A Clinical Trial to Evaluate the Efficacy of α-Viniferin in Staphylococcus aureus – Specific Decolonization without Depleting the Normal Microbiota of Nares

RAHIM, MD ABDUR; SEO, HOONHEE; KIM, SUKYUNG; JEONG, YOON KYOUNG; TAJDOZIAN, HANIEH; KIM, MIJUNG; LEE, SAEBIM; SONG, HO-YEON

doi:10.33073/pjm-2021-011

Figures & Tables

Antimicrobial inhibition. Antibacterial activities, expressed as MICs, of α-viniferin (A), Vancomycin (B) and Methicillin (C) against 20 bacterial isolates. Bacterial suspensions (1 × 105 to 1×106 CFU/ml) were incubated with varying concentrations of α-viniferin, Vancomycin, Methicillin in Mueller Hinton Broth in a 96 well plate for 24 h at 37°C. In vitro MICs were determined by the broth dilution procedures described by the Clinical and Laboratory Standards Institute (CLSI).

Clinical experiment of α-viniferin. General overview of clinical experiment. α-Viniferin was treated three times daily both in the left and right nares using a sterile cotton swab stick for 10 days. Nasal specimens were collected at 0th day (drug-free) and 10th day (α-viniferin treated) and analyzed (A). Skin Moisture Content Measurement. Skin moisture content was also measured during the α-viniferin treatment at day 0, 4, and 8 respectively, using coreometer. After a 4-day and 8-day treatment, the skin moisture content of the area was increased dramatically (p < 0.001) to 21.11% and 41.10% respectively, compared to at day 0 (B). The experiments were performed in triplicate, and the data are represented as mean values and standard deviations. *Statistical significance vs. drug-free control using one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001).

Culture-based quantification. Nasal swab samples of 0 and 10th days were collected and incubated in nutrient agar, staphylococcus agar media 110, and staphylococcus agar media 110 containing methicillin antibiotic for 48 hours at 37°C by using spread plate technique. After this period, antimicrobial activity was determined by the colony forming unit (CFU) method against normal flora (A), S. aureus (B), and MRSA (C), respectively. The experiments were performed in triplicate, and the data are represented as mean values and standard deviations. *Statistical significance vs. drug-free control using unpaired two-tailed Students’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001).

qRT-PCR-based quantification. Nares swab samples of 0 and 10th days were collected, following by DNA extraction and qRT-PCR was performed. Antimicrobial activity of α-viniferin (A, B, C) was determined by comparing the resulting Ct value with the standard curve (D) against normal flora, S. aureus, and MRSA, respectively. The experiments were carried out 3 times, and the data are given as mean values and standard deviations. * Statistical significance vs. drug-free control using unpaired two-tailed Students’ t-test (*p < 0.05, **p < 0.01, ***p < 0.001).

NGS-based 16S rRNA profiling. To know the whole bacterial composition throughout the α-viniferin treatment, we conducted 16S rRNA amplicon sequencing with 0th (α-viniferin-free) and 10th days (α-viniferin treated) nasal swab samples, and the resulting data was analyzed by Ezcloud software. Staphylococcaceae is the most dominant family, which decreased from 51.04% to 23.99% (A), and Staphylococcus is the most abundant among the genus level group that reduced from 51.03% to 23.99% (B) after the α-viniferin treatment.

Number of (FC cutoff > 2 and 5) specific regulated gene groups in S_ aureus by 1 × and 2 × MIC α-viniferin treatment for 8 hours at 37°C,according to functional classification_

Functional classification	Total number of genes	2-fold				5-fold
		1 × MIC		2 × MIC		1 × MIC		2 × MIC
		up	down	up	down	up	down	up	down
3 Protein synthesis
a tRNA aminoacylation	12	1	1	1	4	0	0	1	1
b Ribosomal proteins: synthesis and modification	25	2	3	5	4	0	0	0	0
c tRNA and rRNA base modification	37	2	0	6	4	0	0	0	0
d Other	85	9	8	16	18	2	0	6	2
e Translation factors	7	0	0	0	2	0	0	0	0
4 Transport and binding proteins
a Amino acids, peptides and, amines	32	5	2	7	4	0	1	4a	1
b Cations and iron carrying compounds	72	8	9	12	10	1	0	2	3
c Carbohydrates, organic alcohols, and acids	27	4	6	7	8	1	1	3	5b
d Anions	18	4	2	5	10	0	1	2	5
e Anions; Other	1	1	0	1	0	0	0	1a	0
f Other	85	9	8	16	18	2	0	6	2
g Nucleosides, purines and, pyrimidines	6	0	3	0	5b	0	1	0	3b
h Unknown substrate	20	3	6	6	6	0	0	1	2
i Cations and iron carrying compounds; Anions	3	0	1	0	2	0	0	0	0
5 Protein fate
a Protein and peptide secretion and trafficking	27	2	2	3	5	0	0	2	0
b Degradation of proteins, peptides, and glycopeptides	18	1	0	5	0	0	0	2	0
c Protein modification and repair	20	0	0	0	3	0	0	0	0
d Protein folding and stabilization	4	2a	1	2	1	2b	1	2a	1
e Degradation of proteins, peptides, and glycopeptides; Protein folding and stabilization	6	0	0	3	0	0	0	0	0
f Degradation of proteins, peptides, and glycopeptides; Protein modification and repair	1	0	0	0	0	0	0	0	0
g Protein and peptide secretion and trafficking; Protein modification and repair	1	0	1	0	1	0	0	0	0
6 Amino acid biosynthesis
a Aspartate family	15	3	1	8b	0	1	0	3a	0
b Serine family	11	3	0	4	2	0	0	1	0
c Glutamate family	7	0	3a	0	2	0	1	0	1
d Pyruvate family	12	5b	1	8	1	0	1	5	1
e Histidine family	8	0	0	2	0	0	0	0	0
f Aromatic amino acid family	9	2	0	3	0	2b	0	2	0
16 Cellular processes; Transport and binding proteins
a Toxin production and resistance; Other	13	3	1	4	1	2a	0	3a	0
b Toxin production and resistance; Unknown substrate	3	2a	1	1	0	0	0	0	0
c Detoxification; Other	1	0	0	0	0	0	0	0	0
19 Biosynthesis of cofactors, prosthetic groups, and carriers
a Glutathione and analogs	4	0	0	1	1	0	0	0	0
b Menaquinone and ubiquinone	17	0	0	3	1	0	0	0	0
c Other	85	9	8	16	18	2	0	6	2
d Heme, porphyrin, and cobalamin	19	3	2	7a	2	0	1	2	1
e Folic acid	6	0	0	0	0	0	0	0	0
f Pyridoxine	2	0	2^a	0	2^a	0	2	0	0
g Riboflavin, FMN, and FAD	6	0	0	2	0	0	0	0	0
h Thiamine	7	0	0	0	2	0	0	0	0
i Lipoate	1	0	0	0	0	0	0	0	0
j Other; Thiamine; Pyridoxine	2	0	0	0	0	0	0	0	0
k Pantothenate and coenzyme A	6	0	1	1	1	0	0	0	0
l Biotin	6	2	0	4^a	0	1	0	2^a	0
m Pyridine nucleotides	2	0	0	0	0	0	0	0	0
n Molybdopterin	4	1	0	2	0	0	0	0	0
26 Cellular processes; DNA metabolism
a Cell division; Chromosome-associated proteins	8	0	2	1	4^a	0	0	0	0

Number of genes (FC cutoff > 2 and 5) regulated in S_ aureus by 1× and 2× MIC α-viniferin treatment for 8 hours at 37°C,according to functional class_

Functional classification	Total number of genes	2-fold				5-fold
		1 × MIC		2 × MIC		1 × MIC		2 × MIC
		up	down	up	down	up	down	up	down
1 DNA metabolism	92	9	2	20	11	2	0	2	0
2 Energy metabolism	126	10	17	22	31	1	5	5	11
3 Protein synthesis	85	5	4^a	12	15	0	0	1	1
4 Transport and binding proteins	195	29^b	32^b	42^a	48^b	4	4	16^b	19
5 Protein fate	77	5	4^a	13	10	2	1	6	1
6 Amino acid biosynthesis	62	13^b	5	25	5^a	3^a	2	11	2
7 Signal transduction	13	0	3	0	3	0	0	0	2
8 Purines, pyrimidines, nucleosides, and nucleotides	37	1	5	2	7	1	0	1	2
9 Regulatory functions; Signal transduction	9	0	2	1	4	0	1	0	1
10 Cellular processes	90	9	11	15	21	0	5	5	8
11 Biosynthesis of cofactors, prosthetic groups, and carriers; Transport and binding proteins	3	0	2	0	2	0	1	0	1
12 Central intermediary metabolism	21	1	4	6	7	0	2	1	3
13 Regulatory functions	55	3	8	6	11	1	1	1	3
14 DNA metabolism; Regulatory functions; Cellular processes	1	0	0	0	0	0	0	0	0
15 Cell envelope	50	4	4	8	11	0	0	1	1
16 Cellular processes; Transport and binding proteins	13	5^b	2	5	1	2^a	0	3^a	0
17 Energy metabolism; Purines, pyrimidines, nucleosides, and nucleotides	2	0	0	0	1	0	0	0	0
18 Transcription	22	1	2	3	3	0	0	0	1
19 Biosynthesis of cofactors, prosthetic groups, and carriers	99	8	7^a	25^a	13^a	1	3	6	2
20 Protein fate; Energy metabolism	4	0	1	1	1	0	0	0	1
21 Fatty acid and phospholipid metabolism	24	0	2	3	3	0	0	0	0
22 Transport and binding proteins; Signal transduction	10	0	2	2	3	0	0	0	1
23 Hypothetical proteins	26	3	3	5	4	1	1	3	3
24 Cell envelope; Central intermediary metabolism	1	0	0	0	0	0	0	0	0
25 DNA metabolism; Mobile and extrachromosomal element functions	3	1	0	1	0	0	0	1	0
26 Cellular processes; DNA metabolism	8	0	2	1	4^a	0	0	0	0
27 Protein fate; Transport and binding proteins	1	0	0	0	0	0	0	0	0
28 Regulatory functions; Purines, pyrimidines, nucleosides, and nucleotides	1	0	0	0	0	0	0	0	0
29 Protein fate; Cellular processes	6	1	0	1	0	0	0	0	0
30 Energy metabolism; Central intermediary metabolism	1	0	0	0	0	0	0	0	0
31 Regulatory functions; Cellular processes	2	0	1	0	1	0	0	0	0
32 Signal transduction; Regulatory functions	1	0	0	0	0	0	0	0	0
33 DNA metabolism; Cellular processes	3	0	1	1	1	0	0	0	0
34 Biosynthesis of cofactors, prosthetic groups, and carriers; Central intermediary metabolism	1	0	0	1	0	0	0	0	0
35 Mobile and extrachromosomal element functions	32	1	1	5	1	0	0	0	0
36 Protein synthesis; Cellular processes; Regulatory functions	1	0	0	0	0	0	0	0	0
37 Cellular processes; Cell envelope	2	0	0	0	0	0	0	0	0
38 Transport and binding proteins; Cellular processes	2	0	0	0	0	0	0	0	0
39 Protein fate; Cell envelope	3	0	1	1	0	0	0	0	0
40 Cell envelope; Cellular processes	4	0	0	1	1	0	0	0	0
41 Cell envelope; Transport and binding proteins	4	1	0	1	0	0	0	0	0
42 Regulatory functions; Transport and binding proteins	2	0	0	0	1	0	0	0	0
43 DNA metabolism; Regulatory functions	3	0	0	0	0	0	0	0	0
44 Cellular processes; Protein fate	4	0	0	0	1	0	0	0	0
45 Mobile and extrachromosomal element functions; Regulatory functions	3	0	1	0	1	0	0	0	1
46 Mobile and extrachromosomal element functions; Hypothetical proteins	2	0	0	0	0	0	0	0	0
47 Protein fate; Signal transduction	3	0	1	1	1	0	0	0	0
48 Regulatory functions; Amino acid biosynthesis	2	0	1	0	0	0	0	0	0
49 Purines, pyrimidines, nucleosides, and nucleotides; Central intermediary metabolism	1	0	0	0	0	0	0	0	0
50 Protein synthesis; Biosynthesis of cofactors, prosthetic groups, and carriers	1	0	0	0	0	0	0	0	0
51 Regulatory functions; Central intermediary metabolism	2	0	1	0	1	0	0	0	0
52 Protein fate; Regulatory functions	3	0	0	2	0	0	0	0	0
53 Regulatory functions; Energy metabolism	1	0	1	0	0	0	0	0	0
54 Mobile and extrachromosomal element functions; Protein fate	1	0	0	1	0	0	0	0	0
55 Regulatory functions; Central intermediary metabolism; Signal transduction	1	0	1	0	1	0	1	0	1
56 Central intermediary metabolism; Cell envelope	1	0	0	0	0	0	0	0	0
57 Cellular processes; Mobile and extrachromosomal element functions	4	0	0	0	0	0	0	0	0
58 Protein fate; Purines, pyrimidines, nucleosides, and nucleotides	1	0	1	0	1	0	0	0	1

Skin irritation test result_

Material’s name	No. responder	30 min. after patch removal (48 hrs.)						24 hrs. after patch removal (72 hrs.)						Mean^a
Material’s name	No. responder	0.5+	1+	2+	3+	4+	mean	0.5+	1+	2+	3+	4+	mean	Mean^a
1. α-viniferin 10	0	–^b	–	–	–	–	0.00	–	–	–	–	–	0.00	0.00
2. α-viniferin 100	0	–	–	–	–	–	0.00	–	–	–	–	–	0.00	0.00
3. α-viniferin 1,000	0	–	–	–	–	–	0.00	–	–	–	–	–	0.00	0.00
4. Negative control	0	–	–	–	–	–	0.00	–	–	–	–	–	0.00	0.00

A Clinical Trial to Evaluate the Efficacy of α-Viniferin in Staphylococcus aureus – Specific Decolonization without Depleting the Normal Microbiota of Nares

Figures & Tables

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

Number of (FC cutoff > 2 and 5) specific regulated gene groups in S_ aureus by 1 × and 2 × MIC α-viniferin treatment for 8 hours at 37°C,according to functional classification_

Number of genes (FC cutoff > 2 and 5) regulated in S_ aureus by 1× and 2× MIC α-viniferin treatment for 8 hours at 37°C,according to functional class_

Skin irritation test result_

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