RESUMEN
Los tests de oxacilina habituales pueden fallar en la detección de ciertos casos de Staphylococcus aureus resistente a meticilina. El objetivo del presente trabajo es evaluar la capacidad del método de difusión en disco de cefoxitina (30 μg) para detectar la resistencia a meticilina en aislados de S. aureus con características atípicas de resistencia. Se estudiaron 53 aislados de S. aureus. La sensibilidad de los mismos se estudió mediante el sistema Vitek, y los tests de oxacilina y de cefoxitina mediante difusión en discos (Clinical and Laboratory Standards Institute [CLSI] 2007); la resistencia a oxacilina se confirmó mediante una reacción en cadena de la polimerasa (PCR) múltiple previamente descrita que permite la identificación de S. aureus y la detección simultánea de resistencia a meticilina. Los aislados mecA positivos que presentaban crecimiento en la zona de inhibición al exponerlos a oxacilina se consideraron heterorresistentes; los que eran mecA negativos pero con sensibilidad intermedia o resistencia a oxacilina se consideraron límite (borderline). Todos los aislados se ensayaron frente al disco de 30 μg de cefoxitina según normas CLSI (sensibilidad: > 22 mm; resistencia: < 21 mm). Las cepas de control para todos los ensayos incluyeron S. aureus resistente a meticilina ATCC 43300 y S. aureus sensible a meticilina ATCC 25923. Los aislados formaron cuatro grupos. Grupo I: 20 aislados multirresistentes, sensibles a oxacilina y mecA negativos; grupo II: 16 aislados con resistencia o sensibilidad intermedia a oxacilina y mecA negativos; grupo III: 11 aislados heterorresistentes y mecA positivos; grupo IV: seis aislados mecA positivos con perfiles atípicos de resistencia (penicilina y oxacilina; ciprofloxacino y eritromicina). Treinta y cinco aislados mecA negativos incluidos en los grupos I y II mostraron zonas de inhibición > 22 mm; un aislado del grupo II mostró un halo de 20 mm. Los 17 aislados mecA positivos de los grupos III y IV mostraron resistencia frente al disco de cefoxitina. Podemos concluir que el método de difusión en disco de cefoxitina de 30 μg es eficiente para la detección de resistencia a meticilina y permite una clara definición de aquellos aislados de S. aureus, incluidos aquellos con características atípicas de resistencia (AU)
Oxacillin tests may fail to detect some methicillin- resistant S. aureus populations. The objective of this study is to evaluate the discriminative capacity of the Clinical and Laboratory Standards Institute (CLSI) disk diffusion method with a cefoxitin 30 μg disk on S. aureus isolates with unusual phenotypic characteristics of antimicrobial resistance. We studied 53 clinical S. aureus isolates. The antimicrobial susceptibility of all isolates was routinely studied by the VITEK 2 System (bioMérieux). Methicillin resistance was also studied by CLSI oxacillin method and confirmed by a previously described multiplex polymerase chain reaction (PCR) method which permits S. aureus identification and simultaneous detection of methicillin resistance. MecA positive isolates presenting a diffuse growth within the zone of inhibition when exposed to oxacillin were considered heteroresistant; mecA negative, oxacillin intermediate or resistant isolates were considered borderline. All the isolates were tested with a cefoxitin 30 μg disk, according to the CLSI guidelines (susceptibility: > 22 mm; resistance: < 21 mm). Control strains for all assays included MRSA ATCC 43300 and MSSA ATCC 25923. The isolates formed four groups. Group I: 20 multiresistant, oxacillin susceptible and mecA negative isolates; group II: 16 resistant or with intermediate oxacillin susceptibility and mecA negative isolates; group III: 11 heteroresistant and mecA positive isolates; group IV: six mecA positive isolates with atypical resistance profiles (penicillin and oxacillin, or ciprofloxa- cin and erythromycin resistance). Thirty-five mecA negative isolates included in groups I and II showed inhibition zones > 22 mm; one isolate from group II showed 20 mm. The 17 mecA positive isolates from groups III and IV showed resistance to cefoxitin disk. The 30 μg cefoxitin disk diffusion method is proposed as an efficient method for the detection of methicillin resistance and permits a clear determination set S. aureus isolates, even those with atypical antimicrobial characteristics (AU)
Asunto(s)
Humanos , Antibacterianos , Cefoxitina , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Pruebas de Sensibilidad Microbiana/métodosRESUMEN
Thirty six fluorescent Pseudomonas isolates were obtained from the rhizosphere of sunflower plants. By antibiosis tests, the six more efficient strains in Sclerotinia sclerotiorum growth inhibition, were selected. Simultaneously, twenty three fluorescent Pseudomonas isolates were recuperated from the rhizosphere of wheat plants and the five most efficient strains in growth inhibition of the fungi Gaeumannomyces graminis were selected. The strains selected from the rhizosphere of sunflower plants had no antagonistic effect on G. graminis and the bacteria isolated from the wheat rhizosphere showed no fungistatic activity on S. sclerotiorum. These results suggest the existence of a certain degree of plant bacteria pathogenic specificity. Among the selected bacteria, the strain FF5 of P. fluorescens originated the major inhibiting halo in vitro against S. sclerotiorum (Figure 1). In liquid culture medium this bacterium produces an antifungal substance that promotes lysis of fungi mycelium (Figure 2) and inhibition of ascospore germination and is not inhibited by the presence of Fe+3 in the culture medium (Table 1). Its synthesis is not associated with the production of fluorescein. Its action is not enzymatic because it is a substance of low molecular weight (< 2000), resistant to autoclave sterilization and photo-stable. The amount of NH4+ and the high pH values produced by the FF5 strain in the liquid culture medium (Table 2) are not responsible for the antifungalal action.
RESUMEN
Thirty six fluorescent Pseudomonas isolates were obtained from the rhizosphere of sunflower plants. By antibiosis tests, the six more efficient strains in Sclerotinia sclerotiorum growth inhibition, were selected. Simultaneously, twenty three fluorescent Pseudomonas isolates were recuperated from the rhizosphere of wheat plants and the five most efficient strains in growth inhibition of the fungi Gaeumannomyces graminis were selected. The strains selected from the rhizosphere of sunflower plants had no antagonistic effect on G. graminis and the bacteria isolated from the wheat rhizosphere showed no fungistatic activity on S. sclerotiorum. These results suggest the existence of a certain degree of plant bacteria pathogenic specificity. Among the selected bacteria, the strain FF5 of P. fluorescens originated the major inhibiting halo in vitro against S. sclerotiorum (Figure 1). In liquid culture medium this bacterium produces an antifungal substance that promotes lysis of fungi mycelium (Figure 2) and inhibition of ascospore germination and is not inhibited by the presence of Fe+3 in the culture medium (Table 1). Its synthesis is not associated with the production of fluorescein. Its action is not enzymatic because it is a substance of low molecular weight (< 2000), resistant to autoclave sterilization and photo-stable. The amount of NH4+ and the high pH values produced by the FF5 strain in the liquid culture medium (Table 2) are not responsible for the antifungalal action.