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Antibacterial Activity of Clonidine and Neostigmine In Vitro

Boselli, Emmanuel MD*; Guillier, Marion MD*; Freney, Jean PharmD, PhD; Mazoyer, Marie-Andrée PharmD; Casoli, Emmanuelle PharmD; Renaud, François R. N. PhD; Rimmelé, Thomas MD*; Chassard, Dominique MD, PhD*; Allaouchiche, Bernard MD, PhD*

doi: 10.1213/01.ANE.0000153500.68193.F7
Anesthetic Pharmacology: Research Report

We conducted an in vitro study to investigate the antibacterial activity of clonidine and neostigmine on common microorganisms encountered during infectious complications after regional anesthesia. Standardized suspensions of Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli were incubated during 1, 3, 6, and 24 h at 37°C with concentrations of 37.5, 75, and 150 μg/mL of clonidine and 125, 250, and 500 μg/mL of neostigmine. After 24 h incubation at 37°C, the colony counts were compared by two-way analysis of variance. The mean colony counts for S. aureus decreased significantly from control as the exposure to clonidine increased (P < 0.05), with a ∼100% kill at 6 h for the largest concentration (150 μg/mL) and at 24 h for the intermediate concentration (75 μg/mL). Similar results were observed for S. epidermidis, with a ∼100% kill at 6 h for the largest concentrations (75 and 150 μg/mL). No bactericidal activity of clonidine was observed for E. coli and no bactericidal activity of neostigmine was observed for any of the tested strains. In the conditions of this experiment, clonidine, but not neostigmine, exhibited a concentration-dependent and time-dependent bactericidal activity in vitro on the microorganisms most frequently encountered in infectious complications after regional anesthesia.

IMPLICATIONS: The results of this study show that clonidine possesses a concentration-dependent and time-dependent bactericidal activity on Staphylococcus aureus and Staphylococcus epidermidis. No bactericidal activity of clonidine was observed on Escherichia coli, and no bactericidal activity of neostigmine was observed on any tested strains.

*Department of Anesthesiology, Hôpital Edouard Herriot, and †Laboratory of Microbiology, EA 3090, Lyon, France

Accepted for publication December 1, 2004.

Address correspondence and reprint requests to Dr Emmanuel Boselli, Service d’Anesthésie-Réanimation, Hôpital Edouard Herriot, 5 place d’Arsonval, 69437 Lyon cedex 03, France. Address e-mail to emmanuel.boselli@chu-lyon.fr.

Regional anesthesia is widely used for the treatment of intraoperative and postoperative pain or during labor. Some infectious complications related to theses techniques have been reported with various degrees of severity, such as peripheral nerve catheter colonization, local skin infection, meningitis, or epidural abscesses after spinal or epidural analgesia (1–4). The incidence of infectious complications after regional analgesia remains unknown but has been reported to vary widely (e.g., 1:53,000 for meningitis after spinal blockade or from 0.6 to 0.77 per 1000 catheter days for spinal epidural abscess after epidural analgesia during labor) (4,5). Staphylococcus aureus is responsible for ∼60% of these complications but other microorganisms, such as Staphylococcus epidermidis and α-hemolytic streptococci, or some Gram-negative pathogens (<15%) such as Escherichia coli have also been reported (1,5–7). The antibacterial activity of local anesthetics against a range of bacteria implicated in these infectious complications has been previously described in many in vitro studies (8–11).

Clonidine and neostigmine are frequently used as adjuncts to local anesthetics to enhance the quality and duration of regional anesthesia (12–14). There is no evidence to suggest that these drugs would increase or facilitate bacterial growth; however, such data are important to ensure that their use does not increase the risk of bacterial contamination or sepsis. Therefore, we conducted an in vitro study to test the hypothesis that various concentrations of clonidine and neostigmine may exhibit an antibacterial activity against common etiologic agents encountered during infectious complications after regional anesthesia.

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Methods

Commercially available preservative-free solutions of clonidine chlorydrate 150 μg/mL (Boehringer Ingelheim France, Paris, France) and neostigmine methylsulfate 500 μg/mL (ICN Pharmaceuticals, Orsay, France) were used in this study. The dilutions were prepared with sterile 0.9% saline to obtain final concentrations in the test solutions of 37.5, 75, and 150 μg/mL for clonidine and 125, 250, and 500 μg/mL for neostigmine, with pH ranges of 6–6.5 for all solutions.

Three separate test cultures of S. aureus (American Type Culture Collection [ATCC] 6538), S. epidermidis (ATCC 14990), and E. coli (ATCC 10536) were grown on tryptic soy agar and incubated overnight at 37°C before the antibacterial assays.

The 18 h cultures (i.e., just at the end of the exponential growth), were diluted in sterile saline 0.9% to a density of 1.8 MacFarland (McF) units for S. aureus, 2.5 McF for S. epidermidis, and 1.5 McF for E. coli (Densimat, bioMérieux SA, Marcy l’Etoile, France), corresponding to an initial concentration of ∼3.108 colony forming units/mL for each strain. For a more precise measurement of the size of the inocula, serial 10-fold dilutions were made with physiological saline, the final dilution being 10−6 of the concentration of the initial solution. This was done to enable colonies to be counted because the number of colonies in the undiluted test mixtures was far too large. Two 100-μL samples were taken from the 10−5 and 10−6 dilutions and each was plated on separate tryptic soy agar plates for colony counts to be recorded after 24 h incubation at 37°C. Standardized suspensions containing ∼106 colony forming units/mL of each strain were then prepared from the initial mixture by adding 10-μL aliquots to 990 μL of each tested solution of clonidine and neostigmine, and 10 μL of the standardized suspensions were also inoculated into 990 μL of sterile 0.9% saline without any added adjunct to act as a control. All bacterial suspensions were exposed to the different concentrations of clonidine and neostigmine for 1, 3, 6, and 24 h at 35–37°C. To inactivate the antimicrobial activity of both adjuncts and to decrease the number of microorganisms that could be counted, three series of 10-fold dilutions were performed for each suspension by adding 100-μL aliquots to 900 μL sterile 0.9% saline. From these three series of diluted suspensions, 100 μL were transferred to tryptic soy agar plates in duplicate and the plates were incubated at 37°C during 24 h for colony count. A sterility control check for clonidine and neostigmine was also performed by plating a sample of each adjunct on tryptic soy agar plates and incubating for 24 h. Each determination was performed as six parallel assays.

The results are expressed as log10 values of the mean colony counts. The differences in the colony counts between the cultures of suspension exposed to different concentrations of clonidine and neostigmine were analyzed using two-way analysis of variance for repeated measures with a value of P < 0.05 considered statistically significant (StatView 5.0, SAS Institute, Cary, NC). The Bonferroni test for post hoc comparisons was performed with a value of P < 0.0083 considered statistically significant once significance was reached with two-way analysis of variance.

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Results

No bacterial growth in the control culture plates was observed for either clonidine or neostigmine. The log10 values of the mean colony counts obtained after exposure to the tested concentrations of clonidine at each time interval are shown respectively in Figure 1 for S. aureus, Figure 2 for S. epidermidis, and Figure 3 for E. coli. For reasons of clarity, no error bars are shown. Statistically significant differences were observed with two-way analysis of variance for both S. aureus and S. epidermidis after exposure to clonidine (P < 0.05), with intergroup and intragroup differences as follows after post hoc analysis (P < 0.0083): the mean colony counts for S. aureus decreased significantly from control as the exposure to clonidine increased, with a ∼1 log10 reduction from baseline at 3 h for each concentration, a ∼2 log10 reduction at 6 h for the smaller and intermediate concentrations of clonidine (37.5 and 75 μg/mL), and a ∼6 log10 reduction at 6 h for the largest concentration (150 μg/mL) and at 24 h for the intermediate concentration (75 μg/mL) corresponding to a ∼100% kill. Similar results were observed for S. epidermidis, with a ∼1 log10 reduction from baseline at 3 h for each concentration of clonidine, a ∼2 log10 reduction at 6 h for the smallest concentration (37.5 μg/mL), and a ∼6 log10 reduction at 6 h and 24 h for the largest concentrations (75 and 150 μg/mL) corresponding to a ∼100% kill. No bactericidal activity of clonidine was observed for E. coli. No bactericidal activity of neostigmine nor bacterial growth was observed for any of the tested strains: the mean colony counts varied from ∼106–107 UFC/mL at 1 h to ∼105–106 UFC/mL at 24 h for both controls and the tested concentrations with neither intragroup nor intergroup statistical differences.

Figure 1

Figure 1

Figure 2

Figure 2

Figure 3

Figure 3

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Discussion

Although the impact of drugs used in anesthesia on bacteria has been extensively studied, this is the first study devoted to the bactericidal properties of common adjuncts used in regional anesthesia (8–11,15–17). Concentration-dependent and time-dependent bactericidal activity varying among strains has been observed for local anesthetics such as 0.5% bupivacaine, reducing the colony counts of S. aureus, S. epidermidis, and E. coli by ∼40%, 100%, and 90%, respectively, at 6 hours at 37°C (18). In our study, clonidine showed a concentration-dependent and time-dependent bactericidal activity on the tested strains of S. aureus and S. epidermidis with a 6-log reduction in the colony counts (∼100% kill) after a 6-hour exposure to the largest concentrations at 37°C, whereas no bactericidal activity was observed on E. coli or for neostigmine on any of the tested strains.

Clonidine, an α2-adrenergic agonist, is widely used as an adjunct to local anesthetics in regional anesthesia to intensify both sensory and motor block (12–14,19). The most reported experience with clonidine for regional anesthesia is with epidural administration in obstetric and pediatric patients and for the treatment of intraoperative and postoperative or chronic pain (12). However, clonidine may also be used as an adjunct to local anesthetics in spinal or peripheral regional analgesia (13,14). The reported ranges of concentration and duration of administration of clonidine vary widely among these studies from single boluses of small doses, usually in combination with local anesthetics, to single intrathecal boluses at concentrations up to 150 μg/mL (the largest concentration chosen in this study) or epidural continuous infusion during the treatment of chronic cancer pain (12,14,19).

The use of neostigmine, an acetylcholinesterase inhibitor, as an adjunct to local anesthetics has been described during spinal, epidural, or peripheral regional anesthesia, with various results in efficacy or side effects, such as a dose-dependent increase in the incidence of nausea and vomiting in patients who received spinal or peripheral neostigmine (13,14,19–23). This dose-dependent increase in side effects usually limits the use of neostigmine to small concentrations as an adjunct to local anesthetics.

In our study, the antimicrobial effects of clonidine and neostigmine were studied at concentrations found in the commercially available solutions (150 μg/mL and 500 μg/mL, respectively) and dilutions up to a fourfold dilution. The microorganisms tested with both adjuncts were diluted in normal saline to avoid the effect of nutritional factors of culture media on microorganisms. We observed neither bactericidal activity nor bacterial growth of clonidine for E. coli and for neostigmine for all the tested strains. A bactericidal activity of clonidine was observed on S. aureus and S. epidermidis, the two microorganisms most frequently encountered in infectious complications after regional anesthesia (1,5).

In this study, the number of microorganisms added to the adjuncts was similar to those used in other studies (10,11,18). However, in clinical usage, it is not expected to see such large microorganism concentrations in regional anesthesia procedures. If contamination occurs with a small amount of microorganisms, an antimicrobial effect may be expected from the clonidine solutions. However, it is not possible to say from the results of this study whether the concentration-dependent and time-dependent bactericidal activity of clonidine would lead to an in vivo decrease in infectious complications after regional anesthesia. Indeed, the antibacterial effect of a drug against bacteria depends on many factors, such as the size of the inoculum, the concentration of the antibacterial drug in vivo, or the host-defense mechanisms. The size of the inoculum in vivo is unlikely to be as large as the one used in this study, the conditions for growth of bacteria might be very different than in saline solution, and the concentrations of clonidine might vary from that used in suspensions. Moreover, clonidine and neostigmine are rarely used as sole drugs in regional anesthesia, and the clinically used concentrations are usually less than those used in this study. Their antibacterial activity when used in combination with local anesthetics at clinical concentrations should be investigated in further studies.

Besides the large inoculum size and the large concentrations chosen, which may not be clinically relevant, another limitation to our study is that it is purely descriptive. Further studies are required to elucidate the mechanisms. However, the lack of bacterial growth observed for all strains at each concentration of clonidine and neostigmine might ensure that the use of these drugs does not increase the risk of bacterial contamination or sepsis.

In conclusion, our results show that in the conditions of this experiment, clonidine possesses a bactericidal activity on S. aureus and S. epidermidis, mostly observed for concentrations ≥75 μg/mL after a 6-h incubation period of time. Neither bactericidal activity nor bacterial growth for clonidine was observed on E. coli or for neostigmine on any tested strains. The bactericidal activity of clonidine in vitro on the microorganisms most frequently encountered in infectious complications after regional anesthesia might be useful to ensure that the use of this drug does not increase the risk of bacterial contamination or sepsis, although this should be confirmed by further clinical studies. Moreover, the mechanisms of the bactericidal activity of clonidine are not known and should be elucidated by further studies.

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