Gas Chromatography-mass Spectrometry analysis, Biotoxicity and Antibiofilm Activities of Syzygium aromaticum against Corynebacterium diphtheriae

Abstract

The objective of this study was to investigate the chemical composition of Syzygium aromaticum essential oil (SAEO), both as its toxicity and biological activities on Corynebacterium diphtheriae. The essential oil (EO) was obtained by hydrodistillation and verified by GC-MS. The main chemical components were eugenol (48.79%), caryophyllene (44.29%) and α-humulene (4.87%). In vitro tests with human red blood cells, blood compatibility. SAEO showed high toxicity for Artemia salina and the bioassay on Tenebrio molitor larvae revealed an average survival rate of 80.00%. The antioxidant activity of SAEO was moderate (40.60%) using the phosphomolybdenum method. SAEO inhibited the growth of all C. diphtheriae strains tested at minimum inhibitory concentrations of 62.5-1000 µg mL^-1 (strong to moderate). The minimum bactericidal concentration of SAEO was observed in two strains. Microorganisms cultivated in the presence of subinhibitory concentrations (subMIC) do not show morphological changes. The subMIC of SAEO inhibited in some strains, but in most cases, increased biofilm formation. In conclusion, the results of this study found that SAEO has an inhibitory effect against C. diphtheriae, representing an alternative antimicrobial therapy, although more in vivo biological studies, alone or in combination with occasional antimicrobials, should be carried out to better evaluate its effects.

Key words
antibacterial activity; antioxidant activity; biofilm; essential oil; clove

INTRODUCTION

Over the past two decades, many parts of the world have experienced outbreaks of emerging and re-emerging infectious diseases with epidemic potential that have led to deaths and growing concern. One of these diseases is diphtheria, listed by the World Health Organization in 2018 as one of the priority infectious diseases that threaten global health security (Zumla & Hui 2019).

Corynebacterium diphtheriae is the main etiological agent of diphtheria (Santos et al. 2015), which is a disease whose cases were partially controlled worldwide after the development of the diphtheria toxoid vaccine. However, thousands of episodes of diphtheria are still reported annually, even in countries with high vaccination coverage (World Health Organization 2021).

Vaccination against diphtheria is performed by applying an inactivated form of the diphtheria toxin (toxoid), which allows asymptomatic colonization and silent transmission of the pathogen to continue, resulting in the need for intense epidemiological surveillance (Hennart et al. 2020). It is noteworthy that C. diphtheriae isolates derived from classical diphtheria and invasive infections may be tolerant or resistant to penicillin and erythromycin, the drugs of choice for therapy (Santos et al. 2015, Schaeffer et al. 2021, Nguyen et al. 2022), besides other antibiotics or carried resistance genes against tetracycline or sulfonamides (Schaeffer et al. 2021).

The genomes of C. diphtheriae isolates from infections that occurred between 2010 and 2019 showed a greater number of antimicrobial resistance genes when compared to isolates of the same species from the previous decade, indicating that C. diphtheriae is acquiring resistance (Will et al. 2021).

In the context of bacterial resistance to antibiotics, the search for drugs or compounds that increase the activity of drugs or reduce cases of microbial resistance found a strong source of research in natural products (extracts, oils and phytoconstituents), particularly in medicinal plants that, in certain circunstances, help in primary health care and as a therapeutic complement compatible with conventional medicine (Kpadonou et al. 2019).

Syzygium aromaticum, popularly known as clove, is marketed as a dry flower bud and is obtained from mature inflorescences collected from the clove tree (Gomes et al. 2018). The S. aromaticum essential oil (SAEO) is obtained from the dried inflorescences of the clove tree and presents analgesic, anticarcinogenic, antimutagenic, insecticidal, antifungal, and antibacterial action, for example (Adams 2005).

Considering the challenge in the treatment of infections caused by antimicrobial-resistant bacteria, as already reported for C. diphtheriae, there is a clear need to find new substances that are better and safer, with antimicrobial properties to be used in combating these microorganisms (Kpadonou et al. 2019). Therefore, this study aims to investigate the chemical composition of SAEO, as well as its biological activities on C. diphtheriae.

MATERIAL AND METHODS

Essential oil isolation

Dehydrated inflorescences of S. aromaticum (cloves), purchased at Mercado Central, a popular commercial establishment in the municipality of São Luís, state of Maranhão (MA), were ground in an electric knife mill (TECNAL, model TE-340) to extract the essential oil (EO) by hidrodistellation process. The crushed material (400 grams) was transferred to a Clevenger apparatus coupled to a round-bottom flask with 4,000 mL of distilled water and a heating blanket at a temperature of 100°C for oil extraction, where it remained for four hours. Subsequently, the resulting oil was dried by using the percolation method in anhydrous sodium sulfate (Na₂SO₄) and the aliquots obtained were stored in glass tubes under refrigeration to avoid possible volatile losses. The yield was calculated using the formula R% = (V d m^-1) x 100, where V = total volume of oil extracted (mL), d = oil density (g mL^-1) and m = plant dry matter (g) (Gomes et al. 2018).

Chemical composition of the essential oil

The analysis of volatile constituents was performed in a gas chromatograph (SHIMADZU) coupled to a GC/MS-QP5050A mass spectrometer equipped with a BPX 5 (5% phenylpolyphenylene-siloxane) capillary column. High purity helium was used as carrier gas (99,9995% purity). The temperature of injector and GC interface with selective detector was maintained at 280°C with a flow rate in column (60 m x 0.25 mm x 0.25µm) of 2.7 mL min^-1, programmed to operate at 50°C. For analyses, SAEO aliquots were injected with a volume of 1µL in ethyl acetate (0.0010 g of EO in 1 mL of chromatographic grade ethyl acetate) under the following conditions: split mode with column pressure of 150 kPa, linear velocity of 59.1 m s^-1, with total carrier gas flow rate of 30 mL min^-1 in the impact of electrons in split 1:10, with a gradient of temperature 40°C (5 min), 240°C (4°C min^-1); 240- 300°C (8°C min^-1, 7 min); t= 60 min. In MS, the sweep mode of 0.5 seconds for scan, mass range of 40-500 Daltons, transfer line at 280°C and filament (off) 0 the 4s (Gomes et al. 2018, Martins et al. 2020). We identify the components of the oil from the comparison of these with the data obtained from authentic substances existing in reference libraries Wiley 229 Spectroteca and the Automated Mass Spectral Deconvolution & Identification System (AMSDIS) (Adams 2005).

In vitro hemolysis assay

The study was reviewed and approved by the Human Research Ethics Committee of the Universidade CEUMA, Brazil (CEP-UNICEUMA) under protocol number 1.732.522 and was performed in accordance with the Declaration of Helsinki 1975, as revised in 2008. Blood sample was collected from a human voluntary with no recent history of antibiotic therapy or with anti-inflammatory drugs and/or infectious diseases or inflammatory three weeks before the collect of the sample. Briefly, 0.5 mL aliquots of human erythrocytes 1% in saline solution (0.9%) were added to 0.5 mL of EO in concentrations ranging from 3000 to 50 µg mL^-1. After preparation, the solutions were incubated for 60 minutes at 37ºC and centrifuged at 3000 rpm for 5 minutes. From the supernatant fluid, 100 μL were transferred to a flat bottom microtiter plate and absorbance was measured using a spectrophotometer (550 nm; MICROPLATE READER, model TP-READER NM). To eliminate the EO inference in absorbance, control solutions (white) were prepared without addition of the red blood cells solution. Saline and distilled water were included as minimal and maximal haemolytic controls, respectively. The experiment was carried out in duplicate, twice (Vinhal et al. 2012).

In vivo cytotoxicity assays

Toxic potential in Artemia salina

Eggs of A. salina, acquired from the ornamental fish farm trade in São Luís - MA, Brazil, were subjected to ideal conditions for hatching and subsequent larval development until reaching the metanauplius stage (24 hours), being then transferred to plastic containers, where groups of ten individuals were separated for each container. The experiment was carried out in duplicate. The SAEO was diluted in dimethyl sulfoxide (DMSO, 0.01%) to aliquots at concentrations of 10, 100, 1000 µg mL^-1 that were added to the containers with the larvae. The control negative was done with DMSO (0.01%) in saline solution (0.9% w v^-1, NaCl). As a positive control, 10 µg mL^-1 of potassium dichromate solution (K₂Cr₂O₇) in saline (0.9% w v^-1, NaCl) was used. After 24 hours of contact, dead microcrustaceans were counted (Meyer et al. 1982, Rosa et al. 2016).

Effect in Tenebrio molitor

T. molitor larvae, acquired from an insect biofactory in Recife (PE), Brazil, were maintained at a temperature of approximately 15°C inside a plastic container containing an appropriate substrate for feeding were selected so that they would have similar appearance in size, being subsequently sanitized with 70% alcohol. Then, the insects were distributed in experimental groups containing ten individuals each in disposable and sterile Petri dishes. The larvae of each group were inoculated in second sternit above tail (in the ventral portion) with 10 µL of SAEO previously diluted in DMSO (0.01%) at concentrations ranging from 1000 to 50 µg mL^-1, being subsequently conditioned at a temperature of 37°C. Control groups received saline (0.9% w v^-1, NaCl) and 0.01% DMSO. The mortality in each group was observed at 24 hours intervals for five days after SAEO administration by visually analyzing melanization and the response to physical stimuli by gently touching the larvae (Martínez et al. 2018). The experiments were performed twice, in duplicate.

Antioxidant activity by phosphomolybdenum reduction method

To evaluate the formation of the phosphomolybdenum complex, a reactive (20 mL) containing 0.1 mol L^-1 sodium phosphate (28 mL), 0.03 mol L^-1 ammonium molybdate (12 mL) and 3 mol L^-1 sulfuric acid was prepared, completing the volume with water until reaching 100 mL. SAEO and ascorbic acid solutions (for controlling the maximum antioxidant activity) were prepared at a concentration of 200 μg mL^-1. An 0.4 mL aliquot of each solution was transferred to a test tube containing 4 mL of the reagent. The control of minimum antioxidant activity was prepared by adding 0.4 mL of distilled water to 4 mL of the reagent. The hermetically sealed tubes were incubated for 90 minutes at 95°C and then cooled until reaching room temperature. Then, absorbance readings at 695 nm were performed in a spectrophotometer (MICROPLATE READER, model TP-READER NM). The antioxidant action of the strains was expressed in relation to ascorbic acid, considering that its absorbance value corresponds to 100% of antioxidant action. The percentage of antioxidant activity (%AA) was calculated using the following equation: %AA= A_strain – A_blank/A_standard – A_blank x 100, where A_strain is the absorbance of each strain, A_blank is the absorbance of the blank and A_standard is the absorbance of the standard (acid ascorbic) (Emerenciano et al. 2015). Analyses were performed twice, in duplicate.

Antibacterial activity

Origin of diphtheria bacilli strains and culture conditions

All microorganisms used in the study (Table I) were obtained from cultures of clinical samples collected with swabs from the nasopharynx and throat from five patients and were identified by phenotypic analyses and multiplex polymerase chain reaction technique in the Laboratório de Difteria e Corinebacterioses de Importância Clínica at UERJ (LDCIC-UERJ) (Santos et al. 2015). The strains were provided by the LDCIC and stored in 20% glycerol (GC) medium at the Laboratório de Ciências Biomédicas at Universidade CEUMA. For the experiments, the strains were cultured for 48 hours at 37°C on tryptic soy broth (TSB), tryptic soy agar (TSA), and/or sheep blood agar and tellurite-chocolate agar (Viana et al. 2020).

Table I
Corynebacterium diphtheria strains partially investigated.

Strains	Biovar	Origin	tox gene#
†MA 19	intermedius	Diphtheria/Pharynx Maranhão/Brazil	+
†MA 23	intermedius	Diphtheria/Pharynx Maranhão/Brazil	+
†MA 52	intermedius	Diphtheria/Pharynx Maranhão/Brazil	+
†MA 131	intermedius	Diphtheria/Pharynx Maranhão/Brazil	+
†MA 150	intermedius	Diphtheria/Pharynx Maranhão/Brazil	+
* ‡ATCC 27010 / C7s	mitis	Diphtheria/Pharynx United States of America	-
* ‡ATCC 27012	mitis	Diphtheria/Pharynx United States of America	+

• Corynebacterium diphtheria strains partially investigated. MA: Maranhão; ATCC: Americam Type Culture Colletion; *: Homologoun strains; #: Diphtheria toxin gene; -: Negative; +: Positive. †: Clinical sample. ‡: Standard strain.

Minimum inhibitory concentration (MIC) by broth microdilution assay

Microdilution assays were performed using microbial suspensions in saline solution (1.5 x 10⁸ CFU mL^-1) diluted at the 1:10 ratio in Mueller-Hinton (MH) (Difco) broth culture medium. Subsequently, aliquots were transferred to 96-well microplates containing the SAEO serially diluted at concentrations from 3000 µg mL^-1 to 31,25 µg mL^-1 in a ratio of 1:2. The microplates were incubated at 37°C for 48 hours and the assay was revealed by the addition of 30 μL resazurin (0.03%) in each well of the microplate, which was again incubated for 30 minutes. The controls used in the experiment were the negative one, containing only the culture medium, and the positive one, consisting of the culture medium with chloramphenicol (30 μg), to which the bacterial suspensions were included. The MIC was the lowest concentration of SAEO in which color change from blue to pink was not detected (Palomino et al. 2002). The effect of the addition of 1% DMSO as an oil solubilizer on MIC was investigated and it was found that this concentration did not interfere with bacterial growth.

The classification of the antimicrobial activity of SAEO followed the proposal by Aligiannis et al. (2001) and, according to the authors, ‘strong’ activity should be considered if the MIC is at concentrations from 50 to 500 µg mL^-1, ‘moderate’ activity should be considered for MIC between 600 and 1500 µg mL^-1, and ‘weak’ for MIC above 1500 µg mL^-1. The experiment was carried out twice, in triplicate.

Minimum bactericidal concentration (MBC)

To determine MBC, a 10 μL aliquot was removed from the wells that did not show visible microbial growth in the MIC experiment and was inoculated in a plate with MH agar culture medium (Difco). Then, the plates were incubated at 37°C for 48 hours. The MBC was the lowest concentration of SAEO in which there was no bacterial growth on the surface of the culture medium in visual verification. SAEO was considered a bacteriostatic agent when the MBC/MIC ratio > 4 and a bactericidal agent when the MBC/MIC ratio ≤ 4. The assays were performed twice, in duplicate (Santurio et al. 2007).

Bacterial morphology and aggregation

Strains of C. diphtheriae standardized at an optical density (OD) of 0.3 and wavelength of 570 nm were cultivated in MH broth culture medium (Difco) with the addition of MIC 2^-1 of SAEO and incubated at 37°C. After 24 and 48 hours, 10 µL aliquots of the culture from each strain were stained by the Gram method and observed under an optical microscope and using immersion objective lens. In order to verify the occurrence of changes in the morphology of microorganisms and the effect on bacterial aggregation, ten fields containing between 40 and 70 microorganisms were considered. The experiment was carried out twice (Gomes et al. 2013).

Biofilm formation by crystal violet staining

Biofilm formation was determined in 96-well polystyrene microtiter plates (Stepanovic et al. 2000). Aliquots of bacterial suspensions in Trypticase Soy Broth (TSB) culture medium at OD 0.2 and wavelength of 570 nm were arranged in 96-well microplates together with the MIC 2^-1 of SAEO. Wells containing only TSB (negative control) and microorganisms in dimethylsulfoxide (DMSO, positive control) at 1% were also prepared. After incubation for 48 hours at 37ºC, the contents of the wells were carefully aspirated, with the wells being subsequently washed with 0.9% saline solution. Fixation with 99% methanol and staining with 2% crystal violet was conducted for the microorganisms that remained adhered. The dye was solubilized with 160 μL glacial acetic acid and the OD of the solution was measured at 550 nm (MICROPLATE READER, model TP-READER NM) (Gomes et al. 2013).

Biofilm formation was qualitatively classified according to categories previously proposed by Stepanovic et al. (2000) as follows: non-adherent/non-biofilm former (-) when OD ≤ ODc (control); weakly adherent/poor biofilm former (+) when ODc< OD ≤ 2xOD_C; moderately adherent/moderate biofilm former (++) when 2xODc< OD ≤ 4xODc; strongly adherent/strong biofilm former (+++) when 4xODc ≤ OD. Mean absorbance values of each strain were calculated and compared with the mean values of controls. The experiments were performed twice, in triplicate.

Statistical analysis

All data analyses were performed using the software Graphpad Prism 6. Data from lethal concentration and toxicity assays were expressed as mean ± standard deviation. In the analysis of data obtained in the T. molitor survival and antibiofilm activity assays, p<0.05 was considered significant to verify the differences between the groups. The survival curve in the assay with T. molitor larvae was obtained by applying the Kaplan-Meier tests and analyzing the results with the Log-rank test (Mantel-Cox). For data obtained from the biofilm assay, Tukey test was applied, p<0.05 was considered significant to verify the differences between the groups.

RESULTS AND DISCUSSION

SAEO yield and GC-MS analysis

Yield of SAEO obtained by steam distillation technique was 3.64%. The main compounds (with relative % peak area) identified by chromatographic analysis in the SAEO are listed in Table II. Fifteen components were identified (Figure 1), with the major component being eugenol (48.00%), a phenylpropene, which is reported to have antiviral, antibacterial, antifungal, anticancer, antioxidant and anti-inflammatory action (Adams 2005) followed by the compounds caryophyllene (44.00%), a bicyclic sesquiterpene, has hypocholesterolemic, antioxidant and antimicrobial activity (Yoo & Jwa 2018, Harb et al. 2018) and α-humulene (4.87%), a monocyclic sesquiterpene found in EO, has antineoplastic, anti-inflammatory and cytotoxic effect for hepatocellular carcinoma cells, besides having antibacterial and insecticidal properties (Chen et al. 2019).

Figure 1
Ion-chromatogram (GC-MS) profiles of peak retention of components of Syzygium aromaticum essential oil. The majority components were eugenol, caryophyllene and α-humulene respectively. TIC: Total Ion Chromatogram.

Table II
Chemical composition of essential oil extracted from dehydrated inflorescences of Syzygium aromaticum (clove).

Peak	Retention Times(min)	Relative area (%)	Chemical compounds
1	9,46	44.29	Caryophyllene
2	9,75	0.72	Copaene
3	9,95	0.02	Caryophyllene Oxide
4	10,17	0.04	Isocaryophyllene
5	10,34	48.79	Eugenol
6	10,70	0.03	α-Cebeben
7	10,73	4.87	α-Humulen
8	11,03	0.16	γ- Murolene
9	11,19	0.10	α-farnesene
10	11,30	0.11	Panaxene
11	11,45	0.10	β-Patchoulane
12	11,51	0.08	1-(2-Bromoethyl) Adamantane
13	11,60	0.29	Δ-Cadinene
14	11,73	0.25	Eugenol Acetate
15	12,38	0.15	Hydroazulene

Several authors also reported eugenol as the major compound of the SAEO, corroborating our results (Andrade et al. 2014, Gomes et al. 2018). Silvestri et al. (2018) reported the presence of eugenol (90.30%), followed by caryophyllene (4.83%) and eugenol acetate (1.87%) and Radünz et al. (2019) identified eugenol (56.06%) and caryophyllene (39.63%), among other chemical compounds (Radünz et al. 2019). Differences in the percentages of chemical compounds present in the SAEO observed by the authors can be attributed to the different geographic areas of plant collection, as well as biotic and abiotic factors related to seasonality, development stage, plant age and climatic conditions (Andrade et al. 2014).

Blood compatibility

The hemolytic concentration of SAEO able of lysing 50% of red blood cells (HC₅₀) was 514 µg mL^-1 ± 0.007230. This concentration represents a relatively low hemolytic capacity when compared to the control of maximum hemolysis (hypotonic solution), suggesting the possibility of therapeutic use (Vinhal et al. 2012). In a previous study, a commercial EO of S. aromaticum also showed low hemolytic activity when compared to minimal hemolytic control (Skalickova et al. 2020). The major component eugenol was reported as a compound with little lytic activity for erythrocytes (He et al. 2007), since the percentage of hemolysis at the concentration of 4,000 µg mL^-1 was 4.4%.

Toxic potential against Artemia salina and bioassay with Tenebrio molitor larvae

In this study, the lethal concentration of the SAEO capable of killing 50% of A. salina (LC₅₀) was 377.25µg mL ^-1 ± 3.897293. Considering the parameters described in the literature (Meyer et al. 1982, Amarante et al. 2011), the SAEO is toxic/moderately toxic, suggesting that this EO has biological properties, including antibacterial. Gomes et al. (2018) also characterized the SAEO as toxic to A. salina.

The lethal concentratios (LC₅₀) in a test with the microcrustacean A. salina is usually ten times less than the concentration necessary to inhibit 50% of cell growth in antitumor tests (Mclaughlin et al. 1998), reinforcing that this assay is an alternative to tests performed on animal (Rosa et al. 2016). In general, according to Rahman et al. (2001), the bioactive compounds are toxic to A. salina. The EO rich in phenylpropanoids, such as SAEO, are reported in the literature to present greater toxicity in tests on A. salina than their majority compounds tested alone in these organisms, as observed by Cansian et al. (2017). This fact may be related to the synergistic effect of the substances present in the EO (Betim et al. 2019, Mesquita et al. 2021).

The bioassay with T. molitor larvae in this study revealed a mean survival rate of 80.00% after SAEO administration at five different concentrations (Figure 2). T. molitor larvae are used as an alternative model for in vivo studies of pathogenicity and toxicity due to its ease of handling and short life cycle (Lozoya-Pérez et al. 2021, Hong et al. 2020). Martínez et al. (2018), who studied commercial SAEO, whose major component was also eugenol, sought to find the concentration and lethal time of that EO on T. molitor in the pupa, larva and adult stages, obtaining higher survival rates for T. molitor in the larval stage.

Figure 2
Survival rates of Tenebrio molitor larvaes inoculated with Syzygium aromaticum essential oil estimated using the Kaplan–Meier log-rank test. Negative control was 0.01% DMSO (dimethyl sulfoxide) in saline. p<0.05 was considered significant. The experiments were performed twice, in duplicate.

Reduction of phosphomolybdenum complex

The antioxidant activity of the EO was calculated in relation to standard ascorbic acid. SAEO demonstrated a middle antioxidant activity of 40.60% at a concentration of 200 μg mL^-1, compared to the standard (100.00%), after the phosphomolybdenum assay. The antioxidant activity of SAEO demonstrated in this study may be related to the presence of phenolic compounds here identified, which are known to be found in cloves, playing an important role as antioxidants and being dependent on the environmental conditions of the plant habitat to be produced (Barboza et al. 2018). It is worth mentioning that eugenol and caryophyllene, phenolic compounds described in this research as the majority, have appreciable antioxidant properties (Barboza et al. 2018, Harb et al. 2018).

The results obtained with the DPPH (2,2-diphenyl-1-picrylhydrazyl) elimination method by SAEO were 40.00% at a concentration of 100 μg mL^-1 for Nagaraju et al. (2021), 94.86% at a concentration of 484.7 μg mL^-1 for Chen et al. (2019) and 92.82% at a concentration of 10000 μg mL^-1 for Sebaaly et al. (2015). Due to the diversity of methods with varied fundamentals and interferences, the comparison of the results of antioxidant activity becomes difficult (Sucupira et al. 2012).

SAEO inhibits the growth and can kill C. diphtheriae

Analyzing the inhibition potential of SAEO, it was observed that all isolates were inhibited with the MIC ranging from 1000 to 62.5 µg mL^-1. According to the classification proposed by Aligiannis et al. (2001), SAEO showed an antimicrobial activity pattern from moderate to strong over C. diphtheriae (Table III). It was possible to determine the MBC of the EO with the concentrations evaluated for two strains, and the ratio between MBC and MIC indicated bacteriostatic and bactericidal activity on strains MA 150 and ATCC 27010, respectively. It is important to highlight that at the used concentration, DMSO (1% in PBS) did not affect bacterial growth.

Table III
Antimicrobial activity of Syzygium aromaticum essential oil against Corynebacterium diphtheriae.

Microrganisms	Syzygium aromaticum
	MIC†	Antimicrobial activity pattern††	MBC‡	¥ MBC/MIC
MA 19	500	Strong	ND	ND
MA 23	1000	Moderate	ND	ND
MA 52	250	Strong	ND	ND
MA 131	1000	Moderate	ND	ND
MA 150	62.5	Strong	2000	Bacteriostatic
*ATCC 27010/C7s	1000	Moderate	2000	Bactericidal
*ATCC 27012	1000	Moderate	ND	ND

• Antimicrobial activity of Syzygium aromaticum essential oil against Corynebacterium diphtheria strains. MA: Maranhão; ATCC: American Type Culture Collection; *: Homologous strains. Minimum inhibitory concentration (MIC, †) and minimum bactericidal concentration (MBC, ‡) are expressed in µg mL^-1 with chloramphenicol as positive control. ND: not determined with the concentrations evaluated. ††: Antimicrobial activity pattern: strong- 50to 500 µg mL^-1, moderate- 600 to 1500 µg mL^-1, weak- above 1500µg mL^-1. ¥Bacteriostatic activity- MBC/MIC ratio > 4, Bactericidal activity- MBC/MIC ratio ≤ 4. The data were expressed as arithmetic mean of two independent experiments carried out in triplicate.

The SAEO was able to inhibit all C. diphtheriae isolates evaluated, regardless of the presence of the tox gene. The concentrations for inhibition of the homologous strains ATCC 27010 (tox -) and ATCC 27012 (tox +) were equal (1000 µg mL^-1).

No studies that demonstrated the antibacterial action of SAEO against C. diphtheriae were found, although there are records for other Gram-positive bacteria. Radünz et al. (2019) demonstrated effective inhibition of Listeria monocytogenes and S. aureus at a concentration of 0.304 mg mL^-1 of SAEO. It is important to highlight that, in this study, lower concentrations of SAEO were able to inhibit C. diphtheriae strains, which allowed classifying the action of the EO against the species, according to Aligiannis et al. (2001), as moderate or strong. Despite this, for some strains the MIC was higher than the HC₅₀ and LC₅₀. Alternatives for modulating antibacterial activity can be suggested, such as the use of SAEO in association with conventional therapies.

Assays performed with nosocomial pathogens Acinetobacter baumannii and Klebsiella pneumoniae strains revealed that the addition of SAEO to colistin significantly reduced the MIC of the EO and of the antibiotic (Vázquez-Ucha et al. 2020). Another alternative is the oil nanoencapsulation. Nagaraju et al. (2021) evaluated the biological properties of nanoencapsulated clove oil and found high antioxidant property and low cytotoxicity and hemolytic activity. All results with nanoencapsulated oil were better when compared to native oil. These differences can have a substantial impact on their biological activities and potential health benefits. In S. aureus, the antibacterial activity of the clove bud oil was lower than that of its nanoscale emulsion. The authors reported a greater loss of cell contents due to the interaction between S. aureus and the emulsion (Nirmala et al. 2019).

Regarding the bactericidal effect of the SAEO, it was possible to establish the MBC using the concentrations evaluated only for two strains of C. diphtheriae, even though the MIC was determined for all strains. The same MBC (2000 µg mL^-1) was found for C. diphtheriae strains MA 150 (tox +) and ATCC 27010 (tox -). It is noted that the SAEO was not bactericidal for the homologous ATCC 27012 (tox +). MIC and MBC data suggest that there is no participation of toxicity in mechanisms of sensitivity/resistance to the action of SAEO.

Some studies have described the bactericidal action of SAEO on Gram-positive bacteria, such as the one by Wongsawan et al. (2019), who reported a very low MBC of 0.1% for all 15 Streptococcus suis multidrug-resistant isolates.

Alibi et al. (2020) used the ratio between bactericidal and minimal inhibitory concentration to verify whether S. aromaticum had bacteriostatic or bactericidal effects and observed that the oil was effectively bactericidal for Gram-negative and Gram-positive bacteria, diverging from the results obtained in this study, whose analysis of the same ratio showed that the SAEO was bactericidal for C. diphtheriae (Gram-positive species) without the tox gene (ATCC 27010) and bacteriostatic for C. diphtheriae with tox gene (MA 150).

Santos et al. (2015), after analysis by pulsed-field gel electrophoresis, classified the isolates from Maranhão into pulse types. C. diphtheriae strains MA 19, MA 23, MA 52 and MA 131 were grouped into the pulse type Ia, while the strain MA 150 was classified into the pulse type Ib. The similarity coefficient obtained was ≥ 95% and pulse types Ia and Ib were considered genetically related. Regardless of this relationship, the antibacterial action of the SAEO against C. diphtheriae occurred distinctly and to varying degrees, but it was only possible to define the MBC for the strain MA 150 (pulse type Ib).

The differences in the results obtained in the susceptibility studies and the difficulty in comparing studies that verify the antimicrobial activity of medicinal plants and their derived products can be attributed to changes in environmental conditions at the time of collection of plant material and to the procedures and protocols followed for this purpose regarding the plant to be studied, the physicochemical characteristics of EO, and the form of extraction, among other factors. Differences in data obtained by different authors may also occur in response to variables inherent to the experiments, such as the culture medium used, incubation time and temperature, and the volatility of the EO. The cell wall composition of microorganisms and even the structural differences observed in the same species also influence the results (Auricchio & Bacchi 2003).

The subMIC levels of SAEO do not affect bacterial morphology

We also evaluated whether the subinhibitory concentration of SAEO (MIC 2^-1) was able to affect bacterial morphology by optical microscopy. The smear microscopy of bacteria stained by the Gram method demonstrated that after interaction between the SAEO and C. diphtheriae strains, there was no significant change in bacterial cell morphology as a function of the concentration tested, as seen in Figure 3. Microorganisms cultured in the presence of 1% DMSO did not present morphological alterations or increase in the bacterial grouping.

Figure 3
Photomicrographs of Gram-stained cells of Corynebacterium diphtheriae strain MA 23 grown a) the absence (control) or b) in the presence of MIC 2^-1 of Syzygium aromaticum essential oil by 24h or c) 48h. MIC: Minimum Inhibitory Concentration. Magnification x1000.

The experimental context described here indicates a probable dose-dependent action of the SAEO, besides suggesting that its mechanism of antibacterial action can damage the cell membrane through extravasation of cellular components, without changing the morphology or determining the loss of viability of the microorganism (Nazzaro et al. 2013). However, it cannot be said that there were no structural changes since some modifications are only noticeable when evaluated with greater microscopic resolution. Kovács et al. (2016) evaluated the morphology of Campylobacter jejuni after two hours of treatment with 330 µg mL^-1 (1.65 x MIC) of the SAEO by electron microscopy and found that bacterial cells shortened and presented a less intense curvature.

The action of the EO on the morphology of corynebacteria has already been reported. Viana et al. (2020) described the filamentation of C. ulcerans, as well as self-aggregation of bacteria in the presence of subinhibitory concentrations (subMIC) of A. zerumbet. Gomes et al. (2013) observed filamentation and reduction in the size of C. diphtheriae when cocultured with the subMIC of the antibiotics penicillin and erythromycin, respectively.

The subMIC levels of SAEO affect biofilm formation

We attempted to analyze the effects of subinhibitory concentrations of SAEO (MIC 2^-1) on the ability of C. diphtheriae to form biofilm. Following the methodological proposal by Stepanovic et al. (2000) for the qualitative classification of biofilm formation, it was observed that the C. diphtheriae strain MA 23, MA 52, and MA 131 and the standard strains ATCC 27010 and ATCC 27012 presented poor biofilm formation in polystyrene microplates. When in the presence of SAEO, C. diphtheriae strains MA 52, MA 131, and ATCC 27012 maintained the same classification for biofilm formation, while C. diphtheriae strains MA 19 and MA 150, which were non-adherent, began to adhere weakly. C. diphtheriae strains MA 23 and ATCC 27010 had the ability to form biofilm inhibited, as shown in Table IV.

Table IV
Effect of Syzygium aromaticum essential oil on biofilm formation in Corynebacterium diphtheriae strains.

Microrganisms	#Control bacteria in DMSO 1% without SAEO	¥MIC 2-1 SAEO
MA 19	-	+
MA 23	+	-
MA52	+	+
MA 131	+	+
MA 150	-	+
*ATCC 27010/C7s	+	-
*ATCC 27012	+	+

• Effect of Syzygium aromaticum essential oil in inhibition of Corynebacterium diphtheriae biofilme formation on polystyrene surface. MA: Maranhão; ATCC: American Type Culture Collection; *: Homologous strains. #Bacteria in dimethylsulfoxide 1% without Syzygium aromaticum essential oil (SAEO). ¥MIC 2^-1, Sub-inhibitory concentration tested. -: Non adherent/ non biolfilm former; +: weakly adherent/poor biofilme former. Experiments were performed twice, in triplicate.

In Figure 4, it is observed that SAEO altered the biofilm formation by microorganisms. The C. diphtheriae strain MA 23 presented a significant reduction in biofilm formation (p < 0.05, Tukey test) in the presence of the MIC 2^-1 of SAEO when compared to the control, without oil addition. C. diphtheriae strains MA 150 and ATCC 27012 presented a statistically significant increase in biofilm formation in the presence of SAEO. It was observed that 1% DMSO did not influence biofilm formation in the analyzed strains.

Figure 4
Effect of Syzygium aromaticum essential oil (MIC 2^-1) on Corynebaterium diphtheriae strains biofilm formation in polystyrene microplates. *p<0.05, **p <0.005, ***p <0.0001 compared with DMSO (dimethyl sulfoxide) 1%-treated bacteria (Tukey test). MIC: Minimum Inhibitory Concentration. Experiments were performed twice, in triplicate. Results are presented as mean ± standard deviation. The absorbance values were obtained in nm. MA: Maranhão. ATCC: American Type Culture Collection.

Studies about the antibiofilm activity of EO on C. diphtheriae, the etiologic agent of diphtheria, were not found in the literature. Biofilm assays with Helicobacter pylori suggest that there may be differences in the membrane composition of strains that form biofilms to different degrees of intensity (Hewelt-Belka et al. 2015). In addition, the lipid composition of bacteria also influences susceptibility to antimicrobials, probably due to changes in membrane penetration to antimicrobials (De Marco et al. 2017).

Budri et al. (2015) showed a significant inhibition of S. aureus biofilm production by SAEO on polystyrene. However, its major component, eugenol, was less effective. Streptococcus mutans biofilm was reduced and completely inhibited by 0.16% and 0.32% of the caryophyllene, respectively (Yoo & Jwa 2018).

The use of compounds with antibiofilm activity has been reported as a possible alternative for the treatment of various infections as a solution for microbial resistance (Santhakumari & Ravi 2019). The fact that SAEO decreased biofilm formation in a strain of C. diphtheriae indicate that the oil may be useful in biofilm formation intervention strategies.

Here, SAEO significantly increased biofilm production of two C. diphtheriae strains. The increase in biofilm formation can be attributed to the destabilization of the bacterial cell wall due to the oil components being found at subinhibitory concentrations, stimulating the production of extracellular polymeric substance. These compounds at low concentrations would be activating cellular responses against the stress caused by these molecules. Thus, exopolysaccharide production could be regulated with the aim of increasing biofilm formation. Additionally, cells comprising the biofilm reduce the metabolism and the use of nutrients and oxygen in the presence of an antimicrobial agent at a low concentration, which represents one of the reasons for the greater resistance of biofilm cells to antibacterial agents (Mah & O’toole 2001).

The increase in biofilm formation in polystyrene has already been reported by Gomes et al. (2013) for C. diphtheriae in a study using subinhibitory concentrations of the antibiotic erythromycin (inhibits protein synthesis) and may contribute to the failure of antimicrobial therapy against infections by specie.

The statistically significant reduction in the biofilm formed by the MA 23 strain may be related to the presence of bacterial persister cells, wich are a subpopulation of a genetically sensitive bacterial population showing transient tolerance to antibiotics and that are often slow-growing or growth-arrested. These cells remain alive in the presence of the antibiotic and they are thought to be responsible for relapse of many persistent infections (Cui et al. 2018). This subpopulation can be induced by bacteriostatic antibiotics (Cui et al. 2018), however it was not possible to define whether the SAEO is bactericidal or bacteriostatic for MA 23.

The inactivity of the SAEO and eugenol in polystyrene biofilm formation by Escherichia coli has already been reported (Kim et al. 2016). These results partially corroborate the present study, which demonstrates that there was no significant antibiofilm action under the SAEO action for some of the tested strains, despite the effective inhibitory action of the EO. Clove oil when it has been microencapsulated showed a better antibiofilm effect on stainless steel surfaces in the multiresistant S. aureus (Borborema et al. 2022).

There is little research on plants that have antibiofilm activity in Corynebacteria. Viana et al. (2020) demonstrated that the subMIC of A. zerumbet EO was able to attenuate biofilm production by C. ulcerans. Santos et al. (2020) reported that the EO of Stryphnodendron coriaceum Benth increased biofilm production by C. ulcerans. Propolis extracts were effective in interfering with the formation of the Corynebacterium pseudotuberculosis biofilm but had little activity on the consolidated biofilm (Santos et al. 2021).

CONCLUSIONS

The analysis by GC-SM of the SAEO identified eugenol as the major compound, which is a substance already described as having several biological actions, including antioxidant and antibacterial activity. The SAEO presented antibacterial and antibiofilm potential against C. diphtheriae, as relatively low concentrations were effective. However, low doses of SAEO were also able to favor biofilm formation by C. diphtheriae. The results of this research highlight the promising role of SAEO, prompting the development of additional studies to determine the effectiveness of this natural compound in other analyses, including exploring its combination with conventional antibiotics or other EO to verify the ability to control the virulence factors presented by pathogenic bacteria, such as C. diphtheriae. Studies with nanocapsules have been very promising. Although the survival rate of T. molitor larvae in the presence of the SAEO was considerable and in terms of blood compatibility assessment, the results have indicated a low haemolysis, detailed investigations related to the safety margin are necessary.

ACKNOWLEDGMENTS

We thank the Fundação de Amparo à Pesquisa e Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA), for the financial contribution spent on the Biomedical Sciences Laboratory where the research was developed.

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  13 Dec 2024
• Date of issue
  2024

History

• Received
  30 Mar 2023
• Accepted
  16 Sept 2024