Robert D. Wojtyczka

Susceptibility of Staphylococcus aureus Clinical Isolates to Propolis Extract Alone or in Combination with Antimicrobial Drugs

The objective of this study was to assess in vitro the antimicrobial activity of ethanolic extract of Polish propolis (EEPP) against methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates. The combined effect of EEPP and 10 selected antistaphylococcal drugs on S. aureus clinical cultures was also investigated. EEPP composition was analyzed by a High Performance Liquid Chromatography (HPLC) method. The flavonoid compounds identified in Polish Propolis included flavones, flavonones, flavonolols, flavonols and phenolic acids. EEPP displayed varying effectiveness against twelve S. aureus strains, with minimal inhibitory concentration (MIC) within the range from 0.39 to 0.78 mg/mL, determined by broth microdilution method. The average MIC was 0.54 ± 0.22 mg/mL, while calculated MIC50 and MIC90 were 0.39 mg/mL and 0.78 mg/mL, respectively. The minimum bactericidal concentration (MBC) of the EEPP ranged from 0.78 to 3.13 mg/mL. The in vitro combined effect of EEPP and 10 antibacterial drugs was investigated using disk diffusion method-based assay. Addition of EEPP to cefoxitin (FOX), clindamycin (DA), tetracycline (TE), tobramycin (TOB), linezolid (LIN), trimethoprim+sulfamethoxazole (SXT), penicillin (P), erythromycin (E) regimen, yielded stronger, cumulative antimicrobial effect, against all tested S. aureus strains than EEPP and chemotherapeutics alone. In the case of ciprofloxacin (CIP) and chloramphenicol (C) no synergism with EEPP was observed.

The Antibacterial Effect of Ethanol Extract of Polish Propolis on Mutans Streptococci and Lactobacilli Isolated from Saliva

Dental caries occurrence is caused by the colonization of oral microorganisms and accumulation of extracellular polysaccharides synthesized by Streptococcus mutans with the synergistic influence of Lactobacillus spp. bacteria. The aim of this study was to determine ex vivo the antibacterial properties of ethanol extract of propolis (EEP), collected in Poland, against the main cariogenic bacteria: salivary mutans streptococci and lactobacilli. The isolation of mutans streptococci group bacteria (MS) and Lactobacillus spp. (LB) from stimulated saliva was performed by in-office CRT bacteria dip slide test. The broth diffusion method and AlamarBlue assay were used to evaluate the antimicrobial activity of EEP, with the estimation of its minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The biochemical composition of propolis components was assessed. The mean MIC and MBC values of EEP, in concentrations ranging from 25 mg/mL to 0.025 mg/mL, for the MS and LB were found to be 1.10 mg/mL versus 0.7 mg/mL and 9.01 mg/mL versus 5.91 mg/mL, respectively. The exposure to an extract of Polish propolis affected mutans streptococci and Lactobacillus spp. viability, exhibiting an antibacterial efficacy on mutans streptococci group bacteria and lactobacilli saliva residents, while lactobacilli were more susceptible to EEP. Antibacterial measures containing propolis could be the local agents acting against cariogenic bacteria.

Berberine Enhances the Antibacterial Activity of Selected Antibiotics against Coagulase-Negative Staphylococcus Strains in Vitro

Synergistic interactions between commonly used antibiotics and natural bioactive compounds may exhibit therapeutic benefits in a clinical setting. Berberine, an isoquinoline-type alkaloid isolated from many kinds of medicinal plants, has proven efficacy against a broad spectrum of microorganisms. The aim of the presented work was to assess the antibacterial activity of berberine chloride in light of the effect exerted by common antibiotics on fourteen reference strains of Staphylococccus spp., and to evaluate the magnitude of interactions of berberine with these antistaphylococcal antibiotics. In our study minimum inhibitory concentrations (MIC) of berberine chloride against CoNS ranged from 16 to 512 µg/mL. The most noticeable effects were observed for S. haemolyticus ATCC 29970, S. epidermidis ATCC 12228, S. capitis subsp. capitis ATCC 35661, S. galinarium ATCC 700401, S. hominis subsp. hominis ATCC 27844, S. intermedius ATCC 29663 and S. lugdunensis ATCC 49576. The most significant synergistic effect was noticed for berberine in combination with linezolid, cefoxitin and erythromycin. The synergy between berberine and antibiotics demonstrates the potential application of compound combinations as an efficient, novel therapeutic tool for antibiotic-resistant bacterial infections.

Caffeic Acid Phenethyl Ester and Ethanol Extract of Propolis Induce the Complementary Cytotoxic Effect on Triple-Negative Breast Cancer Cell Lines

Chemotherapy of breast cancer could be improved by bioactive natural substances, which may potentially sensitize the carcinoma cells’ susceptibility to drugs. Numerous phytochemicals, including propolis, have been reported to interfere with the viability of carcinoma cells. We evaluated the in vitro cytotoxic activity of ethanol extract of propolis (EEP) and its derivative caffeic acid phenethyl ester (CAPE) towards two triple-negative breast cancer (TNBC) cell lines, MDA-MB-231 and Hs578T, by implementation of the MTT and lactate dehydrogenase (LDH) assays. The morphological changes of breast carcinoma cells were observed following exposure to EEP and CAPE. The IC50 of EEP was 48.35 µg∙mL−1 for MDA-MB-23 cells and 33.68 µg∙mL−1 for Hs578T cells, whereas the CAPE IC50 was 14.08 µM and 8.01 µM for the MDA-MB-231 and Hs578T cell line, respectively. Here, we report that propolis and CAPE inhibited the growth of the MDA-MB-231 and Hs578T lines in a dose-dependent and exposure time-dependent manner. EEP showed less cytotoxic activity against both types of TNBC cells. EEP and, particularly, CAPE may markedly affect the viability of breast cancer cells, suggesting the potential role of bioactive compounds in chemoprevention/chemotherapy by potentiating the action of standard anti-cancer drugs.

Biofilm Formation and Antimicrobial Susceptibility of Staphylococcus epidermidis Strains from a Hospital Environment

The hospital environment microflora comprise a wide variety of microorganisms which are more or less pathogenic and where staphylococci are one of the most common types. The aim of the presented study was to evaluate the prevalence of the biofilm forming coagulase-negative staphylococci (CoNS) in a hospital environment as a risk factor for nosocomial infections. Among 122 isolated and tested strains of CoNS the most frequent were: S. epidermidis—32 strains, S. haemolyticus—31 strains, S. capitis subsp. capitis—21 strains, S. hominis—11 strains, S. cohnii subsp. cohnii—nine strains. In case of CoNS, the main molecule responsible for intercellular adhesion is a polysaccharide intercellular adhesin (PIA), encoded on the ica gene operon. The analysis revealed the presence of the icaADBC operon genes in 46.88% of S. epidermidis isolates. IcaA and icaD were present in 34.38% and 28.13% of strains respectively while IcaC gene was present in 37.50% of strains. IcaB gene was found in 21.88% of S. epidermidis strains. In 15 (63%) strains all icaADBC operon genes were observed. The assessment of antibacterial drugs susceptibility demonstrated that analyzed CoNS strains were highly resistant to macrolides and lincosamides and more sensitive to rifampicin and linezolid. Our data indicates that the hospital environment can be colonized by biofilm forming coagulase-negative staphylococci and transmission of these strains can cause an increased risk of serious nosocomial infections.

In Vitro Antimicrobial Activity of Ethanolic Extract of Polish Propolis against Biofilm Forming Staphylococcus epidermidis Strains

The aim of the presented study was to examine the antimicrobial activity of ethanol extract of Polish propolis (EEPP) against biofilm-forming CoNS strains in vitro. Our results revealed that EEPP displayed varying degrees of activity against CoNS with MIC values ranging from 1.56 to 0.78 mg/mL. The average MIC was 1.13 ± 0.39 mg/mL while calculated MIC50 and MIC90 values were 0.78 mg/mL and 1.56 mg/mL, respectively. The biofilm formation ability by all tested S. epidermidis strains was inhibited at EEPP concentrations ranging from 0.39 to 1.56 mg/mL. The degree of reduction of AlamarBlue was directly associated with the proliferation of S. epidermidis strains. The increased proliferation of S. epidermidis strains was observed after 12 and 24 hours of incubation in the presence of EEPP concentrations ranging from 0.025 to 0.39 mg/mL. These results suggest that antimicrobial activities of EEPP against S. epidermidis expressed as the reduction of bacterial growth, reduction of biofilm formation ability, and the intensity of proliferation were significantly affected by incubation time and EEPP concentration used as well as the interactions between these factors.

The Ethanol Extract of Polish Propolis Exhibits Anti-Proliferative and/or Pro-Apoptotic Effect on HCT 116 Colon Cancer and Me45 Malignant Melanoma Cells In Vitro Conditions.

BACKGROUND Propolis is a natural product widely consumed in folk medicine. Different biological activities, such as anticancer, antioxidant, anti-inflammatory, antibiotic and antifungal effects have been reported for propolis and its constituents. OBJECTIVES An in vitro study focused on an evaluation of the biological activity of EEPP, including its anti-proliferative influence on selected neoplastic cells, considering qualitative-quantitative chemical characterization of Polish propolis. MATERIAL AND METHODS Cytotoxicity was evaluated by means of the MTT and LDH assays. The apoptosis was determined using fluorescence microscopy with annexin V-FITC. Additional EEPP composition was analyzed by a High Performance Liquid Chromatography (HPLC) method. The antimicrobial activity was evaluated by minimal inhibitory concentrations (MIC) against Streptococcus aureus, Enetecoccus faecalis, Escherichia coli, Pseudomonas aeruginosa and Candida albicans.

. RESULTS The total content of flavonoids per quercetin in the examined propolis extract amounted to 0.442±0.091 mg/mL. The flavonoid compounds identified in Polish propolis included flavones, flavonones, flavonolols, flavonols and phenolic acids. The multi-directional interactions among the various chemical compounds in propolis seem to be the essential biological activities when considering its anticancer effects. The results showed that in case of Me45 and HCT 116 cell lines, the ethanol extract of propolis could inhibit cell growth as well as cell size reduction. Regarding antimicrobial activity, EEPP showed MICs ranging from 0.39 to 6.25 mg/mL. CONCLUSIONS Ethanol extract of propolis from Poland obtained in the study exhibits anti-proliferative activity in different carcinoma cells.

Caffeic acid reduces the viability and migration rate of oral carcinoma cells (SCC-25) exposed to low concentrations of ethanol.

Alcohol increases the risk of carcinoma originated from oral epithelium, but the biological effects of ultra-low doses of ethanol on existing carcinoma cells in combination with natural substances are still unclear. A role for ethanol (EtOH), taken in small amounts as an ingredient of some beverages or mouthwashes to change the growth behavior of established squamous cell carcinoma, has still not been examined sufficiently. We designed an in vitro study to determine the effect of caffeic acid (CFA) on viability and migration ability of malignant oral epithelial keratinocytes, exposed to ultra-low concentrations (maximum 100 mmol/L) EtOH. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-dimethyltetrazolium bromide) and LDH (lactate dehydrogenase) assays were used to assess the cytotoxic effect of EtOH/CFA and the viability of squamous carcinoma SCC-25 cells (ATCC CRL-1628, mobile part of the tongue). Tested EtOH concentrations were: 2.5, 5, 10, 25, 50, and 100 mmol/L, along with an equal CFA concentration of 50 μmol/L. Carcinoma cells’ migration was investigated by monolayer “wound” healing assay. We demonstrated that very low concentrations of EtOH ranging between 2.5 and 10 mmol/L may induce the viability of oral squamous cell carcinoma cells, while the results following addition of CFA reveal an antagonistic effect, attenuating pro-proliferative EtOH activity. The migration rate of oral squamous carcinoma cells can be significantly inhibited by the biological activity of caffeic acid.

Catechin Hydrate Augments the Antibacterial Action of Selected Antibiotics against Staphylococcus aureus Clinical Strains

Synergistic effects between commonly used antibiotics and natural substances may be an alternative to conventional antibacterial therapies. The objective of the presented study was to assess the in vitro antibacterial activity of catechin hydrate (CH) and evaluate the interactions of CH with selected antibiotics using Staphylococcus aureus clinical and reference strains. CH displayed diverse activity towards examined S. aureus strains, with minimal inhibitory concentrations (MICs) ranging from 256 to 2048 µg/mL. The interaction between CH and antibiotics was assessed by an E-test. The most significant synergistic effects were noticed for CH in combination with clindamycin and erythromycin. For cefoxitin and vancomycin a decrease of MIC values in the presence of CH was also observed, but it did not reach statistical significance. The obtained results demonstrate that CH shows antimicrobial activity against Staphylococcus aureus clinical strains. What is more, we proved a synergistic effect of CH with erythromycin and clindamycin.

Migration Rate Inhibition of Breast Cancer Cells Treated by Caffeic Acid and Caffeic Acid Phenethyl Ester: An In Vitro Comparison Study

One of the deadliest cancers among women is a breast cancer. Research has shown that two natural substances occurring in propolis, caffeic acid (CA) and caffeic acid phenethyl ester (CAPE), have significant anticancer effects. The purpose of our in vitro study was to compare cytotoxic activity and migration rate inhibition using CA and CAPE (doses of 50 and 100 µm) against triple-negative, MDA-MB-231 breast adenocarcinoma line cells, drawn from Caucasian women. Viability was measured by XTT-NR-SRB assay (Tetrazolium hydroxide-Neutral Red-Sulforhodamine B) for 24 h and 48 h periods. Cell migration for wound healing assay was taken for 0 h, 8 h, 16 h, and 24 h periods. CAPE displayed more than two times higher cytotoxicity against MDA-MB-231 cells. IC50 values for the XTT assay were as follows: CA for 24 h and 48 h were 150.94 µM and 108.42 µM, respectively, while CAPE was 68.82 µM for 24 h and 55.79 µM for 48 h. For the NR assay: CA was 135.85 µM at 24 h and 103.23 µM at 48 h, while CAPE was 64.04 µM at 24 h and 53.25 µM at 48 h. For the SRB assay: CA at 24 h was 139.80 µM and at 48 h 103.98 µM, while CAPE was 66.86 µM at 24 h and 47.73 µM at 48 h. Both agents suspended the migration rate; however, CAPE displayed better activity. Notably, for the 100 µM CAPE dose, motility of the tested breast carcinoma cells was halted.