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

MD ABDUR RAHIM
MD ABDUR RAHIM
, 
HOONHEE SEO
HOONHEE SEO
, 
SUKYUNG KIM
SUKYUNG KIM
, 
YOON KYOUNG JEONG
YOON KYOUNG JEONG
, 
HANIEH TAJDOZIAN
HANIEH TAJDOZIAN
, 
MIJUNG KIM
MIJUNG KIM
, 
SAEBIM LEE
SAEBIM LEE
 and 
HO-YEON SONG
HO-YEON SONG
   | Mar 19, 2021
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Article Category: original-paper
Published Online: Mar 19, 2021
Page range: 117 - 130
Received: Dec 28, 2020
Accepted: Feb 08, 2021
DOI: https://doi.org/10.33073/pjm-2021-011
Keywords
© 2021 Md Abdur Rahim et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig. 1.

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).
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).

Fig. 2.

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).
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).

Fig. 3.

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).
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).

Fig. 4.

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).
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).

Fig. 5.

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.
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 classificationTotal number of genes2-fold5-fold
1 × MIC2 × MIC1 × MIC2 × MIC
updownupdownupdownupdown
3 Protein synthesis
   a tRNA aminoacylation12  1  1  1  4  0  0  1  1
   b Ribosomal proteins: synthesis and modification25  2  3  5  4  0  0  0  0
   c tRNA and rRNA base modification37  2  0  6  4  0  0  0  0
   d Other85  9  81618  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, amines32  5  2  7  4  0  1    4a  1
   b Cations and iron carrying compounds72  8  91210  1  0  2  3
   c Carbohydrates, organic alcohols, and acids27  4  6  7  8  1  1  3    5b
   d Anions18  4  2  510  0  1  2  5
   e Anions; Other  1  1  0  1  0  0  0    1a  0
   f Other85  9  81618  2  0  6  2
   g Nucleosides, purines and, pyrimidines  6  0  3  05b  0  1  0    3b
   h Unknown substrate20  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 trafficking27  2  2  3  5  0  0  2  0
   b Degradation of proteins, peptides, and glycopeptides18  1  0  5  0  0  0  2  0
   c Protein modification and repair20  0  0  0  3  0  0  0  0
   d Protein folding and stabilization  42a  1  2  12b  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 family15  3  18b  0  1  0    3a  0
   b Serine family11  3  0  4  2  0  0  1  0
   c Glutamate family  7  03a  0  2  0  1  0  1
   d Pyruvate family125b  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  02b  0  2  0
16 Cellular processes; Transport and binding proteins
   a Toxin production and resistance; Other13  3  1  4  12a  0    3a  0
   b Toxin production and resistance; Unknown substrate  32a  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 ubiquinone17  0  0  3  1  0  0  0  0
   c Other85  9  81618  2  0  6  2
   d Heme, porphyrin, and cobalamin19  3  27a  2  0  1  2  1
   e Folic acid  6  0  0  0  0  0  0  0  0
   f Pyridoxine  2  02a  02a  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  04a  0  1  02a  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  14a  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 classificationTotal number of genes2-fold5-fold
1 × MIC2 × MIC1 × MIC2 × MIC
updownupdownupdownupdown
  1 DNA metabolism  92    9    2  20  11    2    0    2    0
  2 Energy metabolism126  10  17  22  31    1    5    5  11
  3 Protein synthesis  85    5    4a  12  15    0    0    1    1
  4 Transport and binding proteins195  29b  32b  42a  48b    4    4  16b  19
  5 Protein fate  77    5    4a  13  10    2    1    6    1
  6 Amino acid biosynthesis  62  13b    5  25    5a    3a    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    5b    2    5    1    2a    0    3a    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    7a  25a  13a    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    4a    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 nameNo. responder30 min. after patch removal (48 hrs.)24 hrs. after patch removal (72 hrs.)Meana
0.5+1+2+3+4+mean0.5+1+2+3+4+mean
1. α-viniferin 100–b––––0.00–––––0.000.00
2. α-viniferin 1000–––––0.00–––––0.000.00
3. α-viniferin 1,0000–––––0.00–––––0.000.00
4. Negative control0–––––0.00–––––0.000.00
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