3.1. Healthcare workers presented high prevalence levels of S. aureus colonization with assoiated divergent resistant phenotypes and EGCG efefcts
The prevalence of S. aureus in the population studied in samples collected from healthcare workers (n=38) was 42.1% (16/38), of which 18.4% (7/38) were MSSA and 23.7% (9/38) MRSA.
3.1.1. S. aureus (MSSA and MRSA) commensal strains presented divergent resistant phenotypes against imipenem, tetracycline, gentamycin and amoxicillin.
Phenotyping of the antibiotic resistance profile of strains isolated from samples collected from volunteers with healthcare occupational exposure, was performed regarding sensitivity to imipenem 10µg, tetracycline 30µg, gentamicin 30µg and amoxicillin 25µg in order to identify divergent profiles. After incubation (24h at 37ºC), the antibiotic inhibition zones were measured for each strain, and the sensible (S) and resistant (R) characterization was performed according to the values established by EUCAST and the results obtained are recorded in Table 3. Data showed that all strains are sensible to tetracycline and gentamicin. MSSA strains encoded as CC4, MB1, MB6, MB10 and MRSA strains encoded with MB2, MB4, VFXB14, VFXB15, VFXB16 were resistant to amoxicillin. The coded strain MB12 was resistant to both imipenem and amoxicillin.
Table 3 - Resistance profile divergence of isolated MSSA and MRSA strains to Imipenem (10µg), Tetracycline (30 µg), Gentamycin (30 µg) and Amoxicillin (25 µg).
|
Code
|
IMIP 10
|
TETRA 30
|
GENTA 30
|
AMOX 25
|
|
CC3
|
S
|
S
|
S
|
S
|
M
|
CC4
|
S
|
S
|
S
|
R
|
S
|
MB1
|
S
|
S
|
S
|
R
|
S
|
MB6
|
S
|
S
|
S
|
R
|
A
|
MB10
|
S
|
S
|
S
|
R
|
|
MB17
|
S
|
S
|
S
|
S
|
|
5-7S
|
S
|
S
|
S
|
S
|
|
|
|
|
|
|
|
MB2
|
S
|
S
|
S
|
R
|
|
MB4
|
S
|
S
|
S
|
R
|
M
|
MB5
|
S
|
S
|
S
|
S
|
R
|
MB12
|
R
|
S
|
S
|
R
|
S
|
1-2S
|
S
|
S
|
S
|
S
|
A
|
VFXB7
|
S
|
S
|
S
|
S
|
|
VFXB14
|
S
|
S
|
S
|
R
|
|
VFXB15
|
S
|
S
|
S
|
R
|
|
VFXB16
|
S
|
S
|
S
|
R
|
3.1.2. Exposure to EGCG induce differential synergistic effects in MSSA and MRSA resistant phenotypes
In order to assess potential synergism divergence between EGCG and the different antibiotics tested, isolated strains were inoculated at different concentrations of EGCG (250 µg / ml, 100 µg / ml, 50 µg / ml and 25 µg / ml) and antibiotic resistance tests performed.
Regarding the results obtained with imipenem, the MRSA strain encoded as MB12, which showed a resistant phenotype (Table 3), when exposed to different concentrations of EGCG, the phenotype went from resistant to sensible. EGCG potentiated the action of imipenem and the phenotype reversal was observed after co-exposure with EGCG at final concentrations of 250 µg/ml, 100 µg/ml, 50 µg/ml and 25 µg/ml.
On the other hand, amoxicillin MSSA and MRSA resistant strains namely CC4, MB1, MB6, MB10, MB2, MB4, MB12, VFXB14 VFXB15, VFXB16 after exposure with EGCG presented divergent resistance phenotypes which are summarized in Table 4. Strains encoded as MB6, MB2, MB4, when exposed to EGCG in different concentrations, the resistance phenotype reverted to sensible, indicating that EGCG potentiated the action of amoxicillin in these strains. Strains encoded as MB1 and VFXB16 only demonstrated the reversion of the resistance to sensible phenotype at 250µg / ml and 100µg / ml EGCG concentrations. Strains encoded as MB10, MB12, VFXB14 AND VFXB15 maintained the resistance phenotype, that is, in these strains there was no synergism between EGCG and amoxicillin.
Table 4- Resistance profile of isolated MSSA and MRSA strains to resistant to Amoxicillin (25 µg) after co-exposure with EGCG at final concentrations of 250 µg/ml, 100 µg/ml, 50 µg/ml and 25 µg/ml.
|
Code
|
AMOX
|
AMOX + 250 µg/ml EGCG
|
AMOX + 100 µg/ml EGCG
|
AMOX + 50 µg/ml EGCG
|
AMOX + 25 µg/ml EGCG
|
M
|
CC4
|
R
|
S
|
S
|
S
|
R
|
S
|
MB1
|
R
|
S
|
S
|
R
|
R
|
S
|
MB6
|
R
|
S
|
S
|
S
|
S
|
A
|
MB10
|
R
|
R
|
R
|
R
|
R
|
|
|
|
|
|
|
|
|
MB2
|
R
|
S
|
S
|
S
|
S
|
M
|
MB4
|
R
|
S
|
S
|
S
|
S
|
R
|
MB12
|
R
|
R
|
R
|
R
|
R
|
S
|
VFXB14
|
R
|
R
|
R
|
R
|
R
|
A
|
VFXB15
|
R
|
R
|
R
|
R
|
R
|
|
VFXB16
|
R
|
S
|
S
|
R
|
R
|
3.1.3. Imipenem, tetracycline, gentamycin and amoxicillin inibition zones values are affected by EGCG exposure
Regarding time exposure effects in EGCG co-exposure with the tested antibiotics, we observed significant divergence in MSSA with Imipenem and Amoxicillin between 18 h and 24 h exposure (p=0,00190135 and p=1,5058E-05, respectively) and 24h and 48 h (p=0,014651 and p=0,000525, respectively).
MRSA between 18 h and 24 h exposure in Imipenem and Amoxicillin (p= 3,27033E-05 and 0,049234613 respectively) and 24h to 48 h only in Amoxicillin (p= 0,000159). On the other hand, at the same exposure time we also reported differences between MSSA and MRSA strains namely after 18h EGCG co-exposure with Imipenem and Amoxicillin (p= 2,87778E-07 and p= 0,000201292, respectively) and Imipenem for 24h and 48h exposures (p= 8,04449E-07 and 6,5191E-06, respectively).
3.1.4. Epigenetic and drug resistance modulators expression patterns differ in divergent resistance phenotypes strains after EGCG exposure
Expression analysis of staphylococci methyltransferase (orfx) and drug resistance (spdC and WalKR) genes was performed in selected MSSA and MRSA strains with described divergent resistance phenotypes obtained after EGCG exposure. Results are summarized in table 5. MSSA strain MB10 and MRSA strain VFXB14 were the most resistant strains and so utilized for transcriptional expression comparation in relation to the other selected strains.
Table 5 - MSSA and MRSA selected strains code and resistant phenotype regarding (imipenem (IMIP), tetracycline (TETRA), gentamycin (GENTA) and amoxicillin (AMOX)) selected for transcriptional analysis.
Code
|
Resistance phenotype
|
CC4
|
IMIP S, TETRA S, GENTA S, AMOX R reversion up to 50 µg/ml EGCG
|
MB6
|
IMIP S, TETRA S, GENTA S, AMOX R total reversion with ECGC
|
MB10
|
IMIP S, TETRA S, GENTA S, AMOX R no reversion
|
MB17
|
IMIP S, TETRA S, GENTA S, AMOX S
|
MB2
|
IMIP S, TETRA S, GENTA S, AMOX R total reversion with ECGC
|
MB12
|
IMIP R total reversion, TETRA S, GENTA S, AMOX R no reversion
|
MB5
|
IMIP S, TETRA S, GENTA S, AMOX S
|
VFXB14
|
IMIP S, TETRA S, GENTA S, AMOX R no reversion
|
VFXB16
|
IMIP S, TETRA S, GENTA S, AMOX R reversion up to 100 µg/ml EGCG
|
3.1.5. Orfx, spdC and WalKR expression levels are affected by co-exposure with EGCG in selected MSSA and MRSA strains
EGCG exposure particularly altered expression patterns of the analyzed genes with higher sensible phenotypes when comparing with the most resistant strains.
Regarding MSSA selected strains (Figure 1 A), MB10 strain was considered the most resistant due to the fact that, like the other strains was sensible to imipenem, tetracycline and gentamicin, but resistant to amoxicillin and, after EGCG exposure, no reversion was observed in the phenotype. Conversely MB17 strain was sensible to all assessed antibiotics and after EGCG exposure we observed a significant increase in transcriptional expression of all analyzed genes, namely spdC, WalR and orfx (4.98 ± 1.14, 17.29 ± 1.9, 19.21 ± 1.99, p<0.01 respectively). Also, MB6 with total reversion of amoxicillin resistance spdC, WalR and orfx mRNA were upregulated (1.62 ± 0.26, 1.85 ± 0.1, 1.5 ± 0.1, p<0.01 respectively). On the other hand, after EGCG exposure CC4 strains which presented a reversion of the resistant phenotype for amoxicillin up to 50 µg/ml, a downregulation of spdC expression was observed (-10.865 ± 0.16 p<0.001) and an increase in WalR and orfx mRNA levels (7.88 ± 0.1 p<0.001 and 3.9 ± 0.2 p<0.01, respectively).
In MRSA selected strains the same pattern was observed (Figure 1 B). For MRSA strains VFXB14 was selected for comparation as it presented the same resistant phenotype as MB10 namely, sensible to imipenem, tetracycline and gentamicin, but maintained resistance to amoxicillin after EGCG exposure. In concordance with MSSA strains, MB5 strain, the most sensible selected MRSA which was sensible to all antibiotics. Transcriptional analysis demonstrated an increase in mRNA levels of spdC and orfx (4.74 ± 0.14, 4.23 ± 0.65, p<0.01 respectively) and more significantly WalR (6.8 ±0.28, p<0.01).
On the other hand, in MB2 strains which presented total reversion of amoxicillin resistance only spdC, was significantly upregulated (20.7± 6.4 p<0.001).
In MB12 strain analysis, which was initially resistant to imipenem but the phenotype was reversed after EGCG exposure and no effect was reported for the amoxicillin resistance spdC, WalR and orfx genes were upregulated (2.74 ± 0.24, 2.4 ± 0.12, 3.86 ± 0.22, p<0.01 respectively). Additionally, VFXB16 strain which presented a reversion of the resistant phenotype for amoxicillin up to 100 µg/ml of EGCG exposure also presented increased mRNA levels of spdC, WalR and orfx (3.43 ± 0.13, p<0.01, 3.15 ± 0.17, 5.99 ± 0.14, p<0.001 respectively).
3.1.6. Orfx, spdC and WalKR expression patterns after 24h subculture of MSSA and MRSA strains with previous co-exposure with EGCG
Transcriptional analysis performed in the selected strains after 24h of subculture with no EGCG exposure, demonstrated the overall maintenance of the expression patterns observed immediately after EGCG exposure (Figure 2). CC4 strain maintained the downregulation of spdC expression (-10.69 ± 0.29 p<0.01) and the WalR and orfx upregulation (0.73 ± 0.17 p<0.01 and 3.9 ± 0.15 p<0.001, respectively). However, in MB6 only orfx mRNA upregulation was maintained (1.77 ± 0.17, p<0.001). Additionally, in MB17 strain the increase in transcriptional expression WalR and orfx was not altered (4.48 ± 2.55, 3.23 ± 0.35, p<0.01 respectively) but a downregulation of spdC expression was reported (-7.35 ± 0.16, p<0.01) (Figure 2 A).
Regarding MRSA strains transcriptional analysis, for MB2 strain we observed the stability of spdC, upregulation (13.1±12 p<0.001) and additionally, VFXB16 strain also maintained the previous pattern with associated upregulation of spdC, WalR and orfx mRNA levels (2.11 ± 0.24, p<0.01, 3.02 ± 0.12, p<0.01 5.99 ± 0.01, p<0.001 respectively). Moreover, MB5, the most sensible selected MRSA strain, also maintained the upregulation of WalR and orfx (1.06 ± 0.07, 4.47 ± 0.22, p<0.001 respectively) (Figure 2 B).
However, conversely in MB12 strain transcriptional analysis, spdC, WalR and orfx genes were downregulated (-3.47 ± 0.1, p<0.01 -3.35 ± 0.007, p<0.001 -2.45 ± 0.15, p<0.01 respectively).
3.2. EGCG oral intake eliminated MRSA phenotype in nasal colonization in the community
Analyzed data from the performed interventional, uncontrolled, prospective, longitudinal and of individual analysis study demonstrated that all samples presented normal commensal flora of gram positive coccus Staphylococcus spp. and Streptococcus spp. We identified a prevalence of S. aureus 33.3% in which 70% were MSSA (23.3% total) and 30% MRSA (10% total) at the beginning of the study (Table 6). After 90 days of 225mg EGCG oral exposure, S. aureus prevalence is maintained (36.6%) however, regarding MSSA strains, 50% of the colonized individuals maintained the colonization, 10% eliminated the colonization and 3 new colonization’s were observed, while in MRSA 33.3% of the colonized individuals eliminated the bacteria while in 66.6% the resistance phenotype was reversed (Table 6).
Table 6 - MSSA and MRSA prevalence (%) in commensal flora of the participants enrolled in the study before (T0) and after (T90) 90 days of 225mg EGCG oral exposure.
ID
|
T0 prevalence
|
T90 prevalence
|
1
|
MSSA, Staphylococcus spp. and Streptococcus spp
|
MSSA, Staphylococcus spp. and Streptococcus spp
|
2
|
Staphylococcus spp. and Streptococcus spp
|
Staphylococcus spp. and Streptococcus spp
|
3
|
Staphylococcus spp. and Streptococcus spp
|
Staphylococcus spp. and Streptococcus spp
|
4
|
Staphylococcus spp. and Streptococcus spp
|
Staphylococcus spp. and Streptococcus spp
|
5
|
MSSA, Staphylococcus spp. and Streptococcus spp
|
Staphylococcus spp. and Streptococcus spp
|
6
|
Staphylococcus spp. and Streptococcus spp
|
Staphylococcus spp. and Streptococcus spp
|
7
|
Staphylococcus spp. and Streptococcus spp
|
MSSA, Staphylococcus spp. and Streptococcus spp
|
8
|
Staphylococcus spp. and Streptococcus spp
|
MSSA, Staphylococcus spp. and Streptococcus spp
|
9
|
Staphylococcus spp. and Streptococcus spp
|
Staphylococcus spp. and Streptococcus spp
|
10
|
Staphylococcus spp. and Streptococcus spp
|
MSSA, Staphylococcus spp. and Streptococcus spp
|
11
|
Staphylococcus spp. and Streptococcus spp
|
Staphylococcus spp. and Streptococcus spp
|
12
|
Staphylococcus spp. and Streptococcus spp
|
Staphylococcus spp. and Streptococcus spp
|
13
|
Staphylococcus spp. and Streptococcus spp
|
Staphylococcus spp. and Streptococcus spp
|
14
|
Staphylococcus spp. and Streptococcus spp
|
Staphylococcus spp. and Streptococcus spp
|
15
|
Staphylococcus spp. and Streptococcus spp
|
Staphylococcus spp. and Streptococcus spp
|
16
|
MSSA, Staphylococcus spp. and Streptococcus spp
|
MSSA, Staphylococcus spp. and Streptococcus spp
|
17
|
MRSA, Staphylococcus spp. and Streptococcus spp
|
MSSA, Staphylococcus spp. and Streptococcus spp
|
18
|
MRSA, Staphylococcus spp. and Streptococcus spp
|
Staphylococcus spp. and Streptococcus spp
|
19
|
MRSA, Staphylococcus spp. and Streptococcus spp
|
MSSA, Staphylococcus spp. and Streptococcus spp
|
20
|
Staphylococcus spp. and Streptococcus spp
|
Staphylococcus spp. and Streptococcus spp
|
21
|
MSSA, Staphylococcus spp. and Streptococcus spp
|
MSSA, Staphylococcus spp. and Streptococcus spp
|
22
|
Staphylococcus spp. and Streptococcus spp
|
Staphylococcus spp. and Streptococcus spp
|
23
|
Staphylococcus spp. and Streptococcus spp
|
Staphylococcus spp. and Streptococcus spp
|
24
|
Staphylococcus spp. and Streptococcus spp
|
Staphylococcus spp. and Streptococcus spp
|
25
|
MSSA, Staphylococcus spp. and Streptococcus spp
|
MSSA, Staphylococcus spp. and Streptococcus spp
|
26
|
MSSA, Staphylococcus spp. and Streptococcus spp
|
MSSA, Staphylococcus spp. and Streptococcus spp
|
27
|
Staphylococcus spp. and Streptococcus spp
|
Staphylococcus spp. and Streptococcus spp
|
28
|
MSSA, Staphylococcus spp. and Streptococcus spp
|
MSSA, Staphylococcus spp. and Streptococcus spp
|
29
|
Staphylococcus spp. and Streptococcus spp
|
Staphylococcus spp. and Streptococcus spp
|
30
|
Staphylococcus spp. and Streptococcus spp
|
Staphylococcus spp. and Streptococcus spp
|