Green propolis as an adjuvant against nontuberculous mycobacteria

Abstract

Natural products have been touted as important tools because of their vast potential for the development of compounds with antimicrobial activity and the possible inhibitory activity and/or adjuvant resistance mechanisms. Propolis has been empirically used for many years for the treatment of diseases, mainly due to its antioxidant, anti inflammatory and antimicrobial activities. This study aimed to evaluate the in vitro antimycobacterial activity of the ethanol extract of propolis alone and in combination with rifampicin (RIF), amikacin (AMI) and ciprofloxacin (CIP). The ethanol extract of propolis showed antibacterial activity against Mycobacterium chelonae and M. kansasii and was capable of increasing AMI, RIF and CIP activity in combination. On the other hand, compared to M. absecessus, M. fortuitum and M. avium, the extract was not active at 200 µg/mL and did not show pronounced adjuvant capacity when evaluated in association with the drugs. Based on these results, it can be concluded that the ethanol extract of propolis could be an alternative in the development of new drugs and can be used complementary with the current mycobacteriosis treatment.

Key words
additivity; antimicrobial; Mycobacterium sp.; propolis

Resumo

Os produtos naturais têm sido apontados como ferramentas importantes devido ao seu vasto potencial para o desenvolvimento de compostos com atividade antimicrobiana e a possível atividade inibitória e/ou adjuvante de mecanismos de resistência. O própolis é utilizado empiricamente há muitos anos no tratamento de doenças, principalmente devido às suas atividades antioxidantes, anti-inflamatórias e antimicrobianas. Este estudo teve como objetivo avaliar a atividade antimicobacteriana in vitro do extrato etanólico de própolis isoladamente e em associação com rifampicina (RIF), amicacina (AMI) e ciprofloxacina (CIP). O extrato etanólico de própolis mostrou atividade antibacteriana frente Mycobacterium chelonae e M. kansasii e foi capaz de aumentar a atividade de AMI, RIF e CIP em associação. Por outro lado, frente a M. absecessus, M. fortuitum e M. avium, o extrato não foi ativo a 200 µg/mL e não apresentou capacidade adjuvante pronunciada quando avaliado em associação com os fármacos. Com base nesses resultados, pode-se concluir que o extrato etanólico de própolis pode ser uma alternativa no desenvolvimento de novos fármacos e pode ser utilizado complementarmente com o atual tratamento das micobacterioses.

Palavras-chave
aditividade; antimicrobiano; Mycobacterium sp.; própolis

Introduction

The genus Mycobacterium consists of a wide variety of organisms, including obligate, opportunistic pathogens and saprophytic species (Falkinham 2016). Nontuberculous mycobacteria (NTM) are opportunistic microorganisms and may occasionally cause serious diseases in humans, being the most frequent and commonly related to patients with preexisting pulmonary infections or compromised immune systems and attributed mainly to members of the Mycobacterium avium complex and M. abscessus complex (Wu et al. 2018).

Treatment of NTM usually occurs with the same antimicrobials used to treat tuberculosis since the diagnosis of NTM is often difficult and is supposedly treated as TB, although there are differences in the symptoms (Egelund et al. 2015). However, NTM is generally resistant to conventional tuberculostatic drugs, which may compromise the therapeutic response since the mechanisms of drug susceptibility in NTM are distinct from M. tuberculosis, and variations in the susceptibility of some antimycobacterial agents may occur with the species (Wu et al. 2018).

In addition, Ali et al. (2018) demonstrated that extracts of propolis have inhibitory activity against Mycobacterium tuberculosis, potentializing the effect of the main drugs used for the treatment of TB. Historically, the discovery of antimycobacterial drugs and preclinical testing efforts have been almost uniquely centered on M. tuberculosis, with virtually no concentrated effort toward extended-spectrum agents that cover NTM, yet represents a therapeutic challenge, as current and innovative treatment options for NTM are limited or unavailable (Kasperbauer & De Groote 2015; Wu et al. 2018).

This study aimed to evaluate the in vitro antimicrobial activity of the ethanolic extract of green propolis and its combined effect with rifampicin, amikacin, and ciprofloxacin against five mycobacterial species that cause pulmonary infection.

Material and Methods

Propolis sample and Propolis Extract Preparation

The green propolis sample was obtained by Nectar Farmacêutica Ltda. in Minas Gerais (Brazil) and conditioned at -20 ºC. The extract was prepared previously as described by Paulino et al. (2002). The sample was frozen and macerated with extraction solution containing absolute ethanol and stirred at 37 °C for seven days. After the solvent was evaporated, the dry matter was dissolved in PBS (pH 6.2) at a final concentration of 40 mg/mL.

Isolation and preparation of the inoculum

The experiments were conducted at the Medical Microbiology Research Center at the Federal University of Rio Grande (FURG), Rio Grande/RS, Brazil. The strains of Mycobacterium chelonae (ATCC 946), M. abscessus (ATCC 19977), M. fortuitum (ATCC 35931), M. avium (ATCC 03057 HC) and M. kansasii (ATCC 12478) were kept in Ogawa-Kudoh medium for approximately 14 days. Bacterial suspensions were prepared in sterile water tubes containing glass beads. The suspension was homogenized by vortexing, and the turbidity was adjusted according to scale 1 McFarland (3.2 × 10⁶ CFU/mL). The inoculum was prepared with a 1:20 bacterial suspension in 7H9 medium (Middlebrook) (Palomino et al. 2002).

Evaluation of minimum inhibitory concentration (MIC) of extract and antibiotics

The method used to determine the antimycobacterial activity was the Resazurin microtiter assay (REMA), using 96-well microplates (Palomino et al. 2002). At the periphery, 200 μL of sterile distilled water was added to avoid evaporation during the incubation period (7–9 days). Then, 100 μL of 7H9 medium enriched with 10% OADC (oleic acid, albumin, dextrose and catalase) was added to the each well, and 100 μL of the propolis ethanolic extract at the initial concentration of 200 μg/mL or antibiotics amikacin, rifampicin and ciprofloxacin at the initial concentration of 10 μg/mL were added. A 1:2 microdilution was performed, where the concentrations ranged from 200 μg/mL to 6.25 μg/mL for propolis and 10 μg/mL to 0.03 μg/mL for antibiotics. After microdilution, 100 μL of the bacterial inoculum was added as described above. The microplate was incubated according to the growth time of each strain: for the slow-growing mycobacteria (M. kansasii and M. avium), seven days, and for the fast growing bacteria (M. abscessus, M. chelonae and M. fortuitum), five days. After the incubation period, 30 μL of 0.02% resazurin, which acts as an indicator of cell viability, was added, and incubated again for 48 h. MIC was defined as the minimum concentration capable of inhibiting bacterial growth.

Determination of the interaction between the ethanolic extract of green propolis with antibiotics by the checkerboard method

After MIC determination, the effect of the combination of ethanolic propolis extract with rifampicin, amikacin and ciprofloxacin was determined based on the checkerboard method described by Caleffi-Ferracioli et al. (2013). For each bacterial isolate (M. chelonae, M. abscessus, M. fortuitum, M. avium and M. kansasii), a microplate was used with the association of the propolis extract and an antimicrobial: rifampicin, amikacin or ciprofloxacin. Fifty microliters of 10% OADC-enriched 7H9 medium was added to all test wells; 50 μL of the antimicrobial was added, at the initial concentration according to the MIC of each one, and a serial dilution 1:2 was performed in the Y axis. Subsequently, 50 μL of the propolis extract previously diluted 1:2, at the initial concentration of 200 μg/mL, was added in each column of X axis. Finally, 100 μL of the bacterial inoculum was added. The plate was incubated in a bacteriological incubator for 5–7 days, and then 30 μL of 0.02% resazurin, which acts as an indicator of cell viability through an oxi-reduction reaction, was added and incubated again for 48 h. The fractional inhibitory concentration index (FICI) was defined as the lowest concentration at which the extract and the antimicrobial in combination were able to inhibit bacterial growth. The interpretation of the results of the checkerboard was performed through the FICI index obtained by the following formula: FICI = (MIC of the combined extract/MIC of the extract alone) + (MIC of the combined antimicrobial/MIC of the antimicrobial alone).

The FICI results were interpreted as follows: FICI ≤ 0.5 = SYNERGISM; 0.5 < FICI ≤ 1 = ADDITIVITY; 1 < FICI ≤ 2 = INDIFFERENCE and FICI > 2 = ANTAGONISM. In addition, the modulatory factor was calculated as follows: MIC of antibiotic alone/MIC of antibiotic in combination with propolis (Roell et al. 2017).

Determination of total phenolic content (TPC)

The quantification of total phenols was carried out according to the methodology described by Pires et al. (2017), in microplate, using the Folin-Ciocalteau reagent. In brief, the extract sample was diluted with methanol from 100 to 0.78 μg/mL in microtiter plates. For TPC analysis, a calibration curve was established using gallic acid, and the absorbance was measured at λ = 760 nm. The TPC results was expressed as milligrams of gallic acid equivalent per gram of propolis sample.

Results and Discussion

The antimycobacterial activity of green propolis against NTM (M. kansasii, M. avium, M fortuitum, M. abscessus, M. chelonae) was different according to the species of mycobacteria evaluated (Tab. 1). When evaluated against M. avium, M. abscessus and M. fortuitum, the extract showed no antimycobacterial activity (MIC > 200 μg/mL); however, the extract was active against M. chelonae and M. kansasii, with an MIC of 25 μg/mL. In addition, at 200 μg/mL, the alcoholic extract of green propolis was not active against strains of Escherichia coli and Klebsiella pneumoniae and did not present adjuvant activity when evaluated in combination with cephalosporins (data not shown). The antimicrobial activity of different propolis extracts has been widely investigated against several bacteria, especially against strains of Staphylococcus aureus (Fernandes et al. 2005; Lavinas et al. 2019); these studies indicate that the activity of propolis extracts may have a narrow spectrum of related microbial species, mainly gram-positive and mycobacterial microorganisms (Al-Waili et al. 2012; Fernandes et al. 2005; Stepanović et al. 2003; Wojtyczka et al. 2013), similar to that reported in our study.

Table 1
Minimum inhibitory concentration (µg/mL) of antibiotics and ethanolic extract of green propolis against five mycobacteria.

Minimum inhibitory concentration (µg/mL)
	Amikacin	Rifampicin	Ciprofloxacin	Propolis extract
Mycobacterium chelonae	≤ 0.5	1	1	25
Mycobacterium abscessus	16	> 128	32	> 200
Mycobacterium fortuitum	0.5	2	0.03	> 200
Mycobacterium kansasii	0.06	0.015	0.5	25
Mycobacterium avium	2	≤ 0.25	2	> 200

The ethanolic extract of green propolis showed antimicrobial activity, with MIC = 25 µg/mL, against M. chelonae and M. kansasii, when tested alone, and a modulatory effect capable of decreasing the MIC values of amikacin and ciprofloxacin eight and four times, respectively. However, when combined with the antimicrobials evaluated, propolis had an additive effect against M. abscessus (amikacin/propolis), M. chelonae and M. avium (rifampicin/propolis). These results indicate that despite not having synergistic effects, the ethanolic extract of green propolis presented modulating activity, potentiating the antimicrobial activity of these drugs.

Together, these data suggest that propolis may potentiate the effect of some antibiotics, especially those whose mechanism of action is associated with genetic processes that interfere with the maintenance of microbial species. In addition, amikacin has been recommended in adjuvant therapy for NTM infections with significant bactericidal activity (Davis et al. 2007), and in this work, amikacin and the extract displayed only positive associations (indifferent or additive) independent of the evaluated mycobacterial species; therefore, green propolis extract is a good model for the development of new therapeutic alternatives to be used with the current NTM treatment to reduce doses and toxicity and to enable the introduction of a new chemical class in the available pharmacological arsenal. In addition, studies of the chemical composition, activity studies and in vivo toxicity of these substances would be of great interest as a continuation of this work.

The antimicrobial activity found could be related to phenolic compounds that have been identified as one of the main constituents of Brazilian propolis and responsible for the bioactive activity of this natural product against a series of pathogenic microorganisms (Zabaiou et al. 2017). The ethanolic extract of green propolis had a total phenolic content (measured as gallic acid equivalents) of 448.80 mg/g (Fig. 1). The findings of this study coincide with that described by the literature for propolis, since phenolic acid has been identified as the main classes of secondary metabolites extracted in propolis ethanolic and the antimicrobial activity may be due to the action of the phenolic compounds detected (Lavinas et al. 2019; Machado et al. 2016), being appointed as responsible for inhibit NTM growth in previous studies by Mickymaray et al. (2020) and Przybyłek & Karpiński (2019).

Figure 1
Values of ethanolic extract concentration (µg/mL) and total phenols (µg of gallic acid/mL of the extract) contents of Brazilian green propolis.

The chemical constituents of propolis, as well as its varied activity, especially green propolis, have been described in the literature; however, the antimycobacterial activity against NMT has not yet been investigated (Al-Waili et al. 2012; Fernandes et al. 2005; Franchin et al. 2018; Pasupuleti et al. 2017; Scheller et al. 1998, 1999, Stepanović et al. 2003; Wojtyczka et al. 2013; Yildirim et al. 2004). Some phenolic compounds, such as isoflavanoids and phenolic acids, have demonstrated the potential to inhibit the mycobacterial efflux system in NTMs (Gröblacher et al. 2012; Lechner et al. 2008).

In addition, it was previously identified that the antibacterial activity of propolis could be influenced by the variation in chemical composition, which is closely related to the geographic, climatic aspects and the associated plant species (Al-Waili et al. 2012; Zabaiou et al. 2017). This fact could be corroborated by the findings of Monzote et al. (2012), which identified the antimicrobial activity of three Cuban propolis, known as brown, red and yellow, against different microorganisms, among them S. aureus and E. coli. This fact was in contrast to the results found in our study, where the object of study was green propolis, which presented activity strictly related to nontuberculous mycobacteria.

Antimicrobial activity of aqueous extracts of Turkish propolis has been identified by in vivo assays using guinea pigs infected with M. tuberculosis (Yildirim et al. 2004), whereas Scheller et al. (1998) identified similar anti-TB activity in ethanolic extracts, corroborating our findings regarding the genus Mycobacterium.

Mycobacterium kansasii continues to be the most easily treatable NTM lung disease pathogen, and unlike most NTM, there is a good correlation between in vitro and in vivo susceptibility in response to a variety of antimicrobial agents, including rifampicin, macrolides and fluoroquinolones Griffith et al. (2007), which was also evidenced in our study, where M. kansasii presented the lowest MIC values for amikacin (0.06 µg/mL) and rifampicin (0.015 µg/mL) between the NTM species evaluated.

Different species within the same genus of microorganisms differ in their susceptibility to antimicrobials. This susceptibility is a reflection of the genetics of each species and environmental pressures (Fogelson et al. 2019). As can be seen in Table 2, rifampicin increases its inhibitory effect in M. chelonae and M. avium when combined with the propolis ethanolic extract. In contrast, the opposite occurs with M. fortuitum, where we observed an antagonistic effect in this same combination. The fact is that the first two species mentioned are phylogenetically distant from M. fortuitum (Tortoli et al. 2017), which may be linked to the differences observed in the susceptibility pattern. Still in Table 2, we can see that the reverse occurs for the combination ciprofloxacin + propolis, where it is antagonistic against the growth of M. chelonae and M. avium, but demonstrates an additivity profile when tested against M. fortuitum.

Table 2
Combined minimum inhibitory concentration (MIC), fractional inhibitory concentration index (FICI) and modulatory factors (MFs) of antibiotics and ethanolic green propolis extract against fast-growing and slow-growing mycobacteria (µg/mL).

Strains	MIC (µg/mL) Amikacin/Propolis	FICI	MF	MIC (µg/mL) Rifampicin/Propolis	FICI	MF	MIC (µg/mL) Ciprofloxacin/Propolis	FICI	MF
Mycobacterium chelonae	0.06 / 25	1.12	8.33	0.5 / 25	1	2	0.5 / 50	3	1
Mycobacterium abscessus	8 / 100	1	2	> 128 / > 200	2	1	> 128 / > 200	5	0.25
Mycobacterium fortuitum	0.5 / 25	1.1	1	> 128 / > 200	5	0.25	0.03 / 6.25	1.0	1
Mycobacterium kansasii	0.03 / 12.5	1.5	2	0.015 / 6.25	1.2	1	0.12 / 25	1.2	4.16
Mycobacterium avium	2 / 25	1.1	1	≤ 0.25 / 6.25	1.0	1	4 / 50	2.3	0.5

• MIC = minimum inhibitory concentration; FICI = fractional inhibitory concentration index; MF = modulatory factor; FICI ≤ 0.5 = synergism; 0.5 < FICI ≤ 1 = additivity; 1 < FICI ≤ 2 = indifference; FICI > 2 = antagonism.

In relation to the antimicrobial FICI and the ethanolic extract of green propolis (Table 2), amikacin had an additive effect only when evaluated in combination with M. abscessus. However, analysis of modulatory factors (MFs) indicated that the MIC value of this antibiotic against M. chelonae was reduced 8.3 times, unlike M. abscessus and M. kansasii, which had a reduction of only half the value.

Compounds that, through rational synergism, act to facilitate the action of antimycobacterial drugs with actions at the intracellular level, interfering in processes critical for maintenance and viability, such that, for example, they aid in the permeability of these drugs, are needed (Falkinham 2018). A similar effect of synergism between propolis and antimicrobial drugs that act on the bacterial ribosome (such as amikacin) has been demonstrated in a previous study (Maurer et al. 2014; Orsi et al. 2012), in addition to the capacity of ethanolic extract of Brazilian green propolis to increase the immunological response acting as adjuvant in the fight against inflammatory processes (Franchin et al. 2018).

Rifampicins have broad antimicrobial coverage and are frequently used in the treatment of NTM infections (Ramis et al. 2018), presenting an additive interaction with the ethanolic extract of green propolis for M. chelonae and M. avium, similar to the findings reported by (Scheller et al. 1999), which showed a positive interaction between the association of this antimicrobial with the ethanol extract of propolis against other mycobacteria.

Ciprofloxacin belongs to the group of fluoroquinolones, which have been used in the treatment of NTM infections (Egelund et al. 2015). Based on results of the present study, the combination of ciprofloxacin and the ethanolic extract of green propolis, even with the indifference identified by FICI for M. kansasii, there seems to be a positive association since the modulatory factor was 4.16.

Acknowledgements

This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) under Grant CNPq 474767∕2013-2.

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Edited by

Area Editor: Dr. Leopoldo Baratto

Publication Dates

• Publication in this collection
  03 Dec 2021
• Date of issue
  2021

History

• Received
  19 Aug 2020
• Accepted
  16 Nov 2020