In vitro evaluation of antimicrobial features of sugammadex^☆

Abstracts

Background:

Drugs administered by intravenous routes may be contaminated during several stages of production or preparation. Sugammadex is a modified gamma cyclodextrin. While research into the antibacterial effects of varieties of cyclodextrin is available, there are no studies focusing on the antibacterial effects of sugammadex. This study investigates the in vitro antimicrobial activity of sugammadex.

Materials and methods:

The in vitro antimicrobial activity of sugammadex was investigated using the broth microdilution method. The pH of the test solution was determined using a pH meter. The test microorganisms included Staphylococcus aureus ATCC 29213, Enterococcus fecalis ATCC 29212, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853. In the second phase of the study 100 mg/mL sugammadex (50 μg) was contaminated with test microorganisms (50 μg), including S. aureus ATCC 29213, E. fecalis ATCC 29212, E. coli ATCC 25922 and P. aeruginosa ATCC 27853, left to incubate for 24 h and then the bacterial production in sugammadex was evaluated.

Results:

The pH of the test solutions ranged between 7.25 and 6.97. Using the microdilution method, sugammadex had no antibacterial effect on S. aureus, E. fecalis, E. coli and P. aeruginosa at any concentration. In the second phase of the study bacterial production was observed after 24 h in 100 mg/mL sugammadex contaminated with the test microorganisms S. aureus, E. fecalis, E. coli and P. aeruginosa.

Conclusions:

Sugammadex had no antimicrobial effect on the test microorganisms, S. aureus, E. fecalis, E. coli and P. aeruginosa. Care should be taken that sterile conditions are maintained in the preparation of sugammadex; that the same sugammadex preparation not be used for more than one patient; and that storage conditions are adhered to after sugammadex is put into the injector.

Sugammadex; Antimicrobial effect; S. aureus ; E. fecalis ; E. coli ; P. aeruginosa

Justificativa e objetivo:

os medicamentos administrados por via intravenosa podem ser contaminados durante as várias fases de produção ou preparação. Sugamadex é uma gama-ciclodextrina modificada. Embora muitas pesquisas sobre os efeitos antibacterianos de uma variedade de ciclodextrinas estejam disponíveis, não há estudos dos efeitos antibacterianos de sugamadex. Este estudo investigou a atividade antimicrobiana in vitro de sugamadex.

Materiais e métodos:

a atividade antimicrobiana in vitro de sugamadex foi investigada pelo método de microdiluição em meio de cultura. O pH da solução de ensaio foi determinado com o uso de um medidor de pH. Os microrganismos-teste analisados incluíram Staphylococcus aureus ATCC 29213, Enterococcus fecalis ATCC 29212, Escherichia coli ATCC 25922 e Pseudomonas aeruginosa ATCC 27853. Na segunda fase do estudo, 100 mg/mL de sugamadex (50 μg) foram contaminados com microrganismos-teste (50 μg), incluindo S. aureus ATCC 29213, E. fecalis ATCC 29212, E. coli ATCC 25922 e P. aeruginosa ATCC 27853, incubados por 24 horas e, em seguida, a produção bacteriana foi avaliada.

Resultados:

o pH das soluções da análise variaram entre 7,25 e 6,97. Com o uso do método de microdiluição, sugamadex não apresentou efeito antibacteriano contra S. aureus, E. fecalis, E. coli e P. aeruginosa em qualquer concentração. Na segunda fase do estudo, a produção bacteriana foi observada após 24 horas em 100 mg/mL de sugamadex contaminados com os microrganismos-teste S. aureus, E. fecalis, E. coli e P. aeruginosa.

Sugamadex; Efeito antimicrobiano; S. aureus; E. fecalis; E. coli; P. aeruginosa

Justificación y objetivo:

Los medicamentos administrados por vía intravenosa pueden ser contaminados durante las diversas fases de producción o preparación. El sugammadex es una gamaciclodextrina modificada. Aunque estén disponibles muchas investigaciones sobre los efectos antibacterianos de una variedad de ciclodextrinas, no existen estudios de los efectos antibacterianos del sugammadex. Este estudio investigó la actividad antimicrobiana in vitro del sugammadex.

Materiales y métodos:

La actividad antimicrobiana in vitro del sugammadex fue investigada por el método de microdilución en medio de cultivo. El pH de la solución de ensayo fue determinado usando un medidor de pH. Los microorganismos testados analizados incluyeron Staphylococcus aureus (S. aureus) (ATCC 29213), Enterococcus faecalis (E. faecalis) (ATCC 29212), Escherichia coli (E. coli) (ATCC 25922) y Pseudomonas aeruginosa (P. aeruginosa) (ATCC 27853). En la segunda fase del estudio, se contaminaron 100 mg/mL de sugammadex (50 µg) con microorganismos testados (50 µg), incluyendo S. aureus (ATCC 29213), E. faecalis (ATCC 29212), E. coli (ATCC 25922) y P. aeruginosa (ATCC 27853), incubados durante 24 h e inmediatamente se calculóla producción bacteriana.

Resultados:

El pH de las soluciones del análisis varió entre 7,25 y 6,97. Usando el métodode microdilución, el sugammadex no tuvo ningún efecto antibacteriano contra S. aureus, E. faecalis, E. coli y P. aeruginosa en ninguna concentración. En la segunda fase del estudio, la producción bacteriana fue observada después de 24 h en 100 mg/mL de sugammadex contaminados con los microorganismos testados S. aureus, E. faecalis, E. coli y P. aeruginosa.

Conclusiones:

El sugammadex no presentó ningún efecto antimicrobiano sobre los microorganismos testados S. aureus, E. faecalis, E. coli y P. aeruginosa. Se debe tener cuidado de que las condiciones estériles se mantengan en la preparación del sugammadex, de que la misma preparación de sugammadex no se use en más de un paciente y de que las condiciones de almacenaje se respeten después de la colocación del sugammadex en un inyector.

Sugammadex; Efecto antimicrobiano; Staphylococcus aureus ; Enterococcus faecalis ; Escherichia coli ; Pseudomonas aeruginosa

Introduction

Some anesthetic agents such as propofol are known to support the growth of microorganisms,^1-5 while other anesthetic agents such as morphine sulphate, thiopental sodium, fentanyl citrate, dexmedetomidine and midazolam inhibit microbial growth.^3-7 Anesthetic agents may be contaminated by microorganisms at various stages during preparation for use.² It is important for this reason that the antibacterial properties, or the ability to enhance bacterial production, of anesthetic agents in a contaminated situation be known.⁸

Sugammadex is a modified gamma cyclodextrin.^9-11 Cyclodextrins are water soluble cyclic oligosaccharides with a lipophilic core. Sugammadex has quickly found a place in clinical use as a selective neuromuscular blockade reverser.^9-11 Sugammadex quickly encapsulates steroidal neuromuscular blockers, increasing the amount of encapsulated steroidal neuromuscular blockers in plasma and separating the blockers from the nicotinic acetylcholine receptors.^9-11

Cyclodextrins are molecules that are often used in the food and pharmaceutical industries. They are commonly used to convert lipophilic medications to hydrophilic forms. Other applications of cyclodextrins include the field of microbiology. Some cyclodextrins, such as dimethyl-b-cyclodextrin, have been used to increase production of Helicobacter pylori,¹² while others, like hydroxypropyl-b-cyclodextrin, have been reported to prevent bacterial production when used to coat vascular prostheses.¹³ However there are no studies evaluating the effect of sugammadex, a modified gamma cyclodextrin molecule lately being used in anesthesiology, on bacterial production.

The aim of this study was to evaluate the antimicrobial effects of sugammadex on the test microorganisms. The test microorganisms chosen were Staphylococcus aureus American Type Culture Collection (ATCC) 29213, Enterococcus fecalis ATCC 29212, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853.

Materials and methods

The antibacterial activity of sugammadex was investigated using the broth microdilution method according to the procedures outlined by the Clinical and Laboratory Standards Institute (CLSI).¹⁴

Briefly, sugammadex was diluted with 0.9% sterile saline to final concentrations of 512 μg/mL, 256 μg/mL, 128 μg/mL, 64 μg/mL, 32 μg/mL, 16 μg/mL, 8 μg/mL, 4 μg/mL, 2 μg/mL, 1 μg/mL and 0.5 μg/mL. For each neuromuscular blocking drug, the pH values of all the dilutions were determined with a pH meter (Sartorius pH Meter PB-11). S. aureus ATCC 29213, E. fecalis ATCC 29212, E. coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as control microorganisms. The bacteria (5 × 10⁵ colony-forming units per milliliter; (CFU/mL)), MHB (Mueller-Hilton broth) and the sugammadex in the specified concentrations were incubated in wells on microplates at 35 °C for 20 h. The minimal inhibitory concentrations (MIC) were determined by observing the lowest concentration of the agent that inhibited visible growth of the bacterium. Haze or turbidity in the wells was an indicator of bacterial growth.

In the second stage of the study 100 mg/mL sugammadex was contaminated with the test organisms, S. aureus ATCC 29213, E. fecalis ATCC 29212, E. coli ATCC 25922 and P. aeruginosa ATCC 27853. Bacteria, 50 μL (5 × 10⁵ colony-forming units per milliliter; (CFU/mL)), and 50 μL sugammadex (100 mg/mL) were incubated at 35 °C for 24 h. After 24 h the bacterial production in the sugammadex was evaluated.

Results

Using the microdilution technique, sugammadex had no antibacterial effect on S. aureus, E. fecalis, E. coli and P. aeruginosa at any concentration.

In the second part of the study, after 24 h incubation 100 mg/mL sugammadex contaminated with S. aureus, E. fecalis, E. coli and P. aeruginosa, bacterial growth was observed.

The pH of the test solutions ranged between 7.25 and 6.97. The pH values are listed in Table 1.

Table 1
The pH values of tested dilutions of sugammadex.

Dilution of sugammadex (μg/mL)		pH
512 μg/mL		7.25
256 μg/mL		7.22
128 μg/mL		7.14
64 μg/mL		7.09
32 μg/mL		7.04
16 μg/mL		7.04
8 μg/mL		7
4 μg/mL		6.99
2 μg/mL		6.98
1 μg/mL		6.97
0.5 μg/mL		6.97
Physiological serum 0.9%		6.8

Discussion

In this study, we found that sugammadex does not have antimicrobial properties with regard to the test organisms, S. aureus, E. fecalis, E. coli and P. Aeruginosa.

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 diaphragms, needles and infusion sets.

Anesthetic agents may be contaminated by microorganisms during preparation. For this reason, the antimicrobial effects of the used agents are important.⁸ It is known that propofol supports the growth of microorganisms.^{2-4,7,8,15-18} On the other hand, morphine sulphate, thiopental sodium, fentanyl citrate, dexmedetomidine, atracurium, rocuronium and midazolam have antimicrobial effects.^3,5-8

Sugammadex is a modified gamma cyclodextrin.^9-11 Cyclodextrins are molecules that are often used in the food and pharmaceutical industries. They are commonly used to convert lipophilic medications to hydrophilic forms. Cyclodextrins are water soluble cyclic oligosaccharides with a lipophilic core. Other applications of cyclodextrins include the field of microbiology. Some cyclodextrins, such as dimethyl-b-cyclodextrin, have been used to increase production of H. pylori.¹² When added to agar gels cyclodextrins such as alpha- and beta-cyclodextrin/hexadecane are suitable foodbeds for the growth of microorganisms such as Candida lipolytica and C. tropicalis.¹⁹ Research has shown that cyclodextrin molecules, such as beta-cyclodextrin, when added to liquid cultures neutralize potential toxic combinations and increase the growth of microorganisms such as H. pylori.^20-22 Solid cultures including modified cyclodextrins have been used for selective isolation of microorganisms such as Bordetella pertussis.^23-26

However other cyclodextrins, such as hydroxypropyl-b-cyclodextrin, have been reported to prevent bacterial production when used to coat vascular prostheses.¹³ Previous studies have reported methyl-beta-cyclodextrins inhibiting the growth of bacillus types.²⁷ Researchers found that methyl-beta-cyclodextrins crossed the cell membranes of bacillus species and caused cell lysis; however they emphasized that this activity was not observed for other gram negative and positive bacteria.²⁷ Another study found that cyclodextrin derivatives acted like antimicrobial peptid polymixin B and could inhibit bacterial proliferation.²⁸

There are no studies evaluating the effect of sugammadex, a modified gamma cyclodextrin molecule lately being used in anesthetic practice, on bacterial production. In our study, we found that sugammadex did not have antimicrobial properties with respect to the growth of S. aureus, E. coli, P. aeruginosa and E. fecalis.

Most bacteria prefer a fairly narrow pH range, between 6 and 8, for survival.^3,17 The growth of S. aureus (ATCC 25923), E. coli (ATCC 25922) or P. aeruginosa (ATCC 27853) was not affected by growth conditions with a pH between 5.0 and 8.0.²⁹ The bactericidal properties of thiopental are thought to be related to its high pH.³⁰ Similarly, the pH range of midazolam was shown to be responsible for its bacterial inhibitory effect.^5,7,31 In our study, prior to performing the recommended dilution, the pH of sugammadex was approximately 7.5. The diluted sugammadex had pH in a narrow range between 6.97 and 7.25. These pH values are within the range for proliferation of the test microorganisms S. aureus (ATCC 25923), E. coli (ATCC 25922) and P. aeruginosa (ATCC 27853).

In conclusion, sugammadex had no antibacterial effect on S. aureus, E. fecalis, E. coli and P. aeruginosa.

• ☆
  A part of this manuscript was presented as a poster presentation at the 46th Annual TARK Congress, Cyprus, 7-11 November 2012.

Referências

• ¹
  Heldmann E, Brown DC, Shofer F. The association of propofol usage with postoperative wound infection rate in clean wounds: a retrospective study. Vet Surg. 1999;28:256-9.
• ²
  Henry B, Plante-Jenkins C, Ostrowska K. An outbreak of Serratia marcescens associated with the anesthetic agent propofol. Am J Infect Control. 2001;29:312-5.
• ³
  Crowther J, Hrazdil J, Jolly DT, Galbraith JC, Greacen M, Grace M. Growth of microorganisms in propofol, thiopental, and a 1:1 mixture of propofol and thiopental. Anesth Analg. 1996;82:475-8.
• ⁴
  Sosis MB, Braverman B, Villaflor E. Propofol, but not thiopental, supports the growth of andida albicans. Anesth Analg. 1995;81:132-4.
• ⁵
  Keleş GT, Kurutepe S, Tok D, Gazi H, Dinç G. Comparison of antimicrobial effects of dexmedetomidine and etomidate-lipuro with those of propofol and midazolam. Eur J Anaesthesiol. 2006;23:1037-40.
• ⁶
  Ayoglu H, Kulah C, Turan I. Antimicrobial effects of two anaesthetic agents: dexmedetomidine and midazolam. Anaesth Intensive Care. 2008;36:681-4.
• ⁷
  Graystone S, Wells MF, Farrell DJ. Do intensive care drug infusions support microbial growth? Anaesth Intensive Care. 1997;25:640-2.
• ⁸
  Hanci V, Cömert F, Ayoglu H, Kulah C, Yurtlu S, Turan IO. Evaluation of the antimicrobial effects of atracurium, rocuronium and mivacurium. Antimicrobial effects of muscle relaxants. Drugs Ther Stud. 2011;1:e2, http://dx.doi.org/10.4081/dts.2011.e2.
  » http://dx.doi.org/10.4081/dts.2011.e2
• ⁹
  Naguib M, Sugammadex:. another milestone in clinical neuromuscular pharmacology. Anesth Analg. 2007;104:575-81.
• ¹⁰
  Brull SJ, Naguib M. Selective reversal of muscle relaxation in general anesthesia: focus on sugammadex. Drug Des Dev Ther. 2009;3:119-29.
• ¹¹
  Rex C, Bergner UA, Pühringer FK. Sugammadex: a selective relaxant-binding agent providing rapid reversal. Curr Opin Anaesthesiol. 2010;23:461-5.
• ¹²
  Joo JS, Park KC, Song JY, et al. Thin-layer liquid culture technique for the growth of Helicobacter pylori. Helicobacter. 2010;15:295-302.
• ¹³
  Jean-Baptiste E, Blanchemain N, Martel B, Neu C, Hildebrand HF, Haulon S. Safety, healing, and efficacy of vascular prostheses coated with hydroxypropyl-b-cyclodextrin polymer: experimental in vitro and animal studies. Eur J Vasc Endovasc Surg. 2012;43:188-97.
• ¹⁴
  Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. Document M100-S15. Wayne, PA: Clinical and Laboratory Standards Institute; 2005.
• ¹⁵
  Langevin PB, Gravenstein N, Doyle TJ, et al. Growth of Staphylococcus aureus in Diprivan and Intralipid: implications on the pathogenesis of infections. Anesthesiology. 1999;91:1394-400.
• ¹⁶
  Durak P, Karabiber N, Ayoglu H, Yilmaz TH, Erdemli Ö. Investigation on antibacterial activities of atracurium, lidocaine, propofol, thiopentone, and midazolam. Acta Anaesth Ital. 2001;52:39-43.
• ¹⁷
  Arduino MJ, Bland LA, McAllister SK, et al. Microbial growth and endotoxin production in the intravenous anesthetic propofol. Infect Control Hosp Epidemiol. 1991;12:535-9.
• ¹⁸
  Sosis MB, Braverman B. Growth of Staphyloccoccus aureus in four intravenous anaesthetics. Anesth Analg. 1993;77:766-78.
• ¹⁹
  Bar R. A new cyclodextrin-agar medium for surface cultivation of microbes on lipophilic substrates. Appl Microbiol Biotechnol. 1990;32:470-2.
• ²⁰
  Douraghi M, Kashani SS, Zeraati H, Esmaili M, Oghalaie A, Mohammadi M. Comparative evaluation of three supplements for Helicobacter pylori growth in liquid culture. Curr Microbiol. 2010;60:254-62.
• ²¹
  Marchini A, d'Apolito M, Massari P, Atzeni M, Copass M, Olivieri R. Cyclodextrins for growth of Helicobacter pylori and production of vacuolating cytotoxin. Arch Microbiol. 1995;164:290-3.
• ²²
  Olivieri R, Bugnoli M, Armellini D, et al. Growth of Helicobacter pylori in media containing cyclodextrins. J Clin Microbiol. 1993;31:160-2.
• ²³
  Ohtsuka M, Kikuchi K, Shundo K, et al. Improved selective isolation of Bordetella pertussis by use of modified cyclodextrin solid medium. J Clin Microbiol. 2009;47:4164-7.
• ²⁴
  Letowska I, Chodorowska M, Kaczurba E, Kuklinska D, Tyski S. Bacterial growth and virulence factors production by different Bordetella pertussis strains. Acta Microbiol Pol. 1997;46:45-55.
• ²⁵
  Imaizumi A, Suzuki Y, Ono S, Sato H, Sato Y. Heptakis(2,6-O-dimethyl)beta-cyclodextrin: a novel growth stimulant for Bordetella pertussis phase I. J Clin Microbiol. 1983;17:781-6.
• ²⁶
  Suzuki Y, Imaizumi A, Ginnaga A, Sato H, Sato Y. Effect of heptakis (2,6-0-dimethyl)beta-cyclodextrin on cell growth and the production of pertussis toxin and filamentous hemagglutinin in Bordetella pertussis. Dev Biol Stand. 1985;61:89-92.
• ²⁷
  Zhang HM, Li Z, Uematsu K, Kobayashi T, Horikoshi K. Antibacterial activity of cyclodextrins against Bacillus strains. Arch Microbiol. 2008;190:605-9.
• ²⁸
  Yamamura H, Suzuki K, Uchibori K, et al. Mimicking an antimicrobial peptide polymyxin B by use of cyclodextrin. Chem Commun (Camb). 2012;48:892-4.
• ²⁹
  Gudmundsson A, Erlendsdottir H, Gottfredsson M. Impact of pH and cationic supplementation on in vitro postantibiotic effect. Antimicrob Agent Chemother. 1991;35:2617-24.
• ³⁰
  Clinton LW, warriner CB, McCormack JP, Alison MC. Reconstituted thiopentone retains its alkalinity without bacterial contamination for up to four weeks. Can J Anaesth. 1992;39:504-8.
• ³¹
  Farrington M, McGinnes J, Matthews I, Park GR. Do infusions of midazolam and propofol pose an infection risk to critically ill patients? Br J Anaesth. 1994;72:415-7.

Publication Dates

• Publication in this collection
  Mar-Apr 2014

History

• Received
  10 Sept 2012
• Accepted
  10 June 2013