Interaction between bark extract of Anadenanthera colubrina var. cebil (Griseb.) Altschul with antibiotics against methicillin-resistant Staphylococcus aureus (MRSA)

Anadenanthera colubrina var. cebil (Griseb.) Altschul, a plant often found in areas of the Caatinga in northeastern Brazil, is widely used in unconventional medicine for the treatment of infections and inflammations. Thus, the aim of the present study was to evaluate the antibacterial activity of A. colubrina bark extracts against methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates, to investigate if intact and regenerated bark extracts have the same effect against MRSA and to determine the interaction between these extracts and antibiotics. The antibacterial activity was performed by the determination of the minimum inhibitory concentration (MIC) according to the Clinical and Laboratory Standards Institute (CLSI) and the interaction assay was performed by the checkerboard method. A. colubrina extracts showed bacteriostatic activity (MIC = 8-32 mg/L) against MRSA clinical strains and no difference was found in antibacterial activity between intact and regenerated barks, suggesting that even after regeneration, the barks of this species have the same antibacterial activity. Moreover, the in vitro interaction of A. colubrina extracts with ciprofloxacin or erythromycin was additive (FICI = 0.52). Thus, the bark extracts of Anadenanthera colubrina exhibit antibacterial activity and can be used alone or in combination with antibiotics against MRSA clinical isolates.


Introduction
Staphylococcus aureus is a microorganism of great interest in studies assessing antibacterial activity given this bacterium is one of the main etiological agents of hospital-acquired infections. Also, the ability of S. aureus to develop resistance to antibiotics is a major concern in the community( (Faria et al., 2005;Mairi, Touati, & Lavigne, 2020) and has prompted researchers in the search for novel therapeutic options such as natural products.
Anadenanthera colubrina var. cebil (Griseb.) Altschul, (synonymous: Mimosa colubrina Vell.; Piptadenia colubrina (Vell.) Benth; Piptadenia macrocarpa Benth.) popularly known as "angico" and "angico de caroço" is a plant that is part of the Fabaceae family. This species is found in areas of the Caatinga, in Northeast region of Brazil. Its bark is very sought after for use in leather tannery. In addition, it is one of the botanical species most cited by the local population due to its medicinal properties (Agra, Baracho, Nurit, Basílio, & Coelho, 2007;A. R. N. Lima et al., 2020;Weber et al., 2011).
Phenolic compounds are the vast majority of substances extracted from the bark of A. colubrina (A. R. N. Lima et al., 2020). These compounds are responsible for conferring their antimicrobial and antibiofilm properties (T. A. de S. Araújo et al., 2008;Katalinic et al., 2013;Kluczynik et al., 2010;R. D. F. Lima et al., 2014;Palmeira et al., 2010). However, there is a lack of studies that are able to evaluate the antibacterial activity of these extracts against antibiotic-resistant microorganisms, such as methicillin-resistant Staphylococcus aureus (MRSA).
Furthermore, there are studies that explore the potential of intact and regenerated A. colubrina bark for the bioprospection of medicines, since natural compounds have been proposed as promising strategies for the treatment of infections (Hiramatsu, 2001;Holetz et al., 2002;Jandú, Silva, Silva, & Correia, 2015;Marques et al., 2018;S. W. C. Silva et al., 2019), especially because it contains three large phenolic compounds (epigallocatechin gallate, tohiperina and gallate) (T. A. de S. Araújo et al., 2015). Additionally, the association of substances with antimicrobial properties represents another important tool for infection treatment caused by resistant bacteria, such as MRSA, since combination therapy can help prevent the selection of resistant isolates and broaden the spectrum of action of antimicrobial agents (Cavalcanti et al., 2018;Jackson, Agboke, & Nwoke, 2009;J. L. da Silva, Mesquita, & Ximenes, 2009).
Thus, the aim of the present study was to ascertain the antibacterial activity of A. colubrina bark extracts against MRSA clinical isolates, to verify if intact and regenerated bark extracts have the same effect against MRSA and to determine the interaction between these extracts and antibiotics.

Plant material and extract preparation
The plant material was collected from the rural area of the city of Altinho, Northeast Brazil (08°35'13.5" S and 36°05'34.6" W). This location is characterized by Caatinga scrub land, highly irregular rainfall, a hot, semi-arid climate (Bsh) and an average temperature exceeding 26 °C (R. M. S. De Araújo et al., 2012). A voucher specimen (48633) was deposited in Herbário Geraldo Mariz, Universidade Federal de Pernambuco (UFPE) and the plant was identified by Dra. Viviany Teixeira do Nascimento, Universidade do Estado da Bahia (UNEB).
Intact and regenerated bark was collected from the same tree specimens, giving a total of 12 samples in order to rule out genetic interference. The bark was also taken from a similar region of the trunk so as to reduce environmental effects such as sunlight exposure and mechanical impact.
The material was dried at an ambient temperature of 25 ± 2 ºC and ground in a Wiley cutting mill to obtain a granulometry of 20 Mesh. The powder was submitted to extraction using 80% methanol at the proportion of 1:20 (w/v) for 72 hours and then filtered. The liquid extracts were evaporated in a rotary evaporator at 40 ± 2 °C under reduced pressure to obtain a dry solid extract.

Antibacterial activity
The antibacterial activity of intact and regenerated bark extracts of A. colubrina and the antibiotics, ciprofloxacin (CIP) and erythromycin (ERY), was performed by the broth microdilution method according to Clinical and Laboratory Standards Institute guidelines (CLINICAL AND LABORATORY STANDARDS INSTITUTE (CLSI)., 2020). Initially, 96well microlitres plates were filled with Müeller-Hinton broth (MHB) and then each extract or antibiotic were added to obtain different extract and CIP concentrations ranging from 0.5 to 250 mg/L and ERY concentrations ranging from 2 to 1024 mg/L. Subsequently, a bacterial suspension, suitably diluted to achieve a final concentration of 10 5 CFU/mL in each well, was added.
The microplates were then incubated at 35 ± 2°C for 24 h. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of the drug that causes complete inhibition by spectrophotometry (Ultrospec® 3000 pro -Amersham pharmacial biotech) at 630 nm. Minimum bactericidal concentration (MBC) was determined from the well where MIC results showed no bacterial growth, which was then seeded in Müeller-Hinton agar and incubated at 35 ± 2°C for 24 h. MBC was defined as the lowest concentration of the drug that resulted in > 99.9% decrease in the initial bacterial inoculum. All the experiments were performed in triplicate.

In vitro interaction assay
The in vitro interaction between A. colubrina extracts and antibiotics was performed by the checkerboard method (Sopirala et al., 2010). The clinical isolates used in this study were the five most resistant to ERY and CIP. Initially, the 96well microplates were seeded by dispersing MHB into each well. Next, it was dispensed in the X-axis of the 96-well microdilution plates the serially diluted antibiotics (ERY or CIP) and in the Y-axis the testing extracts to obtain a final concentration equal to the MIC or dilutions lower than the MIC of the respective drugs. Finally, each plate received the adjusted bacterial suspension (10 5 CFU/mL) and they were then incubated at 35 ± 2°C for 24 h. tested alone, respectively. The interaction is considered synergic for FICI ≤ 0.5; additive (0.5 < FICI ≤ 1), indifferent (1 < FICI ≤ 2) and antagonistic (FICI > 2) (Cavalcanti et al., 2018).

Antibacterial activity
All the A. colubrina extracts exhibited a bacteriostatic effect (MIC = 8 -32 mg/L), but none showed bactericidal effect (MBC > 250 mg/L) against MRSA clinical strains (Table 1) colubrina extracts exhibited good activity against MRSA. In addition, no difference in MIC values was found between extracts derived from intact or regenerated barks, suggesting that even after regeneration, the barks of this species have the same antibacterial activity (Table 1). Study of intact and regenerated bark about antibacterial activity can be valuable for the local population and the pharmaceutical industry by demonstrating whether regenerated bark has the same pharmacological effects, given that its metabolic composition may change following regrowth. If similar action of the bark is observed, this will confirm the species as a renewable resource for use in the manufacture of antibacterial medications.
In studies with extracts of traditional medicinal plants from the Brazilian flora against MRSA strains, it was found that ethanol extracts of Punica granatum (Romãzeira) and Handroanthus impetiginosus (Ipê-roxo) exhibit MICs ranging from 125 to 250 mg/L (Machado et al., 2003). Sousa (2019)  Regarding the susceptibility of these isolates to erythromycin and ciprofloxacin, MIC values ranged from 64 to 1024 mg/L for ERY and 4 to 32 mg/L for CIP. These same values were also found in the others studies (Macêdo et al., 2013;Segatore et al., 2012).
It is important mention that, despite the proven antibacterial activity of A. colubrine, its effect against the MRSA was only bacteriostatic limiting its use in the treatment of infections caused by this microorganism. Thus, the interaction studies with antibacterial agents are an option to propose the use of this extract in therapy. Research, Society andDevelopment, v. 10, n. 6, e14210615469, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i6.15469 7

In vitro interaction assay
The combination of the intact and regenerated A. colubrina bark extracts with ERY or CIP resulted in an additive effect for most of the isolates (FICI = 0.52), except for the combination of A03 and AR03 extracts with ERY and CIP, which produced a synergic effect against most of the MRSA isolates (FICI = 0.27) ( Table 2), allowing that previously ineffective antibiotics can acting against these bacteria.
Antibacterial therapy based on a combination of drugs has shown positive results in preventing the emergence of bacterial resistance and producing beneficial effects in the treatment of infections caused by bacteria. The interaction between antibiotics and bioactive plant extracts is a relatively recent concept and one which can yield beneficial effects such as synergistic or additive interaction (Albano et al., 2016;Gibbons, 2004;Manekeng et al., 2018). This strategy, utilization of plants and drugs in a technique that combines mono-or multi-extracts, is known as "herbal shotgun" or "synergistic multieffect targeting". This method is capable of interact with not only a single target but various targets, in which the different therapeutic components act together in a synergistic way . Comparatively, natural products can vary and have an antibacterial activity or resistance-modifying activity, when considering the existence of variabilities in polarity and secondary metabolites, which are related to affinities for biological action . The mechanisms by which the natural compounds can interfere with the growth of microorganisms are diverse and can be related to the chemical nature of some components. In consequence, phytochemicals can demonstrate a greater interaction with the lipid bilayer of the cell membrane, acting on the respiratory chain and energy production, or even make the cell more permeable to antibiotics, leading to the interruption of vital cellular activity (Menezes et al., 2015). Thus, the combination of antibiotics with A. colubrina bark extract can be an alternative to minimize the side effects of these antibiotics, since the association leads to a synergistic effect, significantly reducing the MIC of these drugs, decreasing the dose needed for therapeutic usage (Fernando G. Figueredo et al., 2013). Braga et al. (2005) assessed the interaction between Punica granatum and the antibiotics chloramphenicol, gentamicin, ampicillin, tetracycline and oxacillin against MSSA and MRSA clinical isolates. The combinations showed synergistic and indifferent effects (Braga et al., 2005). Silva et al. (2019) studied the antibacterial activity of the dichloromethane fraction (DCMF) from the stem bark of Mimosa caesalpiniifolia and its effect on the activity of conventional antibiotics against Staphylococcus aureus strains overexpressing specific efflux pump genes. They concluded that such compounds could be used as adjuvants of norfloxacin, ciprofloxacin or tetracycline for treatment of infections caused by S. aureus strains overexpressing efflux pumps (S. W. C. Silva et al., 2019).
A possible action mechanism for natural compounds in antibiotic-resistant microorganisms is the interaction with efflux pumps, which are energy-dependent proteins that promote the elimination of antimicrobial agents into the extracellular environment faster than plasma membrane diffusion to aid bacterial resistance. In S. aureus, NorA, NorB, NorC and Tet38 are chromosome-encoded efflux pumps of which the overexpression can confer resistance to multiple drugs (MDR), quinolones and other compounds (Nor pumps) or tetracyclines (Tet38). Natural products such as silybin, terpinene, tannic acid, or polyphenols such as gallic and caffeic acids have been shown to inhibit NorA efflux in S. aureus and, thereby, restore sensitivity to antibiotics in MRSA (Fernando Gomes Figueredo et al., 2020).
Thus, in our study, the combination of A. colubrina extracts with the antibiotics ERY and CIP substantially reduced the MIC of these drugs, increased their activity and also reduced the toxic effects of the antibiotics given that lower doses can be used. Research, Society and Development, v. 10, n. 6, e14210615469, 2021 (CC BY 4.

Conclusion
A. colubrina bark extracts exhibited bacteriostatic activity and a highly effective therapeutic option against MRSA.
The intact and regenerated bark showed the same affect against MRSA reiterating that the process of bark regeneration of this plant had no negative impact on the antibacterial activity of the extract against MRSA. A. colubrina bark extracts potentiated the antibacterial activity of antibiotics against MRSA clinical isolates. Thus, the results suggest that A. colubrina intact and regenerated bark extracts offer potential as an antibacterial agent and may, in the future, be used alone or in combination with antibiotics for the treatment of infections caused by resistant microorganisms, especially MRSA.