Today in most German hospitals various ready-to-use or ready-to-administer parenteral products are prepared in the hospital pharmacy departments. The portfolio of products encompasses cytotoxic as well as non-cytotoxic preparations. Typical non-cytotoxic product types are parenteral nutrition solutions, electrolytes, emergency drugs, analgesics, antibiotics, and antifungals. But the pharmacy based preparation service is never all-encompassing and a lot of preparations are still to be reconstituted in clinical areas. The quality-assured aseptic processing within pharmacies is performed following national (e. g. Apothekenbetriebsordnung , ADKA Guideline ) and international regulations and guidelines (PIC/S PE 010-3 , Ph.Eur.7.7 Monograph Pharmaceutical Preparations , Council of Europe Resolution CM/ResAP (2011)1 , USP Monograph <797> Pharmaceutical Compounding – Sterile Preparations ). By pharmacy-based aseptic processing in well-controlled environments and quality assurance driven procedures the risk of preparation errors and microbial contamination is considerably reduced. Sterility tests of extemporaneously and batchwise prepared products are limited by singularity of each preparation and time restraints. However the results of sterility tests, media fills, and environmental monitoring programs are useful for process validation. Preparation in uncontrolled environments such as clinical areas is associated with a higher potential for microbiological contamination and an increased risk of systemic infection after administration. For the individual product types different factors influencing the risk of microbial contamination and infection are discussed. The specific factors are complexity of the preparation, time between preparation and administration, extended periods of administration, elevated temperatures during administration by infusers and other ambulatory devices and last not least the growth potential of the preparation. Multiple additions of multiple components, mixing of large volumes and growth promoting qualities of the mixed components increase the risk of microbial contamination of the preparations and the risk of infection for the patients. If preparations are contaminated with microorganisms, viability and growth of the specific microorganisms in the specific preparations determine the infection risk for the patient. Previously we investigated the growth of microorganisms in antineoplastic drug preparations and found out that most preparations lack antimicrobial properties and monoclonal antibody preparations do not stimulate the growth of microorganisms [7–11]. From the literature it is known, that lipid emulsions support bacterial and fungal growth and bear an increased risk for iatrogenic infections [12–20]. Species-specific growth inhibition is reported for heparin 100 U/mL , midazolam  and local anaesthetics . Recently the lack of growth inhibition of some vasopressor infusion solutions was published . For a number of parenteral products prepared in our central intravenous additive service information about growth promotion or inhibition is missing. Moreover tests are done under different experimental conditions and extrapolation of the results is hardly possible. Therefore the aim of the study was to evaluate the ability of four different pathogens related to hospital infections (Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecium, Candida albicans) to grow in 17 ready-to-use solutions typically prepared in the aseptic preparation unit of the Pharmacy Department of the University Medical Center Mainz. The chosen test conditions promoted the growth of germs and simulated daily practice.
Material and methods
The viability of the selected microbes was tested by inoculation of 17 non-cytotoxic ready-to-use aseptic preparations belonging to the portfolio of the pharmacy based aseptic preparation service. The experiments were carried out in four consecutive series. In the first series the antimicrobial activity of caspofungin 35 mg or 70 mg in 250 mL 0.9 % NaCl solution (NS), micafungin 50 mg in 100 mL 0.9 % NS, heparin sodium 1 IE/mL in NS and 50 % w/v glucose solution was tested. In the second series adrenaline (epinephrine) 0.02 mg/mL in G5, noradrenaline (norepinephrine) 0.01 mg/mL in G5, trace elements in G5 1:1, and vancomycin 5 mg/mL in G5 were examined. In the third series trace elements 1:1 in G10, midazolam1 mg/mL, lipid emulsion 200 mg/mL and vancomycin 5 mg/mL in G10 were inspected. In the fourth series phenylephrine 0.1 mg/mL, 0.8 mmol/mL potassium chloride solution, 1 % propofol injection emulsion, and tranexamic acid injection solution 100 mg/mL was tested. Details about the material used and characteristics of the test solutions are given in Table 1.
Pure vehicle solutions (0.9 % NaCl solution infusion solution 100 mL (Free Flex Isotonische KochsalzLösung NaCl 0.9, Fresenius Kabi, lot 13HLS251, expiration date 10/2016), glucose 5 % infusion solution and glucose 10 % infusion solution prepared from glucose 70 % (Glucose 70 %, 500 mL, B Braun, lot 144238062), water for injection (Ampuwa® 1,000 mL, Fresenius Kabi, lot 14H129, expiration date09/2017)) were used as control solutions.
Preparation of inocula
The microorganisms used in this study were Staphylococcus aureus ATCC strain 6538, Pseudomonas aeruginosa ATCC strain 15442, Enterococcus faecium ATCC strain 6057, and Candida albicans ATCC strain 10231. The strains were cultivated at the Institute of Medical Microbiology and Hygiene of the University Medical Center Mainz, Germany and cultured on agar plates (BD Trypticase™ Soy Agar, BD Medical, Heidelberg, Germany, Lot 5019173, expiration date 12/04/2015) at 37 °C for 24 h in the case of bacteria. C. albicans was cultured for 72 h at 37 °C. The cultures were collected and suspended in 0.9 % NaCl solution. After that the density of bacterial suspensions was adjusted by using McFarland standards (0.5 for S. aureus and E. faecium, 0.2 for P. aeruginosa and 2.5 for C. albicans) to obtain suspensions of approximately 108 CFUs per milliliter. To achieve suspensions with a density of 105 CFU/mL, the suspensions were diluted with 0.9 % sodium chloride solution.
Preparation of samples and analysis
The tested product solutions were aseptically prepared in the pharmacy based centralized aseptic preparation unit at the University Medical Center Mainz, Germany in a cleanroom environment following Good Preparation Practice Guidelines. Physical and chemical stability of the preparations is proven for at least 5 days (see Table 1). Nine milliliters of each freshly prepared test solution were aseptically transferred in quadruplicate to a sterile empty 15 mL centrifuge tube with screw cap (VWR International Randor PA, USA, Lot No-827CB-27C) and inoculated with 1 mL suspension of bacteria or fungus (S. aureus, P. aeruginosa, E. faecium, C. albicans) to achieve a concentration of 104 CFU/mL. The inoculated test solutions were stored at 22 °C. An aliquot of one mL was withdrawn immediately and at predetermined time intervals (1, 3, 5, 24, 48, and 144 h) and diluted 1:10 three times by using tubes prefilled with 0.9 % NaCl solution (NaCl (phys.) 9 mL 2084r-100p, Heipha Dr. Müller GmbH, lot 125309, expiration date 16/10/2014 or lot 129336,expiration date 02/08/2015). From each degree of dilution, 0.1 mL aliquots were withdrawn and transferred to tryptic soy agar plates (BD Trypticase™ Soy Agar, BD Medical, Heidelberg, Germany, lot 5019173, expiration date 12/04/2015, and BD BBL™ Stacker ™ Plates, lot 5027214, expiration date19/04/2015) in duplicate (n=6). The plates were incubated at 37 °C and the colony forming units counted after 24 h of incubation for bacteria and 72 h for C. albicans. The 6 results counted for each species and time interval were checked for plausibility. A representative value was selected and given as CFU log/mL in table format. For each combination of the 17 aseptically prepared products and the 4 different microorganisms the growth curve was constructed by plotting the number of CFUs per milliliter (expressed as logarithm) against the time interval post inoculation.
Most of the tested aseptic preparations affected the growth of the test organisms in the same manner as the control solutions (water for injection, NS, G5, G10). The number of CFUs remained unchanged (E. faecium, C. albicans), decreased (S. aureus) or increased (P. aeruginosa) over a period of five days (see Tables 2–5).
These results reflect the species-specific capability of the microorganisms to survive and grow in nutrient-deficient solutions. However, the tested microorganisms lost viability in preparations containing vancomycin, phenylephrine, and midazolam after a period of a few hours or few days (see Figures 1 and 2).
C. albicans rapidly lost viability in caspofungin and micafungin containing test solutions (see Figure 3). Thereby the proven antimicrobial activity of vancomycin and the echinocandins, which are used as an antibiotic and antifungals, respectively, confirmed the validity of the experimental design and the results.
Species-specific antibacterial activity was observed in tranexamic acid solutions. Only P. aeruginosa lost viability 48 h post inoculation, while the other strains tested remained viable. Glucose 50 % w/v solution also generated species-specific antibacterial activity against P. aeruginosa. Antifungal activity of glucose 50 % against C. albicans got already obvious 1 hour post inoculation. Caspofungin and micafungin exhibited strong antifungal activity as expected. Noteworthy, caspofungin containing test solutions in both concentrations exhibited antibacterial activity against S. aureus and E. faecium. Viability of P. aeruginosa was not affected (see Figure 3). Moreover, micafungin did not inhibit bacterial growth. In some of the test solutions C. albicans tends to grow (compare Table 5).
The lipid containing formulation of 1 % propofol emulsion and the 20 % SMOFlipid emulsion served as nutritive media for all selected microorganisms and the number of CFUs increased rapidly (see Figure 4).
Viability and growth of microorganisms in pharmaceutical preparations is directed by extrinsic (e. g. temperature, oxygen) and intrinsic factors (e. g. type and concentration of ingredients). It is well known that refrigeration retards the growth of microorganisms. The experiments were performed to increase our knowledge about the intrinsic factors of ready-to-use parenteral preparations compounded in the pharmacy department except the drugs used in anticancer therapy. The portfolio of products encompasses different indications, e. g. parenteral nutrition, catecholamines, antibiotics, antifungals, anaesthesia and the use in different patient groups (paediatric patients, intensive care patients, operating theatre patients). These preparations contain different active substances and excipients and have different physicochemical characteristics. The experimental conditions of the intrinsic factors were chosen according to clinical practice. All preparations contained liquid water and some of them contained nutrient sources, minerals or trace elements. The extrinsic factors were the same throughout the study. As a reasonable compromise of typical temperature conditions in clinical practice and optimum growth temperature for pathogenic bacteria (37 °C) the experiments were carried out at room temperature (22 °C). The inoculum size was kept unchanged and simulated low level contamination. The experiments were performed in duplicate for each ready-to-use preparation at each time interval. Each sample was tested in three degrees of dilution (in total 6 experiments). This allows the detection of the influence of the CFU concentration and of process errors. Because these are biological experiments, not the average but a representative value was chosen to be presented as result.
The growth inhibition detected in some preparations is not related to the active substance, but to the physicochemical parameters such as pH and osmolality. Bacterial growth is inhibited by low pH, while the optimum pH for the growth of most fungi is pH 5. Therefore, low pH values are most probably the reason for growth inhibition in midazolam injection solutions [20, 22]. The pH values of diluted vancomycin infusion solutions amount to pH 3–5. That might be the reason for the growth inhibition of P. aeruginosa and C. albicans recognized in our experiments.
The phenylephrine injection preparation (pH 5) contains citric acid and sodium metabisulfite. The comparatively high concentration of sodium metabisulfite (2 mg/mL) reduces the redox potential of the phenylephrine preparations, what may explain the observed antimicrobial activity. This assumption is confirmed by the fact that inhibition of microbial growth did not occur in the adrenaline and noradrenaline containing preparations containing small amounts of sodium metabisulfite (0.072 mg/mL). Similar findings were reported by Bostan et al. . Among the tested catecholamine preparations only dobutamine preparations showed antimicrobial activity because of high sodium metabisulfite concentrations compared to adrenaline and noradrenaline preparations . Notably, the stimulation of bacterial growth by catecholamines [25, 26] is eliminated by the antioxidative excipients in the medicinal products. High osmolality is also known to inhibit microbial growth what explains the antimicrobial of glucose 50 % preparations [13, 27]. Antifungal activity of glucose 50 % against C. albicans got already obvious 1 hour post inoculation and is commonly known.
To our knowledge there is no information available about the antimicrobial activity of tranexamic acid containing preparations. The fact that growth inhibition was only given for P. aeruginosa suggests that the activity is substance specific.
According to the studies of Rosett et al. the antimicrobial activity of heparin is a result of the reduction of divalent cations from the growth media . The lack of antimicrobial activity of the heparin 1 IE/mL containing preparations is to be explained by the low heparin concentration and the experimental conditions used. Obviously the low amount of heparin in our preparations was insufficient to bind the cations essential for bacterial growth. During in vivo experiments also high concentrations of heparin sodium lacked antibacterial activity against S. aureus . The same explanation is applicable for the missing antimicrobial activity of the 0.8 molar potassium chloride preparations. Different concentrations and experimental designs lead to inconsistent results [29, 30].
Fat emulsions like 1 % propofol injection and 20 % lipid emulsion are known to serve as growth medium for a number of microorganisms and as origin of bloodstream infections [31, 32]. The lack of antimicrobial activity is related to the high value of pH (7–8.5) and the high lipid content serving as nutrient source. Fat emulsion serves as an excellent non-nitrogen energy source for a number of microorganisms, including bacteria and fungi. When microbial growth in five different commercially available lipid emulsions was tested no difference in growth patterns due to the nature of the oil or its concentration was observed . SMOFlipid 20 % consists of fish oil, soybean oil, medium chain triglycerides, and olive oil and supported the growth of the selected bacteria and yeast in a similar manner. In both settings the number of CFUs rapidly increased to ≥106 over a period of less than 24 h after inoculation. Pure fat emulsions and lipid containing total parenteral nutrition solutions are the most vulnerable preparations and should always be prepared under strict aseptic conditions and should not be stored or infused more than 12 h after preparation [33, 27].
The species-specific growth promoting activity of the trace elements mixed with glucose solutions is not yet reported in the literature. But plausibility is given as P. aeruginosa and C. albicans grow in nutrient deficient solutions and microorganisms require also trace elements for their growth .
As most of the tested parenteral preparations did not generate antimicrobial activity, preparation should be done under strict aseptic conditions in order to avoid any microbial contamination. Furthermore the insufficient antimicrobial properties of ready-to-use solutions should be considered while determining the shelf-life of the products. Lipid containing preparations should be kept refrigerated whenever possible to inhibit the multiplication of any contaminating organism.
We are thankful to T. Brand and Dr. W. Kohnen at the Institute for Microbiology and Hygiene, University Medical Center, Johannes Gutenberg-University, Mainz for their advice and support.
1. Verordnung über den Betrieb von Apotheken, Apothekenbetriebsordnung (ApBetrO) Juni 2012. Available from: http://www.bgbl.de/Xaver/start.xav?startbk=Bundesanzeiger_BGBl&start=//*[@attr_id=‘bgbl112s1254.pdf‘], latest access May 2015.
2. Herbig S, Vanessa K, Jürgen M, Lenka T, Judith T, Irene K. ADKA-Leitlinie: Aseptische Herstellung und Prüfung applikationsfertiger Parenteralia. Version vom 12.12.2012. Krankenhauspharmazie 2013;34:93–106. Google Scholar
3. Pharmaceutical Inspection Convention/Pharmaceutical Inspection Cooperation Scheme: PIC/S Guide to good practices for preparation of medicinal products in healthcare establishments – PE 010-4 (March 2014). Available from:http://www.picscheme.org.
7. Karstens A, Krämer I. Viability of micro-organisms in novel anticancer drug solutions. EJHP Sci 2007;13:27–32. Google Scholar
8. Krämer I. Viability of microorganisms in novel antineoplastic and antiviral drug solutions. J Oncol Pharm Pract 1998;4:32–7.Google Scholar
9. Krämer I, Wenchel H. Wachstumsverhalten ausgewählter Mikroorganismen in Zytostatika-Zubereitungen. Krankenhauspharmazie 1988;11:439–42.Google Scholar
10. Krämer I, Wenchel H. Viability of microorganisms in antineoplastic drug solutions. Eur J Hosp Pharm 1991;1:14–19.Google Scholar
11. Sarakbi I, Federici M, Krämer I. Viability of microorganisms in in novel chemical and biopharmaceutical anticancer drug solutions. Eur J Parent Pharm Sci 2015;20:5–12. Google Scholar
12. Crocker KS, Noga R, Filibeck DJ, Krey SH, Markovic M, Steffee WP. Microbial growth comparisons of five commercial parenteral lipid emulsions. JPEN J Parenter Enteral Nutr 1984;8:391–5. Google Scholar
13. Gilbert M, Gallagher SC, Eads M, Elmore MF. Microbial growth patterns in a total parenteral nutrition formulation containing lipid emulsion. JPEN J Parenter Enteral Nutr 1986;10:494–7. Google Scholar
14. Reiter P. Sterility of intravenous fat emulsion in plastic syringes. Am J Health Syst Pharm 2002;59:1857–9.Google Scholar
15. Sosis MB, Braverman B, Villaflor E. Propofol, but not thiopental, supports the growth of Candida albicans. Anesth Analg 1995;81:132–4.Google Scholar
16. Berry C, Gillespie T, Hood J, Scott N. Growth of micro‐organisms in solutions of intravenous anaesthetic agents. Anaesthesia 1993;48:30–2.Google Scholar
17. Kuwahara T, Shimono K, Kaneda S, Tamura T, Ichihara M, Nakashima Y. Growth of microorganisms in total parenteral nutrition solutions containing lipid. Int J Med Sci 2010;7:101–9.Google Scholar
18. Avila-Figueroa C, Goldmann DA, Richardson DK, Gray JE, Ferrari A, Freeman J. Intravenous lipid emulsions are the major determinant of coagulase-negative staphylococcal bacteremia in very low birth weight newborns. Pediatr Infect Dis J 1998;17:10–17.Google Scholar
19 Graystone S, Wells MF, Farrell DJ. Do intensive care drug infusions support microbial growth? Anaesth Intensive Care 1997;25:640–2.Google Scholar
20. Farrington M, McGinnes J, Matthews I, Park G. Do infusions of midazolam and propofol pose an infection risk to critically ill patients? Br J Anaesth 1994;72:415–17. Google Scholar
21. Rosett W, Hodges GR. Antimicrobial activity of heparin. J Clin Microbiol 1980;11:30–4. Google Scholar
22. Ayoglu H, Kulah C, Turan I. Antimicrobial effects of two anaesthetic agents: dexmedetomidine and midazolam. Anaesth Intens Care 2008;36:681–4. Google Scholar
23. Johnson SM, Saint John BE, Dine AP. Local anesthetics as antimicrobial agents: a review. Surg Infect 2008;9:205–13. Google Scholar
24. Bostan H, Tomak Y, Karaoglu SA, Erdivanli B, Hanci V. In vitro evaluation of antimicrobial features of vasopressors. Braz J Anesthesiol 2014;64:84–8. Google Scholar
25. Lyte M, Ernst S. Catecholamine induced growth of gram negative bacteria. Life Sci 1992;50:203–12. Google Scholar
26. Neal CP, Freestone PP, Maggs AF, Haigh RD, Williams PH, Lyte M. Catecholamine inotropes as growth factors for Staphylococcus epidermidis and other coagulase-negative staphylococci. FEMS Microbiol Lett 2001;194:163–9. Google Scholar
27. Austin PD, Hand KS, Elia M. Factors influencing Escherichia coli and enterococcus durans growth in parenteral nutrition with and without lipid emulsion to inform maximum duration of infusion policy decisions. JPEN J Parenter Enteral Nutr 2015; 39:953–65. doi: . Epub 2014 Jun 25. CrossrefWeb of ScienceGoogle Scholar
28. Capdevila JA, Gavalda J, Fortea J, Lopez P, Martin MT, Gomis X, et al. Lack of antimicrobial activity of sodium heparin for treating experimental catheter-related infection due to Staphylococcus aureus using the antibiotic-lock technique. Clin Microbiol Infect 2001;7:206–12. Google Scholar
30. Highsmith AK, Greenhood GP, Allen JR. Growth of nosocomial pathogens in multiple-dose parenteral medication vials. J Clin Microbiol 1982;15:1024–8. Google Scholar
31. Bennett SN, McNeil MM, Bland LA, Arduino MJ, Villarino ME, Perrotta DM, et al. Postoperative infections traced to contamination of an intravenous anesthetic, propofol. N Engl J Med 1995;333:147–54. Google Scholar
32. Scott EM, Gorman SP, Wyatt TD, Magill EA. Growth of microorganisms in total parenteral nutrition mixtures and related clinical observations. J Clin Pharm Ther 1985;10:79–88. Google Scholar
33. Crocker KS, Noga R, Filibeck DJ, Krey SH, Marcovic MS, Steffee WP. Microbial growth comparisons of five commercial parenteral lipid emulsion. JPEN J Parenter Enteral Nutr 1984;8:391–5. Google Scholar
34. Weinberg ED. Infectious diseases influenced by trace element environment. Ann N Y Acad Sci. 1972;199:274–84. Google Scholar
36. Tsiouris M, Ulmer M, Yurcho JF, Hooper KL. Stability and compatibility of reconstituted caspofungin in select elastomeric infusion devices. Int J Pharm Compd 2010;14:436–9. Google Scholar
38. Wright A, Hecker J. Long term stability of heparin in dextrose‐saline intravenous fluids. Int J Pharm Pract 1995;3:253–5. Google Scholar
39. Quay I, Tan E. Compatibility and stability of potassium chloride and magnesium sulfate in 0.9 % sodium chloride injection and 5% dextrose injection solutions. Int J Pharm 2000;5:323–4. Google Scholar
40. Briot T, Vrignaud S, Lagarce F. Stability of micafungin sodium solutions at different concentrations in glass bottles and syringes. Int J Pharm 2015;492:137–40. Google Scholar
41. Pramar Y, Loucas V, El-Rachidi A. Stability of midazolam hydrochloride in syringes and i.v. fluids. Am J Health Syst Pharm 1997;54:913–15. Google Scholar
42. Gupta VD, Stewart K, Nohria S. Stability of vancomycin hydrochloride in 5% dextrose and 0.9% sodium chloride injections. Am J Health Syst Pharm 1986;43:1729–31. Google Scholar
About the article
Iman Sarakbi received her diploma degree in pharmacy and pharmaceutical chemistry in 2007 from the faculty of pharmacy, Al-Baath University, Homs, Syria. She is currently preparing her PhD thesis at the Pharmacy Department of the University Medical Center, Johannes Gutenberg-University, Mainz, Germany on “Stability and compatibility of selected novel ready-to-use cytotoxic preparations”. Her research interests include the stability and compatibility of admixtures of drug-loaded beads and different types of non-ionic contrast media as well as the viability of microorganisms in cytotoxic or non-cytotoxic parenteral solutions, which are aseptically prepared in pharmacy-based aseptic preparation units.
Rita Marina Heeb studied pharmacy at Johann Wolfgang Goethe University in Frankfurt, Germany. Since 2012 she is Head of Quality Control at the Department of Pharmacy of Johannes Gutenberg-University Medical Center Mainz, Germany. She has completed her doctoral thesis in Clinical Pharmacy entitled: “Compliance – and Quality of life measurements at dialysis and liver cirrhosis patients before transplantation” at Johannes-Gutenberg-University Mainz. Her research interests include monitoring of medication compliance and physicochemical stability of pharmaceuticals.
Judith Thiesen is working at the Pharmacy Department of the University Medical Center of Johannes Gutenberg-University Hospital in Mainz since 1997. In 2001 she completed her doctoral thesis entitled: Evidence-based optimization of parenteral drug application for oncological patients: incompatibilities-reducing infusion schemes, stability of ready-to-use parenteral solutions of camptothecin-derivatives and taxanes. Her special interests and research projects include aseptic drug preparation, quality control, total quality management as well as physicochemical and microbiological stability of parenteral drug solutions.
Irene Krämer is currently Director of the Pharmacy Department, University Medical Center, Johannes Gutenberg-University Hospital, Mainz and is also a Professor for clinical pharmacy at the Pharmacy School of Johannes Gutenberg-University. She completed her postdoctoral thesis in Pharmaceutical Technology entitled: Development, quality assurance, and optimization of ready-to-use parenteral solutions in the integrated cancer care concept. Her special interests include oncology pharmacy, infectious diseases, and aseptic drug preparation. She is doing research projects in the field of physicochemical and microbiological stability of cytotoxic drugs, compatibility of admixtures of nebulizer solutions, and monitoring of medication compliance.
Published Online: 2016-02-06
Published in Print: 2016-03-01
Conflict of interest statement: The authors state no conflict of interest. All authors have read the journal’s Publication ethics and publication malpractice statement available at the journal’s website and hereby confirm that they comply with all its parts applicable to the present scientific work.