Antibacterial effects of lidocaine and adrenaline

Abstract

The most commonly used local anaesthetics (LAs) for postoperative analgesia and surgical anaesthesia are lidocaine and bupivacaine. Adrenaline is a vasopressor agent, which is widely used in anaesthesia for many purposes. This study aims to determine the antibacterial efficacy of lidocaine, mupirocin, adrenaline, and lidocaine + adrenaline combination. In our study, the in vitro antimicrobial effect of 1 mL of sterile saline, 20 mg/mL mupirocin, 20 mg/mL lidocaine, 1 mg/mL adrenaline, 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, classified as Group C (control), Group M (mupirocin), Group L (lidocaine), Group A (adrenaline), and Group LA (lidocaine+adrenaline), respectively. S. aureus ATCC 29213, P. aeruginosa ATCC 27853, and E. coli ATCC 25922 were cultured on 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. In terms of inhibition zone diameters, S. aureus ATCC 29213 values obtained after 12 and 24 hours of incubation were significantly different between groups (P < .001). According to inhibition zone diameters, Group M > Group LA > Group L > Group C = Group A. P. Aeruginosa ATCC 27853 values obtained after 12 and 24 hours of incubation were significantly different between groups (P < .001). According to inhibition zone diameters, Group M > Group LA > Group L = Group C = Group A. E. coli ATCC 25922 values obtained after 12 and 24 hours of incubation were significantly different between groups (P < .001). According to inhibition zone diameters, Group M > Group LA > Group L > Group C = Group A. It is known that LAs have antimicrobial effect potential in addition to their anaesthetic, analgesic, antiarrhythmic, and anti‐inflammatory effects. There are also studies showing the antimicrobial effects of vasopressor agents, which are frequently used, particularly in intensive care unit (ICUs). However, it has been observed in the present study that adrenaline alone did not have any antimicrobial effect. Adrenaline, when used in combination with lidocaine, provides a stronger and broad‐spectrum antimicrobial activity, suggesting that its combined use in proper indications will be clinically significant. Because the prevention and treatment of wound infections make a positive contribution to wound healing, the potential of antimicrobial effect of LAs can provide successful results in the prevention and treatment of ICU and wound infections. Thus, an important contribution can be made in terms of reducing the costs of antibacterial treatment and reducing morbidity.

1. INTRODUCTION

The most commonly used local anaesthetics (LAs) for postoperative analgesia and surgical anaesthesia are lidocaine and bupivacaine.1 Adrenaline is a vasopressor agent, which is widely used in anaesthesia for many purposes.2 It has been reported to have a potential antimicrobial effect when used alone or to increase the antimicrobial effect potential when used in combination with LAs.1, 2 Considering the fact that the same LAs show different antibacterial activity in different studies in the literature, the antimicrobial effects of LAs are still controversial today.1

This study aims to determine the antibacterial efficacy of lidocaine, mupirocin, adrenaline, and lidocaine + adrenaline combination.

2. MATERIALS AND METHODS

2.1. Determination of in vitro antimicrobial effect

In our study, the in vitro antimicrobial effect of 1 mL sterile saline (Izotonik, Biosel, Istanbul), 20 mg/mL mupirocin (Bactroban, Bilim, Kocaeli), 20 mg/mL lidocaine (Jetokain Sımplex, Adeka, Samsun), 1 mg/mL adrenaline (Adrenalin, Biofarma, Istanbul), and 20 mg/mL lidocaine and adrenaline (Jetokain, Adeka, Samsun), were tested against Staphylococcus aureus American‐type culture collection (ATCC) 29 213, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli ATCC 25922, classified as Group C (control), Group M (mupirocin), Group L (lidocaine), Group A (adrenaline), and Group LA (lidocaine+adrenaline), respectively. S. aureus ATCC 29213, P. aeruginosa ATCC 27853, and E. coli ATCC 25922 were cultured on 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 (Oxoid) 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 mupirocin, 20 mg/mL lidocaine, 1 mg/mL adrenaline, and 20 mg/mL lidocaine plus adrenaline were evaluated by the disc diffusion method with the determination of 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 the 12th and 24th hours. 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 (Oxoid, UK) using 96‐well microplates in accordance with the Clinical and Laboratory Standards Institute guidelines (CLSI, 2015). 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, adrenaline, and mupirocin were tested; 100 μL of mupirocin, lidocaine, adrenaline, and lidocaine plus adrenaline were added to the wells of a 96‐well microplate, each containing bacteria from overnight culture (adjusted to 1.5 × 10⁵ CFU/mL). Each microwell was assessed twice. Microwell plates were incubated at 35°C in a microplate incubator shaker. The OD600 (wavelength of 600 nm) was measured after 24 hours of incubation using an Epoch spectrophotometer (BioTek Inst. Inc., Winooski, Vermont). Wells without anaesthetic agents were used as growth control, and wells with Mueller‐Hinton broth alone served as a negative control. The percentage of viable cells was normalised to 100% for the growth control.3

To determine the minimal bactericidal concentration (MBC), each well exhibiting no visible growth (viability) after 18 hours was tested for viable organisms by sub‐culturing 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.4

2.2.1. Statistical analysis

The normality of continuous variables was investigated by Shapiro‐Wilk's test. Descriptive statistics were presented using mean and SD for normally distributed variables and median (and minimum‐maximum) for the non‐normally distributed variables. Non‐parametric statistical methods were used for values with skewed distribution. For comparison of two non‐normally distributed independent groups, the Mann‐Whitney U test was used, or for the comparison of two non‐normally distributed dependent groups, the Wilcoxon test was used. For the comparison of more than two non‐normally distributed independent groups, the Kruskall‐Wallis test was used. Statistical analysis was performed using the MedCalc Statistical Software version 12.7.7 (MedCalc Software bvba, Ostend, Belgium; http://www.medcalc.org; 2013).

3. RESULTS

Distribution of inhibition zone diameters in groups against bacteria in Mueller‐Hinton plates (mm) is presented in Table 1.

Table 1.

Distribution of inhibition zone diameters in groups against bacteria in Mueller‐Hinton plates (mm)

	Group C (control)	Group M (mupirocin)	Group L (lidocaine)	Group A (adrenaline)	Group LA (lidocaine+adrenaline)
Staphylococcus aureus ATCC 29213
12th hour	0.00	31.60	11.60	0.00	13.60
24th hour	0.00	32.40	12.00	0.00	14.80
Pseudomonas aeruginosa ATCC 27853
12th hour	0.00	11.60	0.00	0.00	4.00
24th hour	0.00	12.40	0.00	0.00	4.40
Escherichia coli ATCC 25922
12th hour	0.00	22.20	15.40	0.00	18.40
24th hour	0.00	22.80	15.60	0.00	19.00

In terms of inhibition zone diameters, S. aureus ATCC 29213 values obtained after 12 and 24 hours of incubation were significantly different between groups (P < .001). According to inhibition zone diameters, Group M > Group LA > Group L > Group C = Group A.

P. aeruginosa ATCC 27853 values obtained after 12 and 24 hours of incubation were significantly different between groups (P < .001). According to inhibition zone diameters, Group M > Group LA > Group L = Group C = Group A.

E. coli ATCC 25922 values obtained after 12 and 24 hours of incubation were significantly different between groups (P < .001). According to inhibition zone diameters, Group M > Group LA > Group L > Group C = Group A.

Within all groups, there was a statistically significant difference only in Group M in terms of inhibition zone diameter for S. aureus ATCC 29213 measured after 24 hours of incubation compared with those measured after 12 hours of incubation ( P = .046). In the other groups, no statistically significant difference was observed in terms of S. aureus ATCC 29213, P. aeruginosa ATCC 27853, and E. coli ATCC 25922 values measured after 24 hours of incubation compared with those measured after 12 hours of incubation (P > .05). The comparison of inhibition zone diameters between groups is presented in Table 2.

Table 2.

Comparison of inhibition zone diameters between groups

	Group C (control)	Group M (mupirocin)	Group L (lidocaine)	Group A (adrenaline)	Group LA (lidocaine + adrenaline)
	Mean + SD Med. (Min‐Max)	Mean + SD Med. (Min.‐Max)	Mean + SD Med. (Min.‐Max)	Mean + SD Med. (Min.‐Max)	Mean+SD Med. (Min.‐Max)	P *
Staphylococcus aureus 12th hour	0 (constant)	31.6 ± 0.532 (31‐32)	11.6 ± 0.512 (11‐12)	0 (constant)	13.6 ± 1.513 (12‐16)	<.001
S. aureus 24th hour	0 (constant)	32.4 ± 0.933 (31‐33)	12 ± 0.712 (11‐13)	0 (constant)	14.8 ± 0.815 (14‐16)	<.001
P	1,000	0.046	0.157	1,000	0,059
Pseudomonas aeruginosa 12th hour	0 (constant)	11.6 ± 0.911 (11‐13)	0 (constant)	0 (constant)	4 ± 0.74 (3‐5)	<.001
P. Aeruginosa 24th hour	0 (constant)	12.4 ± 0.913 (11‐13)	0 (constant)	0 (constant)	4.4 ± 0.54 (4‐5)	<.001
P	1,000	0.102	1,000	1,000	0.157
Escherichia coli 12th hour	0 (constant)	22.2 ± 0.822 (21‐23)	15.4 ± 1.115 (14‐17)	0 (constant)	18.4 ± 0.518 (18‐19)	<.001
E. coli 24th hour	0 (constant)	22.8 ± 0.423 (22‐23)	15.6 ± 1.116 (14‐17)	0 (constant)	19 ± 0.719 (18‐20)	<.001
P	1.000	0.180	0.317	1.000	0.083

Note: All statistically significant values are provided in bold.

^* Kruskal‐Wallis test, Wilcoxon test.

When the MIC values were examined using the microdilution method, adrenaline was found to be effective on S. aureus at 20 mg/mL. However, it did not affect E. coli and P. aeruginosa. Distribution of MIC values and the effect of different concentrations in groups against S. aureus, P. aeruginosa, and E. coli strains are shown in Table 3.

Table 3.

Distribution of minimum inhibitory concentration (MIC) values and the effect of different concentrations in groups against Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli strains

Mupirocin (mg/mL)	0.078	0.156	0.313	0.625	1.25	2.50	5.00	10.00	20.00
S. aureus ATCC 29213	+	+a	+	+	+	+	+	+	−a
P. aeruginosa ATCC 27853	+	+	+	+	+	+	+	+	+
E. coli ATCC 25922	+	+	+	+	+	+	+	+	+
Lidocaine (mg/mL)	0.078	0.156	0.313	0.625	1.25	2.50	5.00	10.00	20.00
S. aureus ATCC 29213	+	+	+	+	+	+	+	+	−a
P. aeruginosa ATCC 27853	+	+	+	+	+	+	+	+	+
E. coli ATCC 25922	+	+	+	+	+	+	−a	−	−
Lidocaine + adrenaline (mg/mL)	0.078	0.156	0.313	0.625	1.25	2.50	5.00	10.00	20.00
S. aureus ATCC 29213	+	+	+	+	+	+	+	−a	−
P. aeruginosa ATCC 27853	+	+	+	+	+	+	+	+	−a
E. coli ATCC 25922	+	+	+	+	+	−a	−	−	−
Adrenaline (mg/mL)	0.078	0.156	0.313	0.625	1.25	2.50	5.00	10.00	20.00
S. aureus ATCC 29213	+	+	+	+	+	+	+	+	−a
P. aeruginosa ATCC 27853	+	+	+	+	+	+	+	+	+
E. coli ATCC 25922	+	+	+	+	+	+	+	+	+

^aMIC values.

MBC values were not detected for anaesthetic agents in any of the bacterial strains at the concentrations studied.

4. DISCUSSION

Although the antimicrobial mechanism of the action of LAs is not clear, their broad spectrum of antibacterial activity has been reported in recent studies.1, 5 Antimicrobial efficacy demonstrated in the studies on LAs and vasopressor agents in the literature can greatly contribute to the prevention of wound and intensive care infections.1, 2 These results increase the interest in and need for studies on LAs and vasopressor agents.

During local and regional anaesthesia application, infection may develop because of a patient's skin flora or because of ear, nose, and throat flora of the anaesthetist. As a result, postoperative wound infections may negatively affect wound healing and increase both the cost of treatment and morbidity.1, 6 Nosocomial infections are another factor that increases morbidity and mortality rates. In such infections, microbial contamination occurs because of various sources and invasive procedures. Invasive procedures are frequently performed in intensive care unit (ICUs), leading to an increase in the risk of microbial contamination.2, 7 Vasoactive agents such as adrenaline are commonly used in ICUs. Microbial contamination may develop during the preparation and infusion of these drugs.2

Besides their anaesthetic, analgesic, antiarrhythmic, and anti‐inflammatory effects, LAs are also known to have antimicrobial effects.8 In our study, lidocaine used as a LAs and antiarrhythmic showed an antimicrobial effect on S. aureus and E. coli; however, it had no antimicrobial effect against P. aeruginosa. When lidocaine was combined with adrenaline, an increase was observed in its antimicrobial effect against S. aureus and E. coli, and it showed an antimicrobial effect against P. aeruginosa. However, it was concluded that adrenaline alone showed no antibacterial activity but increased the antibacterial potency and spectrum of lidocaine. The results of this study were found to be compatible with the study by Kesici et al.1 There are a limited number of studies on the antibacterial efficacy of adrenaline, which is one of the vasopressor agents. In the literature, adrenaline and other vasopressor agents have been shown to have antimicrobial effects.2, 9 However, our study showed that adrenaline alone did not have any antimicrobial effect, unlike the results of other studies in the literature. In line with the results of the present study, making use of the antimicrobial potentials of LAs can significantly contribute to the prevention and treatment of ICU and wound infections.

5. CONCLUSION

It is known that LAs have antimicrobial effect potential in addition to their anaesthetic, analgesic, antiarrhythmic, and anti‐inflammatory effects. There are also studies showing the antimicrobial effects of vasopressor agents, which are frequently used, particularly in ICUs. However, it has been observed in the present study that adrenaline alone did not have any antimicrobial effect. Adrenaline, when used in combination with lidocaine, provides a stronger and broad‐spectrum antimicrobial activity, suggesting that its combined use in proper indications will be clinically significant. Because the prevention and treatment of wound infections make a positive contribution to wound healing, the potential of antimicrobial effect of LAs can provide successful results in the prevention and treatment of ICU and wound infections. Thus, an important contribution can be made in terms of reducing the costs of antibacterial treatment and reducing morbidity.

CONFLICTS OF INTEREST

All the authors have no conflicts of interest and have received no financial support.

AUTHOR CONTRIBUTIONS

All authors contributed in investigation, methodology, validation, writing, reviewing and editing the original manuscript.

Kesici S, Demirci M, Kesici U. Antibacterial effects of lidocaine and adrenaline. Int Wound J. 2019;16:1190–1194. 10.1111/iwj.13182