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

Abstract

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.

Keywords: Antibacterial; Antifungal; Efflux pump inhibitors; Essential oil; Fungal dimorphism; Piper caldense.

Fig.1

Majority constituents of essential oil of Piper caldense

Fig. 2

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

Fig. 3

MIC values of Norfloxacin (Nor) (A) and Ethidium Bromide (EtBr) (B) against S. aureus K2068 (mepA) in absence or presence of the essential oil from the leaves of P. caldense (EOPC) or Chlorpromazine (CPZ). Each result represents the geometric mean of three simultaneous experiments. (***) Statistically significant values (p < 0.0001)

Fig. 4

MIC values of Ethidium Bromide (EtBr) against S. aureus K4100 (qacC) in absence or presence of the essential oil from the leaves of P. caldense (EOPC) or Chlorpromazine (CPZ). Each result represents the geometric mean of three simultaneous experiments. (***) Statistically significant values (p < 0.0001)

Fig. 5

Morphological transition of C. albincans in presence of subinhibitory concentrations of Piper caldense C.D.C. essential oil; 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