key: cord-1054431-u6ghhtwu
authors: Leal, Antonio Linkoln Alves Borges; Bezerra, Camila Fonseca; Confortin, Camila; da Silva, Luiz Everson; Marinho, Emanuelle Machado; Marinho, Márcia Machado; Vasconcelos, Mayron Alves; da Silva, Terezinha Gonçalves; Marinho, Emmanuel Silva; Teixeira, Alexandre Magno Rodrigues; Coutinho, Henrique Douglas Melo; Barreto, Humberto Medeiros; dos Santos, Hélcio Silva
title: Chemical composition and potentiating action of Norfloxacin mediated by the essential oil of Piper caldense C.D.C. against Staphylococcus aureus strains overexpressing efflux pump genes
date: 2021-05-29
journal: Arch Microbiol
DOI: 10.1007/s00203-021-02393-5
sha: eb36e3e3ef38f3f3e2758357da219bd63bc46ff3
doc_id: 1054431
cord_uid: u6ghhtwu

Infectious diseases caused by multidrug-resistant microorganisms has increased in the last years. Piper species have been reported as a natural source of phytochemicals that can help in combating fungal and bacterial infections. This study had as objectives characterize the chemical composition of the essential oil from Piper caldense (EOPC), evaluate its potential antimicrobial activity, and investigate the synergistic effect with Norfloxacin against multidrug-resistant S. aureus overproducing efflux pumps, as well as, verify the EOPC ability to inhibit the Candida albicans filamentation. EOPC was extracted by hydrodistillation, and the chemical constituents were identified by gas chromatography, allowing the identification of 24 compounds (91.9%) classified as hydrocarbon sesquiterpenes (49.6%) and oxygenated sesquiterpenes (39.5%). Antimicrobial tests were performed using a 96-well plate microdilution method against C. albicans ATCC 10231, Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 standard strains, as well as against multidrug-resistant strains S. aureus SA1199B (overexpressing norA gene), S. aureus K2068 (overexpressing mepA gene) and S. aureus K4100 (overexpressing qacC gene). The oil showed activity against C. albicans ATCC 10231 (≥ 512 µg/mL) and was able to inhibit hyphae formation, an important mechanism of virulence of C. albicans. On the other hand, EOPC was inactive against all bacterial strains tested (≤ 1,024 µg mL). However, when combined with Norfloxacin at subinhibitory concentration EOPC reduced the Norfloxacin and Ethidium bromide MIC values against S. aureus strains SA1199B, K2068 and K4100. These results indicate that EOPC is a source of phytochemicals acting as NorA, MepA and QacC inhibitors.

Infectious diseases caused by multidrug-resistant microorganisms has become a global public health concern (Guschin et al. 2015) . Multidrug-resistant bacteria have been isolated with high frequencies from community or hospital-acquired infections (Klein et al. 2020; Xiang et al. 2020) , as well as, from infections of veterinary importance (Lima et al. 2012 ). On the other hand, infections caused by resistant fungi are more and more prevalent in hospitals, being known as a sleeping giant, for never having completely solved the problem of ineffective antifungals (Veríssimo et al. 2016) . In fact, antimicrobial resistance it was predicted to reach pandemic proportions by 2050, accounting for 10 million deaths annually (Sugden et al. 2016) .

Antibiotic resistance can acquired by a mutation that generate a change in a target protein with lower for affinity for antibiotic or through acquisition of resistance placed in plasmids, transposons or phage DNA that often involves inactivation, antibiotic degradation or extrusion by active efflux pumps (Santos et al. 2007) . Efflux pumps are transmembrane proteins that become both bacteria and fungi resistant, because they are able to extrude antibiotics and biocide reducing their intracellular concentrations using ATP or proton gradient as energy source (Du et al. 2018) .

Traditionally used antibiotics have been losing their effectiveness because of increased microbial resistance (Mayers et al. 2009 ). Epidemiological studies warn of the importance of further research for discovery of new drugs or compounds with therapeutic properties (Balouiri et al. 2016) . Another important point, to combat infections caused by resistant microorganisms, is the discover of compound acting as efflux pump inhibitors to provide new ways to reverse bacterial and fungal resistance (Rezende-Júnior et al. 2020) . In this way, alternative forms such as in vitro methods in antimicrobial tests to assess the potential of extracts, essential oils, and isolated compounds have been intensified, revealing potential antimicrobial agents or even enhancing the activity of antibiotics used in clinical practice (Balouiri et al. 2016; Coutinho et al. 2008 Coutinho et al. , 2010 Matias et al. 2013; Costa et al. 2017; Siebra et al. 2018) . The association of natural products as well as the use of these combined with antimicrobials, has been widely discussed in the scientific literature, given the ability to provide antimicrobial efficacy in previously ineffective doses due to the antimicrobial resistance presented by some strains (Guimaraes et al. 2010; Silva et al. 2014; Tulgar et al. 2018) .

The inhibition of the production of virulence factors by pathogenic microorganisms is also an approach that can be useful in the treatment of infectious diseases. Candida species, produce infections ranging from skin and mucous infections, to deep and generalized infections, they make use of alternative mechanisms to invade the host organism and develop virulence factors, where they colonize significantly, leaving the responses to aggression, weakened (Egue et al. 2018) . C. albicans is the species responsible for about 50-70% of the cases of invasive candidiasis (Castro et al. 2016) . C. albicans is a yeast species capable of producing hyphae or pseudo-hyphae able to invade host tissues leading to candidiasis (Fernandes et al. 2020; Sellam and Whiteway 2016) . The ability to alter structural morphology from yeast to filamentous cells, is known as one of the main mechanisms of fungal virulence (Hou et al. 2011; Sánchez-Martıńez and Pérez-Martıń 2001) .

Natural products with antimicrobial properties represent an alternative to the use of traditional antimicrobial agents. Piper caldense C.D.C. (Piperaceae) is a shrub popularly known as "pimenta d'agua" that has been used in some Brazilian regions as a sedative agent (Cardozo Júnior and Chaves 2003) . This study aimed to characterize the chemical composition of the essential oil from the leaves of Piper caldense (EOPC), as well as, its antimicrobial activity against bacteria and yeast strains. The ability of the EOPC to inhibit cell dimorphism in C. albicans was also assessed. Futhermore, the effect of the EOPC on the activity of Norfloxacin against multidrug-resistant S. aureus SA1199B, S. aureus K2068 and S. aureus K4100 strains was assessed to evaluate its potential as inhibitor of the NorA, MepA or QacC efflux pumps.

Plant material was collected in the dense lowland rainforest, Antonina, PR 2015/2106, Brazil, under coordinates 25° 17′ 51.9′′ S and 48° 40′ 51.4′′ W at 14 m elevation, during the spring. Material collection was carried out under license from the Environmental Institute of the Paraná State, number 284/11, and an exsiccata was deposited at the Herbarium of the Municipal Botanical Museum (MBM) in Curitiba, PR, under No. 267636 . Fresh leaves of P. caldense were used to extract the oil without any indication of contamination by parasites.

The oil was prepared by hydrodistillation using a Clevenger graduated device. The proportion of fresh leaves was 100 g/L of distilled water, following the methodology described by Wasicky (1963) . An electric dryer model-FANEM 320 SE was used, with air circulation at 40º C for 24 h to dry the leaves. Following to determine the moisture content at the time of extraction, samples of 20 g were collected in triplicates, submitted to drying in an electric dryer model FANEM 320 SE with air circulation at 65 °C until reaching constant weight. After extraction, the samples were collected with a precision pipette and placed in a freezer where they remained until the time of the chromatographic analysis.

Evaluation of the intrinsic antimicrobial activity of the EOPC was performed against standard microbial strains Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 and Candida albicans ATCC 10231, as well as, against multidrug-resistant strains S. aureus SA1199B overexpressing the norA gene , S. aureus K2068 overexpressing the mepA gene (Kaatz et al. 2005 ) and S. aureus K4100 overexpressing the qacC gene (Littlejohn et al. 1991) . Assays for evaluation of the modulating effect on drug resistance were performed only with drug-resistant S. aureus SA1199B, K2068 and K4100 strains. Assays for evaluation of the ability to inhibit the cellular dimorphism were conducted against C. albicans ATCC 10231 strain. Bacterial and yeast strains were maintained on Brain Heart Infusion Agar (BHIA, Himedia, India) slants at 4 °C, and prior to assay the cells were grown overnight at 37 °C in Brain Heart Infusion (BHI, Himedia, India). Subsequently, an aliquot of each microbial culture was removed and diluted in 3 mL of 0.9% saline, and the turbidity adjusted to the McFarland 0.5 scale.

Norfloxacin (Nor), Ethidium Bromide (EtBr) and Chlorpromazine (CPZ) were obtained from Sigma Chemical Corp., St. Louis. With the exception of Nor that was dissolved in a mixture of 1 M NaOH and sterile distilled water (1:9 proportion), EtBr and CPZ were dissolved in sterile water.

Initially, the essential oil was diluted in dimethylsulfoxide (DMSO) at a concentration of 10,000 µg/mL. Subsequently, this solution was diluted in sterile distilled water to a concentration of 1024 µg/mL. The minimum inhibitory concentration of EOPC against bacteria and fungi strains was determined using the 96-well plate microdilution technique. Initially, 100 µL of essential oil was diluted serially in BHI 10% medium in concentrations that ranged from 1024 to 8 µg/mL. Subsequently, 100 µL of the microbial suspension (bacteria or fungi) in BHI 10% was added. For the control of microbial growth, BHI 10% was composed and microbial suspension in the absence of essential oil (Leal et al. 2019a ) The plates were incubated at 37 °C for 24 h, and the MIC was identified by adding 20 μL of sodium resazurin to each well and after 1 h visually checking the presence or absence of colorimetric change.

Fungal viability was achieved by adding a sterilized stem to each well that did not show fungal growth and subculturing in an Sabouraud Dextrose Agar (SDA, Himedia, India) petri dish. The plates were incubated at 37 °C for 24 h and, subsequently, the development or suppression of fungal colonies (Morais-Braga et al. 2016) was verified.

To evaluate if the EOPC was able to modulate antibiotic resistance in S. aureus strains overexpressing specific efflux pump genes, antibiotic MICs were determined in the presence or absence of subinhibitory concentrations of EOPC (1/8 or 1/4 MICs). Antibiotic concentrations ranged from 0.125 to 128 μg/mL. The microtiter plates were incubated at 37 °C for 24 h and readings were performed with resazurin as described above. To verify whether the modulation of drug resistance occurred due to inhibition of the efflux pump, the modulation assay was performed by substituting antibiotics for EtBr, which is a known substrate of efflux pumps (Markham et al. 1999) , used herein as inhibition of the pump. Control trials were also performed, replacing each natural product with CPZ, which is a known efflux pump inhibitor (Neyfakh et al. 1993 ).

The tests were performed according to Sidrim and Rocha (2010) , with some modifications. In this test, the effect of the EOPC on the suppression of the filament of C. albicans was verified. Depleted Potato Dextrose Agar (PDA) was placed inside of Falcon tubes and, then the EOPC was added varying the concentration according to MIC as follow: MIC (512 μg/mL), MIC 1/2 (256 μg/mL) and MIC 1/4 (128 μg/ mL). Then, PDA was immediately poured in a slide. Petri dishes with a blade were inoculated by a calibrated loop (1 μg), where two parallel streaks were made in the medium already solidified and then covered by a sterile microscopic sheet. After incubation at 24 h (37 °C), the culture was visualized by optical microscopy. A control without the EOPC was made for the growth of yeasts whose hyphae emission was stimulated by the impoverishment of the medium, once stress caused by starvation leads to hyphae emission.

Data were analyzed using the statistical program GraphPad Prisma 6.0. Differences between treatment with Norfloxacin and treatment with Norfloxacin combined with EOPC (or Chlorpromazine) were analyzed using a two-way ANOVA test, using the geometric mean of the triplicates as the central data and standard deviation. Then, a post hoc Bonferroni test was performed and p values < 0.05 were considered significant.

The content of the EOPC was 14.74% and 24 constituents (91.9%) were identified (Table 1) . Chemical compounds were separated into groups, according to their chemical classification as hydrocarbon sesquiterpenes (49.6%), oxygenated sesquiterpenes (39.5%) and hydrocarbon monoterpenes (2.9%). Oxygenated monoterpenes were not found in the EOPC. Caryophyllene oxide (11.9%) was the major constituent found, followed by δ-Cadinene (9.6%) and Spathulenol (9.1%) (Fig. 1) . Chemical composition of the EOPC used in the present study (from Antonina, PR at 14 m elevation) was different to that verified by the EOPC from Piraquara, PR (528 m elevation), with nine different compounds as follow: trans-Caryophyllene, β-Copaene, Trans-α-bergamotene, α-Amorfene, β-Selinene and Valencene (Bezerra et al. 2020) . Variations in the chemical composition of the EOPC are probably associated with different edaphomatic and rainfall conditions in the region where the specimen was found (Viana et al. 2019) . Seasonality, circadian rhythm, temperature and brightness, as well as the stage of development and nutritional factors, are all likely to influence the chemical composition of essential oils.

MIC values above 1000 µg/mL for plant extracts have been considered as clinically irrelevant, because it may be impracticable to extrapolate the dose from that giving activity in vitro, to that which would be required in equivalence for the size of a human adult (Houghton et al. 2007 ). Using this value as a cutoff point, the EOPC did not show antimicrobial activity against bacterial strains tested (Table 2) . Similar results were verified for other Piper species, such as, P. rivinoides (Leal et al. 2019b) and Piper cernuum (Leal et al. 2019a ) that also did not present activity against both (Rocha et al. 2016) . EOPC tested in the present showed a weak antifungal activity against C. albicans ATCC 10231 (Table 2) . This antifungal activity could be related with the presence of some constituents such as Caryophyllene oxide, a compound described as showing antifungal activity against C. albicans and dermatophyte fungi (Ghavam et al. 2020; Schmidt et al. 2010; Yang et al. 1999 ). However, the Caryophyllene oxide content found in the oil tested in the present study was lower (11.9%) than that verified for the EOPC from Piraquara, PR (13.9%), which did not show activity against C. albicans INCQ40006 and C. albicans URM4387 (Bezerra et al. 2020 ). Thus, we cannot rule out that other oil constituents acting isolatelly or synergistically could be related with the antifungal activity showed by the EOPC tested in the present study. As an example, Spathulenol, a sesquiterpene reported to be between major volatile components of the essential oils of several aromatic Piperaceae species, has some biological activities, including antimicrobial activity (Dib et al. 2017) . Coming from the cyclization of nerolidine pyrophosphate, δ-cadinene presents a cadinane skeleton, which the precursor is the farnesyl pyrophosphate (RuizReyes and Suarez 2015). Among the activities reported in the literature for δ-cadinene can be mentioned the antitumor activity (Wright and Sutherland 2007) , antimicrobial activity (Skaltsa et al. 2003) . Thus, the antifungal activity observed in our approach could be associated to the levels of the compounds related to the local environments and conditions to which the assayed plant was exposed, acting possibly by a synergistic effect.

However studies with essential oils with species of Piper cernuum and P. caldense made by Duarte et al. (Duarte et al. 2016) and Constantin et al. (Costantin et al. 2001 ) present compounds that present several properties, among them antifungal, antibacterial and even capable of potentiating antibiotic effect. However, Costantin et al. (2001) who used the essential oil of P. cernnum and the compound Eugenol, demonstrated antifungal activity on C. albicans. The oil presented an inhibition halo of 12.2 ± 0.6 mm in diameter, and the Eugenol isolate 28.9 ± 1.3 mm.

EOPC potentiated the activity of Norfloxacin against S. aureus SA1199B overproducing NorA, as well as, against S. aureus K2068 overproducing MepA at subinhibitory concentrations. MIC values for Norfloxacin against SA1199B decreased eightfold in the presence of the oil from 64 to 8 µg/mL ( Fig. 2A) . For K2068 strain, MIC values for Norfloxacin also were reduced eightfold when the EOPC was added to the growth medium from 16 to 2 µg/mL (Fig. 3A) . These results indicate that EOPC contains phytochemicals able to modulate the Norfloxacin resistance, probably by inhibition of the NorA and MepA efflux pumps. To verify if the modulating effect is related with efflux pump inhibition, assays were performed replacing Norfloxacin by EtBr. As the only known mechanism of bacterial resistance to EtBr is mediated by efflux pumps (Markham et al. 1999) , this DNA-intercalating dye have been used to evaluate the potential efflux pump inhibitory activity of natural products (Silva et al. 2021a) , isolated phytochemicals (Silva et al. 2021b ) and synthetic compounds (Faillace et al. 2021; Costa et al. 2016) . Results showed that EOPC was also able to modulate the resistance to EtBr in both SA1199B (Fig. 2B) and K2068 (Fig. 3B) strains. These results indicate the presence of compounds acting as NorA and MepA inhibitors which could be used against Norfloxacin-resistant S. aureus strains overproducers of efflux pumps, leading to a higher antibiotic accumulation in the bacterial cell.

NorA is a proton motive-dependent efflux pump belongs to Major Facilitator Superfamily (MFS), meanwhile MepA is a member of the Multidrug and Toxin Extrusion (MATE) superfamily, which is dependent of H + or Na + gradient to extrude Norfloxacin or EtBr (Schindler et al. 2014) . To guarantee the integrity and functioning of NorA, there must be a continuous proton-driving force, integrity of DNA zones responsible for the synthesis of membrane proteins and balance in the permeability of substances inside the cell (Thai et al. 2015) . The modulating effect verified for EOPC could be provoked by interaction of their hydrophobic monoterpenes and sesquiterpenes constituents, leading to plasma membrane damage and dissipation of the proton gradient (Souza et al. 2013; Ahmad et al. 2013) , resulting in the NorA and MepA inhibition. As a consequence, the intracellular concentrations of Norfloxacin becomes enough to inactivate their intracellular targets increasing its antibacterial effect against the S. aureus strains tested. Thus, the EOPC could be used in combination to Norfloxacin aiming to enhancing the effectiveness of this antibiotic against multidrug-resistent S. aureus overproducers of NorA or MepA. Similar results were found for S. aureus K4100 overexpressing the qacC gene (Littlejohn et al. 1991) once the EOPC potentiate the activity of EtBr, indicating that the oil is able to inhibit QacC. MIC values for EtBr against K4100 decreased eightfold in the presence of the oil from 32 to 4 µg/ mL (Fig. 4) . QacC is a proton motive-dependent efflux pump that confer resistance to EtBr and quaternary ammonium compounds (QACs), as Benzalkonium Chloride (Schindler et al. 2014) . Biocide agents such as QACs are widely used in the disinfection of surfaces mainly in hospitals, outpatient clinics, dentist offices, as well as, veterinary clinics, being including effectiveness in the SARS-CoV inactivation (Kamp et al. 2020) . This intensive use can contribute for emergence of QAC-resistant S. aureus strains overproducers of QacC (Schindler et al. 2014) . Thus, the EOPC could be used in association with QACs aiming to eliminate these multidrug strains present in surfaces preventing outbreaks of S. aureus infections in hospital environments.

Assays for evaluation of morphological transition of C. albicans were performed in the presence and absence of subinhibitory concentrations of EOPC. The fungus grown in the absence of oil showed a clear morphological transition, being possible to observe the presence of blastospores and hyphae (Fig. 5) . On the other hand, EOPC showed potential to inhibit fungal dimorphism at 512 µg/mL (MIC) and at subinhibitory concentration 256 µg/mL (MIC 1/2). However, at 128 µg/mL, it was possible to observe the presence of pseudo-hyphae. Thus, besides antifungal activity, EOPC demonstrating a clear inhibition of the morphological transition in C. albicans ATCC 10231. Results found for EOPC tested in the present study were better than those found by Bezerra et al. (2020) once they verified inhibition of hyphae projection just at 2048 µg/mL.

Hyphae projection by fungi such as C. albicans is an adaptation process to environmental changes such as temperature, pH or nutrient depletion, and filamentous forms generally promote invasive diseases and hinder the action of antifungals (Wightman et al. 2004; Barbedo and Sgarbi 2010; Veses and Gow 2014) . Development of pseudo-hyphae by C. albicans increases its ability to adhere to the host tissues, increasing its contact surface, leading to a higher propagation and developing of virulence factors (Giolo and Svidzinski 2010) . Under stress, C. albicans change from yeast form to hyphae, that it is closely interconnected with its pathogenicity, given that filamentous fungi present better adherence to the tissue since at the end of hyphae, there are enzymes that, when secreted, can degrade proteins, lipids Micrography was performed through 40X objective optical microscopy; photographic image captured in digital camera with 4X zoom and resized for better computer resolution. Growth control (A); P.caldense essential oil at 128 µg/mL (B); 256 µg/mL (C) and 512 µg/mL and other components of host cells, facilitating the invasion process (Costa et al. 2011) .

The emission of hyphae is considerably relevant in what concerns the mechanism of candidiasis virulence since the pathogenicity of the fungus is due to the fact that it presents dimorphism, this appears as an aptitude method in the development of superficial and systemic infections that hyphae spread, promoting the pathogenicity of the infectious form .

Although there are several antifungals available on the market, the high frequency of resistant strains becomes worrying and borderline in several clinical conditions. Thus, the EOPC tested in the present study could be applied as an agent able to inhibit the conversion of commensal yeast cells of C. albicans in filamentous and pathogenic forms, in the treatment or prevention of candidiasis. It is important to note that cell differentiation is regulated by intracellular signaling pathways, including cAMP-PKA and MAPK pathways (Han et al. 2011) . Regarding the inhibition of the filamentous form of fungi, it is suggested that inhibition of the Efg1 transcription factor associated with the PKA pathway, which stimulates the transcription of genes that participate in the formation of hyphae (Stoldt et al. 1997; Nobile et al. 2006 ).

EOPC showed antifungal action and it was able to inhibit the hyphae projection by C. albicans ATCC 10231, indicating a promise use in the treatment of candidiasis. In addition, the essential oil potentiating the activity of Norfloxacin decreasing its MIC values for resistant strains overexpressing NorA or MepA efflux pumps. Futhermore, EOPC promoted a significant reduction of the MIC values for EtBr indicating that the modulating effect could be related to its ability to cause inhibition of this efflux pumps. These results point to a potential of the EOPC as an adjuvant of Norfloxacin in the treatment of infections caused by S. aureus strains overproducing NorA or MepA efflux pumps, a protein overexpressed in S. aureus SA1199B. Besides NorA and MepA, EOPC was also to act as a QacC efflux pump inhibitor compound indicating a potential technological application in association with quaternary ammonia compounds for elimination of QacC overproducing S. aureus strains in both human and veterinary clinical environments.

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