The catheters (Fig. 2) Impregnated by the EUE ( with the concentration of 25, 50, 100, 200 and 300 mg/ml), the ROE (with the concentration of 100, 200 and 300 mg/ml), the GTE (with the concentration of 50, 100, 200 and 300 mg/ml) and the ZIE (with the concentration of 50, 100 and 200, mg/ml) were tested on the nutrient agar encoutering Staphylococcus aureus and Escherichia coli (both 1× 106 CFU/ml); the zone of inhibition is shown in Fig. 3 and Table 1. The number of bacteria grown by the dilution method on the different agar nutrients (N.A, Merk US) related to the impregnated catheters and no impregnated catheters sample groups were obtained on the 1st, 3rd, 7th, 14th, and 21st days of the experiment and are illustrated by Fig. 4 and 5. The Graph Pad Prism 8.0.1 software was used to analyze and calculate and the data’s mean ±SD.
Table 1 The diameter of the inhibition zone of two plants in two strain bacteria
Type of
Extract
|
Bacterial Isolates
(1×106 cfu/ml)
|
Concentration of extract (mg/ml)
|
25
|
50
|
100
|
200
|
300
|
Clean zone diameter (mm)
|
ROE-IMC
|
S. aureus
|
-
|
-
|
23
|
23
|
24
|
EUE-IMC
|
15
|
15
|
22
|
24
|
25
|
GTE-IMC
|
-
|
-
|
-
|
-
|
-
|
ZIE-IMC
|
-
|
-
|
-
|
-
|
-
|
ROE-IMC
|
E. coli
|
-
|
-
|
22
|
24
|
32
|
EUE-IMC
|
20
|
20
|
24
|
25
|
34
|
GTE-IMC
|
-
|
-
|
-
|
-
|
-
|
ZIE-IMC
|
-
|
-
|
-
|
-
|
-
|
(-) No clean zone diameter
|
As shown in the Fig. 3, 4 and 5 and Table 2, 3, 4 and 5, after the comparison of the effects the two bacteria on the different concentrations of EUE-IMC samples during the experiment period, it was observed that the 100 mg/ml EUE-IMC sample could not have the bactericidal effect after 24 hours; however, their growth was prevented. Conversely, the tests on the 3rd, 7th, 14th, and 21st days showed that the 100 mg/ml EUE-IMC sample could extinguish the bacteria (no colonies observed on the plate). The tests also showed that the 200 and 300 mg/ml concenterated EUE-IMC samples and all the concentrations of ROE-IMC (100, 200 and 300 mg/ml) could extinguish the bacteria on the different test days. As observerd in the experiment, as the concentration of the herbal extracts went higher, their antibacterial effects increased, too. The EUE-IMC sample was more effective than the ROE-IMC sample in preventing E. Coli. compared with S. Aureus. So, EUE-IMC yielded better results, compared to that of the ROE-IMC. The figures 4 and 5 and the tables 2, 3, 4 and 5 indicates that the obtained results are significantly close to the mean but they show some dispersion. The different concentrations of EUE-IMC showed a significant antibacterial effect (p<0.0001) on the bacteria which is consistent with the results of Bhalodia et al[3] Also, the ROE-IMC samples showed the same results (P<0.0001). According to the figures 4 and 5, while being in the body, the extractions penetrated into the samples GTE-IMC (P˂0.0001) and ZIE-IMC (p˂0.0001) released and eliminated the bacteria, while the obtained results of the GTE-IMC (100 mg/ml) and GTE-IMC (200 mg/ml) were not significant. As a result, the modified IMC samples can extinguish the bacteria over time compared to the NON-IMC control samples. The number of bacteria on the control samples of catheters increased day by day during the experiment.
The green tea extract has natural aromatic polyphenol compounds such as catchiness that include hydroxyl[30]. A small amount of green tea extract was used in the impregnation step of this experiment. The hydroxyl monoterpenes of ziziphora compounds are carvacerol and thymol which were the main factor of antimicrobial activity. These ingredients are non-polar phenolic compounds which are in the ziziphora extract and they are slightly solvable in water[18]. The effectiveness of the non-phenolic compounds depends on the type of the alkyl group; the alkenyl, however, is more active than alkyl[31]. The hydroxyl groups (-OH) are active compounds having high antibacterial effects which alkenyl (-CH=CH-) increases their efficiency compared to alkyl (-C=C-)[32].
Table 2 The mean±SD of the number of colonies formed with E.coli on EUE-IMC, ROE-IMC, Control-Catheter, and Control-Bacteria in 21 days
Colonization of E.coli (CFU/ml)
|
|
|
EUE-IMC (concentration mg/ml)
|
|
ROE-IMC (concentration mg/ml)
|
Day
|
|
100
|
200
|
300
|
Control-Catheter
|
Control-Bacteria
|
|
100
|
200
|
Control-Catheter
|
Control-Bacteria
|
|
1
|
|
0.00±0.00
|
0.00±0.00
|
0.00±0.00
|
5.30±0.08
|
5.40±0.10
|
|
0.00±0.00
|
0.00±0.00
|
5.40±0.08
|
5.30±0.08
|
|
3
|
|
0.00±0.00
|
0.00±0.00
|
0.00±0.00
|
5.43±0.05
|
5.60±0.08
|
|
0.00±0.00
|
0.00±0.00
|
5.60±0.05
|
5.43±0.05
|
|
7
|
|
0.00±0.00
|
0.00±0.00
|
0.00±0.00
|
5.56±0.05
|
5.70±0.10
|
|
0.00±0.00
|
0.00±0.00
|
5.70±0.05
|
5.56±0.05
|
|
14
|
|
0.00±0.00
|
0.00±0.00
|
0.00±0.00
|
5.70±0.00
|
5.73±0.05
|
|
0.00±0.00
|
0.00±0.00
|
5.73±0.00
|
5.70±0.00
|
|
21
|
|
0.00±0.00
|
0.00±0.00
|
0.00±0.00
|
5.70±0.00
|
5.73±0.05
|
|
0.00±0.00
|
0.00±0.00
|
5.73±0.00
|
5.70±0.00
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Table 3 The mean±SD of the number of colonies formed with E.coli on GTE-IMC, ZIE-IMC, Control-Catheter, and Control-Bacteria in 21 days.
Colonization of E.coli (CFU/ml)
|
|
|
|
|
GTE-IMC (concentration mg/ml)
|
|
ZIE-IMC (concentration mg/ml)
|
Day
|
|
50
|
100
|
200
|
300
|
Control-Catheter
|
Control-Bacteria
|
|
50
|
100
|
200
|
Control-Catheter
|
Control-Bacteria
|
1
|
|
4.70±0.00
|
4.70±0.00
|
4.60±0.05
|
4.60±0.00
|
5.30±0.08
|
5.40±0.10
|
|
4.56±0.05
|
4.50±0.00
|
4.60±0.00
|
5.30±0.08
|
5.40±0.10
|
3
|
|
4.63±0.05
|
4.50±0.00
|
4.53±0.05
|
4.40±0.00
|
5.43±0.05
|
5.60±0.08
|
|
4.56±0.05
|
4.50±0.00
|
2.86±2.48
|
5.43±0.05
|
5.60±0.08
|
7
|
|
4.63±0.05
|
4.40±0.00
|
4.40±0.00
|
4.00±0.00
|
5.56±0.05
|
5.70±0.10
|
|
0.00±0.00
|
0.00±0.00
|
0.00±0.00
|
5.56±0.05
|
5.70±0.10
|
14
|
|
4.23±0.05
|
4.10±0.05
|
4.00±0.00
|
3.70±0.00
|
5.70±0.00
|
5.73±0.05
|
|
0.00±0.00
|
0.00±0.00
|
0.00±0.00
|
5.70±0.00
|
5.73±0.05
|
21
|
|
4.30±0.05
|
4.10±0.05
|
4.00±0.00
|
3.60±0.00
|
5.70±0.00
|
5.73±0.05
|
|
0.00±0.00
|
0.00±0.00
|
0.00±0.00
|
5.70±0.00
|
5.73±0.05
|
Table 4 The mean±SD the number of colonies formed with S. aureus on EUE-IMC, ROE-IMC, Control-Catheter, and Control-Bacteria in 21 days.
Colonization of S.aureus (CFU/ml)
|
|
|
IMC-EUE (concentration mg/ml)
|
|
IMC-ROE (concentration mg/ml)
|
Day
|
|
100
|
200
|
300
|
Control-Catheter
|
Control-Bacteria
|
|
100
|
200
|
Control-Catheter
|
Control-Bacteria
|
|
1
|
|
1.36±0.00
|
0.00±0.00
|
0.00±0.00
|
5.30±0.08
|
5.30±0.08
|
|
0.00±0.00
|
0.00±0.00
|
5.30±0.08
|
5.30±0.08
|
|
3
|
|
0.00±0.00
|
0.00±0.00
|
0.00±0.00
|
5.43±0.05
|
5.43±0.05
|
|
0.00±0.00
|
0.00±0.00
|
5.43±0.05
|
5.43±0.05
|
|
7
|
|
0.00±0.00
|
0.00±0.00
|
0.00±0.00
|
5.56±0.05
|
5.56±0.05
|
|
0.00±0.00
|
0.00±0.00
|
5.56±0.05
|
5.56±0.05
|
|
14
|
|
0.00±0.00
|
0.00±0.00
|
0.00±0.00
|
5.70±0.00
|
5.70±0.00
|
|
0.00±0.00
|
0.00±0.00
|
5.70±0.00
|
5.70±0.00
|
|
21
|
|
0.00±0.00
|
0.00±0.00
|
0.00±0.00
|
5.70±0.00
|
5.70±0.00
|
|
0.00±0.00
|
0.00±0.00
|
5.70±0.00
|
5.70±0.05
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Table 5 The mean±SD of the number of colonies formed with S. aureus on GTE-IMC, ZIE-IMC, Control-Catheter, and Control-Bacteria in 21 days.
Colonization of E.coli (CFU/ml)
|
|
|
GTE-IMC (concentration mg/ml)
|
ZIE-IMC (concentration mg/ml)
|
Day
|
|
50
|
100
|
200
|
300
|
Control-Catheter
|
Control-Bacteria
|
|
50
|
100
|
200
|
Control-Catheter
|
Control-Bacteria
|
1
|
|
4.83±0.05
|
4.70±0.00
|
0.00±0.00
|
0.00±0.00
|
5.30±0.08
|
5.30±0.08
|
|
4.56±0.05
|
4.50±0.00
|
4.36±0.00
|
5.30±0.08
|
5.30±0.08
|
3
|
|
4.83±0.05
|
4.70±0.00
|
0.00±0.00
|
0.00±0.00
|
5.43±0.05
|
5.43±0.05
|
|
4.56±0.05
|
4.50±0.00
|
0.00±0.00
|
5.43±0.05
|
5.43±0.05
|
7
|
|
4.80±0.83
|
4.40±0.00
|
0.00±0.00
|
0.00±0.00
|
5.56±0.05
|
5.56±0.05
|
|
0.00±0.00
|
0.00±0.00
|
0.00±0.00
|
5.56±0.05
|
5.56±0.05
|
14
|
|
4.80±0.83
|
4.36±0.05
|
0.00±0.00
|
0.00±0.00
|
5.70±0.00
|
5.70±0.00
|
|
0.00±0.00
|
0.00±0.00
|
0.00±0.00
|
5.70±0.00
|
5.70±0.00
|
21
|
|
4.40±0.00
|
4.30±0.05
|
0.00±0.00
|
0.00±0.00
|
5.70±0.00
|
5.70±0.00
|
|
0.00±0.00
|
0.00±0.00
|
0.00±0.00
|
5.70±0.00
|
5.70±0.05
|
The Bacteria in the environment are killed within 21 days, which can indicate that the release of the extract from the catheter over time could be effect on planktonic bacteria[33].(Fig 6.)
Attenuated total reflectance Fourier transform infrared (ATR-FTIR) was used to indicate the penetration of the extracts into the catheters. The ATR-FTIR analysis was used to show the amount of the extracts penetrated into the polymer as well as the chemical properties of the coated catheters (Fig 7.).
Accordingly, the ATR-FTIR spectra of the EUE-IMC, ROE-IMC, GTE-IMC, and ZIE-IMC catheters are shown in Fig. 4 and Table 6. As it can be seen, , the peak of O-H group blended in the hydroxyl compounds of EUE-IMC, GTE-IMC, ROE-IMC and ZIE-IMC catheters is located at 3335, 3346, 3350 and 3330 cm-1[34-36]. Moreover, the carboxylic group (C=O) of the different extracts related to EUE-IMC, ROE-IMC, GTE-IMC and ZIE-IMC is located at1712, 1689, 1691 and 1707 cm-1[37-39]. The decrease of peak in 2917, 2916, 1280 and 1006 cm-1 EUE-IMC spectra indicates that the EUE extract coating on the polymer surface[39]. Based on four spectra the EUE has more absorption on the surface of catheters than other extracts. The aromatic rings of ROE-IMC, GTE-IMC AMD ZIE-IMC were located on 681, 686 and1515 cm-1[40-41].(Table 6.) and (Fig 8.)
Table 6 Characteristics of ATR-FTIR absorption bands in Silicone catheter, EUE-IMC, ROE-IMC.GTE-IMC and ZIE-IMC
Wave number (cm-1)
|
Bond
|
Reference
|
1445-1455
|
Ar OH
|
[35]
|
3200-2800
|
OH stretch in a carboxylic acid
|
[42]
|
2966-2962
|
CH in CH3
|
[43]
|
1715-1705
|
RCO-OH
|
[38]
|
1645-1637
|
C = C
|
[35]
|
1695-1685
|
C = C – CO-OH
|
[37]
|
1600-1580
|
C = C
|
[36]
|
1520-1513
|
C-H (CH3)
|
[36]
|
1440-1390
|
CH3 in Si – CH3
|
[43]
|
1420-1330
|
OH
|
[35]
|
1280-1240
|
Si (CH3)3
|
[43]
|
1100-1000
|
Si-O-Si
|
[43]
|
870-856
|
Si (CH3)3
|
[43]
|
840-790
|
Si (CH3)2
|
[43]
|
700
|
Si (CH3)2
|
[43]
|
The hydrophobic surface of the silicone rubber caused the bacteria to adhere to the surface of them, leading to the formation of biofilms. Accordingly, the silicone rubber hydrophilicity should be increased to prevent the adhesion of bacteria[10]. The physicochemical interactions include electrostatic, van der Waals, and acid bases interplay. This interplay depends on the substance and the bacterial surface's physicochemical attributes: such as hydrophobicity[44].
As shown in Fig. 9, the water contact angle of the impregnated catheter by Eucalyptus, Rosemary, Green tea and Ziziphora's extracts decreased, so the hydrophilicity increased, too, due to the coating of the IMC surface by the herbal extracts. Moreover, the bacteria adhesion to the surface depends on the natural bacteria and the target surface. The hydrophilic surface is more resistant to the sticking bacteria[9].
Because of the hydrophilic groups of the rosemary ethanolic extract available at the top surface of ROE-IMC samples, the wettability effect of the surface was obtained. The rosmarinic acid has 4 (OH) groups and 1 (COOH) group, so it shows more polar and hydrophilic properties. Accordingly, in this ethanol-water solution, ROE released rosmarinic acid more[45]. The contact angle for the droplets on the surface of the silicone catheters impregnated by green tea and ziziphora extracts became less than that of the silicone Foley catheter which indicates their hydrophilic effect. The (-)-epigallocatechin gallate is the major compound in the green tea extract which is a hydrophilic natural compound[46]. The hydroxyl group position was different in the similar structure of thymol and carvacrol. In the Thymol, the hydroxyl group is close to the short-chain of isopropyl. On the other hand, in the carvacrol, the hydroxyl group is close to the methyl group. So, thymol is more non-polar than carvacrol[47].
In this research, we may have extracted carvacrol more than thymol. The surface topography of the EUE-IMC, GTE-IMC and NON-IMC catheters was examined by AFM. As it can be seen in Fig. 10, the surface modification had a detrimental effect on the surface property of catheters.
The IMC samples were soaked in the solution for 1 hour. While the NON-IMC surface (not soaked) had a smooth morphology, the roughness of the EUE-IMC and GTE-IMC surface increased a little (illustrated by Fig. 10). The increased roughness can be due to the swelling of the catheters and changes in polymer volume[48].
The amount of the bacterium Staphylococcus aureus on the NON-IMC samples was compared with those of EUE-IMC, ROE-IMC, GTE-IMC and ZIE-IMC samples. As it can be observed in Figure 10, the amount of the bacteria sticking to the surface of the NON-IMC catheter is higher compared with the bacteria adhesion to the other four impregnated catheters because of their hydrophilic surfaces. The phenolic compounds react to the protein and cell wall structures of bacteria; they may damage the cytoplasmic membranes, reduce membrane fluidity, and inhibit nucleic acid synthesis, cell wall synthesis, or energy metabolism[49].
The mechanical test results before and after soaking the catheters in the P.B.S is also demonstrated in Table 7
Table 7 The results of the mechanical test of catheters before and after 21 days in the P.B.S.
Before 21 days in P.B.S
|
Samples
|
Max force [N]
|
Max elongation [mm]
|
Yield stress [Mpa]
|
Break strain [%]
|
Young modulus [Mpa]
|
NON-IMC
|
100.94
|
2.37
|
0.300
|
651.400
|
19.625
|
EUE-IMC
|
84.28
|
2.26
|
0.277
|
618.233
|
19.313
|
ROE-IMC
|
88.20
|
2.20
|
0.207
|
523.567
|
19.419
|
GTE-IMC
|
99.12
|
2.30
|
0.202
|
485.900
|
19.390
|
ZIE-IMC
|
92.120
|
2.33
|
0.222
|
682.167
|
19.466
|
After 21 days in P.B.S
|
NON-IMC
|
165.62
|
2.07
|
0.186
|
822.500
|
20.901
|
EUE-IMC
|
162.68
|
2.33
|
0.233
|
747.976
|
18.999
|
ROE-IMC
|
163.22
|
2.03
|
0.218
|
835.400
|
19.736
|
GTE-IMC
|
166.40
|
2.45
|
0.231
|
831.767
|
18.843
|
ZIE-IMC
|
163.44
|
2.50
|
0.230
|
820.830
|
19.322
|
Based on the results obtained from the tensile test, it can be said that the break strain in silicone catheters before and after impregnation was not different. The Break strain before and after swilling catheters in the phosphate buffer is not much change. As shown in Table 7, Young modulus of silicone catheters is not change before and after impregnation; the Young module of catheters increased after the swilling in p.b.s. When a thermodynamically compatible solvent is in contact with uncross-linked amorphous glassy polymer, it will be plasticizated by the solvent diffusion[50]. When some solvent remaines in the polymer (after its swelling in the solvent), it plays the role of a plasticizer. Consequently, due to the flexibility of the film, the Young module increases. Initially, the modulus of elasticity decreases due to the opening of the coiled chain. But after the chains are opened, the elastic deformation increases as the bonds are pulled, resulting in an increase in the modulus of elasticity[51].