Anesth Analg 2003;97:409-411
© 2003 International Anesthesia Research Society
ANESTHETIC PHARMACOLOGY
Ropivacaine 0.1% with Sufentanil 1 µg/mL Inhibits In Vitro Growth of Pseudomonas Aeruginosa and Does Not Promote Multiplication of Staphylococcus Aureus
Sandra Kampe, MD*,
Carsten Poetter, MD
,
Shariah Buzello, MD*,
Hans-Martin Wenchel, MD
,
Matthias Paul, MD*,
Peter Kiencke, PhD
, and
Stefan-Mario Kasper, MD*
*Department of Anesthesiology,
Infection Control Laboratory,
Department of Hospital Infection Control, and
Department of Medical Statistics, University of Cologne, Cologne, Germany
Address correspondence and reprint requests to Sandra Kampe, MD, Department of Anesthesiology, University of Cologne, Joseph-Stelzmann-Str. 9, 50931 Cologne, Germany. Address e-mail to Sandra.Kampe{at}medizin.uni-koeln.de
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Abstract
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We investigated the effect of ropivacaine combined with sufentanil, a mixture frequently used for postoperative epidural analgesia, on the growth of Staphylococcus aureus and Pseudomonas aeruginosa at room temperature. Aliquots of suspension of S. aureus and P. aeruginosa in saline were transferred into test tubes containing either a mixture of ropivacaine 0.1% and sufentanil 1 µg/mL (R+S) or saline (SA), with the latter serving as control. At 0, 3, 6, 24, and 48 h after inoculation, 1 mL of each solution was spread over standard blood agar. The plates were incubated at 22°C for 48 h, and the numbers of colony-forming units (cfu) were counted. The growth ratio for both bacterial strains was calculated as cfu time (tn)/cfu baseline (t0). The primary efficacy variable was the area under the curve (AUC) in (cfu tn/cfu t0) x time, based on the growth ratios. The AUC for P. aeruginosa was significantly less in R+S than in SA (P = 0.028). Multiplication of P. aeruginosa (growth ratio >1) was observed for at least 6 h after inoculation in SA. Growth of P. aeruginosa was significantly less in R+S than in SA at 3 h (P = 0.043) and 24 h (P = 0.012) after inoculation. The AUC for S. aureus did not differ significantly between R+S and SA (P = 0.74). Neither R+S nor SA promoted multiplication of S. aureus. Forty-eight hours after inoculation, growth of S. aureus was significantly less in R+S than in SA (P < 0.0001). We conclude that R+S inhibited growth of P. aeruginosa and did not promote multiplication of S. aureus when compared with SA.
IMPLICATIONS: This laboratory study demonstrated that compared with saline, ropivacaine 0.1% with 1 µg/mL of sufentanil inhibited growth of Pseudomonas aeruginosa and did not promote multiplication of Staphylococcus aureus at room temperature. With respect to bacterial infection with these two strains, the mixture seems to be safe for continuous epidural administration if prepared under aseptic conditions and after alcohol hand rub.
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Introduction
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Epidural space infection associated with epidural anesthesia for peri- and postoperative analgesia is a rare but serious complication (1). It has been demonstrated recently that ropivacaine 1% inhibited growth of Staphylococcus aureus and Escherichia coli, whereas E. coli grew in ropivacaine 0.2% (2).
For postoperative epidural analgesia, it has become very popular to use small-dose mixtures of ropivacaine combined with sufentanil for continuous epidural infusion or patient-controlled epidural analgesia (3,4). Because these mixtures are not commercially manufactured, clinicians often have to resort to preparing their own mixtures. However, no data are available on the effect of such mixtures on bacterial growth.
We investigated in vitro the effect of ropivacaine 0.1% with sufentanil 1 µg/mL, a combination highly effective for lumbar epidural analgesia (5,6), on bacterial growth over 48 h at room temperature. We designed our study to imitate the setting during the preparation and administration of these drugs by continuous infusion through the same administration set. Two clinically relevant bacterial agents, S. aureus and Pseudomonas aeruginosa, which are common causes of catheter-related infections and sepsis, were used (1,7,8).
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Methods
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The tests were performed in an accredited laboratory by experienced personnel using approved materials. Two separate test cultures of S. aureus (American Type Culture Collection [Mannassas, VA] No. 25923) and P. aeruginosa (American Type Culture Collection No. 27853) were grown on standard blood agar and incubated overnight.
A sample of each test culture (<20 h old) was suspended in sterile saline (SA) (NaCl 0.9%) solution. By washing and spinning the cells twice, a nutrient-free environment was ensured. Dilution of plain ropivacaine hydrochloride (AstraZeneca, Wedel, Germany) was prepared by adding 48 mL of SA and 2 mL of sufentanil citrate (100 µg; Ratiopharm GmbH, Ulm, Germany) to 50 mL of ropivacaine 0.2%. The pH of the solution was 5.7. We did not adjust pH because we wanted to reproduce conditions during the preparation and administration of the drugs. Under aseptic conditions (Laminar-Air-Flow) and after alcohol hand rub (9), 10 test tubes were filled with 10 mL of ropivacaine 0.1% and 1 µg/mL of sufentanil (R+S), and another 10 tubes were filled with 10 mL of sterile preservative-free SA to assess bacterial viability.
One-milliliter aliquots were then transferred from the SA/bacteria suspension into the R+S solution and the SA control and were well mixed by vortex. After mixing, 1 mL was taken from each inoculum at 0, 3, 6, 24, and 48 h after inoculation, diluted, and spread over standard blood agar. The plates were incubated at 22°C for 48 h. The test cultures were identified morphologically and by using an Oxidase test. The numbers of colony-forming units (cfu) were counted.
Statistical analysis was performed with the SPSS 11.0 statistical package (SPSS Inc., Chicago, IL). The growth ratio for both bacterial strains was calculated by the following quotient: cfu time (tn)/cfu baseline (t0).
The primary efficacy variable was the area under the curve (AUC) in (cfu tn/cfu t0) x time, based on the growth ratios of S. aureus and P. aeruginosa in the R+S and the SA solution. The AUC, based on repeated measurements up to 48 h, was calculated by using the trapezoidal rule (Stata Corp., College Station, TX). Two-tailed Student’s t-tests were used to compare bacterial growth in R+S and SA solutions at each assessment point. Significance was set at the P < 0.05 level. Because bacterial growth ratios after 3, 6, 24, and 48 h of inoculation were compared for explorative purpose only, the P values were not corrected for multiple testing. Unless otherwise indicated, data are presented as means ± SD.
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Results
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Bacterial growth is presented in Figure 1 for P. aeruginosa and in Figure 2 for S. aureus. The AUC for bacterial growth of P. aeruginosa was significantly less in the R+S solution than in the SA control solution [R+S: 21 ± 3 (cfu tn/cfu t0) x time versus SA: 27 ± 6 (cfu tn/cfu t0) x time; P = 0.028]. There was no statistical difference in AUC for S. aureus between R+S and SA [R+S: 19 ± 6 (cfu tn/cfu t0) x time versus SA: 20 ± 8 (cfu tn/cfu t0) x time, P = 0.74].
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Figure 1. Growth of P. aeruginosa, calculated as growth ratio, over an observation period of 48 h. ( ) Saline, (•) ropivacaine 0.1% plus 1 µg/mL of sufentanil. Data are presented as means ± 95% confidence interval; 3 h, P = 0.043; 24 h, P = 0.012. cfu = colony-forming unit.
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Figure 2. Growth of S. aureus, calculated as growth ratio, over an observation period of 48 h. () Saline, (•) ropivacaine 0.1% plus 1 µg/mL of sufentanil. Data are presented as means ± 95% confidence interval; 48 h, P = 0.0001. cfu = colony-forming unit.
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P. aeruginosa did multiply in SA for at least 6 h after inoculation (Fig. 1). Growth of P. aeruginosa was significantly less in R+S than in SA at 3 h (P = 0.043) and 24 h (P = 0.012) after inoculation. S. aureus did not multiply in either solution, as indicated by growth ratios <1.0. After 48 h of inoculation, growth for S. aureus was significantly less in R+S than in SA (P < 0.0001; Fig. 2).
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Discussion
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Bupivacaine has been reported to have a significant antibacterial effect, which improves with increasing concentrations (10). Although ropivacaine and bupivacaine have the same chemical structure, except for the alkyl group, small changes in the length of the alkyl chains influence the antibacterial activity of hydrophobic inhibitors (11).
Antibacterial effects have been described recently for ropivacaine 1%, which inhibits the growth of S. aureus and E. coli, an effect more evident at body temperature than at room temperature (2). Available data on smaller concentrations of ropivacaine showed that the antibacterial effect was reduced with decreased concentrations and that ropivacaine was a weaker inhibitor of bacterial growth than bupivacaine (2,10).
We found that the combination of ropivacaine 0.1% with 1 µg/mL of sufentanil, compared with SA, produced a greater inhibitory effect on growth of P. aeruginosa, a representative of the Gram-negative family capable of growing in a nutrient-poor environment and even surviving in disinfectant solutions. S. aureus did not multiply in ropivacaine with sufentanil either, and, 48 hours after inoculation, the inhibitory effect was greater than in the SA control. Recent data investigating diluted solutions of commercially available ropivacaine hydrochloride found no inhibitory effect of ropivacaine 0.1% (0.938 mg/mL) on bacterial growth of S. aureus and P. aeruginosa (10). The contradictory results to our study are not entirely explained by a different methodology (adsorbence of light at a wavelength of 540 nm, incubation for 18 hours at 37°C, and adjustment of pH to 7.2). We postulate an additive effect of sufentanil citrate on the growth inhibition of S. aureus and P. aeruginosa.
Few data are available on the bacterial activity of mixtures of local anesthetics with opioids, a combination favored in the clinical setting of postoperative epidural analgesia. Neither fentanyl 2 µg/mL nor sufentanil 0.3 µg/mL, when combined with bupivacaine, inhibited bacterial growth in culture (12). Pure morphine hydrochloride did not inhibit growth of any bacterial strain tested (13). In contrast, data from 1964 (14) showed an inhibitory effects of drugs of the morphine series on the growth of E. coli. The more potent the drug, the more effective the inhibitory effect on bacterial growth (levallorphan > dextrorphan > nalorphine > morphine). Sufentanil has a 100- to 300-fold relative potency compared with morphine, but until now no data were available on the possible antimicrobial effects of sufentanil 1 µg/mL.
In conclusion, we demonstrated that R+S inhibited in vitro growth of P. aeruginosa and did not promote multiplication of S. aureus over 48 hours at room temperature. With respect to the literature, it is unlikely that ropivacaine 0.1% alone produced the observed inhibitory effect; we postulate that sufentanil 1 µg/mL had an additive effect. Further laboratory studies will be necessary to evaluate this hypothesis. However, for the clinical setting, the mixture of ropivacaine 0.1% with 1 µg/mL of sufentanil seems to be safe for the two investigated bacteria, given that preparation takes place under aseptic conditions, with emphasis on hand hygiene by using an alcoholic hand rub to prevent transmission of microbial pathogens.
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Acknowledgments
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Supported in part by a grant from AstraZeneca, Wedel, Germany.
The authors gratefully acknowledge the expert technical assistance of Michaela Weber, Ursula Mermagen, and Angelika Mertens, Infection Control Laboratory, Department of Hospital Infection Control, University of Cologne.
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Footnotes
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Presented in part at the 28th Annual Spring ASRA Meeting, April 2003, San Diego, CA.
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Accepted for publication March 4, 2003.
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Abstract
Introduction
