Abstract
After the introduction of cocaine to the medical practice, local anaesthetics (LA) became essential in pain control. LA infiltration along the incision may be used to provide surgical anaesthesia or postoperative analgesia. This study aimed to compare the antimicrobial effects of the topical antimicrobial agent mupirocine with those of the LA lidocaine and the combination of lidocaine and adrenalin. In our study, the in vitro antimicrobial effects of 1 mL sterile saline, 20 mg/mL mupirocine, 20 mg/mL Lidocaine, and 20 mg/mL Lidocaine and Adrenaline were tested against Staphylococcus aureus American type culture collection (ATCC) 29213, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli ATCC 25922 as Group C (Control), Group M (Mupirocine), Group L (Lidocaine), and Group LA (Lidocaine + adrenaline), respectively. S aureus ATCC 29213, P aeruginosa ATCC 27853, and E coli ATCC 25922 were cultured onto Mueller–Hinton agar (Oxoid, UK) plates for 18 to 24 hours at 37°C. Colonies from these plates were suspended in sterile saline and a 0.5 McFarland turbidity standard suspension (corresponding to 1.5 × 108 CFU/mL) of each isolate was prepared. S Aureus ATCC 29213 inhibition zone diameter values of Group M, Group LA, and Group L were significantly higher compared with the group C (P ˂ 0.05). P aeruginosa ATCC 27853 inhibition zone diameter values of Group M and Group LA were significantly higher compared with the group C (P ˂ 0.05). E coli ATCC 25922 inhibition zone diameter values of Group M, Group LA, and Group L were significantly higher compared to the group C (P ˂ 0.05). LA infiltration along the incision may be used to provide surgical anaesthesia or postoperative analgesia. Considering that LAs show antimicrobial effects besides their analgesic effects, they may contribute to preventing the development and reducing the rate of surgical infections, decreasing the requirement to administer antibiotics. However, caution should be exercised not to antagonise the effective treatment of surgical infections, remembering that controversy on the antimicrobial effects of LAs remains in the literature. Therefore, further comprehensive studies with larger patient populations are warranted to demonstrate the antimicrobial effects of LAs.
Keywords: adrenaline, antimicrobial, lidocaine, local anaesthetics, mupirocine
1. INTRODUCTION
After the introduction of cocaine to the medical practice, local anaesthetics (LAs) became essential in pain control.1 LA infiltration along the incision may be used to provide surgical anaesthesia or postoperative analgesia.2 The most frequently used LAs for these purposes are lidocaine and bupivacaine.3 Although their antimicrobial mechanism of action has not been completely clarified yet, studies in recent decades have demonstrated that LAs have a wide spectrum of antimicrobial effects.1, 4 It is reported that LAs inhibit the growth of live bacteria, reduce the membrane‐dependent enzymatic activity of living cells, cause lysis in protoplasts, change the membrane permeability, and lead to ultrastructural alterations; however, these findings have not been completely established.5
This study aimed to compare the antimicrobial effects of the topical antimicrobial agent mupirocine with those of the LA lidocaine and the combination of lidocaine and adrenalin.
2. METHOD
2.1. Determination of the in vitro antimicrobial effect
In our study, the in vitro antimicrobial effects of 1 mL sterile saline, 20 mg/mL mupirocine, 20 mg/mL Lidocaine, and 20 mg/mL Lidocaine and Adrenaline were tested against Staphylococcus aureus American type culture collection (ATCC) 29 213, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli ATCC 25922 as Group C (Control) (Biofleks, Osel, Istanbul, Turkey), Group M (Mupirocine) (Bactroban %2, Glaxo Smith Kline, Istanbul, Turkey), Group L (Lidocaine) (Jetokain simplex, Adeka, Istanbul, Turkey), and Group LA (Lidocaine+adrenaline) (Jetokain, Adeka, Istanbul, Turkey), respectively. S aureus ATCC 29213, P aeruginosa ATCC 27853, and E coli ATCC 25922 were cultured onto Mueller–Hinton agar (Oxoid, UK) plates for 18 to 24 hours at 37°C. Colonies from these plates were suspended in sterile saline and a 0.5 McFarland turbidity standard suspension (corresponding to 1.5 × 108 CFU/mL) of each isolate was prepared. Each labelled Mueller–Hinton agar plate was uniformly seeded with a test organism by means of sterile swab rolled in the suspension and streaked on the plate surface. in vitro antimicrobial activity of 1 mL sterile saline, 20 mg/mL mupirocine, 20 mg/mL Lidocaine, and 20 mg/mL Lidocaine plus Adrenaline was evaluated by the disc diffusion method by determining the inhibition zones. Each sterile disc (Merck, Germany) was impregnated with 20 μL of the anaesthetics (corresponding with 20 mg/mL) and was placed and incubated on Mueller–Hinton Agar for 24 hours at 37°C. The zone of inhibition was measured for 12th and 24th hour. Each experiment was performed five times.
2.2. Determination of minimum inhibitory concentration and minimal bactericidal concentration
The broth microdilution method was used to determine the minimum inhibitory concentration (MIC) values on Mueller–Hinton broth using 96 well microplates in accordance with the Clinical and Laboratory Standards Institute guidelines.6 In this study, the anaesthetic agents were prepared by twofold serial dilution in Mueller–Hinton broth and 20, 10, 5, 2.5, 1.25, 0.625, 0.312, 0.156, and 0.078 mg/mL concentrations of anaesthetic agents of lidocaine, lidocaine plus adrenaline, and mupirocine were tested. A volume of 100 μL of mupirocine, lidocaine, and lidocaine plus adrenaline was added to the 96 well microplates each containing bacteria obtained from overnight culture (adjusted to 1.5 × 105 CFU/mL). Each microwells were performed two times. Microwell plates were incubated at 35°C in a microplate incubator shaker. The OD600 (wavelength of 600 nm) was measured after 24 hour incubation by using Epoch spectrophotometer (BioTek Inst. Inc. Vermont, USA). Wells without anaesthetic agents were used as growth control and wells with Mueller–Hinton broth alone served as negative control. The percentage of viable cells was normalised to 100% for the growth control.7
To determine the minimal bactericidal concentration (MBC), each well exhibiting no visible growth (viability) after 18 hours was tested for viable organisms by subculturing 10 μL samples of each well onto Mueller–Hinton agar. The plates were incubated at 35°C to observe the growth of any colony after 24 hours.6
2.3. Statistical methods
Mean, standard deviation, median, minimum, maximum value frequency, and percentage were used for descriptive statistics. The distribution of variables was checked with the Kolmogorov–Simirnov test. Kruskal–Wallis and Mann–Whitney U tests were used for the comparison of quantitative data. The Wilcoxon test was used for the repeated measurement analysis. SPSS 22.0 was used for statistical analyses.
3. RESULTS
With regard to the inhibition zone diameters; the 12‐hour and 24‐hour S aureus ATCC 29213 values were significantly higher in group M compared with the groups C, L, and LA (P ˂ 0.05). The 12‐hour and 24‐hour S aureus ATCC 29213 values in the group LA were significantly higher compared with the groups C and L (P ˂ 0.05). The 12‐hour and 24‐hour S aureus ATCC 29213 values in the group L were significantly higher compared with the group C (P ˂ .05) (Group M > Group LA > Group L > Group C).
In the group M, the 12‐hour and 24‐hour P aeruginosa ATCC 27853 values were found to be significantly higher than those found in the groups C, L, and LA (P ˂ .05). The 12‐hour and 24‐hour P aeruginosa ATCC 27853 values were significantly higher in the group LA compared with the groups C and L (P ˂ .05). The 12‐hour and 24‐hour P aeruginosa ATCC 27853 values in the group L were not significantly different compared with those found in the group C (P ˃ .05) (Group M > Group LA > Group L = Group C).
In the group M, the 12‐hour and 24‐hour E coli ATCC 25922 values were significantly higher compared with the groups C, L, and LA (P ˂ 0.05). The 12‐hour and 24‐hour E coli ATCC 25922 values in the group LA were significantly higher compared with the groups C and L (P ˂ 0.05). The 12‐hour and 24‐hour E coli ATCC 25922 values in the group L were significantly higher compared with the group C (P ˂ .05) (Group M > Group LA > Group L > Group C).
The 12‐hour and 24‐hour inhibition zone diameters of S aureus ATCC 29213, P aeruginosa ATCC 27853, and E coli ATCC 25922 were not significantly different within each group (P ˃ 0.05).
Distribution of inhibition zone diameters for anaesthetic drugs against bacteria on Mueller–Hinton plates (mm) is shown in Table 1.
Table 1.
Distribution of inhibition zone diameters for anaesthetic drugs against bacteria on Mueller–Hinton plates (mm)
| Group C (control) | Group M (mupirocine) | Group L (lidocaine) | Group LA (lidocaine + adrenaline) | P | |||||
|---|---|---|---|---|---|---|---|---|---|
| Mean ± SD | Med | Mean ± SD | Med | Mean ± SD | Med | Mean ± SD | Med | ||
| S. aureus ATCC 29213 | |||||||||
| 12 h | 0.0 ± 0.0 | 0.0a , b , c | 31.8 ± 0.4 | 32.0 | 10.8 ± 0.8 | 11a , c | 13.8 ± 0.8 | 14a | .000d |
| 24 h | 0.0 ± 0.0 | 0.0a , b , c | 31.0 ± 0.7 | 31.0 | 10.4 ± 0.5 | 10a , c | 13.2 ± 0.8 | 13a | .000d |
| Intra‐group difference p | 1.00e | 0.102e | 0.157e | 0.180e | |||||
| P. aeruginosa ATCC 27853 | |||||||||
| 12 h | 0.0 ± 0.0 | 0.0a , c | 10.4 ± 0.5 | 10.0 | 0.0 ± 0.0 | 0.0a , c | 3.8 ± 0.8 | 4a | .000d |
| 24 h | 0.0 ± 0.0 | 0.0a , c | 9.8 ± 0.4 | 10.0 | 0.0 ± 0.0 | 0.0a , c | 3.4 ± 0.5 | 3a | .000d |
| Intra‐group difference p | 1.00e | 0.083e | 1.000e | 0.157e | |||||
| E coli ATCC 25922 | |||||||||
| 12 h | 0.0 ± 0.0 | 0.0a , b , c | 21.6 ± 0.5 | 22.0 | 15.2 ± 0.8 | 15a , c | 18.2 ± 0.8 | 18a | .000d |
| 24 h | 0.0 ± 0.0 | 0.0a , b , c | 20.8 ± 0.8 | 21.0 | 14.4 ± 0.9 | 15a , c | 17.8 ± 0.8 | 18a | .000d |
| Intra‐group difference p | 1.00e | 0.102e | 0.102e | 0.157e | |||||
Difference with Group M (mupirocine) P < .05.
Difference with Group M (mupirocine) P < .05.
Difference with Group LA (lidocaine + adrenaline) P < .05.
Kruskal–Wallis (Mann–Whitney U test).
Wilcoxon test.
No MIC values were found for mupirocine in P aeruginosa and E coli strains, but a MIC value of 20 mg/mL for mupirocine was found in S aureus strains. MIC values of 20 and 5 mg/mL for Lidocaine were found in S aureus and E coli strains, respectively. On the other hand, Lidocaine plus adrenalin reduced these MIC values (10 mg/mL) and (2.5 mg/mL) in S aureus and E coli respectively. No MIC value for Lidocaine was detected in P aeruginosa strain but a MIC value of 20 mg/mL was found for Lidocaine plus adrenaline in P aeruginosa strain. Distribution of MIC values and the effect of different concentrations of anaesthetic agents against S aureus, P aeruginosa and E coli strains are shown in Table 2.
Table 2.
Distribution of minimum inhibitory concentration (MIC) values and the effect of different concentrations of anaesthetic agents against S aureus, P aeruginosa, and E coli strains
| Mupirocine (mg/mL) | 0.078 | 0.156 | 0.312 | 0.625 | 1.25 | 2.5 | 5 | 10 | 20 |
|---|---|---|---|---|---|---|---|---|---|
| S aureus ATCC 29213 | + | + | + | + | + | + | + | + | −a |
| P aeruginosa ATCC 27853 | + | + | + | + | + | + | + | + | + |
| E coli ATCC 25922 | + | + | + | + | + | + | + | + | + |
| Lidocaine (mg/mL) | 0.078 | 0.156 | 0.312 | 0.625 | 1.25 | 2.5 | 5 | 10 | 20 |
| S aureus ATCC 29213 | + | + | + | + | + | + | + | + | −a |
| P aeruginosa ATCC 27853 | + | + | + | + | + | + | + | + | + |
| E coli ATCC 25922 | + | + | + | + | + | + | −a | − | − |
| Lidocaine plus adrenaline (mg/mL) | 0.078 | 0.156 | 0.312 | 0.625 | 1.25 | 2.5 | 5 | 10 | 20 |
| S aureus ATCC 29213 | + | + | + | + | + | + | + | −a | − |
| P aeruginosa ATCC 27853 | + | + | + | + | + | + | + | + | −a |
| E coli ATCC 25922 | + | + | + | + | + | −a | − | − | − |
MIC values.
MBC values were not detected for anaesthetic agents in any of the bacterial strains at the concentrations studied.
4. DISCUSSION
Local and regional anaesthesia techniques are commonly used intraoperatively or in the postoperative period to control pain. Infections have been reported as complications after the administration of these techniques in patients and the development of these infections has been associated with the cutaneous flora of patients and the ear‐nose‐throat flora of anaesthetists.8 The antibacterial effects of LAs were first reported in 1909. After the demonstration of the toxic effects of tetracaine on pseudomonas, further studies were conducted on the antimicrobial effects of LAs.9, 10
The studies in the literature indicate that the debate on the antimicrobial effects of LAs remains. Although several different studies reported the various spectra of antimicrobial effects with the same LAs, further studies are required to investigate this issue.4, 11 In this study, we aimed to demonstrate the antimicrobial efficacy of a commonly used LA, lidocaine, and the combination of lidocaine and adrenalin in comparison to an antibacterial agent, mupirocine. A study by Srisatjaluk et al4 reported that lidocaine demonstrated antimicrobial effects on E coli; however, these effects were not reported for S aureus. A study by Aydin et al8 reported that lidocaine showed antimicrobial effects on S aureus, E coli, and P aeruginosa. In the present study, we determined the antimicrobial effects of lidocaine on E coli and S aureus but not on P aeruginosa. These various antimicrobial effects reported by several different studies in the literature are associated with the dose of LA. Therefore, we are of the opinion that further large‐scale studies are required to determine the effective antimicrobial doses of these agents. In this present study, MIC values of lidocaine for S aureus and E coli were found to be 20 and 5 mg. respectively; and these values were found to decrease to 10 and 2.5 mg when lidocaine and adrenaline were administered in combination. A study by Sedef et al12 determined that mupirocine demonstrated antimicrobial effects on E coli, P aeruginosa, and S aureus but these effects were not demonstrated on P aeruginosa. Our present study results are consistent with these previously reported results in the latter study. However, we found that lidocaine showed antimicrobial effects on P aeruginosa when it was administered in combination with adrenalin.
Antimicrobial effects of LAs have been demonstrated by in vitro and experimental studies in the literature. In experimental studies; Lu et al,13 Stratford et al,14 and Kose et al15 reported that lidocaine showed antimicrobial effects on S aureus.
Ephedrine and adrenalin are vasopressor agents commonly used for several patient management purposes in the anaesthesia practice.16, 17 A study by Tulgar et al11 determined the antimicrobial effects of ephedrine on S aureus when used alone or in combination with propofol but it did not show these effects on E coli and P aeruginosa. In our study, we determined that lidocaine in combination with adrenalin showed antimicrobial effects on S aureus, E coli, and P aeruginosa; and these antimicrobial effects were found out to be statistically significant compared with the group, in which lidocaine was administered alone. These results and the results from the previous studies suggest that LA may be administered in combination with vasopressor agents, such as ephedrine or adrenaline, in order to show antimicrobial efficacy.
As it has been well‐established, surgical wound infections are the most common type of complications in the postoperative period.13 Surgical wounds are classified clean, clean‐contaminated, contaminated, infected, or dirty, and the surgical site infection (SSI) rate was 2.1%, 3.3%, 6.4%, and 7.1%, respectively.18 Wound infections adversely affect wound healing.19This situation results in longer hospital stays, higher treatment costs, and increased morbidity rates.13 Therefore, beneficial antimicrobial effects of LAs may significantly contribute to the prevention of surgical infections, wound healing, and reduction of treatment costs. In our opinion; choosing LAs with high antimicrobial activity may be very important for SSI and wound healing for all surgical wounds. A study by Adler et al20 reported that administration of antibiotics can be questioned when LA infiltration was performed. These findings warrant that large‐scale clinical and experimental studies to be carried out in the future to demonstrate the antimicrobial effects of LAs and to determine their efficacious antimicrobial doses. In the present study, we determined the MIC values of the LA agent, lidocaine, demonstrating its antimicrobial efficacy at lower doses when used in combination with adrenalin.
5. CONCLUSION
LA infiltration along the incision may be used to provide surgical anaesthesia or postoperative analgesia. Considering that LAs show antimicrobial effects besides their analgesic effects, they may contribute to preventing the development and reducing the rate of surgical infections, decreasing the requirement to administer antibiotics. However, caution should be exercised not to antagonise the effective treatment of surgical infections, remembering that controversy on the antimicrobial effects of LAs remains in the literature. Therefore, further comprehensive studies with larger patient populations are warranted to demonstrate the antimicrobial effects of LAs.
AUTHOR CONTRIBUTIONS
Investigation was conducted by Ugur Kesici and Sevgi Kesici, methodology was prepared by Ugur Kesici and Mehmet Demirci, Ugur Kesici and Sevgi Kesici validated the study, original draft was written by Ugur Kesici, and Ugur Kesici, Sevgi Kesici, and Mehmet Demirci wrote, reviewed, and edited the article.
CONFLICT OF INTEREST
The authors have no conflict of interest and have no financial support.
Kesici U, Demirci M, Kesici S. Antimicrobial effects of local anaesthetics. Int Wound J. 2019;16:1029–1033. 10.1111/iwj.13153
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