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. 2023 Jun 29;13(7):255. doi: 10.1007/s13205-023-03681-1

Chemical composition of Piper gaudichaudianum Kunth essential oil and evaluation of its antimicrobial and modulatory effects on antibiotic resistance, antibiofilm, and cell dimorphism inhibitory activities

Antonio Linkoln Alves Borges Leal 1,5, Matheus Carvalho da Silva 5, Andressa Kelly Ferreira e Silva 5, Avilnete Belem de Souza Mesquita 5, Camila Fonseca Bezerra 2, Ana Rafaela Freitas Dotto 4, Wanderlei do Amaral 3, Érika de Araujo Abi-chacra 5, Luiz Everson da Silva 4, Humberto Medeiros Barreto 5, Hélcio Silva dos Santos 1,6,7,✉
PMCID: PMC10310684  PMID: 37396469

Abstract

Essential oils extracted from many plant species have different biological activities, among which microbial activity stands out. Species of the genus Piper have antimicrobial potential against different species of bacteria and fungi. In this sense, the present study aimed to determine the chemical composition of the essential oil from the leaves of Piper gaudichaudianum (EOPG), as well as to investigate their antimicrobial activity and their modulatory effect on the Norfloxacin resistance in the Staphylococcus aureus SA1199B strain overproducer of the NorA efflux pump. Furthermore, their inhibitory activities on the biofilm formation as well as on the cellular differentiation of C. albicans were evaluated. Gas chromatography analysis identified 24 compounds, such as hydrocarbon sesquiterpenes (54.8%) and oxygenated sesquiterpenes (28.5%). To investigate the antimicrobial potential of EOPG against S. aureus, E. coli, and C. albicans, a microdilution assay was performed, and no intrinsic antimicrobial activity was observed. On the other hand, the oil potentiated the activity of Norfloxacin against the SA1199B strain, indicating that EOPG could be used in association with Norfloxacin against S. aureus strains resistant to this antibiotic. EOPG also inhibited S. aureus biofilm formation, as evidenced by the crystal violet assay. In the dimorphism assay, EOPG was able to inhibit the cell differentiation process in C. albicans. Results indicate that EOPG could be used in association with Norfloxacin in the treatment of infections caused by resistant S. aureus strains overproducing the NorA efflux pump. Furthermore, its ability to inhibit the formation of hyphae by C. albicans suggests that EOPG could also be applied in the prevention and/or treatment of fungal infections.

Keywords: Piper gaudichaudianum, Antimicrobial activity, Essential oil, Chemical composition, Resistance to antimicrobials

Introduction

Aromatic plants are an important group of medicinal plants from which essential oils can be extracted, many of which have already been shown to have antimicrobial properties (Barreto et al. 2014a, b). The Piperaceae family is predominantly tropical and includes 5 to 8 genera and approximately 2000 species. This family is represented in Brazil by about 500 species, occurring in forest areas, mainly in the Atlantic Forest. Chemical studies on species of the genus Piper have resulted in the isolation of many biologically active natural products, such as pyrenes, lignans, neolignans, terpenes, propenylphenols, chalcones, flavones, benzopyrenes, chromenes, lactones, and amides (Puhl et al. 2011). It has been demonstrated that many Piper species have important biological activities, including strong molluscicide activity against Biomphalaria glabrata (Morandim-Giannetti 2010), cytotoxic, insecticide, and antimicrobial activities (Leal et al. 2019a).

Treatment of infectious diseases has become a challenge in face to high prevalence of multidrug-resistant microorganisms (Casanova and Costa 2017). Infections caused by multidrug-resistant S. aureus are prevalent in humans including intensive care unit patients (Oliveira et al. 2018). On the other hand, the prevalence of fungal infections caused by Candida species has increased considerably in countries around the world, affecting mainly immunosuppressed patients (Mayer et al. 2013; Neufeld 2004).

Thus, there is an urgent need for new strategies for the prevention, treatment, and control of multidrug-resistant microorganisms. Different technological strategies have been suggested to alleviate this problem, including the search for new antimicrobial agents as well as drug resistance modulators able to inhibit resistance mechanisms potentiating the action of traditional antibiotics (Costa et al. 2016; Coutinho et al. 2010).

The present work aimed at obtaining and determining the chemical composition of the essential oil obtained from the leaves of P. gaudichaudianum (EOPG), investigating its intrinsic antimicrobial activity, as well as its inhibitory activity on biofilm formation and fungal dimorphism, and evaluating the reversion effect of Norfloxacin resistance in a strain of S. aureus (SA1199B) resistant by overexpression of the norA gene.

Materials and methods

Plant material

The leaves of P. gaudichaudianum were collected in the Bom Jesus Biological Reserve, located in the municipality of Guaraqueçaba, PR (S 25º 13.939' and W 08º 34.813'), Paraná State, southern Brazil, in the spring of 2015, under license from the Environmental Institute of the State of Paraná, number 284/11. An exsiccate was prepared and deposited at the Herbarium Municipal Botanical Museum (MBM) under number 396403 (UPCB).

Essential oil isolation and analysis methodology

The essential oil was obtained from 100 g of fresh or 50 g of the dried sample by hydrodistillation over 4 h using a Clevenger-type apparatus. The dried samples were obtained after drying the plant material for 24 h in an electric dryer FANEM (320 SE Mod) with air circulation at 40 °C. The extracted oil was stored in dark vials at  – 20 °C until analysis. GC–MS analysis was performed using 1.0 µL of the samples in 1% dichloromethane which was injected with a split ratio of 1:20 in an Agilent 6890 gas chromatograph (Palo Alto, CA) coupled to an Agilent 5973 N selective mass detector. The injector temperature was maintained at 250 °C. Separation of the constituents was obtained with a HP5MS capillary column (5% phenyl–95% dimethylpolysiloxane, 30 m × 0.25 mm × 0.25 μm) using helium as the carrier gas (1.0 mL min−1). The incubation temperature was programmed from 60 to 240 °C at a rate of 3 °C min−1. The mass detector was operated in the electronic ionization mode (70 eV) at a rate of 3.15 scan s−1 and a mass range from 40 to 450 u. The transfer line was maintained at 260 °C, the ion source at 230 °C and the analyzer (quadrupole) at 150 °C. For quantification, the diluted sample was injected into an Agilent 7890A chromatograph equipped with a flame ionization detector (FID), operated at 280 °C. The same column and analytical conditions described above were employed, using hydrogen at a flow rate of 1.5 mL min−1 as the carrier gas. The percentage composition was obtained by electronic integration of the FID signal with the division area of each component in the total area. The chemical constituent’s identification was obtained by comparing their mass spectra with libraries and by linear retention indices, calculated after the injection of a homologous series of hydrocarbons (C7–C26) and compared with the literature data Wiley library spectra and according to Adams (Adams 2012). An analysis of variance for the essential oil yield as well as the Scott–Knott test (P < 0.05) of the mean comparison procedures were performed using ASSISTAT, release 7.6 Beta.

Evaluation of the antimicrobial activity and modulation of antibiotic resistance

Antimicrobial activity of the EOPG was evaluated against standard microbial strains Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and C. albicans ATCC 10231, as well as against S. aureus SA1199B, which is resistant to Norfloxacin due to overexpression of the norA gene encoding the NorA efflux pump (Kaatz and Seo 1997). The SA1199B strain was provided by Dr. Glenn W. Kaatz, John Dingell VA Medical Center, Detroit, MI, USA. Evaluation of the antimicrobial activity and evaluation of the modulatory effect on resistance to antibiotics were performed according to previously described methodologies (Kaatz and Seo 1997; Leal et al. 2019b). The known efflux pump inhibitor chlorpromazine (CPZ), in place of EOPG, was used in control experiments.

Evaluation of the inhibitory effect of EOPG on the formation of bacterial biofilms

Bacterial suspensions were made in BHI medium at 108 CFU/mL. In the first test, the bacteria (107) were pre-treated with EOPG (10, 25, 50, and 100 µg/mL) for 24 h before forming the biofilm. In the second, the EOPG was incubated with the bacteria (107) during the 24 h of adhesion to the polystyrene plate, and in the third, after the biofilm was formed (24 h), the system was treated with the EOPG for an additional 24 h. Afterwards, the wells were washed three times with PBS and incubated with 0.4% crystal violet dye at room temperature for 5 min. Then, the systems were washed three times with distilled water to remove excess dye, and absolute ethanol was added for 5 min. The bleach solution was transferred to another 96-well plate so that absorbance was measured at 570 nm in a microplate reader (EZ Read 400, Biochroom Ltd., Cambridge, England) (Ravi et al. 2009).

Evaluation of the inhibitory effect of EOPG on fungal dimorphism

To observe the morphological changes of Candida albicans, the strain ATCC 10231 was used in the microculture technique for yeasts according to the previous methodology reported by Brito (Brito et al. 2015).

Statistical analysis

Statistical analyses were performed using GraphPad Prism, version 6.0. Differences between treatment with antibiotic (or EtBr) alone or associated with EOPG or CPZ were examined using one-way analysis of variance (ANOVA). The differences mentioned above were analyzed by the Bonferroni posttest, and p < 0.05 was considered statistically significant.

Results and discussion

Characterization of the chemical composition of the EOPG

The results of the phytochemical characterization of the EOPG are presented in Table 1. It can be verified that the EOPG presents a higher concentration of sesquiterpenic compounds, both oxygenated and hydrocarbons. Among these, it was possible to identify 33 compounds (83.4%), among which sesquiterpenes δ-Cadinene (9.83%) and Germacrene B (8.96%) were the most prevalent (Fig. 1).

Table 1.

Chemical constituents of the essential oil (≥ 1%) from samples of Piper gaudichaudianum (Piperaceae) leaves

Constituents *Al Al IK %
α-Copaene 1381 1374 1376 2.09
β-Elemene 1397 1389 1390 1.31
α-Santalene 1421 1416 1417 1.26
γ-Elemene 1438 1434 1436 3.07
α-Humulene 1460 1452 1454 3.46
Aromadendrene 1467 1458 1460 1.77
(E)-Caryophyllene 1478 1464 1466 8.48
γ-Himachalene 1491 1481 1482 1.44
Viridiflorene 1500 1496 1496 1.25
Pseudowiddrene 1502 1498 1498 1.07
β-Macrocarpene 1504 1499 1499 1.22
β-Dihydro agarofuran 1508 1503 1503 2.38
α-Farnesene 1513 1505 1505 1.63
α-Selinene 1526 1520 1522 4.30
δ-Cadinene 1530 1522 1523 9.83
α-Calacorene 1551 1544 1545 2.61
Germacrene B 1566 1559 1561 8.96
(E)-Nerolidol 1569 1561 1563 2.01
β-Calacorene 1572 1564 1565 1.10
Sesquisabinene hydrate < trans- >  1583 1577 1579 1.58
β-Capaen-4-α-ol 1587 1590 1590 1.54
Globulol 1593 1590 1590 2.15
Guaiol 1607 1600 1600 1.10
Bisaboladien-4-ol < 2,7Z 1620 1618 1619 1.34
Eudesmol 10-epi-γ 1627 1622 1623 1.25
Cadin-4-em-7-ol-cis 1639 1635 1636 1.50
Muurolol epi-α- 1653 1640 1642 1.93
α-Muurolol 1657 1644 1646 1.65
Intermedeol neo 1665 1658 1660 1.81
Khusinol 1681 1679 1680 4.98
Germacrone 1688 1693 1693 1.80
Cedr-8(15)-en-9-α-ol, acetate 1750 1741 1742 1.53
Oxygenated sesquiterpenes 28.55
Hydrocarbon sesquiterpenes 54.85
Total identified % 83.40
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*Al Calculated retention rate, AL Literature retention rate, IK Kovats index. Methods of Identification: RI-Retention index calculated using C7-C30 n-alkane standard solution in an HP-5 MS UI Agilent (30 m × 0.250 mm × 0.25 µm) column. RIa-Relative retention index found in literature in capillary column HP-5 and comparison of the retention indices and/or mass spectra from literature. Identification based on comparison with Wiley library mass spectra. %—values of areas

Fig. 1.

Fig. 1

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Major constituents of essential oil from Piper gaudichaudianum

Evaluation of the intrinsic antimicrobial activity

The results of the trials for the evaluation of intrinsic antibacterial activity are presented in Table 2. EOPG showed no intrinsic activity against the strains tested at concentrations below 1024 µg/mL. Essential oils are rich in lipophilic compounds, which can interact with the bacterial cytoplasmic membrane, causing damage that can increase its permeability (Bertini et al. 2005). Thus, the antimicrobial activity of essential oils as well as their isolated components has been mainly attributed to lesions in the structure of the cytoplasmic membrane (Souza et al. 2013; Luz et al. 2014; Sikkema et al. 1994). However, the EOPG showed no intrinsic antimicrobial activity in the concentrations tested against S. aureus, E. coli, or C. albicans species.

Table 2.

Minimum inhibitory concentrations (MICs) of the essential oil from the leaves of P. gaudichaudianum against different microorganisms

Microbial strains MIC (μg/mL)
Staphylococcus aureus SA1199B  ≥ 1024
Staphylococcus aureus ATCC 25923  ≥ 1024
Escherichia coli ATCC 25922  ≥ 1024
Candida albicans ATCC 10231  ≥ 1024
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A recent study showed that the essential oil extracted from P. rivinoides leaves was also inactive against S. aureus and E. coli species; however, it showed antifungal activity against C. albicans at a concentration of 512 µg/ml. On the other hand, the essential oil of P. cernuum leaves was active against S. aureus and C. albicans species but was inactive against the E. coli strains (Leal et al. 2019a). The different results observed in each study are probably related to differences in the chemical composition of the different oils tested, which may be influenced by several factors, such as seasonality, soil type, collection period, extraction method, extract drying temperature, and genotype (Diniz et al. 2007; Schindler et al. 2018; Alencar et al. 2016).

Evaluation of the modulatory effect on the resistance to Norfloxacin

The ability to potentiate the action of antibiotics against resistant microorganisms has been demonstrated for different natural products of plant origin, including essential oils (Ribeiro et al. 2012; Silva et al. 2019). The essential oil of P. rivinoides leaves enhanced the activity of gentamicin against strains of S. aureus and E. coli that are multidrug resistant and was also able to enhance the action of erythromycin against a strain of multidrug-resistant E. coli (Leal et al. 2019b).

The modulating effect of the EOPG on the resistance of the SA1199B strain to norfloxacin can be seen in Fig. 2A. The MIC value of norfloxacin for this strain is 64 μg/mL and the addition of EOPG in subinhibitory concentrations to the growth medium caused a reduction of these values to 32 and 8 μg/mL, respectively. The modulating effect presented by the EOPG was like the modulating effect observed for CPZ, which is cited in the literature as an inhibitor of the NorA efflux pump (Neyfakh et al. 1993). On the other hand, the EOPG was unable to reduce the MIC value of EtBr against the SA1199B strain in any of the subinhibitory concentrations tested (Fig. 2B).

Fig. 2.

Fig. 2

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MIC values of Norfloxacin (Nor) (A) and Ethidium Bromide (EtBr) (B) against S. aureus SA1199B (norA) in the absence or presence of the essential oil from the leaves of P. gaudichaudianum (EOPG) or chlorpromazine (CPZ). Each result represents the geometric mean of three simultaneous experiments. (***) Statistically significant values (p < 0.0001)

The essential oil obtained from P. cernuum leaves was also able to potentiate the activity of gentamicin against strains of S. aureus and multiresistant E. coli (Leal et al. 2019a). The results obtained in the present study showed that the EOPG showed a modulating effect of Norfloxacin resistance in a strain of S. aureus that overexpresses NorA. The antibacterial effect of Norfloxacin is due to its ability to inhibit the enzyme DNA gyrase, resulting in blocking DNA replication, and for this action to occur, the antibiotic needs to reach sufficient intracellular concentrations (Hooper and Jacoby 2016). Lipophilic phytochemicals such as sesquiterpenes present in the EOPG may be related to the modulating effect observed since such compounds can cause damage to the bacterial cytoplasmic membrane, increasing its permeability to the antibiotic.

Another mechanism that could explain the greater sensitivity of the SA1199B strain to norfloxacin in the presence of subinhibitory concentrations of the EOPG against the SA1199B strain would be a possible inhibition of the NorA efflux pump by the sesquiterpenes present in the EOPG (Coutinho et al. 2009). Since the interaction of lipophilic compounds with the cytoplasmic membrane can dissipate the proton gradient, proton-motive force-dependent efflux pumps such as NorA could be inhibited, leading to an increase in intracellular concentrations of the antibiotic (Oliveira et al. 2006). To test this hypothesis, tests were performed in which norfloxacin was replaced by EtBr, a biocidal agent capable of causing DNA damage whose only described resistance mechanism occurs through efflux pumps (Markham 1999). However, the results showed that the addition of EOPG to the growth medium in subinhibitory concentrations was not able to potentiate the inhibitory effect of EtBr against the SA1199B strain, suggesting that the modulating effect of Norfloxacin resistance demonstrated by EOPG does not involve inhibition of the NorA efflux pump.

Evaluation of the inhibitory effect of EOPG on the formation of bacterial biofilms

The results of the biofilm inhibition tests with S. aureus ATCC 25923 can be seen in Fig. 3. Results presented in Fig. 3A show that pre-treatment of bacterial suspension with EOPG significantly decreased biofilm formation by the S. aureus ATCC 25923 strain at concentrations of 100 to 25 μg/mL. In Fig. 3B, the EOPG was also able to inhibit biofilm formation during the growth of the tested strain and, therefore, during biofilm formation, but only at a concentration of 100 μg/mL. On the other hand, the EOPG was able to disaggregate the biofilm produced by the ATCC 25923 strain at concentrations of 100 to 25 μg/mL (Fig. 3C).

Fig. 3.

Fig. 3

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Effect of EOPG on the biofilm formation of the standard strain of S. aureus (ATCC 25923). Effect of EOPG on the biofilm aggregation (pre-treatment) (A), on the biofilm formation (during) (B) and on the breakdown of the already formed biofilm (post-treatment) (C). The cells of S. aureus were treated with 10, 25, 50 and 100 µg/ml of EOPG and the inhibition was measured by the violet crystal assay. The values represent the means ± standard deviation corresponding to three experiments performed in triplicate. The results are expressed as a percentage of inhibition. *Significant values in relation to the control (cells without treatment), p < 0.005-test one-way ANOVA

The results of the biofilm inhibition tests with Escherichia coli ATCC 25922 can be seen in Fig. 4. The effect of EOPG on the aggregation or biofilm formation of the standard strain of E. coli ATCC 25922 did not show significance at any stage. Biofilm formation is an important virulence factor for different bacteria (Moormeier and Bayles 2017). The bacterial biofilm is formed by multicellular aggregates surrounded by an extracellular matrix composed of exopolysaccharides, proteins, carbohydrates, teichoic acids, and/or extracellular DNA (Flemming and Wingender 2010), which allows the survival of these microorganisms in hostile environments and under extreme conditions. In addition, the biofilm produced by bacteria of medical importance allows these microorganisms to evade the host’s immune response and increases their ability to resist antibiotics commonly used in the clinic (Bhattacharya et al. 2018; Hosseini et al. 2020).

Fig. 4.

Fig. 4

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Effect of EOPG on the biofilm formation of the standard strain of Escherichia coli ATCC 25922. Effect of EOPG on the biofilm aggregation (pre-treatment) (A), on the biofilm formation (during) (B) and on the breakdown of the already formed biofilm (post-treatment) (C). The cells were treated with 10, 25, 50 and 100 µg/ml of E. coli EOPG and the inhibition was measured by the violet crystal assay. The values represent the means ± standard deviation corresponding to three experiments performed in triplicate. The results are expressed as a percentage of inhibition. *** Significant values in relation to the control (cells without treatment), p < 0.0001-test one-way ANOVA, and **p < 0.001

S. aureus can cause chronic infections due to its ability to resist antibiotics used in therapy, forming biofilms in medical-surgical instruments such as implanted artificial heart valves, catheters, and prostheses (Ribeiro et al. 2012; McConoughey et al. 2014). The biofilm produced by S. aureus is 10–1000 times less susceptible to a vast number of antimicrobials (Mohammed et al. 2018). Thus, infections related to the biofilm produced by S. aureus are associated with high rates of morbidity and mortality, surgical removal of infected medical devices, and increased hospitalization time, which causes high costs for the public health system (Parvizi et al. 2010; Song et al. 2015).

According to the results obtained in the present study, EOPG was able to inhibit biofilm formation in the pre-treatment (25, 50, and 100 µg/mL) steps, during (100 µg/mL), and post-treatment (25, 50, and 100 µg/mL). These results demonstrate the therapeutic potential of EOPG and its use in the development of drugs that can help fight infections caused by bacteria.

Adhesion and biofilm formation in S. aureus are mediated by an autolysin protein called AtlA (Foster 1995) and by surface components that recognize adhesive matrix molecules and intracellular adhesion (Patti et al. 1994; Speziale et al. 2009). These components include β-1,6-linked glucosaminoglycans, biofilm-associated proteins (Bap) (Cucarella et al. 2001; O’Gara 2007), SasG (Geoghegan et al. 2010), and fibronectin-binding proteins (FnBPA and FnBPB) (O’Neill et al. 2008) and are essential for the stages of initial adhesion, cell aggregation, formation of multicellular layers, and biofilm maturation (Mack et al. 2004). Possibly, EOPG inhibited one of these components or their biosynthesis, decreasing the total biomass of the S. aureus biofilm. However, further studies need to be done to define which components or biosynthesis pathways are inhibited by EOPG in the formation of the S. aureus biofilm. The Gram-negative strain, for presenting a more complex cell wall, was possibly one of the factors that caused the positive reduction of the results on all stages of the bacterial biofilm assays.

Action of Piper gaudichaudianum essential oil on Candida micromorphology

The microculture technique was employed to determine the effect of the EOPG on the morphological transition of Candida samples. Microscopic analysis of fungal growth in the absence of EOPG revealed the presence of several pseudohyphae (Fig. 5A). On the other hand, the addition of the EOPG at a concentration of 256 µg/mL caused a significant reduction in the amount and size of the pseudohyphae (Fig. 5B), whereas from the concentration of 512 µg/mL total inhibition of the morphological transition can be observed (Fig. 5C and D).

Fig. 5.

Fig. 5

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Action of the essential oil of Piper gaudichaudianum (EOPG) at subinhibitory concentrations on the cellular dimorphism of C. albicans. Fungal growth in the absence of EOPG (A). Fungal growth on medium containing EOPG at subinhibitory concentrations 512 µg/mL (B), and 256 µg/mL (C), and 128 µg/mL (D). The micrography was performed through optical microscopy in a 40X objective; photographic image captured on a digital camera with 4X zoom and scaled for better computer resolution

The ability of fungi to move between the different morphological stages is considered essential for virulence (Palková and Váchová 2016). This property can promote tissue penetration, allowing forms such as yeasts or conidia to be more successful in their dissemination through peripheral blood. Filamentous forms can play important roles during infection, invading endothelial and epithelial cells, causing tissue damage by releasing hydrolytic enzymes, and evading the host's immune response (Sudbery 2011).

We observed in our study that EOPG did not have an antifungal effect against C. albicans; however, EOPG was able to inhibit the cellular differentiation of C. albicans. The process of cell differentiation is regulated by intracellular signaling pathways, including the cAMP-PKA pathway and the MAPK pathway (Han et al. 2011). The transcription factor Efg1 associated with the PKA pathway stimulates the transcription of specific hyphae genes (Nobile et al. 2006; Sharkey et al. 1999; Stoldt et al. 1997) and the CH1 transcription factor of the MAPK pathway also participates cell differentiation process (Lo et al. 1997). EOPG possibly inhibited these transcription factors or enzymes that participate in the growth and biosynthesis of the fungal cell wall. However, other studies need to be carried out to determine the mechanism of EOPG inhibition in the cell differentiation process in C. albicans.

Conclusion

Although the essential oil of P. gaudichaudianum did not show significant intrinsic antimicrobial activity against the tested strains, it was able to potentiate the action of norfloxacin against a S. aureus strain, and it inhibited the biofilm formation by this pathogen, suggesting a possible use as an adjuvant in the treatment of infections caused by S. aureus. In addition, the EOPG inhibited the cellular differentiation of C. albicans, which could suggest a possible use of this essential oil against this pathogen.

Acknowledgements

This study was supported by CAPES, CNPq, FUNCAP and by the Piauí Research Foundation (FAPEPI). The authors acknowledge the financial support from CNPq (Grant 306008/2022-0), and financial support from the Piauí Research Foundation (FAPEPI) under Grant (Grant#: 050/2019).

Declarations

Conflict of interest

The authors declared the absence of conflict of interest.

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