Bacterial inhibition efficiency of prilocaine and bupivacaine

Abstract

This study aimed to demonstrate the antibacterial effects of bupivacaine and prilocaine on Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. In our study, the in vitro antimicrobial effects of 20 mg/mL prilocaine and 5 mg/mL bupivacaine were tested against a S. aureus American‐type culture collection (ATCC) 29213, P. aeruginosa ATCC 27853, and E. coli ATCC 25922, divided into Group P (Prilocaine) and Group B (Bupivacaine), respectively. S. aureus ATCC 29213, P. aeruginosa ATCC 27853, and E. coli ATCC 25922 were cultured on Mueller Hinton agar (Oxoid, Basingstoke, UK) plates for 18 to 24 hours at 37°C. In terms of inhibition zone diameters, inhibition of S. aureus ATCC 29213 was observed in both groups at the 12th and 24th hours. The 12th‐ and 24th‐hour S. aureus ATCC 29213 value was significantly higher in Group P compared with Group B (P = .008). At the 12th and 24th hours, inhibition of E. coli ATCC 25922 was observed in both groups. The 12th‐ and 24th‐hour E. coli ATCC 25922 value was significantly higher in Group P compared with Group B (P = .008). In our study, it was seen that prilocaine and bupivacaine had an antimicrobial effect on S. aureus and E. coli. In the comparison between these two local anesthetics (LAs), this effect was found to be significantly higher in prilocaine than bupivacaine. Therefore, we are of the opinion that antimicrobial effect potentials should also be taken into account in the selection of an LA agent in order to prevent the complications of an infection that might develop during LA infiltration and might lead to serious morbidity.

1. INTRODUCTION

Local and regional anaesthesia practices are frequently used during surgery or postoperatively for pain management.1 One of the complications observed during these practices is localised infections.2 Raedler et al3 demonstrated the contamination of epidural and spinal needles after lumbar puncture. In various studies in the literature, different findings on the antimicrobial effects of bupivacaine and prilocaine have been presented.1, 4, 5 Therefore, further studies are required to establish the effects of these local anaesthetics.

This study aimed to demonstrate the antibacterial effects of bupivacaine and prilocaine on Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa.

2. METHODS

2.1. Determination of in vitro antimicrobial effect

In our study, the in vitro antimicrobial effects of 20 mg/mL prilocaine and 5 mg/mL bupivacaine were tested against a S. aureus American‐type culture collection (ATCC) 29 213, P. aeruginosa ATCC 27853, and E. coli ATCC 25922, divided into Group P (prilocaine) and Group B (bupivacaine), respectively. S. aureus ATCC 29213, P. aeruginosa ATCC 27853, and E. coli ATCC 25922 were cultured on Mueller Hinton agar (Oxoid, Basingstoke, 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, UK) plate was uniformly seeded using a test organism by means of a sterile swab rolled in the suspension and streaked on the plate surface. In vitro antimicrobial activity of the 20 mg/mL prilocaine and 5 mg/mL bupivacaine was evaluated using the disc diffusion method with the determination of inhibition zones. Each sterile disc (Merck, Darmstadt, 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 prilocaine and bupivacaine were tested; 100 μL of prilocaine and bupivacaine were added to the wells of 96‐well microplates 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. Vermont, USA). 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.6

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.7

2.3. Statistical analysis

The normality of continuous variables was investigated using Shapiro–Wilk's test. Descriptive statistics were presented using mean and standard deviation 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 the 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 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

The distribution of inhibition zone diameters in groups against bacteria in the Mueller–Hinton plates (mm) is presented in Table 1.

Table 1.

Distribution of inhibition zone diameters for anaesthetic drugs against bacteria on Mueller–Hinton plates (mm)

	Group P (Prilocaine)	Group B (Bupivacaine)
S. aureus ATCC 29213
12th h	11.20	3.20
24th h	12.40	3.60
P. aeruginosa ATCC 27853
12th h	0.00	0.00
24th h	0.00	0.00
E. coli ATCC 25922
12th h	14.00	5.80
24th h	14.80	6.20

In terms of inhibition zone diameters, the inhibition of S. aureus ATCC 29213 was observed in both groups at the 12th and 24th hours. The 12th‐ and 24th hour S. aureus ATCC 29213 value was significantly higher in Group P compared with Group B (P = .008) (Group P > Group B).

No inhibition of P. aeruginosa ATCC 27853 was observed in either group at the 12th and 24th hours.

At 12th and 24th hours, the inhibition of E. coli ATCC 25922 was observed in both groups. The 12th‐ and 24th‐hour E. coli ATCC 25922 value was significantly higher in Group P compared with Group B (P = .008) (Group P > Group B).

Inhibition zone diameters of all groups varied in themselves; Group B demonstrated a significant difference between the 12th and 24th hours in terms of S. aureus ATCC 29213 (P = .041), and Group P demonstrated a significant difference between the 12th and 24th hours in terms of P. aeruginosa ATCC 27853 (P = .046). The comparison of inhibition zone diameters between groups is presented in Table 2.

Table 2.

Comparison of inhibition zone diameters between groups

	Group P (Prilocaine)	Group B (Bupivacaine)
	Mean ± SD Med. (Min.–Max.)	Mean ± SD Med. (Min.–Max.)	P a
S. aureus 12th h	11.2 ± 0.8 11 (10–12)	3.2 ± 0.4 3 (3–4)	0.008
S. aureus 24th h	12.4 ± 1,1 12 (11–14)	3,6 ± 0.9 3 (3–5)	0.008
P	0.063	0.041
P. aeruginosa 12th h	0 (constant)	0 (constant)	—
P. aeruginosa 24th h	0 (constant)	0 (constant)	—
P	—	—
E. coli 12th h	14 ± 0.7 14 (13–15)	5,8 ± 0.8 6(5‐7)	0.008
E. coli 24th h	14.8 ± 0.8 15 (14–16)	6,2 ± 0,8 6 (5–7)	0.008
P	0.046	0.157

^aMann–Whitney U test, Wilcoxon test.

Distribution of MIC values and the effect of different concentrations of anaesthetic agents against S. aureus, P. aeruginosa, and E. coli strains were shown in Table 3.

Table 3.

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

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

^aMIC values.

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

4. DISCUSSION

There are in vivo and in vitro studies in the literature which indicate that LAs have a broad potential of antimicrobial effect besides their capability in pain management.8 However, discussions on the antimicrobial effect potential of the LAs are still relevant as the results of these studies differ. In addition, it is understood that further studies are required in order to determine their effect spectrum, effect potentials, and mechanism of action. The fact that antimicrobial effects vary across the studies is related to the dosage of the LA.2 Therefore, presenting the MIC values in the studies would contribute to the utilisation of antimicrobial effect potential in the clinical practice. In our study, as no antimicrobial effect of bupivacaine and prilocaine on P. aeruginosa was observed, the MIC value could not be determined. However, because the antimicrobial effect potential of prilocaine on S. aureus and E. coli was more prominent compared with bupivacaine, the prilocaine MIC value was observed to be lower. In the study by Rosenberg et al,4 it was reported that bupivacaine did not have an antimicrobial effect on P. aeruginosa. In addition, in the study by Gocmen et al,9 it was reported that the prilocaine/lidocaine combination did not have antimicrobial effect on P. aeruginosa. Consonant to the results of this study, it was detected in our study that neither bupivacaine nor prilocaine had an antimicrobial effect on P. aeruginosa. However, in the study by Aydin et al,1 it was reported that bupivacaine had an antimicrobial effect on P. aeruginosa; yet, the MIC values were not stated. Also in the same study, it was reported that prilocaine had a stronger antibacterial effect compared with bupivacaine. In our study, it was found that prilocaine had a higher antibacterial effect potential on S. aureus and E. coli compared with bupivacaine.

Local and regional anaesthesia procedures are widely used for the purposes of surgical anaesthesia or postoperative analgesia. Bupivacaine and prilocaine are among the local anaesthetics used for this purpose. Infection complications might occur during LA infiltration. Wound infections adversely affect wound healing. This leads to increased treatment costs, morbidity, and mortality.2, 9, 10 Bupivacaine is also used as an epidural infusion for postoperative pain management and labour analgesia.8 Microorganisms most sensitive to bupivacaine are Staphylococcus, enterobacter, E. coli, and Proteus.11 In our study, it was found that bupivacaine had an antimicrobial effect on S. aureus and E. coli. One of the complications encountered during epidural catheterisation is epidural abscess. Even though epidural abscess is a rare complication, it might lead to a serious morbidity that could result in paraplegia. Therefore, it is of paramount importance to adhere to the rules of asepsis during epidural catheterisation; that said, the antibacterial activity of the used LA agent might contribute to minimising the risk of complications.12 In our study, it was observed that bupivacaine had an antibacterial effect on S. aureus and E. coli, and the MIC value for E. coli was 20 mg/mL.

When the studies in the literature are analysed, it can be seen that LAs have a wide antimicrobial effect spectrum, yet P. aeruginosa is resistant to LAs.2, 8, 9 In line with the literature, it was detected in our study that prilocaine and bupivacaine did not have an antimicrobial effect on P. aeruginosa. However, in the literature, some local anaesthetics are seen to have an antimicrobial effect on P. aeruginosa by synergistic effect when combined with vasoactive agents.2, 8 These results demonstrate that combining vasoactive agents and LAs might contribute to decreasing the dosage of LAs and minimising the infection complications by expanding the antibacterial spectrum.

5. CONCLUSION

The fact that LAs have antimicrobial effects along with their analgesic effects might reduce the complications of infections that might develop in LA practices. This might ensure a significant decrease in treatment costs, morbidity, and mortality. In our study, it was seen that prilocaine and bupivacaine had an antimicrobial effect on S. aureus and E. coli. In the comparison between these two LAs, this effect was found to be significantly higher in prilocaine than bupivacaine. Therefore, we are of the opinion that antimicrobial effect potentials should also be taken into account in the selection of LA agent in order to prevent the complications of an infection that might develop during LA infiltration and might lead to serious morbidity.

CONFLICT OF INTEREST

The authors declare no potential conflict of interest.

Kesici S, Demirci M, Kesici U. Bacterial inhibition efficiency of prilocaine and bupivacaine. Int Wound J. 2019;16:1185–1189. 10.1111/iwj.13180