Propofol is widely used for induction and maintenance of anaesthesia, and for sedation in intensive care units (ICU) . Recently, propofol has been used for new therapeutic indications such as traumatic head injury, status epilepticus, delirium tremens and asthma . Propofol is formulated in an emulsion, which contains soybean oil, glycerol and egg lecithin. This formulation is well known to support bacterial growth in extrinsically contaminated propofol. Contamination is associated with post surgical infections including outbreaks of blood and surgical site infections and acute febrile episodes [3-5]. In order to prevent or minimize such infections, propofol is formulated with ethylenediaminetetraacetic acid (EDTA), as an antibacterial agent . The effectiveness of EDTA on bacterial growth is still controversial [7,8] and the available formulation of propofol differs in different parts of the world.
Propofol injection into peripheral veins is associated with high incidence of pain ranging from 28% to 90% . Lidocaine and diphenhydramine have been used successfully to prevent propofol injection pain . There are studies in the literature that assess the bactericidal properties of both lidocaine and diphenhydramine [11,12]. To our best knowledge, there is no report to date about the antibacterial activity of diphenhydramine when added to propofol. This laboratory study was designed to evaluate the effects of diphenhydramine on bacterial growth when added to extrinsically contaminated propofol emulsions.
The organisms used were clinical (hospital) isolates or standardized typed organisms from the American Type Culture Collection (ATCC) as follows: Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (clinical isolate) Escherichia coli (ATCC 35218) and Candida albicans (clinical isolate). Several colonies of each isolate were added to blood agar culture medium and incubated at 35°C for 24 h. Overnight cultures of S. aureus, E. coli, P. aeruginosa and C. albicans were diluted to a density of 0.5 McFarland units with 0.9% sterile non-bacteriostatic saline. Then each organism solution underwent serial dilutions with 0.9% saline until the final concentration of bacteria was 4 × 105 colony-forming units (CFU) mL−1.
The diphenhydramine (Allenik® 1%; Galen, Turkey) and propofol (Pofol® 1%; Dongkook pharmaceutical Co., South Korea) test mixtures were prepared under aseptic conditions. The study organisms were inoculated into the following solutions: 1% propofol, 0.3% diphenhydramine + 1% propofol, 0.2% diphenhydramine + 1% propofol, 0.1% diphenhydramine + 1% propofol, 0.05% diphenhydramine + 1% propofol, 1% diphenhydramine and 0.1% lidocaine + 1% propofol.
A 100 μL of inoculum suspension adjusted for each of the micro-organisms was added separately to each tube and then mixed gently and left at room temperature (20°C). A 10-μL aliquot of each mixture was inoculated onto blood agar medium at 5 and 24 h (5% blood agar; Merck). These plates were incubated at 35°C for 24 h. Each plated medium was read and the number of CFU were counted and recorded.
For each organism, a bactericidal effect was defined as detecting no CFU's and a bacteriostatic effect as no statistical difference in CFU count increase between 5 and 24 h. Two replicates were performed with each organism to complete the study.
Analysis of variance (ANOVA) with a post hoc Tukey HSD test and paired t-tests were used as appropriate. A probability of P < 0.05 was taken to indicate statistical significance.
The number of CFU of the four organisms 5 and 24 h after inoculation is shown in Table 1. There were significant increases in CFU in all organisms except for C. albicans in 1% propofol group 24 h after inoculation when compared with 5 h (P < 0.05). There were progressive reductions in colony counts of all micro-organisms with the increasing concentration of diphenhydramine 24 h after inoculation. Diphenhydramine 1% was bactericidal for all micro-organisms both at 5 and 24 h. The number of CFU was significantly lower for all micro-organisms in 0.1% diphenhydramine + 1% propofol group when compared with 0.1% lidocaine + 1% propofol group at 24 h (P < 0.05).
Table 2 shows the bacteriostatic and bactericidal concentrations of diphenhydramine for the micro-organisms tested. Diphenhydramine 0.1% was bacteriostatic for all of micro-organisms except S. aureus. Bacteriostatic minimal concentration of diphenhydramine was 0.3% for S. aureus.
The results of this study show that propofol supports growth of E. coli, P. aeruginosa and S. aureus at 24 h. Diphenhydramine inhibits bacterial growth in propofol solutions in a dose-dependent manner. At concentration of 1%, diphenhydramine is bactericidal. At similar concentration diphenhydramine is more effective than lidocaine 0.1% in preventing growth of all organisms.
Postoperative nosocomial infections are known to increase patient morbidity and mortality, increasing healthcare costs and reducing hospital management efficiency . It is known that propofol emulsion is an excellent vehicle for supporting the growth of several micro-organisms, including S. aureus, P. aeruginosa, E. coli and C. albicans [3,4,13]. In this study, 1% propofol supported the growth of all organisms except for C. albicans. The increases in CFU's were statistically significant for all organisms except for C. Albicans. Similar to our results previous studies which used blood agar medium also showed slight increase in colony counts [7,14]. The apparent lack of growth of C. albicans may be due to the use of blood agar medium instead of Sabaroud's dextrose agar medium, which is known to be more sensitive for C. albicans growth . The other reason for the lack of growth of C. albicans may be the use of a small amount of inoculum in our study .
In the ICU, emulsions of propofol prepared daily for sedation purposes are exposed to ambient temperatures for up to 24 h. Because of daily prepared syringes, patients in the ICU are at risk of accidental extrinsic contamination of propofol. In spite of repeated warnings and clear safety recommendations, poor aseptic technique is commonplace among healthcare workers in the hospital environment , and there seems to be poor compliance with data sheet recommendations for the use of propofol . Moreover, there are some case reports of infections due to extrinsically contaminated propofol in the literature [5,18]. In an outbreak of S. aureus, bloodstream infection in electroconvulsive therapy is an example of this situation. Nine patients received propofol drawn from the same vial at the same time. The first four remained asymptomatic, while the last five developed S. aureus bloodstream infections and one of them died. Possible extrinsic contamination of propofol was implicated .
In order to reduce the risk of such bacterial contamination, EDTA, which has antimicrobial activity, was added to propofol emulsions introduced to the US market. But effectiveness of this agent is still controversial [7,8], and in some countries propofol is marketed without EDTA. In the present study, propofol solution consisted of diisopropylphenol (10 mg mL−1), in soybean oil (100 mg mL−1), glycerol (22.5 mg mL−1) and egg lecithin (12 mg mL−1) which does not contain EDTA.
Antimicrobial actions of local anaesthetics such as lidocaine are well known  and the addition of lidocaine to propofol for the relief of pain is a common anaesthetic practice . There are previous studies [7,20] that assess the probable effectiveness of adding lidocaine to propofol to prevent bacterial growth. Recommended maximal concentration of lidocaine that does not lead to destabilization of propofol emulsion is 0.1%. But, at this concentration lidocaine is inadequate to prevent bacterial growth . In the present study, 0.1% diphenhydramine was more effective than 0.1% lidocaine in reducing bacterial growth at 24 h. This study also studied higher concentrations of diphenhydramine (0.2%, 0.3% and 1%) and found increasing concentrations to be more effective.
Diphenhydramine is traditionally used as an H1 antagonist with well-known local anaesthetic properties . Diphenhydramine is also known to have antibacterial properties and inhibits the growth of E. coli at a concentration of 0.18%, of S. aureus hemolyticus at 0.39% and of P. aeruginosa at 0.75% . The present study is a laboratory study and the results show that 1% diphenhydramine is bactericidal for S. aureus, E. coli, P. aeruginosa and C. albicans strains and bacteriostatic activity is present at 0.1% and higher concentrations. These concentrations may be achieved by mixing diphenhydramine/propofol in at least a 1/9 ratio. Our previous study showed that intravenous injection of diphenhydramine (pretreatment) before propofol injection can be used as an alternative to lidocaine in reducing propofol injection pain . Because the chemical compatibility of diphenhydramine with propofol was unknown in the previous study, we did not mix the two drugs in the same syringe. We believe that further pharmacological studies on compatibility and safety are needed before mixing the two drugs for antibacterial purposes.
The probable mechanism for the inhibition of bacterial growth with diphenhydramine is unknown. Semenitz  showed that the addition of 0.2% diphenhydramine to a broth with growing bacteria interfered very quickly with cell metabolism and stopped further reproduction. In another study  on phenothiazine, antihistaminics such as trimeprazine, promethazine and fonazine, the antibacterial activity was found to correlate with both the rate of absorption of these drugs on the bacterial surface tension of their solutions. The above-mentioned mechanisms may be involved with the bacteriostatic and bactericidal effects of diphenhydramine in our study.
In conclusion, diphenhydramine inhibits bacterial growth in extrinsically contaminated propofol solutions. It may be considered as an adjuvant for propofol emulsions but chemical compatibility of the two drugs needs to be studied before clinical use.
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