Synergism between molecules derived from Garcinia brasiliensis and antimicrobial drugs on field isolates of Mycoplasma hyopneumoniae

Abstract

Mycoplasma hyopneumoniae (M. hyopneumoniae) is one of the smallest free-living bacteria found in nature; it has an extremely small genome and lacks a cell wall. It is the main etiological agent of porcine enzootic pneumonia (EP), a chronic respiratory disease with worldwide distribution that causes significant losses in swine production. Due to the great economic impact caused by EP, new strategies for treating and controlling this agent are researched. The objective of this study was to verify the anti-M. hyopneumoniae activity of compounds derived from Garcinia brasiliensis and the synergism with the main antimicrobials used in the treatment of EP; this is the first study assessing the synergism between bioactive molecules and antimicrobial compounds in vitro against isolates of M. hyopneumoniae. The minimum inhibitory concentrations (MICs) of the antimicrobials tiamulin, valnemulin, and enrofloxacin, as well as the bioactive compounds guttiferone-A (Gut-A), 7-epiculsone (7-Epic), copper 7-epiculsone (7-Epic-Cu), and benzophenone, were determined. Subsequently, the interactions of antibiotics with the compounds were evaluated using the checkerboard method. Three field M. hyopneumoniae isolates were used, and the J strain was used as a control. The MIC values of the antimicrobials compared to the field isolates were equal to and lower than those of the reference strain J. Among the compounds used, 7-Epic-Cu showed the lowest MIC value. Synergistic association was observed for Gut-A with tiamulin and valnemulin, whereas 7-Epic and 7-Epic-Cu showed synergistic action with enrofloxacin. No synergistic effect was observed for benzophenone. Despite being an initial study, the results suggest that these combinations hold promise for the treatment of infections caused by M. hyopneumoniae.

Introduction

Mycoplasma hyopneumoniae (M. hyopneumoniae) is the etiological agent of porcine enzootic pneumonia (EP) and is considered one of the leading agents involved in the porcine respiratory disease complex (CDRS). This complex is responsible for considerable economic losses in pig farming [1].

The control of infections in herds is carried out with the management of biosafety practices, vaccination, and antimicrobials. However, under field conditions, vaccines against M. hyopneumoniae have shown variable efficacy, leading in practice to the regular use of antimicrobials against their infections [2].

Due to selective pressure, resulting in multidrug-resistant bacteria, alternative antimicrobials are being researched. Plants of the genus Garcinia are rich sources of bioactive compounds [3]. Garcinia spp. is a genus of the tree known as “bacupari mirim”; one of the most abundant bioactive substances isolated from its fruits is guttiferone-A (Gut-A), presenting antimicrobial, anti-HIV, trypanocidal, antispasmodic, antioxidant, and antitumor activity [4, 5]. Other compounds from this tree and semi-synthetic compounds also have antimicrobial activities, among other activities, such as anti-proliferative activity against cancer cells [6, 7]. The molecule 7-epiculsone is derived from the substance Gut-A and displays a broad spectrum of activities, including antimicrobial action against Streptococcus spp. [8]. From 7-epiculsone, copper 7-epiculsone originated, and the association of copper microparticles in bioactive compounds enhances the antimicrobial property of the compound especially for applications where there is humidity for the release of ions present [9, 10]. The compound 2,2′,4-trihydroxybenzophenone is a non-hemolytic compound derived from benzophenone, a synthetic molecule that acts on the cell wall of microorganisms [9]. Its synergistic potential has already been evaluated for other microorganisms Staphylococcus aureus, Bacillus cereus, Streptococcus mutans, and Streptococcus spp. [8, 10, 11] but never against M. hyopneumoniae.

The antibacterial potential of these compounds against M. hyopneumoniae and their synergistic effects with antimicrobials used in the treatment of EP are not known. Thus, this work aimed to verify the anti-M. hyopneumoniae activity of bioactive compounds and their synergism with the main antimicrobials used in the treatment of EP.

Materials and methods

Culture of strains

Origin

The M. hyopneumoniae field isolates used in this study were obtained from lungs with lesions suggestive of M. hyopneumoniae from a slaughterhouse in the Zona da Mata region of the state of Minas Gerais, Brazil, from November to December 2017 [12] as described by [13]. Identification was confirmed by partial sequencing of the 16s gene (Fig. 1). The isolates were denominated UFV01, UFV02, and UFV03 (Fig. 1a). The standard strain J was provided by the Brazilian Company for Research and Agriculture (EMBRAPA) for swine and poultry. The isolates were stored in an ultra-freezer at − 80 °C.

Fig. 1.

Identification of Mycoplasma hyopneumoniae isolates with reference strains deposited in GenBank (a, b), and conserved sequences of the 16S rRNA gene among isolates were performed using the CLC Main Workbench 6.7.1 software (c)

Preparation of compounds and antimicrobials

The bioactive substances studied were obtained from fruits of Garcinia brasiliensis cultivated at the herbarium of the Federal University of Viçosa (UFV), with the voucher specimen of the species deposited under the number VIC2604. The compounds were obtained in the laboratory of the Department of Chemistry.

To dilute Gut-A and 7-Epic, 500 μL of DMSO dimethylsulfoxide (Merck) and 500 μL of phosphate-buffered saline (PBS) were added to 1 mg of each compound. For 7-Epic-Cu, 100 μL of DMSO and 900 μL of PBS diluted in 1 g were used. To 2,2′,4-trihydroxybenzophenone (benzophenone), 50 μL of DMSO was added, followed by 950 μL of PBS for a final concentration of 1 mg/mL. Serial dilutions were used so that the plate concentration ranged from 50 to 0.00001 mg/L. The antimicrobials were weighed and diluted in distilled water, maintaining a concentration of 1280 mg/L for the stock solution. Again, serial dilution was used. Thus, antimicrobial concentrations ranged from 64 to 0.00001 mg/L, placed into the holes in the plates. The antimicrobials used were valnemulin, tiamulin, and enrofloxacin (Sigma).

Minimum inhibitory concentration (MIC)

The antimicrobial susceptibility test was performed using the microbroth dilution method to determine the MIC, as described by [14, 15]. Each isolate was reactivated in FRIIS medium 1x until reaching a count between 10³ and 10⁵ CCU/mL [16]. Subsequently, 100 μL of the culture was added, 10 μL of the isolate and 90 μL of the modified Friis medium were added per well, and the plate was incubated at 37 °C for 1 h. After that, 100 μL of the antibiotics tiamulin, enrofloxacin, and valnemulin were added to the wells at a concentration of 64 to 0.00001 mg/L. The concentrations of the compounds in the plate ranged from 50 to 0.00001 mg/L. The compounds were added to the wells in triplicate until the final tested concentrations of 7-Epic-Cu, 7-Epic, Gut-A, and benzophenone. Strain J was used as a positive control and Friis medium as a sterility control. The plates were sealed with parafilm, covered, and incubated at 37 °C for 14 days.

Checkerboard

The checkerboard method was applied to determine the interactions between antimicrobials and bioactive compounds [17]. The UFV03 isolate and the J strain were selected for performing the technique. The UFV03 isolate was chosen because, among the field isolates, it presented a better growth curve. Solutions containing antimicrobials and compounds were readjusted to 1x concentrations, 0.5x, 0.25x, 0.125x, and 0.06x, where 1x corresponds to the MIC value found. The inocula were homogenized in a sterile channel (10 μL of the stock of isolates, 90 μL of the Friis medium) and added to each well in the 96-well plate. The wells were filled with 50 μL of the antimicrobial solution (Abscissa) and with 50 μL of the bioactive substance solution (ordered) in the following concentrations: 1x, 0.5x, 0.25x, 0.125x, and 0.06x. As sterility control, Friis medium was used.

The results were algebraically evaluated based on the inhibitory concentration fraction index (FICi), using the following equation: MICab/MICa+MICab/MICb, where MICab is the MIC of the antimicrobial in combination in the well, MICa is the MIC of the antimicrobial alone, MICab is the MIC of the bioactive compound in combination in the well, and MICb is the MIC of the compound alone. Synergism values are evaluated considering total synergism (FICI ≤ 0.5), partial synergism (0.5 < FICi ≤ 0.75), no effect (0.75 < FICi ≤ 2), or antagonism (FICi > 2). The results were interpreted from the construction of an isobologram, as described elsewhere [18].

Results

The MIC results are shown in Table 1. The MIC values of the tested compounds and antibiotics showed slight variations among the three isolates and the J strain. The field isolates suffered great selective pressure due to the constant use of antimicrobials (tiamulin, valnemulin, and enrofloxacin). However, the results presented in Table 1 show that they were still sensitive in vitro when compared to isolate J. The high MIC value of benzophenone (6.2 μg/mL) is noteworthy.

Table 1.

Minimum inhibitory concentration (MIC) values of antimicrobials and bioactive compounds used in the antimicrobial susceptibility test in Mycoplasma hyopneumoniae isolates

Antimicrobials/bioactive compounds*	MIC (μg/ml)
	UFV01	UFV02	UFV03	J
Valnemulin	0.001	0.001	0.001	0.001
Tiamulin	0.125	0.06	0.125	0.25
Enrofloxacin	0.03	0.03	0.03	0.06
Guttiferone-A*	3.1	3.1	3.1	3.1
7-epiculsone*	0.39	0.39	0.39	0.39
Copper 7-epiculsone*	0.19	0.19	0.19	0.19
Benzophenone A*	6.2	6.2	6.2	6.2

*Bioactive compounds

As the three field isolates had the same MICs for the respective antimicrobials and compounds, except for the UFV02 isolate, which had a lower MIC value for tiamulin, the UFV03 isolate was selected for the checkerboard method. The results and interpretation of the interaction between bioactive compounds and antimicrobials are shown in Table 2 and Fig. 2.

Table 2.

Inhibitory concentration fraction index (FICi) values of bioactive compounds interacting with antimicrobials showed synergism

Checkerboard	FICi
	UFV03	J
Tiamulin × Gut-A	0.2A	0.34A
Valnemulin × 7-Epic- Cu	0.56B	0.54B
Valnemulin × Gut-A	0.3A	0.4A
Enrofloxacin × 7- Epic	0.5A	0.55B
Enrofloxacin × 7-Epic- Cu	0.3A	0.3A

All other interactions tested did not show a synergistic effect, with interaction index indices in the range of 0.75 < FICi ≤ 2

^ATotal synergism

^BPartial synergism

Fig. 2.

Isolobogram demonstrating the synergism between valnemulin + guttiferone-A using the UFV03 isolate. The FICi was 0.3*

Discussion

The commonly used sensitive, resistant, or intermediate designations have yet to be determined for mycoplasma infections as pharmacological data are scarce [12]. Even international reference guides, such as the Clinical and Laboratory Standards Institute (CLSI), which establishes standards for antimicrobial susceptibility tests, do not provide information on Mycoplasma species of veterinary importance, making it difficult to assess the results and leading to a lack of consensus among the researchers [12]. Due to the scarcity of data regarding reference standards and the fastidious nature of M. hyopneumoniae, few research groups worldwide have mastered its isolation and cultivation technique. The authors of one study [2] determined that the J strain MIC values for tiamulin and valnemulin were 0.125 and 0.008 μg/mL, respectively, whereas another study [19] found values of 0.05 and 0.2 μg/mL for valnemulin and enrofloxacin. Both cited authors used the J strain results as a reference.

In this study, we also used the result of the J strain as a reference, obtaining values of 0.25, 0.06, and 0.001 μg/mL for tiamulin, enrofloxacin, and valnemulin, respectively. Although previous studies used the same strain as reference, some methodological aspects of the technique and the brands of antibiotics used were different, which may explain the variation in the data.

In this study, when we compared the MIC values of tiamulin, enrofloxacin, and valnemulin of the field isolates (UFV01, UFV02, and UFV03), they presented values equal to or lower than those found for the J strain, confirming that these isolates are sensitive to the tested antimicrobials.

The bioactive compounds used in this study were obtained from the fruits of Garcinia brasiliensis [3]. The MIC values of the field isolates were the same as those found in isolate J (Table 1). Notably, only the benzophenone compound showed much higher MIC values when compared to the other compounds. In a study on the action of benzophenone [9], the authors determined that the main action takes place on the cell wall of gram-positive and gram-negative bacteria. However, it is worth mentioning that M. hyopneumoniae is an agent of the class Mollicutes, which phylogenetically originate from gram-positive bacteria but do not have a cell wall. This explains the high MIC value and the fact that all checkerboard tests of benzophenone with the antimicrobials tested revealed no effect. The other compounds tested also have their action described on the cell wall of bacteria, but based on the results, there may still be unknown sites of action and interaction with the cell membrane of M. hyopneumoniae.

Tiamulin and valnemulin showed strong synergism with the compound Gut-A, mainly because both belong to the class of Pleuromutilins and have similar chemical structures and mechanisms of action. Another study [8] also found synergistic interactions of antimicrobials when associated with the compound Gut-A on isolates of Streptococcus spp.

Enrofloxacin acted synergistically when associated with the compounds 7-Epic and 7-Epic-cu. In other studies, [8, 10] 7-Epi was investigated in combination with antimicrobials used for the treatment of bovine mastitis on field isolates of Streptococcus spp. Although the authors observed synergism, 7-Epic-cu showed better results. This result corroborates a previous study [20] which proved that the association of metals, such as copper, to bioactive compounds could increase their antimicrobial activity. However, the inhibition of M. hyopneumoniae multiplication against the compounds used is new and essential information to outline new strategies for PE control. It is worth mentioning that the UFV03 isolate tested in this study is a field isolate that suffered great selective pressure and had a low MIC value compared to the J strain. Yet the compounds were able to inhibit its multiplication.

The synergistic effect found in the interactions of tiamulin + Gut-A, valnemulin + Gut-A, enrofloxacin + 7-Epic, and enrofloxacin + 7-Epic-Cu is a satisfactory result, even if at sub-inhibitory concentrations, commercial antimicrobials showed better antimicrobial action compared to their individual antimicrobial activity, and these associations may become promising for the treatment of PE.

This study has the limitation of being conducted with a limited number of M. hyopneumoniae isolates, primarily due to the challenges associated with obtaining new isolates and the difficulties in in vitro cultivation. However, the obtained results do not invalidate the current research. Instead, they highlight an opportunity for further exploration of novel interactions between natural compounds and antimicrobials. Future studies can expand upon this by including a larger number of M. hyopneumoniae isolates and conducting tests against other Mycoplasma species

Conclusions

The results of this study indicate that the bioactive compounds Gut-A, 7-Epic, and 7-Epic-Cu alone or in combination with the antimicrobials enrofloxacin, valnemulin, and tiamulin are promising in the treatment of M. hyopneumoniae infections.

Author contribution

All authors contributed to the study conception. Material preparation, execution, and analysis were performed by BAEAJL, LTT, JL, FAFR, MB, LFL, NFG, MHS, ASJ, and MASM. The manuscript was written by BAEAJL and LTT and all. All authors read and approved the final manuscript. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Funding

This research was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES; Brasília, Brazil), the Conselho Nacional de Desenvolvimento Científco e Tecnológico (CNPq; Brasília, Brazil) (CNPQ - 307701/2019-0), and the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG; Belo Horizonte, Brazil).

Data availability

The datasets generated or analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Ethical approval

Not applicable.

Conflict of interest

The authors declare no competing interests.