasma membrane-based efflux pumps has been observed as well [18,19]. The enhancement of antibiotic activity or the reversal of antibiotic resistance by natural or synthetic non-conventional antibiotics results in the classification of these compounds as modifiers of antibiotic activity. Aminoglycosides are potent bactericidal antibiotics that target the bacterial ribosome and development of bacterial resistance to aminoglycosides is widely recognized as a serious health threat [18]. In E. coli, the main mechanisms of resistance to aminoglycosides are active drug efflux and enzymatic inactivation [19].
The aim of the present study was to evaluate the decoctions of N. corniger obtained from two different trees (Commiphora leptophloeos Mart J. B. Gillet and Anacardium occidentale L.) as resistance-modifying agents against E. coli.

Strains
The strain used was the clinical isolate Escherichia coli (EC27), resistant to neomycin and gentamicin (low level) and to amikacin and kanamycin. The strain Escherichia coli (EC -ATCC8539) was used as the positive control. All strains were maintained in heart infusion agar slants (HIA, Difco), and prior to assay, the cells were grown overnight at 37°C in brain heart infusion (BHI, Difco).

Preparation of N. corniger decoctions from Commiphora leptophloeos (DCL) and Anacardium occidentale (DAO)
Two hundred grams of termites with nests were collected and powdered. The powdered material was extracted by maceration using 100 mL of sterile water as solvent at room temperature. The mixture was allowed to stand for 72 h at room temperature. Decoctions were then filtered and assayed to determine antibacterial activity.

Drugs
Chlorpromazine, gentamicin, kanamycin, amikacin and neomycin were obtained from SIGMA. All drugs were dissolved in sterile water.

Drug susceptibility test and determination of fractional inhibitory concentration (FIC)
The minimum inhibitory concentrations (MICs) of the decoctions, antibiotics and chlorpromazine (CPZ) were determined in BHI by the microdilution assay using suspensions of 10 5 cfu/mL and a drug concentration range of 1024 to 1 μg/mL (two-fold serial dilutions) [20]. The MIC was defined as the lowest concentration at which no growth was observed. For the evaluation of the decoctions as a modulator of antibiotic resistance, the MICs of the antibiotics were determined in the presence of the decoctions and CPZ at a sub-inhibitory concentration and the FIC calculated. The fractional inhibitory concentration (FIC) was used to interpret the dilution method results and was calculated as follows [21]: FIC of drug A = MIC drug A in combination with decoction or CPZ/MIC drug A alone. Synergy was defined as FIC < 0.5; indifference was defined as 4 > FIC > 0.5; and antagonism was defined as an FIC > 4. The plates were incubated for 24 h at 37°C. CPZ was used as the positive control for efflux pump modulation, due to the fact it affects the function of efflux pumps. All experiments were realized in duplicate.

Results
Both decoctions showed no substantial antibacterial activity at 1024 μg/mL against the strains tested (MIC ≥ 2048 μg/mL). However, when the decoction of termites from C. leptophloeos was added to the growth medium at 256 μg/mL (≥1/8 MIC), a reduction of the MIC for gentamicin and neomycin was observed in the strain E. coli 27 (but not in ATCC 8539), demonstrating a synergistic or additive effect of this natural product with these aminoglycosides ( Table 1). The decoction of termites from A. occidentale L. shown synergism only against neomycin (Table 2), demonstrating the influence of the plant substrate in the pharmacological properties of this natural product of termites.
Synergism between CPZ and gentamicin was not observed, which is suggestive of the occurrence of another resistance mechanism. Another possibility is a pump that can be affected by the termite decoction from C. leptophloeos in the case of gentamicin (Table 1).

Discussion
Evidence of antimicrobial activity of products isolated from termites has been reported. Peptides such as spinigerin and termicin, isolated from Pseudocanthotermes spiniger, showed antifungal and antibacterial activity [22]. Studies on the molecular biology and bioinformatics of the Australian termites of the genus Nasutitermes demonstrated their potential as producers of antimicrobial peptides [23,24]. However, as far as we know, no antimicrobial activity of natural products from N. corniger in terms of synergism with aminoglycosides or any other antibiotic has been reported so far.
Phenothiazines, such as chlorpromazine, act on the plasma membrane of bacteria affecting efflux pumps and causing alterations in permeability, thereby enhancing the activity of antibiotics, including the aminoglycosides [25][26][27]. Efflux pumps are known as resistance mechanisms of E. coli since the 1980s, belonging to the RND family (resistance nodulation division) and representing a mechanism of multidrug resistance (MDR), which has led to antibiotic resistance to aminoglycosides [28,29].
Animals have been methodically tested by pharmaceutical companies as sources of drugs for modern medical science, and the current number of animal sources for producing essential medicines is quite impressive. The chemical constituents and pharmacological actions of some animal products are already known to some extent, and ethnopharmacological studies focused on animal medicines could be very important in clarifying the eventual therapeutic usefulness of this class of biological remedies [30,31]. As pointed out by Alves and Rosa, further ethnopharmacological studies are necessary to increase our understanding of the links between traditional uses of faunistic resources and conservation biology, public  health policies, sustainable management of natural resources and biological prospecting [1].