The antimicrobial effects of ketamine combined with propofol: an in vitro study

Abstracts

BACKGROUND AND OBJECTIVES: Ketamine and propofol are the general anesthetics that also have antimicrobial and microbial growth-promoting effects, respectively. Although these agents are frequently applied together during clinical use, there is no data about their total effect on microbial growth when combined. In this study, we investigated some organisms' growth in a ketamine and propofol mixture. METHOD: We used standard strains including Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans in this study. Time-growth analysis was performed to assess microbial growth rates in 1% propofol. Antimicrobial activity of ketamine, alone and in propofol was studied with microdilution method. RESULTS: In propofol, studied strains grew from 10³-10(4) cfu/mL to >10(5) cfu/mL concentrations within 8-16 hours depending on the type of organism. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) (for candida, minimal fungicidal concentration) of ketamine were determined as follows (MIC, MBC): E.coli 312.5, 312.5 µg/mL; S.aureus 19.5, 156 µg/mL; P.aeruginosa 312.5, 625 µg/mL; and C.albicans 156, 156 µg/ml. In ketamine+propofol mixture, ketamine exhibited antimicrobial activity to E.coli, P.aeruginosa and C.albicans as MBCs at 1250, 625 and 625 µg/mL, respectively. Growth of S. aureus was not inhibited in this mixture (ketamine concentration=1250 µg/mL). CONCLUSION: Ketamine has sustained its antimicrobial activity in a dose-dependent manner against some organisms in propofol, which is a strong microbial growth-promoting solution. Combined use of ketamine and propofol in routine clinical application may reduce the risk of infection caused by accidental contamination. However, one must keep in mind that ketamine cannot reduce all pathogenic threats in propofol mixture.

Antimicrobial activity; Benzethonium chloride; Ketamine; Ketofol; Propofol

EXPERIÊNCIA E OBJETIVOS: Cetamina e propofol são os anestésicos gerais que também exibem efeitos antimicrobianos e promotores do crescimento microbiano, respectivamente. Embora esses agentes sejam frequentemente aplicados em combinação durante o uso clínico, não há dados sobre seu efeito total no crescimento microbiano na administração combinada. Nesse estudo, investigamos o crescimento de alguns microrganismos em uma mistura de cetamina e propofol. MÉTODO: Nesse estudo, utilizamos cepas padronizadas: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa e Candida albicans. Realizamos uma análise de tempo-crescimento para avaliar as taxas de crescimento microbiano em propofol 1%. A atividade antimicrobiana de cetamina, isoladamente e em propofol, foi estudada pelo método de microdiluição. RESULTADOS: Em propofol, as cepas estudadas cresceram de concentrações de 10³-10(4) ufc/mL para > 10(5) ufc/mL, dentro de 8-16 horas, dependendo do tipo de microrganismo. Foram determinadas a concentração inibitória mínima (CIM) e a concentração bactericida mínima (CBM) (para Candida, concentração fungicida mínima) de cetamina, como se segue (CIM, CBM): E. coli 312,5, 312,5 µg/mL; S.aureus 19,5, 156 µg/mL; P. aeruginosa 312,5, 625 µg/mL; e C. albicans 156, 156 µg/mL. Na mistura cetamina + propofol, cetamina exibiu atividade antimicrobiana para E. coli, P. aeruginosa e C. albicans em CBMs a 1250, 625 e 625 µg/mL, respectivamente. O crescimento de S. aureus não foi inibido nessa mistura (concentração de cetamina = 1250 µg/mL). CONCLUSÃO: Cetamina preservou sua atividade antimicrobiana de maneira dose-dependente contra alguns microrganismos em propofol, que é robusta solução promotora de crescimento microbiano. O uso combinado de cetamina e propofol na aplicação clínica de rotina pode diminuir o risco de infecção causada por contaminação acidental. Entretanto, deve-se ter em mente que cetamina não pode reduzir todas as ameaças patogênicas na mistura com propofol.

Atividade antimicrobiana; Cloreto de benzetônio; Cetamina; Cetofol; Propofol

EXPERIENCIA Y OBJETIVOS: La Cetamina y el propofol son los anestésicos generales que también tienen efectos antimicrobianos y son los promotores del crecimiento microbiano, respectivamente. Aunque esos agentes sean frecuentemente aplicados en combinación durante el uso clínico, no hay datos sobre su efecto total en el crecimiento microbiano en la administración combinada. En ese estudio, investigamos el crecimiento de algunos microrganismos en una mezcla de cetamina y propofol. MÉTODO: En este estudio, utilizamos cepas estandarizadas: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa y Candida albicans. Realizamos un análisis de tiempo-crecimiento para evaluar las tasas de crecimiento microbiano en el propofol al 1%. La actividad antimicrobiana de cetamina, aisladamente y en propofol, fue estudiada por el método de microdilución. RESULTADOS: En el propofol, las cepas estudiadas crecieron de concentraciones de 10³-10(4) ufc/mL para #> 10(5) ufc/mL, dentro de 8-16 horas, dependiendo del tipo de microrganismo. Fueron determinadas la concentración inhibitoria mínima (CIM) y la concentración bactericida mínima (CBM) (para Candida, concentración fungicida mínima) de cetamina, como vemos (CIM, CBM): E. coli 312,5, 312,5 µg/mL; S.aureus 19,5, 156 µg/mL; P. aeruginosa 312,5, 625 µg/mL; y C. albicans 156, 156 µg/ml. En la mezcla cetamina + propofol, la cetamina mostró una actividad antimicrobiana para E. coli, P. aeruginosa y C. albicans en CBMs a 1250, 625 y 625 µg/mL, respectivamente. El crecimiento de S. aureus no se inhibió en esa mezcla (concentración de cetamina = 1250 µg/mL). CONCLUSIONES: La cetamina preservó su actividad antimicrobiana de manera dosis-dependiente contra algunos microrganismos en propofol, que es una robusta solución que promueve el crecimiento microbiano. El uso combinado de cetamina y propofol en la aplicación clínica de rutina puede disminuir el riesgo de infección causada por la contaminación accidental. Sin embargo, debemos tener presente que la cetamina no puede reducir todas las amenazas patógenas en la mezcla con el propofol.

Actividad antimicrobiana; Cloruro de benzetonio; Cetamina; Cetofol; Propofol

SCIENTIFIC ARTICLE

The antimicrobial effects of ketamine combined with propofol: an in vitro study^*

Zekine Begec^I; Aytac Yucel^I; Yusuf Yakupogulları^II; Mehmet Ali Erdogan^I; Yucel Duman^II; Mahmut Durmus^I; M. Ozcan Ersoy^I

^IDepartment of Anesthesiology and Reanimation, School of Medicine, Inonu University, Malatya, Turkey

^IIDepartment of Clinical Microbiology, School of Medicine, Inonu University, Malatya, Turkey

Corresponding author

ABSTRACT

BACKGROUND AND OBJECTIVES: Ketamine and propofol are the general anesthetics that also have antimicrobial and microbial growth-promoting effects, respectively. Although these agents are frequently applied together during clinical use, there is no data about their total effect on microbial growth when combined. In this study, we investigated some organisms' growth in a ketamine and propofol mixture.

METHOD: We used standard strains including Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans in this study. Time-growth analysis was performed to assess microbial growth rates in 1% propofol. Antimicrobial activity of ketamine, alone and in propofol was studied with microdilution method.

RESULTS: In propofol, studied strains grew from 10³-10⁴ cfu/mL to ≥10⁵ cfu/mL concentrations within 8-16 hours depending on the type of organism. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) (for candida, minimal fungicidal concentration) of ketamine were determined as follows (MIC, MBC): E.coli 312.5, 312.5 µg/mL; S.aureus 19.5, 156 µg/mL; P.aeruginosa 312.5, 625 µg/mL; and C.albicans 156, 156 µg/ml. In ketamine+propofol mixture, ketamine exhibited antimicrobial activity to E.coli, P.aeruginosa and C.albicans as MBCs at 1250, 625 and 625 µg/mL, respectively. Growth of S. aureus was not inhibited in this mixture (ketamine concentration=1250 µg/mL).

CONCLUSION: Ketamine has sustained its antimicrobial activity in a dose-dependent manner against some organisms in propofol, which is a strong microbial growth-promoting solution. Combined use of ketamine and propofol in routine clinical application may reduce the risk of infection caused by accidental contamination. However, one must keep in mind that ketamine cannot reduce all pathogenic threats in propofol mixture.

Keywords: Antimicrobial activity; Benzethonium chloride; Ketamine; Ketofol; Propofol

Introduction

Propofol is a widely used sedative-hypnotic drug that is administered in the induction and maintenance of anesthesia. Propofol is considered as a good microbial growth-promoting agent due to its rich nutritional content, like soybean oil, glycerol, and egg lecithin.^1,2 Accordingly, severe infections have been reported in patients following the use of contaminated propofol.^3,4

Ketamine is a general anesthetic that has an antagonist effect on the n-methyl d-aspartate receptors. It is characterized by rapid onset of actions, including analgesia, anesthesia, elevated blood pressure, and dilatation in lower airways. Considering its favorable effects on the cardiovascular and pulmonary system, ketamine may be particularly valuable for induction of anesthesia in a hypovolemic patient.^5,6 Additionally, some studies have documented ketamine's antimicrobial activity.^7,8

Combination of ketamine and propofol (ketofol) is shown to be pharmaceutically compatible when applied in the same syringe. Several studies have reported that ketofol has positive regulatory activity on the hemodynamic parameters in human volunteers.^9-11 Regarding the high microbial growth-promoting effect of propofol and antimicrobial activity of ketamine, investigation of the total effect of their combination on the growth of some bacteria and yeast was thought to be valuable. Therefore, we conducted an in vitro study to determine the effect of ketofol mixture on some clinically important organisms that were the significant pathogens of hospital-acquired infections in the world.

Materials and methods

Drugs and microorganisms

We used ketamine (Ketalar^® 50mg/ml Pfizer), and1% propofol (Propofol^® %1 Fresenius) in this study. Drug mixtures were prepared in aseptic conditions.

In this study, we used the following standard strains: Escherichia coli (ATCC 25922) (RSHM/Turkey), Staphylococcus aureus (ATCC 29213) (Oxoid/UK), Pseudomonas aeruginosa (ATCC 27853) (Oxoid/UK) and Candida albicans (ATCC 14053) (Oxoid/UK).

Microbial-growth-promoting activity of propofol

We studied growth rates of the tested microorganisms in time-growth analyses. Briefly, we selected bacterial and fungal colonies grown on nutrient agar plates and suspended in sterile 0.9% physiologic saline at McFarland 0.5 density. These suspensions were re-suspended in propofol to adjust final concentration of organisms to 1-2x10⁴ bacteria per mL, and 4-5x10³ yeast per mL. We incubated these suspensions at 35ºC for 24 hours. In 2-hour periods, subcultures to nutrient agar mediums were performed between 0 and 24 hours. We read the number of colony-forming units (cfu/mL) grown on the plates visually by single investigator.

Antimicrobial activity of ketamine

We investigated the impact of ketamine, alone and in propofol mixture, on microbial growth rates of each organism with microdilution method according to published standards for antimicrobial susceptibility tests by Clinical and Laboratory Standards Institute (CLSI),^12,13 and previously published study by Gocmen et al.⁷ We accepted the ketamine concentration that was associated with 100% inhibition of yeast growth as MIC value for C. albicans.

Antimicrobial activity of ketamine in standard tests

Briefly, we serially diluted ketamine in 100 µL cation adjusted Mueller Hinton Broth (Himedia/India) (for bacterial strains), and in MOPS (Applichem GmbH/Germany)-buffered RPMI 1640 (Sigma/Germany) liquid medium (for candida) in sterile plysteren 96-well plates. Then, we prepared bacterial and fungal suspensions in sterile 0.9% physiologic saline at McFarland 0.5 density. We re-suspended these suspensions in their standard broths. A 100 µL volume of inoculums was distributed into each well. Final ketamine concentrations ranged from 1250 to 1.22 µg•mL^-1 in the wells with 2.5-5x10⁴ cfu•mL^-1 inoculums of bacteria, or 1-5x10³ cfu•mL^-1 inoculums of candida. After 24-h incubation at 35ºC, we determined minimal inhibitory concentrations (MIC) by visual reading. For candida strain, 100% inhibition was accepted as the MIC value regarding the drug-free inoculums control. We determined minimal bactericidal concentrations (for candida, minimal fungicidal concentration) (MBC) by performing subcultures from the wells showing inhibition of visible bacterial of fungal growth to appropriate nutrient agar mediums. We accepted the drug concentration in the well that had 99.9% inhibition of the tested strain as the MBC. We prepared control cultures for microorganisms, broth, and drug solution.

Antimicrobial activity of ketamine in propofol

We serially diluted ketamine in propofol solution in sterile plysteren 96-well plates. The bacterial and fungal suspensions prepared in sterile 0.9 % physiologic saline at McFarland 0.5 density were re-suspended in propofol, and distributed into each well as equal aliquots. Final ketamine concentrations ranged from 1250 to 1.22 µg/mL in the wells with 2.5-5x10⁴ cfu•mL^-1 inoculums of bacteria, or 1-5x10³ cfu•mL^-1 inoculums of candida. After 24-h incubation at 35ºC, we determined MBCs by performing subcultures from the wells onto the appropriate agar mediums as described above. We performed control cultures for microorganisms, propofol and drug solution.

Results

Microbial growth in propofol 1%

Fig. 1 shows growth rates of the tested strains in propofol 1% suspension. At the first two hours of the incubation, we detected no significant growth for E. coli, P. aeruginosa, and S. aureus regarding time zero. But, the concentration of C. albicans doubled (from 5x10³ cfu•mL^-1 to 1x10⁴ cfu•mL^-1) within the same interval. E. coli and P. aeruginosa reached ≥ 1x10⁵ cfu•mL^-1 concentration at 10^th and 8^th hours of incubation, C. albicans at 14^th hours, and S. aureus at 16^th hours.

Antimicrobial activity of ketamine in standard tests, and in propofol mixture

Ketamine showed an in vitro antimicrobial effect against all tested strains in standard antimicrobial susceptibility tests. We determined the lowest ketamine MIC for S. aureus as19.5 µg•mL^-1; and measured the highest MIC for E. coli and P. aeruginosa as 312.5 µg•mL^-1. Regarding the MBCs of ketamine, we detected the lowest value for S. aureus and C. albicansas 156 µg•mL^-1, and determined the highest value for P. aeruginosa as 625 µg•mL^-1.

In propofol mixture, ketamine MBCs for P. aeruginosa and C. albicanswere measured as 625 µg•mL^-1, and for E. coli 1,250 µg•mL^-1. We could not determine MBC of S. aurues (MBC > 1,250 µg•mL^-1), as no inhibition occurred.

Table 1 shows the measured MIC and MBC values of ketamine for each organism.

Discussion

In this study, we determined that propofol was a strong microbial growth promoting solution not only for bacteria but also for funghi. Considering the organisms' types, gram negatives, P. aeruginosa and E. coli, showed the fastest growth index, reaching 1x10⁵ cfu•mL^-1 concentration within 8 and 10 hours, respectively, while C. albicans reached this concentration at the 14^th hour. However, S. aureus had relatively the slowest growth rate among all tested strains (Fig. 1).

Ketamine is a drug that is primarily used for the induction and maintenance of general anesthesia. It is a core medicine which is categorized in the World Health Organization's "Essential Drug List".¹⁴ Ketamine has a wide range of effects on humans, including analgesia, anesthesia, hallucinations, elevated blood pressure, and bronchodilation. In 2008, Gocmen et al.⁷ published an in vitro study reporting that ketamine had antimicrobial activity against some streptococci, staphylococci, E. coli, and P. aeruginosa between 500-2,000 µg•mL^-1 concentrations. As for the fact that anesthetic blood level of ketamine was about 2 µg•mL^-1, they state that this antibacterial activity cannot be seen in humans during anesthesia.

We observed that ketamine had a potential antibacterial and antifungal activity on the tested strains. Regarding the organisms' types, we found P. aeruginosa and E. coli to be more resistant, and S. aureus were the most susceptible to ketamine. We detected that S. aureus had 5 logs lower MIC and 2 logs lower MBC values than the gram negatives. On the other hand, MIC and MBC values of ketamine for C. albicans were equal in this experiment.

In this study, we investigated the antimicrobial activity of ketamine in propofol mixture. This mixture has been used successfully in different clinical situations including monitored care anesthesia, electroconvulsive therapy, procedural sedation, and analgesia in emergency patients.^9-11,15 In the ketofol mixture, we detected that ketamine sustained its antibacterial and antifungal activities at higher MBCs. In our study, we could not measure MICs of ketamine in propofol, due to fact that turbid solution was formed in the microplates following the propofol inclusion, which did not allow for clear visual evaluation. Therefore, we detected only MBCs of ketamine in the ketofol mixture. Regarding the tested strains, we observed twofold increase in the MBC values of ketofol mixture for E. coli and C. albicans. However, a certain MBC of ketofol was not determined for S. aureus, due to fact that its value was over the test's detection limit. Interestingly, MBC of this mixture did not change in comparison to ketamine alone, and remained stable for P. aeruginosa at 625 µg•mL^-1.

Infection is a considerable concern during the clinical use of propofol. Particularly because of its lipid base, propofol provides a preferable media for many classes of microorganisms. Therefore, nosocomial postoperative infections which impose heavy burden of morbidity and mortality and have serious economic consequences can develop due to the contamination of propofol.¹⁶ Mueller et al.⁴ reported an outbreak of sepsis caused by gram negative organisms including Klebsiella pneumoniae and Serratia marcescens in 7 patients, due to contaminated propofol use in minor surgical procedures. Similarly, Henry et al.¹⁷ reported postoperative bacteremia and wound infections caused S. marcescens, following the propofol use in Canada. Additionally, Bennett et al.¹⁸ reported propofol-related infections including bloodstream infections, surgical site infection, and acute febrile episodes in 62 cases, after surgical procedures in seven US hospitals. They identified S. aureus, C. albicans and gram-negative bacteria such as morexella, enterobacter and serratia species to be responsible for these infections. In all these studies, the authors emphasized that extrinsic contamination of propofol, as a result of the lapses in aseptic preparation, handling and storage of this drug, caused these life-threatening infections.

Center of Disease Control and Prevention suggested safe medication practices, including avoiding the use of syringes on multiple patients as well as avoiding single-use medication vials for multiple patients, and strictly adhering to aseptic techniques and infection control practices during propofol application.¹⁹ Additionally, in order to reduce the incidence of propofol-related postoperative infection, antimicrobial preservative-containing (i.e., EDTA- or Na-metabisulphide-containing) emulsions have been manufactured according to discussion with the Food and Drug Administration Agency (FDA). Such formulations are used in the United States today; nevertheless, preservative-free propofol solutions are still being marketed in Europe and in other areas of the world. Jansson et al.¹⁶ reported that the incidence of propofol-related infection reduced from 39 to 9 infections per year in the U.S., after onset of EDTA-containing propofol use in 1996. However, as the infection problem with propofol continued despite of preservative inclusion, they have underlined that addition of EDTA is only an additional safety precaution. Hence, the practitioner must regard to good aseptic practice in any occasion during propofol medication. In this study, we tested preservative-free emulsion of propofol, which was also used in our hospital. Though antimicrobial-added propofol is also being marketed in our country, preservative-free forms are widely preferred possibly due to economic concerns.

Propofol and ketamine are unlicensedly mixed as 1:1 volumes before clinical application, achieving 5 mg•mL^-1 ketamine in this mixture. Since Gocmen et al.⁷ reported that blood levels of ketamine were too low to not to show any antimicrobial effect in the body, we investigated whether it could prevent the microbial growth in propofol. In this study, we observed that MBCs of ketamine in ketofol mixture were between 625 and 1,250 µg•mL^-1 (> 1,250 µg•mL^-1 for S. aureus). Therefore, we thought that ketamine might be useful to reduce growth of some bacterial and fungal pathogens in propofol before application.

In this study, we found that particularly gram-negative bacteria rapidly grew in propofol solution. These results are interestingly parallel with the data that is previously reported from the outbreak studies.^3,4,17,18 We thought that this selective promotion of propofol for the gram-negatives might explain why such bacteria would be the leading pathogens of the nosocomial outbreaks related to propofol use.

In this study, we showed that inclusion of ketamine into propofol might reduce bacterial and fungal growth in this solution and, consequently, provide a safe anesthetic medication for surgical approaches. However, ketamine's activity may vary according to type of organism. Therefore, regardless of this safeguard, we underlined that strict hygienic measurements must be taken in any occasion of propofol use, according to the recommendations of the authorities.

Conflicts of interest

The authors declare no conflicts of interest.

References

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  World Health Organization. WHO Model List of Essential Medicines. 17th List. 2011. Disponível em: http://whqlibdoc.who.int/hq/2011/a95053_eng.pdf
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*

Estudo realizado na Faculdade de Medicina, Universidade Inonu, Malatya, Turquia.

Publication Dates

• Publication in this collection
  18 Dec 2013
• Date of issue
  Dec 2013

History

• Received
  09 Aug 2012
• Accepted
  03 Sept 2012