In vitro evaluation of antimicrobial features of vasopressors

Abstracts

Background:

Drugs administered as intravenous infusion may be contaminated during several stages of production or preparation. However studies focusing on antibacterial effects of vasopressor drugs are very rare. This study investigates the in vitro antimicrobial activity of the clinically used forms of vasopressors.

Materials and methods:

In vitro antimicrobial activities of vasopressor drugs of different concentrations were investigated by using the micro dilution technique. Microorganisms used in the test were Escherichia coli ATCC 25922, Yersinia pseudotuberculosis ATCC 911, Pseudomonas aeruginosa ATCC 10145, Listeria monocytogenes ATCC 43251, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 25923, Bacillus cereus 702 Roma, Mycobacterium smegmatis ATCC607, Candida albicans ATCC 60193, and Saccharomyces cerevisiae RSKK 251. Antibacterial assays were performed in Mueller-Hinton broth at pH 7.3 and antifungal assays were performed in buffered Yeast Nitrogen Base at pH 7.0.

Results:

Two different dopamine preparations showed antimicrobial activity. No other study drug showed any antimicrobial activity.

Conclusions:

In our opinion, dopamine's antibacterial effects may be advantageous for inhibiting the spread of bacterial contamination during the preparation of the infusion solutions. However, it is important that strict guidelines regarding the need for sterile equipment and deliverables be adhered to during all procedures performed in the intensive care units.

Antimicrobial activities; Vasopressor drugs; Drug contamination

Justificativa e objetivo:

os medicamentos administrados como perfusão intravenosa podem ser contaminados durante as várias etapas de produção ou preparação. No entanto, estudos sobre os efeitos antibacterianos de vasopressores são muito raros. Este estudo investiga a atividade antimicrobiana in vitro das formas de vasopressores usados clinicamente.

Materiais e métodos:

atividades antimicrobianas in vitro de substâncias vasopressoras de diferentes concentrações foram investigadas com o uso da técnica de microdiluição. Os microrganismos empregados no teste foram: Escherichia coli ATCC 25922, Yersinia pseudotuberculosis ATCC 911, Pseudomonas aeruginosa ATCC 10145, Listeria monocytogenes ATCC 43251, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 25923, Bacillus cereus 702 Roma, Mycobacterium smegmatis ATCC607, Candida albicans ATCC 60193 e Saccharomyces cerevisiae RSKK 251. Os ensaios antibacterianos foram feitos em caldo de cultura Mueller-Hinton (pH 7,3) e os ensaios antifúngicos em solução tampão de base nitrogenada para levedura (pH 7,0).

Resultados:

duas preparações diferentes de dopamina mostraram atividade antimicrobiana. Nenhuma outra substância do estudo mostrou qualquer atividade antimicrobiana.

Conclusões:

em nossa opinião, os efeitos antibacterianos da dopamina podem ser vantajosos para inibir a propagação de contaminação bacteriana durante a preparação das soluções para perfusão. Contudo, salientamos a importância do seguimento rigoroso das diretrizes de esterilização dos equipamentos e de assepsia durante todos os procedimentos feitos em unidades de terapia intensiva.

Atividade antimicrobiana; Vasopressores; Contaminacão de medicamentos

Justificativa y objetivo:

Los medicamentos administrados como perfusión intravenosa pueden ser contaminados durante las diversas etapas de producción o preparación. Sin embargo, son muy raros los estudios existentes sobre los efectos antibacterianos de los vasopresores. Este estudio investiga la actividad antimicrobiana in vitro de las formas de vasopresores usados clínicamente.

Materiales y métodos:

Actividades antimicrobianas in vitro de sustancias vasopresoras de diferentes concentraciones fueron investigadas con el uso de la técnica de microdilución. Los microrganismos usados en el test fueron: Escherichia coli ATCC 25922, Yersinia pseudotuberculosis ATCC 911, Pseudomonas aeruginosa ATCC 10145, Listeria monocytogenes ATCC 43251, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 25923, Bacillus cereus 702 Roma, Mycobacterium smegmatis ATCC607, Candida albicans ATCC 60193 y Saccharomyces cerevisiae RSKK 251. Los ensayos antibacterianos se hicieron en un caldo de cultivo Mueller-Hinton (pH 7,3) y los ensayos antifúngicos en una solución tapón de base nitrogenada para levadura (pH 7,0).

Resultados:

Dos preparaciones diferentes de dopamina mostraron actividad antimicrobiana. Ninguna otra sustancia del estudio mostró alguna actividad antimicrobiana.

Conclusiones:

En nuestra opinión, los efectos antibacterianos de la dopamina pueden ser ventajosos para inhibir la propagación de la contaminación bacteriana durante la preparación de las soluciones para perfusión. Sin embargo, destacamos la importancia del seguimiento riguroso de las directrices de esterilización de los equipos y de asepsia durante todos los procedimientos realizados en las unidades de cuidados intensivos.

Activid adantimicrobiana; Vasopresores; Contaminación de medicamentos

Introduction

Septic shock is the primary cause of death in critical care units. Shock states are primarily characterized by acute circulatory failure leading to tissue hypoperfusion, and potentially resulting in multi-organ failure. Observed hypotension can be the consequence of three major hemodynamic disorders: hypovolemia, vascular failure, and heart failure.¹ When appropriate fluid administration fails to restore adequate tissue perfusion and arterial pressure, vasopressors are usually necessary to increase mean systemic pressure, cardiac output, and oxygen delivery.²

In vitro studies focusing on catecholamine molecules demonstrated proliferation of bacteria.^3-5 A portion of catecholamines, which are used as vasopressor, are endogenously produced in the body. However, catecholamines used as vasopressor drugs are synthetically produced and infused for the treatment of cardiovascular failure which arises during septic shock. Dopamine, dobutamine, adrenaline and noradrenaline are most frequently used vasopressors prepared synthetically with supplemental chemicals having antioxidant and antimicrobial activity. Sodium metabisulfite, N-acetylcysteine and disodium edetate are the most frequently used antioxidant and antimicrobials for this purpose in drugs commonly found in medical markets (Table 1).

Considering several studies pointing catecholamine molecules’ proliferating effect on bacteria, we investigated commercially prepared catecholamine products’ in vitro effect on proliferation of several yeast and bacterial strains commonly encountered in septic shock.

Table 1
Study drugs and ingredients.

Catecholamine	Ingredients
Epinephrine	In 1 mL Ampoule:
	- Epinephrine 0.5 mg
	- Sodium chloride 8.5 mg
	- Metabisulfite 0.5 mg
	- Water for injection
Norepinephrine	In 4 mL Ampoule:
	- Norepinephrine bitartarate 8 mg (equivalent to 4 mg norepinephrine base)
	- Sodium metabisulfite 4 mg
	- Sodium chloride 34.35 mg
	- Water for injection
Dobutamine	In 20 mL Ampoule:
	- Dobutamine hydrochloride 280 mg (equivalent to 250 mg dobutamine base)
	- Sodium metabisulfite 4.8 mg
	- Water for injection
Dopamine	In 5 mL Ampoule:
	- Dopamine hydrochloride 200 mg
	- N-acetylcysteine 2 mg
	- Disodium edetate 2 mg
	- Water for injection
Dopamine	In 5 mL Ampoule:
	- Dopamine hydrochloride 200 mg
	- Sodium metabisulfite 50 mg

Materials and methods

Microorganisms used in tests were obtained from the Refik Saydam Hifzissihha Institute (Ankara, Turkey) and were as follows: Escherichia coli ATCC 25922, Yersinia pseudotuberculosis ATCC 911, Pseudomonas auroginosa ATCC 10145, Listeria monocytogenes ATCC 43251, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 25923, Bacillus cereus 709 ROMA, Mycobacterium smegmatis ATCC607, Candida albicans ATCC 60193 and Saccharomyces cerevisiae ATCC 60193.

Antimicrobial effects of the drugs were tested quantitatively in appropriate broth media using the double dilution method, and the minimum inhibitory concentration (MIC) values in μg/mL were determined.^6,7 Antibacterial assays were performed in Mueller-Hinton broth (MH) (Difco, Detroit, MI) at pH 7.3 and antifungal assays were performed in buffered Yeast Nitrogen Base (YNB) (Difco, Detroit, MI) at pH 7.0. Each tested drug was prepared in 0.1 mL volumes of sterile MH and YNB broths in concentrations ranging from 5 μg/mL to 5 mg/mL for microdilution. One drop (0.02 mL) of microorganism's suspension (approximately 10⁶ microorganisms per mL) was added to the extract/broth dilutions. After incubation at 35 °C for 18-72 h, the media were examined for growth. MIC is defined as the lowest concentration of drug showing no growth of microorganism. The dilutions without visible growth were used to determine minimum bactericidal concentration (MBC) by spreading 100 μL of the sample across the surface of dried MH and YNB agar plates with sterile glass rods, and then incubating at 35 °C for 18 h. MBC of each extract is defined as the lowest concentration that showed no growth of microorganism on agar plate. Fluconazole, Ampicillin and Streptomycin were used as standard antifungal and antibacterial drugs, respectively.

Ingredients of study drugs widely used as vasopressor in medical market are presented in Table 1.

Results

None of the study drugs containing norepinephrine, epinephrine and dobutamine showed any antimicrobial activity (Table 2). However study drugs containing dopamine showed antimicrobial activity (Table 2), one of which showed no activity against yeast-like microorganisms. The solutions containing dopamine (125-1000 μg/mL), N-acetylcysteine and disodium edetate (1.25-10 μg/mL) showed bacteriostatic activity against Gram-positive and -negative microorganisms. Higher concentration solutions showed bactericidial activity against all microorganisms except Y. pseudotuberculosis, which is capsular. Solutions of different concentrations of dopamine (125-500 μg/mL), N-acetylcysteine and disodium edetate (1.25-5 μg/mL) resulted in similar MIC and MBC values for bacterial strains like Gram-positive bacillus L. monocytogenes, Gram-positive coccus E. faecalis, Gram-negative M. smegmatis, which contain mycolic acid in their cell wall. However, these values were markedly different for each solution. Solutions containing dopamine (125-250 μg/mL), N-acetylcysteine and disodium edetate (1.25-2.5 μg/mL) showed bacteriostatic activity against E. coli, P. aeruginosa and S. aureus. Solutions containing dopamine 2000 μg/mL, N-acetylcysteine and disodium edetate 20 μg/mL showed bactericidial activity against the same microorganisms. Solutions containing dopamine 1000 μg/mL, N-acetylcysteine and disodium edetate 10 μg/mL showed bacteriostatic and bactericidal activity against B. cereus.

Table 2
Antimicrobial activity of the compounds expressed as MIC value in 100 mL volume.

Study drugs	Ingredient	Concentration (μg/mL)	MIC values (100mL)
			Ec		Yp		Pa		Li		Ef		Sa		Bc		Ms		Ca		Sc
Norepinephrine 4 mg/4 mL Ampoule	Norepinephrine	1000	-		-		-		-		-		-		-		-		-		-
	Sodium metabisulfite	1000	-		-		-		-		-		-		-		-		-		-
	Sodium chloride	34.350	-		-		-		-		-		-		-		-		-		-
Adrenalin 0.5 mg/1 mL Ampoule	Epinephrine	500	-		-		-		-		-		-		-		-		-		-
	Sodium metabisulfite	500	-		-		-		-		-		-		-		-		-		-
	Sodium chloride	8500	-		-		-		-		-		-		-		-		-		-
Dobutamine 250 mg/20 mL Ampoule	Dobutamine	12.500	-		-		-		-		-		-		-		-		-		-
	Sodium metabisulfite	240	-		-		-		-		-		-		-		-		-		-
Dopamin 200 mg/5 mL Ampoule	Dopamine	40.000	125		125		250		250		125		125		1000		250		-		-
	N-acetylcysteine	400	1.25		1.25		2.50		2.50		1.25		1.25		10		2.50		-		-
	Disodium edetate	400	1.25		1.25		2.50		2.50		1.25		1.25		10		2.50		-		-
Dopamine 200 mg/5 mL Ampoule	Dopamine	40.000	500		500		500		250		250		500		250		250		500		125
	Sodium metabisulfite	10.000	125		125		125		62.5		62.5		125		62.5		62.5		125		62.5
Ampicillin		10	2		32		>128		2		2		2		<1
Streptomycin		10															4
Fluconazole		5																	<8		<8

• Bc, Bacillus cereus 702 Roma; Ca, Candida albicans ATCC 60193; Ec, Escherichia coli ATCC 25922; Ef, Enterococcus faecalis ATCC 29212; Li, Listeria monocytogenes ATCC 43251; Ms, Mycobacterium smegmatis ATCC607; Pa, Pseudomonas aeruginosa ATCC 10145; Sa, Staphylococcus aureus ATCC 25923; Sc, Saccharomyces cerevisiae RSKK 251; Yp, Yersinia pseudotuberculosis ATCC 911.
  (-): no activity.

Other drugs containing dopamine (125-500 μg/mL) and sodium metabisulfite (62.5-125 μg/mL) showed bactericidial activity against all microorganisms used in the test.

Discussion

In this study, we have found that two different dopamine preparations out of all tested showed antimicrobial activity.

Drugs manufactured for intravenous use should be prepared and administered in sterile conditions. Infectious microorganisms can be introduced into the patient through contaminated containers, rubber diaphragm, needles and infusion sets. Anesthetic agents and vasopressors may be contaminated by microorganisms during the preparation of an infusion. For this reason, the antimicrobial effects of anesthetic agents and vasopressors have been deemed important, and they have been investigated in previous studies.⁸ Notably, propofol is known to support the growth of microorganisms.⁸ On the other hand, previous studies have shown that morphine sulphate, thiopental sodium, fentanyl citrate, dexmedetomidine and midazolam all have antimicrobial effects.^8-13 However, studies on the antimicrobial effects of vasopressors drugs, which are commonly used in intensive care units (ICU), are very few.

Studies demonstrating that catecholamines stimulate growth of microorganisms are increasing.^4,5,14,15 Among the causative factors are binding of catecholamines to transferrin and lactoferrin, enabling bacteria to acquire normally inaccessible ferric-iron¹⁴ and possible α-adrenergic specific response system of some bacteria to recognize catecholamines.¹⁵

On the other hand, additives having antioxidant and antimicrobial properties are commonly added to commercial vasopressor formulas to prevent bacterial contamination.^16-19 However, studies investigating the in vitro antimicrobial activity of clinically used commercial forms of vasopressors are very few. Most commonly used additives in vasopressors are N-acetylcysteine and disodium edetate, which are known as potent antioxidants with antimicrobial properties.^16-19

This study evaluates the antimicrobial properties of most commonly used vasopressors in the medical markets in different concentrations by the micro-dilution method.

Vasopressor drugs are administered as infusion through a preferably central, high caliber vein to ensure a steady state plasma concentration. We used the micro-dilution method to mimic different levels of concentrations since catecholamines interact over a wide dose-response range and exhibit multiple potencies.

Our study revealed antimicrobial activity of both of the dopamine preparates. No other study drug was able to inhibit microorganismal growth at any concentration. This finding could be explained by the high sodium metabisulfite concentration for one of the Dopamine preparate compared to norepinephrine, adrenaline and dobutamine preparates.

Sodium metabisulfite is an oxidizing agent active at low pH. While all the study drugs have effective pH ranges between 2.2 and 5.0, MH broth has a pH value of 7.3 ± 0.1 and buffered YNB used in our study has a pH value of 7.0 at 25 °C (unbuffered medium has a pH value of 5.4 ± 0.2 at 25 °C). Therefore we do not think that sodium metabisulfite may exhibit any antimicrobial activity at the neutral pH. Since human blood has a slightly alkaline pH value of 7.35-7.45, and most pathogenic bacteria prefer a narrow pH range of 6-8,¹¹ we think that this finding is concordant with real-life applications of the drugs.

The other oxidizing additive contained in tested drugs is N-acetylcysteine, which was shown to be a valuable mucolytic agent, capable to aid in antimicrobial treatment if combined with antibiotics. Examples where antimicrobial activity of N-acetylcysteine is seen are lysis of gastric basal mucosal layer, which enables Helicobacter pylori to escape from the acidic gastric secretions²⁰ and decreasing formation of biofilms by reducing production of extracellular polysaccharide matrix and promoting the disruption of mature biofilm.²¹ Both of these activities are augmented by an acidic environment, and the slightly alkaline pH levels established in the study broth environments may have hindered N-acetylcysteine's antimicrobial activity. However, as for metabisulfite, we conclude that a slightly alkaline pH level is more concordant with real-life applications of these drugs.

It is important that strict guidelines regarding the need for sterile equipment and deliverables be adhered to during all procedures performed in the ICU. In some circumstances, vasopressor drugs may be contaminated with microorganisms that can then lead to infections.^8,9 Thus, the antimicrobial effect of vasopressor drugs in these types of settings is of paramount importance.^8,9 In our opinion, dopamine's antibacterial effects may be sufficient to inhibit contamination during the preparation of the infusion solutions.

We have shown that dopamine has antibacterial effects on some microorganisms frequently encountered in hospital settings. We suggest that dopamine preparations should be preferred in septic patients due to their antimicrobial activity against several yeast and bacterial strains commonly encountered in septic shock. However, translating such laboratory researches into recommendations requires delineation of the interactions between catecholamines, several molecules co-existing or co-secreted with them, and microorganisms in their expected environments.

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Publication Dates

• Publication in this collection
  Mar-Apr 2014

History

• Received
  05 Apr 2012
• Accepted
  28 Feb 2013