Small description. Buxus sempervirens was chosen for its promising antibiotic-potentiating activity and evaluated for its ability to control (remove and inactivate) biofilms of S. aureus CECT 976 within 1 h of exposure. Further phytochemical investigation of B. sempervirens was performed. One possible candidate, betulinic acid, along with five similar triterpenoids, were assessed for their antibacterial activity, synergic interaction with the antibiotics by checkerboard technique and, additionally, were also applied against S. aureus biofilms.
Bacterial strains. S. aureus CECT 976. Information on this strain can be found in Section 3.1.4.
Antibiotics. Ampicillin, ciprofloxacin, erythromycin and tetracycline. Preparation and specificities of the antibiotics are described in Section 3.1.5.
Phytochemicals. Betulinic acid, lupeol, betulin, hederagenin, ursolic acid and oleanolic acid.
Preparation and specificities of the triterpenoids are described in Section 3.1.6.
Plant material. Buxus sempervirensL. for. grandiflora (F. Muell.) P. Cout. (leaves). Relevant information about the preparation of this plant can be found in Section 3.1.7.
Biofilm control assay
Biofilms control experiment was developed as described in Section 3.2.2.1. Overnight cultures (~106 cells ml-1) were added to sterile 96-well polystyrene microtiter plates to form biofilms at 37ºC and stirring at 150 rpm for 24 h. Afterwards, the medium was removed and the biofilms were exposed to the antibiotics and to the plant extract individually and in combination at 37ºC and stirring at 150 rpm for 1 h. Antibiotic solutions were applied at MIC and 50 × MIC against biofilms. The MeOH extract of B. sempervirens was applied at concentrations ranging from 0.05 to 5 g l-1. Drug combinations did not exceed 5% v v-1 of the well (200 µl). After incubation, biofilms were washed twice with saline solution (0.85% NaCl), scraped and diluted for CFU counting. The numbers of CFU per unit of adhesion area (CFU cm-2) were assessed in MH agar.
Reduction (%) of the number of CFU cm-2 (compared with DMSO growth control) was also assessed.
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Fractionation of active extract of Buxus sempervirens
Buxus sempervirens leaves (160 g) were extracted with 500 ml of MeOH following the process described previously (Section 3.2.3.1). The MeOH extract was taken to dryness and redissolved in 225 ml of H2O/MeOH (4:1) and successively partitioned with 3 × 112.5 ml of dichloromethane (CHCl2)and buthanol (n-BuOH), respectively. All the fractions were analysed by 1H NMR and tested for both antibacterial and antibiotic-potentiating activities. Afterwards, the n-BuOH fraction (1.2 g) was further submitted to phytochemical investigation. The n-BuOH fraction was subjected to medium pressure liquid chromatography (MPLC, Sepacore, Büchi, Switzerland) in a silica gel column (80 g, 200 × 35 mm, i.d, Büchi), eluted with a gradient of CHCl3 (A): MeOH + HOAc 2% (B) as follow: 10% B for 15 min; linear increase 10-30% B in 5 min; isocratic elution using 30% B for 20 min; linear increase 30-50% B in 5 min, and finally 50% B for 10 min. The flow rate was 20 ml min-1 and the analysis was monitored by UV spectrometer at 220, 254, 280 and 365 nm. Collection was performed by volume where each fraction contained 20 ml, totalizing 53 fractions. The fractions were analysed by analytical TLC and combined into 8 subfractions.
TLC was performed using silica gel TLC plates (Merck, Darmstadt, Germany) with CHCl3:MeOH:HOAc (7.5:2.5:0.2). These eight fractions were analysed by 1H NMR and for their antibacterial-potentiating activity.
NMR analysis
Five hundred microliters of CD3OD were added to dried samples, and the resultant mixtures were vortexed for 10 s and sonicated for 20 min at 42 kHz, followed by centrifugation at 3999 g at room temperature for 5 min. Three hundred microliters of the supernatant were transferred to a 3 mm micro-NMR tube and analysed. 1H NMR spectra were recorded at 25 °C on a 600 MHz Bruker DMX-600 spectrometer (Bruker, Karlsruhe, Germany) operating at a proton frequency of 600.13 MHz. MeOD-d4 was used as the internal lock. 1H NMR experimental parameters were described in Section 3.2.4.5. The resulting spectra were manually phased and baseline corrected, and calibrated to residual CD3OD at 3.3 ppm, using TOPSPIN 3.2 software (Bruker BioSpin GmbH, Rheinstetten, Germany).
Antibacterial susceptibility testing
The MIC of the tested compounds was determined by microdilution techniques according to CLSI (2015a) guidelines as described in Section 3.2.1.2. The highest concentration of DMSO remaining after dilution (5% v v-1) caused no inhibition of bacterial growth. At least three independent experiments were performed.
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Antibiotic-potentiation testing by checkerboard
The checkerboard assay was performed according to CLSI (2015a) guidelines as described in Section 3.2.1.3 between the phytochemical tested (which had a detectable MIC) and antibiotics.
Both antibacterial compounds to be combined yielded final concentrations ranging from 2 × MIC to 1/64 × MIC. Drug combinations did not exceed 5% v v-1 of the well (200 µl). Antibiotic–
phytochemical interactions were classified using the FICI as described in TABLE 3.6 at Section 3.2.1.7.
Statistical analysis
For statistical analysis, the in vitro results were analysed by Student’s t test using the statistical program SPSS version 19.0. Statistical calculations were based on a confidence level ≥ 95% (P <
0.05), which was considered statistically significant.
4. R ESULTS AND D ISCUSSION
n this section, all the results obtained within this thesis are described, analysed and discussed. Comparison with other important studies and results from relevant bibliographic sources is performed and the novelty of the results obtained is emphasised. A brief conclusion of each task is presented, highlighting the main results obtained and suggestions for future work.
C 4 hapter
C hapter
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This chapter contains information that was published or has been submitted in:
Abreu, A. C., Serra, S., Borges, A., Salgado, A., Saavedra, M. J., McBain, A. J., Simões, M. (2015) Combinatorial activity of flavonoids with antibiotics against drug-resistant Staphylococcus aureus. Microb Drug Res; 21: 600-609 (Task 1; Section 4.1).
Abreu, A. C., Serra, S., Borges, A., Salgado, A., Saavedra, M. J., Simões, M. (2014) Evaluation of the best method to assess antibiotic potentiation by phytochemicals against Staphylococcus aureus. Diagn Microbiol Infect Dis; 9: 125-134 (Task 1; Section 4.1).
Abreu A. C., Saavedra, M. J., Simões, L. C., Simões, M. (2016) Combinatorial approaches with selected phytochemicals to increase antibiotic efficacy against Staphylococcus aureus biofilms. Biofouling; 32:
1103-1114 (Task 2; Section 4.2).
Abreu A. C., Coqueiro, A., Sultan, A. R., Lemmens, N. Kim, H. K., Verpoorte, R. van Wamel, W. J. B., Simões, M., Choi, Y. H. (2016) Looking to Nature for new concepts in antimicrobial treatments:
isoflavonoids from Cytisus striatus as antibiotic adjuvants against MRSA. Submitted. (Tasks 4 and 5;
Sections 4.4 and 4.5).
Abreu A. C., Paulet, D., Coqueiro, A., Malheiro, J., Borges, A., Saavedra, M. J., Choi, Y. H., Simões, M.
(2016) Antibiotic adjuvants from Buxus sempervirens to promote effective treatment of drug-resistant Staphylococcus aureus including biofilms. RSC Advances; 6: 95000-95009(Task 3 and 6; Sections 4.3 and 4.6).
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