human pathogens may represent a novel catheter lock
BALAZS ITTZES
1,5, EVA SZENTKIRALYI
1, ZOLTAN SZABO
2, ISTVAN Z. BATAI
3, ORS GYORFFY
1,4, TAMAS KOVACS
2,4, ISTVAN BATAI
2pand MONIKA KERENYI
11Department of Medical Microbiology, Medical School, University of Pecs, Pecs, Hungary
2Department of Anaesthesiology and Intensive Therapy, Medical School, University of Pecs, Pecs, Hungary
3Department of Pharmacology and Pharmacotherapy, Medical School, University of Pecs, Pecs, Hungary
4Department of Anaesthesiology and Intensive Therapy, St. Rafael Hospital, Zalaegerszeg, Hungary
5Department of Anaesthesia and Intensive Therapy, North Devon District Hospital, Barnstaple, Devon, UK
Received: January 20, 2020 • Accepted: April 09, 2020 Published online: July 06, 2020
ABSTRACT
Infection is one of the most feared hospital-acquired complications. Infusion therapy is frequently administered through a central line. Infusions facilitating bacterial growth may be a source of central line-associated bloodstream infections. On the other hand, medications that kill bacteria may protect against this kind of infection and may be used as a catheter lock.
In this study, we examined the impact of amiodarone on bacterial growth. Amiodarone is used for controlling cardiac arrhythmias and can be administered as an infusion for weeks. Standard microbi- ological methods have been used to study the growth of laboratory strains and clinical isolates of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa,Escherichia coli,Klebsiella pneumoniae, and multidrug-resistantAcinetobacter baumanniiin amiodarone. The minimum inhibi- tory concentration (MIC) of amiodarone was determined. Bacterial growth from in use amiodarone syringes and giving sets was also investigated.
Most examined strains were killed within 1 min in amiodarone. The other strains were killed within 1 h. The MICs of amiodarone were <0.5–32mg/mL.
Amiodarone infusion is unlikely to be responsible for bloodstream infections as contaminating bacteria are killed within 1 h. Amiodarone may also protect against central line infections if used as a catheter lock.
KEYWORDS
amiodarone, fast-acting bactericidal, catheter lock
INTRODUCTION
Intravenous drugs may support or inhibit bacterial growth [1] thus, they may have an impact on the risk of nosocomial bloodstream infections [2]. Infusion therapy may also be responsible for bloodstream infections. Description of the risks of intravenous medications is discussed in detail elsewhere [3]. The use of central venous catheters is inevitable for dialysis, parenteral nutrition, cancer therapy or in intensive care. Their use may be associated with infections that increase morbidity, mortality, and health care costs [4–6].
Acta Microbiologica et Immunologica Hungarica
67 (2020) 2, 133-137 DOI:
10.1556/030.2020.01144
© 2020 The Author(s)
ORIGINAL ARTICLE
*Corresponding author.
Tel.: +36 302770599.
E-mail:ibatai@gmail.com
Medications that inhibit bacterial growth may have a role in reducing central line-associated bloodstream infections (CLABSI) not only due to the fact that these infusions do not cause a bacterial load to the patient but also, as they may also be used as a catheter lock when the central line is not in use [7].
Amiodarone is an important therapeutic agent used in the treatment of ventricular and supraventricular arrhyth- mias; initially, it has to be administered as an infusion for 24 h or longer [8]. In a previous study, we investigated bacterial growth from syringes and administration sets collected from a University Hospital Intensive Care Unit. The infusions contained 10 different medications. The overall contami- nation rate was 15% but there was no growth found from amiodarone syringes [9]. In this study, we investigated the impact of amiodarone on the growth of standard laboratory strains and human bacterial pathogens including multidrug- resistant isolates and we studied the bacterial growth from syringes and extension lines that were used for amiodarone administration. We also determined the minimum inhibi- tory concentration (MIC) of amiodarone.
MATERIALS AND METHODS
Bacterial strains
Staphylococcus aureus (ATCC 23923), biofilm producing Staphylococcus epidermidis(ATCC 35984), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumoniae(ATCC13883), clinical isolates of S.
epidermidis, metallo-beta-lactamase producing (MBL) P.
aeruginosa, extended-spectrum beta-lactamase producing (ESBL)E. coli and K. pneumoniae, and multidrug-resistant (MDR)Acinetobacter baumanniiwere investigated.
Investigated medication
Investigated medication was: amiodarone (Cordarone®, 2-butyl-3-[30,50diido-40alpha-diethylaminoethoxybenzoyl]- benzofuran, Sanofi Aventis, Budapest, Hungary, 50 mg/mL).
It was diluted to 0.6 mg/mL in glucose 5% (B. Braun, Mel- sungen, Germany) as recommended by the manufacturer.
In vitro assay for bacterial growth
The growth of different bacterial strains were investigated in amiodarone infusion. Standard microbiological methods were used to grow and dilute the investigated strains to 106 colony forming units (cfu)/mL in 0.9% saline. Ten
m
L ofeach bacterial suspension was inoculated into 1 mL amio- darone 0.6 mg/mL diluted in glucose 5% respectively and kept at room temperature (20oC). The initial bacterial count was 103–104cfu/mL. At 0, 1, 15, 45, 60 min, 2, 3, 4, and 6 h 10
m
L was plated on Mueller–Hinton (MH) agar (Bio-Rad, Marnes-la-Coquette, France). After incubation for 24 h at 37 8C, the cfu was counted. Five parallels were performed for each bacterial strain. The method has been described in detail previously [10]. Glucose 5% and MH broth controls were also applied. Sterility control checks for amiodaroneand glucose 5% were also performed by plating a sample from the investigated ampoules or infusions on MH agar plates and were incubated for 24 and 48 h at 378C.
Determination of the minimal inhibitory concentrations (MIC) of amiodarone in glucose 5%
The method recommended by Clinical and Laboratory Standards Institute (CLSI) [11] was modified so that amio- darone was diluted in glucose 5% instead of MH broth.
Amiodarone causes opacity in MH broth and loses its effect in this medium. Therefore, the method had to be modified and we used a diluent recommended by the manufacturer of amiodarone. Briefly, the bacterial strains were grown on Mueller Hinton agar. The bacteria were suspended in 0.9%
saline solution to MacFarland 0.5 and diluted to∼53 106 cfu/mL. Amiodarone was diluted in glucose 5% on standard 96 wells plates. After the dilution, each well was inoculated with 10
m
L bacterial suspension containing∼5x104cfu. The plates were incubated at 378C for 24 h. To determine the bactericidal or bacteriostatic concentration, 10m
L sub-cultures were performed on MH agar from each well.
Bacterial growth from amiodarone syringes
The syringes and the extension lines of 20 amiodarone treated patients (septic and non-septic) were collected.
Amiodarone was diluted to 12 mg/mL1 (600 mg amio- darone in 50 mL glucose 5%) which is one of the dilutions used in everyday clinical practice.
Before completing intravenous administration of the amiodarone infusions, approximately 1 mL fluid was left in the syringes. Subsequently, the syringes along with their extension lines were collected, sealed with a sterile needle and sent for microbiological analysis. The syringes and extension lines were detached under sterile conditions and 1 drop from both the syringe and extension line was spread onto blood agar, chocolate agar, and eosin-methylene blue agar respectively. Having been incubated for 24 and 48 h at 378C the plates were inspected for bacterial growth.
Statistical analysis was performed by using analysis of variance. Individual comparisons between group means were made with the Scheffe test.P< 0.05 was regarded as significant.
RESULTS
Bacterial growth in amiodarone in vitro
All the examined strains grew in MH broth. There was no growth from the sterility control check for amiodarone and glucose 5%. In glucose 5% control the cfu of all strains decreased but there were viable cells at the end of the experiment (6 h). Amiodarone showed a fast-acting anti- bacterial activity (within 1 min) against the standardE. coli, P. aeruginosa,K. pneumoniaestrains, andP. aeruginosaand A. baumannii clinical isolates. It killed the ATCC S. epi- dermidis and S. aureus and the clinical isolate of S. epi- dermidisandE. coliwithin 15 min and the clinical isolate of
ESBL producingK. pneumoniaewithin 45 min [Table 1]. All cfu numbers in amiodarone at 0, 1, 15 min and later were significantly different from inoculated bacterial cfu and from the cfu number in MH broth or glucose 5% control at the same sampling times.
Amiodarone MIC
The MIC values of amiodarone diluted in glucose 5% were low, <0.5
m
g/mL for theS. aureus(ATCC 23923), multidrug- resistant A. baumannii, 0.5m
g/mL for the E. coli (ATCC 25922), P. aeruginosa (ATCC 27853), 32m
g/mL for K.pneumoniae(ATCC13883), ESBL producing E. coli and K.
pneumoniae strains. Bactericidal concentrations of amio- darone were different for the investigated strains. The above concentration values were bactericidal in the case of S.
aureus and A. baumannii and were bacteriostatic for the other investigated strains.
Bacterial growth from amiodarone syringes and extension lines
There was no bacterial growth from the amiodarone sy- ringes and extension lines.
DISCUSSION
In a previous study, we collected in use syringes from the intensive care unit of a university hospital. Ten different medications were administered via the syringes; the overall contamination rate was 15% but there was no growth from the amiodarone syringes [9]. We designed this study on the basis of the results of the above investigation.
Our results suggest that amiodarone has bactericidal ef- fect in vitro against the investigated human pathogenic strains of S. epidermidis, E. coli, P. aeruginosa, Klebsiella pneumoniaand A. baumannii. Thefirst three of the above microbes are frequently isolated from intravascular catheter- related bacterial bloodstream infections [12]. The bacteri- cidal effect against most of the strains was fast-acting which can be compared to the fast-acting effect of an antiseptic agent [13]. Amiodarone for the in vitro investigations was diluted to 0.6 mg/mL as this is the most diluted form in clinical use. In practice, its concentration in the syringe during administration can be as high as 12 mg/mL.
In accordance with the above findings, no bacteria were recovered from the syringes and extension lines that were in clinical use at an intensive care unit.
In search of the mode of action of amiodarone previous studies have examined the impact of amiodarone on the growth of non-pathogenic bacteria, non-pathogenic and pathogenic fungi, yeast, and viruses. Thesein vitro studies have shown antimicrobial effect against Bacillus stear- othermophilus [14], Saccharomyces cerevisiae, Aspergillus fumigatus, Cryptococcus neoformans, Fusarium oxysporum, Candida albicans [15], and SARS coronavirus [16]. These studies did not give any answer on the speed of bactericidal Table1.Growthoflaboratorystrainsandclinicalisolatesinamiodarone0.6mg/mL Growthinamiodarone0.6mg/mL LaboratorystrainsClinicalisolates Time (min)
S.aureus ATCC 23923S.epidermidisATCC 35984
E.coli ATCC 25922P.aeruginosaATCC 27853K.pneumoniae ATCC13883S. epidermidisP.aeruginosa MDRE.coli ESBLK.pneumonia ESBLA.baumannii MDR 083.6± 11.4138±1413.3±0.633.3±1.551±1265±793±158±4.489.6±6.,0147±4.1 14.6±4.05±000018±3.603.3±2.17.3±4.90 15000000000.6±1.10 450000000000 600000000000 Colonyformingunits(cfu)±SDoflaboratorystrainsandclinicalisolatesfollowing0,1,15,45,or60minincubationatroomtemperature(208C)inamiodarone0.6mg/mL.Eachcfuat1and 15minwassignificantlydifferentfrom0min(P<0.05).
activity, no human pathogenic strains were used and the incubation was at 378C not at room temperature. Recent research has revealed that amiodarone may be effective against the Ebola virus [17]. There are reports of the off label use of amiodarone in Ebola infection [18].
The mode of action has been extensively studied.
Amiodarone exerts its antimicrobial effect on multiple cell components including the phospholipid bilayer structure [14] and intracellular calcium homeostasis [15, 19]. The alcohol content of amiodarone is 20 mg/mL (2%) which has no antibacterial effect.
To the best of our knowledge, this is the first investiga- tion examining the effect of amiodarone against human pathogens including multidrug-resistant isolates, to show the fast-acting bactericidal action and to determine the MIC values of the drug.
There is a continuous demand for intravascular catheters for dialysis, parenteral nutrition, cancer therapy or in intensive care. Their use may be associated with complica- tions. One of them is CLABSI. There have been many re- ports giving detailed figures of infection rates and the effect of these infections on mortality and morbidity that have a significant impact on hospital financing as well [4–6].
There are various ways to reduce CLABSI, including the use of antibacterial materials, antibiotic and non-antibiotic ointments or impregnated sponge dressings at the skin exit site [20]. Another approach is the use of antimicrobial medication with or without anticoagulants to lock the cath- eters when not in use. The antibiotics studied were genta- micin, tobramycin, minocycline, cefotaxime, vancomycin, and cefazolin used as antibiotic lock therapy [21]. There has al- ways been a fear of the emergence of antibiotic-resistant strains with the use of antibiotics as a catheter lock, however, the results are controversial [22, 23]. In search to reduce CLABSI without the possibility of increasing the appearance of antibiotic-resistant strains, different non-antibiotics have been introduced for use as catheter lock. The most studied substances have been heparin, citrate, ethanol, taurolidine, methylene blue, and parabens alone or in combination. Most of them helped to reduce CLABSI but so far none of the above agents have provided a definite solution for the eradication of infection [24]. The above mentioned CLABSI-reducing non- antibiotics may have side effects including an impact upon the clotting system, causing protein precipitation, compromising catheter polymers, or liver damage [25, 26].
Amiodarone is an extensively used antiarrhythmic medication [8, 27]. When administered intravenously the recommended initial dose is 5mg/kg bodyweight and the total dose can be as high as 1.2 g in thefirst 24 h [28]. As the MIC value of amiodarone for the examined human patho- gens is much less (≤32
m
g/mL) than the concentration we use for antiarrhythmic therapy (600–15,000m
g/mL), it may be a safe method to lock intravenous catheters with diluted amiodarone. Even if the possible catheter lock is not with- drawn before using the central line again butflushed into the circulation, the amount given this way is much less than that infused/second at the beginning of amiodarone therapy or much less than the hourly dose.Caution should be taken when other medications may mix with amiodarone. There is a known incompatibility of amiodarone with sodium bicarbonate, heparin, sugamma- dex, meropenem and vaborbactam, and total parenteral nutrient solutions [29–33].
Glyceryl trinitrate is another medication that was used in cardiology and intensive therapy. We reported in 1999 that it had an antibacterial effect [10] and in 2017, thefirst study was published discussing that a nitroglycerin-based catheter lock solution was successful in reducing central venous catheter infections [34].
It is known that other medications used in anaesthesia and intensive care have non-anaesthetic effects that may beneficially influence morbidity [35–37].
The serum amiodarone therapeutic concentration is 0.7– 41
m
g/mL [38]. This means that bactericidal amiodarone concentration is achieved in the serum against a few strains during amiodarone treatment. On the other hand, in the central venous cannula, the concentration is at least 600m
g/mL that is much higher than the MIC for any of the investigated strains.
Further research and clinical studies are needed either to prove or to rule out the effectiveness of an amiodarone based catheter lock.
Ethical committee approval:Not required.
Conflict of interest:None.
ACKNOWLEDGMENT
We thank Ms Lilla A. Horvath for checking and correcting language.
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