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Bacterial Growth in Ropivacaine Hydrochloride

Bátai, Istvan, PhD*; Kerényi, Monika, MD; Falvai, Judit*; Szabó, Gyorgy, PhD

doi: 10.1097/00000539-200203000-00046
Brief Report: Brief Report

*Department of Anesthesia and Intensive Care, †Department of Clinical Microbiology, ‡Department of Orthopedic Surgery, University of Pécs, Pécs, Hungary

Supported, in part, by Ministry of Health of Hungary Grant Number 385/2000/ETT.

Presented, in part, at the 12th World Congress of Anaesthesiologists, June 4-9, 2000, Montréal, Canada.

November 6, 2001.

Address correspondence to Istvan Bátai, PhD, University of Pécs, Department of Anaesthesia and Intensive Care, Pécs, Szigeti u. 12., H-7624 Hungary. Address e-mail to

Drugs used in anesthesia may influence bacterial growth (1). Used ampules and syringes may be contaminated in a busy environment (2). Epidural space infection in association with epidural anesthesia is rare. The antibacterial effects of local anesthetics themselves may contribute to this infrequent incidence. A local anesthetic was first reported to kill bacteria in 1909 (3). Since then antibacterial activity of other local anesthetics has been confirmed (1) but there is no study on the effect of undiluted ropivacaine on bacterial growth at room temperature. We investigated the effect of ropivacaine 2 mg/mL and 10 mg/mL on bacterial growth.

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Materials and Methods

Bacterial strains were isolates of Staphylococcus aureus (ATCC 23923), and Escherichia coli (ATCC 25922). The tested pharmaceutical preparations were ropivacaine hydrochloride 2 mg/mL and 10 mg/mL (Naropin®, AstraZeneca, Macclesfield, UK). Mueller-Hinton broth (Oxoid) and saline 0.9% controls were also applied to assess bacterial viability.

The method is described in detail elsewhere (4). Mueller-Hinton broth (Oxoid) was inoculated with each organism and incubated overnight at 37°C. The cultures were diluted to a density of 0.5 McFarland units (1.5 × 108/mL) with sterile nonbacteriostatic saline 0.9%. Each bacterium solution was further diluted to an approximate initial concentration of 103 colony forming units (cfu)/mL. The final concentration of ropivacaine was 1.98 mg/mL and 9.90 mg/mL. Five vials of each tested drug and control were inoculated. After inoculation the culture vials were incubated at 20°C and 37°C. Each vial was vortexed and a 10 μL sample was then removed and plated on Mueller-Hinton agar (Oxoid) at the following times after inoculation: 0, 3, 6, 12, and 24 h. The plates were then incubated at 37°C for 24 h. The numbers of cfu were counted.

The results are expressed as mean ± sd. Statistical analysis was performed by using analysis of variance. Individual comparisons between group means were made by using the Scheffé test. P < 0.05 was regarded as significant.

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Both strains grew in Mueller-Hinton broth at both 20°C and 37°C. S. aureus did not multiply in saline, although E. coli grew in this control. These results showed the normal growing pattern in these controls.

S. aureus did not multiply in ropivacaine 2 mg/mL or 10 mg/mL at room temperature. At 37°C the 2 mg/mL solution reduced its cfu; ropivacaine 10 mg/mL killed it after 6 h (Table 1). E. coli grew in ropivacaine 2 mg/mL at both temperatures. This strain did not multiply in the 10 mg/mL solution at room temperature, and its cfu was reduced after 6 h at 37°C (Table 2).

Table 1

Table 1

Table 2

Table 2

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Our data suggest that although ropivacaine 10 mg/mL inhibits bacterial growth, E. coli grows in ropivacaine 2 mg/mL. When ropivacaine inhibited bacterial growth, it was more evident at 37°C than at 20°C. This effect of temperature is in accordance with previous studies examining the effects of anesthetics on bacteria (1).

We evaluated bacterial growth at two different temperatures. Concerning infection control, results obtained at room temperature should be considered. Intra- or epidural injections and infusions may cause infections if contaminated during their preparation or administration. In a previous study the incidence of contamination of syringes used during epidural analgesia was almost 5%(5). The incidence of bacterial contamination of spinal and epidural needles can be as frequent as 17%(6). Bacteria can be introduced into ampules during manufacture (intrinsic contamination) or during preparation and administration in the hospital (extrinsic contamination). Contaminated glass particles, rubber diaphragm, or needles may introduce bacteria into the fluids. The likelihood of fluid becoming contaminated during use is directly related to the duration of uninterrupted infusion through the same administration set and the frequency with which the set is manipulated. The mechanism of fluid contamination is described in detail elsewhere (7).

Results obtained at 37°C are interesting for a different reason. The continuous emergence of multiresistant strains makes antibiotic therapy difficult. Nonantibiotic drugs may contribute to the treatment of resistant infection (8). It is unlikely that ropivacaine alone could help treat infections, as its concentration in vivo is far smaller than in this in vitro test. We have scarce knowledge of the interaction between antibiotics and medications used in anesthesia and intensive care but the results available are encouraging. There is evidence of synergistic effects of lignocaine with antibiotics in vitro(9). Whether ropivacaine has any additive or synergistic effect with antibiotics in clinical settings is not clear.

The study by Pere et al. (10) compared the antimicrobial effect of diluted ropivacaine and bupivacaine (0.938–3.75 mg/mL) on bacterial growth at 37°C after 18 h incubation. In their paper the initial bacterium count was 105 cfu/mL and they measured bacterial growth as the absorbance of light at the wavelength of 540 nm. The aim of our study was to evaluate whether ropivacaine poses an infection risk or not if contaminated. To answer our question concerning bacterial growth at room temperature, more frequent sample times (e.g., 0, 3, 6, 12, and 24 h) and a lower initial bacterium count (103 cfu/mL) are needed (11). There is a relevant difference between our results and the findings of Pere et al. (10). They stated that E. coli did not multiply in ropivacaine and bupivacaine 0.938–3.75 mg mL at 37°C. Our results showed that E. coli (we used the same ATCC strain) grows in ropivacaine 2 mg/mL at both room temperature and 37°C so it may cause nosocomial infection if contaminated. The results of Pere et al.’s study (10) on bupivacaine are also different from other studies. Rosenberg et al. (12) found that the same strain of E. coli grew in bupivacaine 1.25 and 2.5 mg/mL at 35°C after 18 h. The different methods of evaluating bacterial growth (direct method in ours and Rosenberg’s study but indirect in Pere et al.’s work) may explain the different results.

We would like to thank Istvan Juricskai for helping with the statistical analysis.

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