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TECHNICAL COMMUNICATION

The Growth of Bacteria in Intravenous Glyceryl Trinitrate and in Sodium Nitroprusside

Istvan Bátai, MD^*,, Monika Kerényi, MD, and Miklos Tekeres, PhD

*Department of Anaesthesia, Royal Liverpool University Hospital, Liverpool, United Kingdom; and Departments of Anesthesia and Intensive Care, and Clinical Microbiology, University of Pécs, Pécs, Hungary

Address correspondence and reprint requests to Istvan Bátai, MD, Royal Liverpool University Hospital, Department of Anaesthesia, 12th floor, Prescot St., Liverpool, England L7 8XP.

	Introduction

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Drugs used in anesthesia may support or inhibit bacterial growth (1), and the ampules and syringes we use may be contaminated in a busy environment (2).

IV glyceryl trinitrates (GTN) and sodium nitroprusside (SNP), have been used for many years (3,4), and their therapeutic value as nitric oxide (NO) donors may increase in the future (5).

NO can be generated during the degradation of GTN and SNP by tissues (6). GTN can also be degraded by bacteria (7), while SNP is photochemically degraded to NO (8). NO with other reactive intermediate nitrates has an antibacterial effect (9).

We investigated the effect of GTN and SNP on bacterial growth.

	Methods

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Bacterial strains were clinical isolates of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa.

The tested pharmaceutical preparations were 100 µg/mL GTN in alcohol 10%, pH 5.5; 100 µg/mL SNP dissolved in 5% glucose, pH 5.9.

The control solutions were selected as the solvents of the tested drugs: glucose 5% (pH 5.9); alcohol 10% (pH 5.5). Sodium chloride 0.9% (pH 5.9) and Mueller-Hinton broth controls were also applied to assess bacterial viability.

Bacterial suspensions were prepared by modifying the method previously described by Berry et al. (10) and Sosis and Braverman (11). Mueller-Hinton broth 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 x 10⁸ mL^-1) with sterile nonbacteriostatic saline 0.9%. Each bacterium solution was further diluted 1:1000 with sterile saline 0.9%. A 0.2-mL aliquot of each diluted bacterial suspension was added to sterile sealed culture vials containing 20 mL of the tested drug solutions or the controls. Five vials of each tested drug and controls were inoculated. It gave an approximate initial concentration of 10³ colony forming units per mL. All diluted suspensions were vortexed for 1 min between each aliquot removal. After inoculation, the culture vials were kept in incubators at both 20°C and 37°C. Each vial was vortexed for 5 min, and a 10-µL sample was then removed and plated on Mueller-Hinton agar at the following times after inoculation: 0, 3, 6, and 24 h. The plates were then incubated at 37°C for 24 h. The numbers of colony forming units 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 using the Scheffe test. P < 0.05 was regarded as significant.

	Results

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All strains grew in Mueller-Hinton broth at both 20°C and 37°C. P aeruginosa and Escherichia coli multiplied in glucose 5% and saline 0.9%, whereas the count of S aureus did not change significantly in these controls.

GTN reduced the number of all bacteria at both temperatures.

SNP had less bactericidal activity. It reduced the bacterial counts of S aureus at both temperatures. Escherichia coli increased in number before declining. It was bacteriostatic against P aeruginosa at 37°C and supported its growth at 20°C (Tables 1–3).

	Discussion

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Our data suggest that while GTN only inhibits, SNP can either inhibit or support bacterial growth. GTN at 37°C had the most pronounced antibacterial effects. However, P aeruginosa grew quickly in SNP at 20°C.

The Gram-negative strains were more resistant to the tested drugs than the Gram-positive bacterium. In both GTN and SNP solutions, the inhibition of bacterial growth 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).

NO is an important component of the host response to infection (9). It may directly kill bacteria (9), or it may play a role in the limitation of the available intracellular iron (12). Both GTN and SNP can serve as NO donors (6–8). In this study, SNP showed less antimicrobial activity than GTN. SNP is photochemically degraded to NO (8), and it was light-protected as is the case in clinical practice. In this way, the spontaneous NO production was reduced, which could explain its reduced antimicrobial activity.

We evaluated bacterial growth at both 20°C and 37°C. For infection control, the results obtained at 20°C should be considered. The potential and the sequelae for infections caused by infusion therapy are discussed in other reviews (13). Graystone at al. (14) demonstrated that both GTN and SNP were bactericidal against S aureus, methicillin-resistant S aureus, Escherichia coli, Enterococcus faecalis, and P aeruginosa strains at 22°C. Our experiments revealed that, at room temperature, SNP supported the growth of the tested P aeruginosa and Escherichia coli strains. The different strains, the different dilutions (SNP 100 µg/mL versus 1 mg/mL), and the different technique [Greystone et al. (14) plated only 1.0 µL-aliquots] may explain the different results. Our results suggest that, while S aureus or P aeruginosa cannot survive in GTN infusion, Escherichia coli can persist in it for at least 24 hours. SNP can even support the growth of bacteria.

Our results at 37°C are interesting for different reasons. The continuous emergence of multi-resistant strains makes effective antibiotic therapy difficult. Nonantibiotic drugs may contribute to the treatment of resistant infection (15). It is unlikely that GTN or SNP alone could help treat infections, as their concentrations in vivo are less than those in this in vitro test. At present, we have scarce knowledge of the interaction between antibiotics and medications used in anesthesia and intensive care, but the few results available are encouraging. There is evidence of synergistic effects of promethazine with gentamicin in vivo (16) and, in the case of lidocaine with other antibiotics, in vitro (17).

Whether GTN or SNP has any additive or synergistic effects with antibiotics in clinical settings requires further investigation.

	References

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