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Antibacterial Effect of Sevoflurane and Isoflurane Ángel Martínez-Monsalve3 María Dolores Crespo- Sánchez1

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Antibacterial Effect of Sevoflurane and Isoflurane Ángel Martínez-Monsalve3 María Dolores Crespo- Sánchez1 Original María Martínez-Serrano1 Manuel Gerónimo-Pardo2 Antibacterial effect of sevoflurane and isoflurane Ángel Martínez-Monsalve3 María Dolores Crespo- Sánchez1 1Servicio de Microbiología y Parasitología. Complejo Hospitalario Universitario de Albacete. 2Servicio de Anestesiología y Reanimación. Complejo Hospitalario Universitario de Albacete. 3Servicio de Cirugía Vascular. Complejo Hospitalario Universitario de Albacete. ABSTRACT property in vivo. This might then allow these agents to be con- sidered as rescue treatment against multidrug resistant patho- Introduction. Multidrug resistant bacteria are increasing gens, including a topical use in infected wounds. worldwide and therapeutic options are limited. Some anaes- Key words: Sevoflurane, Isoflurane, Anaesthetics, Inhala- thetics have shown antibacterial activity before. In this study, tion, Anti-Infective Agents. we have investigated the antibacterial effect of the halogen- ated anaesthetic agents sevoflurane and isoflurane against a range of resistant pathogens. Actividad antibacteriana de sevoflurano e Methods. Two experiments were conducted. In the first, isoflurano bacterial suspensions of both ATCC and resistant strains of Staphylococcus aureus, Escherichia coli and Pseudomonas RESUMEN aeruginosa were exposed to liquid sevoflurane and isoflurane during 15, 30 and 60 minutes. In the second experiment clinical Introducción. Las bacterias multirresistentes están au- resistant strains of E. coli, Klebsiella pneumoniae, Enterobac- mentando en todo el mundo y las opciones terapéuticas son ter cloacae, P. aeruginosa, Acinetobacter baumannii, S. aureus, limitadas. Algunos anestésicos han mostrado actividad an- and Enterococcus faecium were studied. Previously inoculated tibacteriana previamente. En este estudio hemos investigado agar plates were irrigated with the halogenated anaesthet- dicha actividad en los anestésicos halogenados sevoflurano e ic agents and these were left to evaporate before the plates isoflurano frente a un grupo de patógenos resistentes. were incubated. In both experiments colony forming units were counted in resultant plates. Métodos. Se llevaron a cabo dos experimentos. En el primero se enfrentaron suspensiones bacterianas de aislados Results. In the first experiment, isoflurane showed faster clínicos resistentes y cepas de referencia (ATCC) de Staphylo- and higher antimicrobial effect than sevoflurane against all the coccus aureus, Escherichia coli y Pseudomonas aeruginosa a strains studied. Gram-negative organisms were more suscep- sevoflurano e isoflurano en su forma líquida durante 15, 30 tible. In the second experiment, E. faecium was found to be y 60 minutos. Una muestra de la suspensión obtenida se ino- resistant to both halogenated agents; only isoflurane showed culó en agar sólido y se incubó. En el segundo experimento se statistically significant activity against the rest of the strains estudiaron aislados clínicos multirresistentes de E. coli, Kleb- studied. siella pneumoniae, Enterobacter cloacae, P. aeruginosa, Acine- Conclusions. Both halogenated agents, but particularly tobacter baumannii, S. aureus y Enterococcus faecium. Placas isoflurane, showed in vitro antibacterial activity against patho- de agar inoculadas con una cantidad conocida de las cepas gens resistant to conventional antibiotics. Further investigation se expusieron a los anestésicos líquidos, hasta su evaporación is required to determine whether or not they also exhibit this completa, antes de su incubación. En ambos experimentos se determinó el número de unidades formadoras de colonias en Correspondence: María Martínez-Serrano las placas obtenidas. Servicio de Microbiología y Parasitología. Complejo Hospitalario Universitario de Albacete. C/ Hermanos Falcó 47, 02006 Albacete. Resultados. En el primer experimento isoflurano demos- Phone: 967597507 tró una actividad mayor y más rápida que sevoflurano frente Fax: 967597170 E-mail: [email protected] a las cepas estudiadas. Los microorganismos gramnegativos Rev Esp Quimioter 2017;30(2): 84-89 84 M. Martínez-Serrano, et al. Antibacterial effect of sevoflurane and isoflurane resultaron más sensibles. En el segundo E. faecium se mostró thetic. Pure sevoflurane and isoflurane (Abbott) are supplied resistente a ambos agentes y sólo isoflurano mostró diferen- with neither additives nor preservatives. The tubes were then cias significativas en su efecto antimicrobiano frente al resto incubated at 37ºC with continuous shaking. After 15, 30 and 60 de las cepas. minutes of exposure, a 100 μL sample was spread on a sterile Conclusiones. Ambos anestésicos halogenados, especial- blood agar plate (bioMérieux). The plates were incubated for mente isoflurano, mostraron actividad antibacterianain vitro 24 hours at 37ºC (5-10% CO2) and then colonies were counted. frente a patógenos resistentes a los antibióticos convencio- Each experiment was repeated three times. nales. Se necesita mayor investigación para determinar si este Solid-liquid experiment. In a second experiment, we efecto se confirmain vivo. En ese caso se podría considerar a tried to mimic the conditions of topical use on infected ul- estos agentes como una alternativa frente a bacterias multi- cers by pouring liquid sevoflurane and isoflurane over bacteria rresistentes, incluyendo por ejemplo su uso tópico en heridas spread in solid medium. Only multidrug-resistant strains from infectadas. the laboratory collection were included (table 1). Palabras clave: Sevoflurano, Isoflurano, Anestésicos, Inhalación, Agentes Bacterial suspensions adjusted to a density of 0.5 Mc- 8 anti infectivos. Farland (1.5 x 10 CFU/mL) from cultures of 18-24 hours were prepared in sterile physiological saline. Serial 10-fold dilutions INTRODUCTION were performed to give approximately 105 CFU/mL. For each strain, 5 blood agar plates were inoculated, each with 5 μL of Isolation of multidrug-resistant bacteria is increasing the resulting suspension. One was incubated 24 hours at 37ºC worldwide and there is an urgent need for the development of (5-10% CO2) with no other manipulation and used as a growth new antimicrobial drugs. This situation has increased interest in control. Two plates were treated with sevoflurane and two with the potential use of alternative antibacterial agents and older, previously discarded drugs. Research on molecules with possible antimicrobial activity has led to a new group of “non-antibiotic Table 1 Strains included in each experiment. drugs” which includes a number of inhalational anesthetics1-3. However, previous studies have reported contradictory results on the antimicrobial activity of these molecules. Some of these Liquid-Liquid Experiment studies included isoflurane and sevoflurane but, from the data available, they have yet to be tested against resistant strains4-9. Reference Strains: The aim of this study was to investigate the possible in S. aureus (ATCC* 29213) vitro antibacterial effect of sevoflurane and isoflurane on ref- E. coli (ATCC 27853) erence and multidrug-resistant strains of species commonly P. aeruginosa (ATCC 25922) found in hospital settings. Clinical Isolates (antimicrobial resistancea): MATERIALS AND METHODS S. aureus (Met, Ery, Ami, Cip) E. coli (ESBL, Cip) Liquid-liquid experiment. In this experiment, we tested mi- P. aeruginosa (Gent, Tob, Carbap) croorganisms usually associated with ventilator associated pneu- monia (VAP) (table 1). American Type Culture Collection strains of Staphylococcus aureus (ATCC 29213), Pseudomonas aeruginosa Solid-Liquid Experiment (ATCC 27853) and Escherichia coli (ATCC 25922) were studied as Clinical Isolates (antimicrobial resistancea): reference strains. For each species, one multidrug-resistant strain E. coli (ESBL, Amino, FQ, TMP/SMX) isolated from clinical samples was included. Minimal inhibitory concentrations (MIC) were determined by broth microdilution K. pneumoniae (ESBL, Amino, FQ, TMP/SMX) (Microscan Walkaway) and antimicrobial susceptibility profiles E. cloacae (Cepha, Amino, FQ) were set following Clinical and Laboratory Standards Institute P. aeruginosa (Carbap, Amino, FQ) (CLSI) breakpoints. A. baumannii (Carbap, Amino, FQ) One tube with 5 mL of nutrient broth (bioMérieux) was inoculated with each strain. The tubes were then incubated S. aureus (Met, Ery, Amino, FQ) overnight at 37ºC. Half a millilitre of these cultures was added E. faecium (Van, Str, Ery, FQ) to 4.5 mL of fresh nutrient broth and incubated at 37ºC for 90 *ATCC; American Type Culture Collection. minutes. Further dilutions of each suspension were performed aMet: methicillin; Ery: erythromycin; Ami: amikacin; Cip: ciprofloxacin; Gent: 3 in sterile physiological saline to give approximately 10 CFU/mL. gentamycin; Tob: tobramycin; TMP/SMX: trimethoprim-sulfamethoxazole; Van: One-millilitre samples of this suspension were inoculated vancomycin; Str: streptomycin (high-level resistance); Carbap: carbapenems; into sterile evacuated tubes (BD Vacutainer®) containing either Amino: aminoglycosides; FQ: fluoroquinolones; Cepha: cephalosporins; ESBL: 1 mL of sterile saline solution (controls) or 1 mL of the anaes- extended-spectrum beta-lactamase. Rev Esp Quimioter 2017;30(2): 84-89 85 M. Martínez-Serrano, et al. Antibacterial effect of sevoflurane and isoflurane Table 2 Growth of strains after exposure to anesthetics. Liquid-liquid experiment. Strain Drug Exposure time 15 min 30 min 60 min S. aureus ATCC Sevo 8x102 ± 0.5x102 4.8x102 ± 2.2x102* 2.6x102 ± 0.9x102* Control 8.1x102 ± 0.8x102 7.8x102 ± 1.9x102 8.3x102 ± 1.8x102
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