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From the *Equipe de Microbiologie, UPRES-EA 1254, Université de Rennes I, France; Laboratoire de Biochimie Médicale A; and Département d’Anesthésiologie, CHR de Rennes, France.
Address correspondence and reprint requests to Martine Bonnaure-Mallet, Equipe de Microbiologie, UPRES-EA 1254, Université de Rennes I, 2 Avenue du Professeur Léon Bernard, 35043 Rennes Cedex, France. Address e-mail to martine.bonnaure{at}univ-rennes1.fr.
Abstract
BACKGROUND: Tramadol is a synthetic analog of codeine with opioid and local anesthetic properties. It is used as a central-acting analgesic, and recently, in subcutaneous or intradermal injections, as a local anesthetic. We investigated in vitro the antibacterial activity of tramadol in the absence of any local anesthetics against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa pathogens that can cause infectious complications after local or regional anesthesia.
METHODS: Bacterial cultures were grown for 18 h, diluted in sterile physiological saline, and incubated for 6 or 24 h at 37°C with 6.25, 12.5, or 25 mg/mL tramadol. The mixtures were then plated onto blood agar and colony counts were recorded after 24 h incubation at 37°C.
RESULTS: Tramadol had bactericidal activity against E. coli and S. epidermidis compared with controls: at 25 mg/mL for 6 h or at 12.5 mg/mL for 24 h, tramadol decreased by approximately 7 log10 (P < 0.001) the colony counts of E. coli (100% kill). Similar results were obtained with S. epidermidis, with approximately 6 log10 reduction (100% kill) when tramadol was used at 25 mg/mL for 24 h (P < 0.001). The antibacterial effect of 25 mg/mL tramadol was lower against S. aureus and P. aeruginosa, reducing the growth of these strains by approximately 3log10 after 24 h (P < 0.001).
CONCLUSIONS: Tramadol has dose- and time-dependent bactericidal activity against E. coli and S. epidermidis, as well as antibacterial activity against S. aureus and P. aeruginosa. The antibacterial properties of tramadol may be useful for reducing the risk of bacterial infection after local or regional anesthesia.
Some infectious complications, including meningitis, local skin infections, and epidural abscesses related to regional analgesic techniques have been reported (1–3). Both Gram-positive (S. aureus, S. epidermidis) and Gram-negative (E. coli) bacteria have been associated with these infectious complications (4,5). However, the antibacterial properties of local anesthetics (bupivacaine, ropivacaine, and lidocaine, for example) against pathogenic bacterial strains have been described by several in vitro studies (6–8). Many adjunctive drugs, such as epinephrine, clonidine, and opioids, are commonly administered in combination with local anesthetics to enhance the quality and duration of anesthesia and the postoperative analgesia of peripheral nerves blocks (9,10).
Tramadol, a synthetic analog of codeine, binds to µ-opioid receptors and inhibits norepinephrine and serotonin reuptake. Its mechanism of action has some characteristics in common with clonidine and opioids (11). Tramadol is used at different concentrations as an analgesic (12,13), and was shown to have a local anesthetic action on peripheral nerves when administered alone in subcutaneous or intradermal injections (14–16).
To our knowledge, no data regarding the antibacterial activity of tramadol are available. The aim of this study was to investigate the in vitro antibacterial activity of tramadol alone against a range of bacteria implicated in infectious complications after local or regional anesthesia.
METHODS
A commercially available solution of tramadol chlorhydrate (50 mg/mL, Aventis Pharma, France) was diluted with sterile 0.9% saline to produce final tramadol concentrations of 6.25, 12.5, and 25 mg/mL. The tramadol solutions were preservative-free and sterile; no bacterial growth was observed in the control culture. The four bacterial strains included in this study were E. coli CIP 7624, S. aureus ATCC 9144, S. epidermidis CIP 81.55T, and P. aeruginosa CIP 76110. These bacteria commonly present on human skin, are, in part, responsible for nosocomial infections, and are often used for studies on the antibacterial effects of local anesthetics.
Fresh bacterial cultures grown for 18 h (to the end of the exponential-growth phase) in trypticase soy broth were diluted into sterile physiological saline solution to a density of one McFarland unit (Densimat, bioMérieux SA, Marcyl l’Etoile, France), corresponding to an initial concentration of approximately 3 x 108 colony forming units (CFU)/mL (standard inocula). Measurement of the standard inocula was confirmed precisely by counting the cultivable cells. Each standard inocula was serial 10-fold diluted in sterile physiological saline until 10–6. Aliquots (100 µL) of each 10–5 and 10–6 dilutions were separately plated onto blood agar and incubated for 24 h at 37°C.
For antibacterial assays, 20 µL of each standard inocula containing approximately 5 x 106 CFU were added to 980 µL of each tested solution of tramadol. For controls, the same experiments were performed with sterile 0.9% saline without tramadol. Cultures were incubated for 6 or 24 h at 37°C. At the end of the incubation time, to inactivate the tested tramadol solutions via drug dilution method and to make the colony counting easier, five series of 10-fold dilutions in 0.9% saline solution were performed by adding 100 µL of each suspension (tested solution of tramadol + bacteria) to 900 µL sterile 0.9% saline. From these five series of diluted suspensions, 100 µL of each were then transferred onto blood agar plates and incubated for 24 h at 37°C to determine the colony count (17). Each experiment using tramadol solutions was repeated three times, and each dilution series assay was performed in duplicate for all strains in each experiment. The results were expressed as log10 values of the mean colony count. Statistical analysis was performed using two-way analysis of variance and the PLSD-Fisher’s test to compare the mean colony count from cultures treated with 6.25, 12.5, and 25 mg/mL tramadol with the colony count of the control. The comparison between the means of the two groups was made using the nonparametric test of Mann–Whitney. For all tests, a value of P < 0.05 was considered statistically significant.
RESULTS
The mean log10 colony counts of E. coli, S. epidermidis, S. aureus, and P. aeruginosa after 6 and 24 h exposure to the tested concentrations of tramadol are shown in Table 1 and Figures 1–4, respectively.
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DISCUSSION
Some local anesthetics have been reported to have antibacterial effects against bacteria that are commonly present on human skin and which have been implicated in nosocomial infections. Bupivacaine, at the concentration of 0.5%, induced a bactericidal effect in E. coli and S. epidermidis (18). This bactericidal activity was also observed in 2% prilocaine and 5% lidocaine against P. aeruginosa (7). A low concentration of bupivacaine (0.08%) and ropivacaine induced an antibacterial effect on E. coli and S. aureus growth (8). This investigation is the first to report that tramadol has antibacterial activity. Our results have shown that tramadol has in vitro dose- and time-dependent bactericidal activity against E. coli and S. epidermidis, and antibacterial effects on S. aureus and P. aeruginosa as well. At the highest concentration (25 mg/mL), tramadol reduced by approximately 7 log10 the colony counts of E. coli (100% kill) after a 6 h incubation, and by approximately 6 log10 (100% kill) S. epidermidis growth after 24 h incubation. The antibacterial activity of tramadol against S. aureus and P. aeruginosa was lower. The results obtained using E. coli and S. aureus are in agreement with those of our previous study, which showed the antibacterial activity of tramadol against these strains in exponential-phase culture (19).
The antibacterial effects of tramadol may be due to its local anesthetic properties. The exact mechanism of these local anesthetics action is not known, but may be caused by an effect on microbial cell membrane. It was shown that antibacterial activity of lidocaine is associated with depolarization of the cytoplasmic membrane, preceded by the permeabilization of the outer membrane for Gram-negative bacteria (20). Procaine, by interacting with the cell membrane, inhibits the processing of precursors of exported proteins in E. coli (21). Also, some local anesthetics (nupercaine, tetracaine) induced an inhibition of membrane-bound enzymatic activities and a characteristic ultrastructural alterations in Gram-positive bacterial cells (22).
Since tramadol is commonly used in combination with local anesthetics, it would be interesting to know whether it affects the antibacterial properties of local anesthetics. In a previous study, we found that sufentanil had a partial synergistic effect on bupivacaine’s antibacterial activity and a partial antagonistic effect on ropivacaine’s antibacterial activity (8). In the current study, different concentrations of tramadol were prepared in 0.9% saline from a 50 mg/mL commercial solution. All bacterial suspensions were also prepared in 0.9% saline to avoid the effect of nutritional culture media on their growth. Our results were obtained using a rather high concentration of bacteria (106–107 CFU/mL). In a clinical setting, such a high concentration of bacterial contamination after local or regional anesthesia would be unexpected. In addition, final drug concentrations used in this investigation may be higher than those typically used in IV administration, but lower than those used in intradermal or subcutaneous injections (50–65 mg/mL) (15,16). Thus, at clinically relevant doses, tramadol may have antibacterial activity against bacterial strains implicated in infectious complications.
Tramadol is one of the few analgesic adjuvants to show a consistent benefit when added to local anesthetics for peripheral nerve block. Furthermore, its potential antibacterial activity may be a positive factor for decreasing the contamination risk of short-term dwelling catheters. Concerning the possible neural toxicity of drug, it was shown that direct application of tramadol on the sciatic nerve of rats inhibited spinal somatosensory evoked potentials in a dose-dependent manner. This action is reversible without any deleterious neurological effects (23).
In conclusion, this study indicates that tramadol, in a dose- and time-dependent manner, has in vitro bactericidal activity against E. coli and S. epidermidis, and antibacterial activity against S. aureus and P. aeruginosa. These effects were observed for tramadol concentrations equal to or larger than 6.25 mg/mL after 6 or 24 h incubation. The in vitro antibacterial activities of tramadol may be useful for reducing the risk of bacterial contamination after local or regional anesthesia. Further studies are necessary to determine the antibacterial activity of tramadol at clinical doses, alone or in combination with local anesthetics.
ACKNOWLEDGMENTS
The authors thank C. Allaire for editorial assistance and I. Derrien for revising this article.
Footnotes
Accepted for publication April 5, 2007.
This study was supported by Foundation Langlois.
REFERENCES
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