Evaluación de diversos métodos fenotípicos para la detección de carbapenemasas KPC en Klebsiella pneumoniae / Evaluation of phenotypic methods for the detection of KPC carbapenemases in Klebsiella pneumoniae

Se investigó el desempeño de diversos métodos fenotípicos para la detección de carbapenemasas KPC en 44 aislamientos de Klebsiella pneumoniae con sensibilidad disminuida a los carbapenems, 30 productores y 14 no productores de KPC. Se determinó la sensibilidad a imipenem, meropenem y ertapenem por dilución en agar y difusión por discos. Se evaluaron los siguientes métodos fenotípicos: sinergia entre discos de carbapenems y discos con los inhibidores amoxicilina/ácido clavulánico (AMC ) y ácido aminofenilborónico (APB); inhibición por "discos combinados" (según una técnica de predifusión diseñada a tal fin en nuestro laboratorio), comparando el efecto de los carbapenems solos o asociados con los inhibidores mencionados; y el test de Hodge modificado, para el cual se propone una lectura cuantificada. El método de Hodge detectó todos los aislamientos KPC positivos con los tres carbapenems evaluados, mientras que fue negativo para todos los aislamientos KPC negativos con imipenem y meropenem, y produjo dos resultados falsos positivos con ertapenem. Cuantificando la lectura de este método se pudieron discriminar objetivamente los resultados verdaderos positivos (≥ 8 mm) de los falsos positivos (< 5 mm). Se observó sinergia entre carbapenems y APB con todos los aislamientos KPC positivos, aunque esto requirió ajustar la distancia entre discos. En los aislamientos KPC negativos no se observó sinergia entre carbapenems y APB. Empleando el método con discos combinados con imipenem o meropenem más APB se detectaron la mayoría de los aislamientos KPC positivos y no se observaron falsos positivos. Por el contrario, las combinaciones carbapenem más AMC no fueron sensibles ni específicas.

We evaluated phenotypic methods for the detection of KPC carbapenemases in 44 clinical isolates of K. pneumoniae having reduced susceptibility to carbapenems, 30 of which were KPC-positive and 14 KPC-negative. Both the agar dilution and disk diffusion methods were performed for imipenem, meropenem and ertapenem. The following phenotypic methods were assayed: the double disk synergy test, using boronic acid or clavulanic acid as inhibitors, "combined" disks of carbapenem plus inhibitor (boronic acid, clavulanic acid and both boronic plus clavulanic acid), by using a pre-diffusion technique and the modified Hodge test. The double disk diffusion test using boronic acid could detect all KPC-positive isolates, but adjustment of disk distance was necessary for achieving such performance. The simulation of combined disks by our pre-diffusion technique detected all KPCpositive strains for all 3 carbapenems when using boronic acid as inhibitor, clavulanic acid was less susceptible and specific as compared with boronic acid. The modified Hodge test using any carbapenem was clearly positive for all KPC-producing isolates. This test was negative for all KPC-negative strains when imipenem or meropenem were used, but 2/14 isolates yielded a weak positive result when using ertapenem. By measuring the enhanced growth of E. coli ATCC 25922 observed in this test, we could objectively discriminate true-positive (≥ 8 mm) from false-positive results (< 5 mm). Our results show that the use of phenotypic methods is effective for the rapid detection of KPC producers in K. pneumoniae isolates with reduced susceptibility to carbapenems.

Sensibilidad de Acinetobacter a la colistina evaluada mediante los métodos de predifusión y de concentración inhibitoria mínima: Detección de aislamientos heterorresistentes / Comparative evaluation of the sensitivity of Acinetobacter to colistin, using the prediffusion and minimum inhibitory concentration methods: Detection of heteroresistant isolates

El objetivo de este estudio fue evaluar comparativamente los métodos de predifusión y de concentración inhibitoria mínima para establecer la sensibilidad de aislamientos del complejo Acinetobacter calcoaceticus-baumannii (ABC) a la colistina y detectar a aquellos que presenten heterorresistencia a dicho antibiótico. Se estudiaron 75 aislamientos de ABC recuperados de materiales clínicamente significativos. Se determinó su sensibilidad a la colistina por el método de predifusión y de concentración inhibitoria mínima. Todos los aislamientos resultaron sensibles, con CIM = 2 µg/ml y halos de inhibición en el ensayo de la predifusión = 20 mm. Mediante el método de eficiencia de plaqueo se evaluó la presencia de heterorresistencia a la colistina. Se encontraron 14 aislamientos que originaron colonias heterorresistentes; sus CIM aumentaron en algunos casos en más de 8 veces. Con estas colonias seleccionadas se repitió el ensayo de predifusión. Finalmente se confeccionaron los gráficos de dispersión y se realizaron los análisis de regresión lineal, tanto para el conjunto inicial de todos los aislamientos clínicos como para el subgrupo de los aislamientos resistentes generados durante la evaluación de la heterorresistencia. Se obtuvieron coeficientes de determinación (r²) de 0,2017 y 0,604, respectivamente, lo que indica correlación entre los métodos sólo al evaluar aislamientos preseleccionados por su resistencia a este agente.

The objective of this study is to perform a comparative evaluation of the prediffusion and minimum inhibitory concentration (MIC) methods for the detection of sensitivity to colistin, and to detect Acinetobacter baumanii-calcoaceticus complex (ABC) heteroresistant isolates to colistin. We studied 75 isolates of ABC recovered from clinically significant samples obtained from various centers. Sensitivity to colistin was determined by prediffusion as well as by MIC. All the isolates were sensitive to colistin, with MIC = 2µg/ml. The results were analyzed by dispersion graph and linear regression analysis, revealing that the prediffusion method did not correlate with the MIC values for isolates sensitive to colistin (r² = 0.2017). Detection of heteroresistance to colistin was determined by plaque efficiency of all the isolates with the same initial MICs of 2, 1, and 0.5 µg/ml, which resulted in 14 of them with a greater than 8-fold increase in the MIC in some cases. When the sensitivity of these resistant colonies was determined by prediffusion, the resulting dispersion graph and linear regression analysis yielded an r² = 0.604, which revealed a correlation between the methodologies used.

Antibiotic Susceptibility Test of Neisseria gonorrhoeae by Disc Diffusion Method and Its Correlaction with Agar Plate Dilution Method / 대한피부과학회지

In this study, we compared the MIC by agar plate dilution method with the inhibition zone diameters by ordinary disc diffusion method and 24 hr-prediffusion disc method on five antibiotics, respectively. The results were summarized as follows; 1. The agar plate dilution method and the ordinary disc diffusion method gave a satisfactory degree of correlation by linear regression. The correlation coeffecients were highly significant for penicillin (r= -0.88) and ampicillin(r = -0.91) but less significant for tetracyclin(r= -0.34), kanamycin(r= -0.55) and spectinomycin (r = -0.46). 2. Between the agar plate dilution method and the 24 hour-prediffusion disc method the correlation coeffecients were highly significant for penicillin(r= -0.9) and ampicillin (r= -0.91) but less so for tetracyclin(r= -0.4), kanamycin (r=-0.57) and spectinomycin(r=-0.5), 3. In relation to the MIC values by agar pate dilution method, the correlation coefficients were slightly higher and the slopes of linear regression were steeper for the 24 hour-prediffusion disc method than the ordinary disc diffusion method, but the differences were statistically not significant.

Surveillance for Spectinomycin Resistant Neisseria Gonorrhoeae / 대한피부과학회지

In 1982, we reported a case of recalcitrant urethritis caused by a strain of spectinomycin resistant PPNG. It caused a great fear that it might be the signal of an end of spectinomycin era, The disk-prediffusion method was used on all isolates from the Choong-Ku VD Clinic between August 1984 and December 1985 to screen out strains of N. gonorrhoeize resistant to spectinomycin, with disks containing 100pg of spectinomycin, Strains with inhibition zone greater tha.n 25 mm were considered susceptible, We could not disclose a single strain which wa.s resistant to spectinornycin, The reason why we do not see any more of spectinomycin. N. gonorrhoeae requires some speculation, It is possible that the mutant which had resistant to spectinornycin might not have teen quite stable and, after series of passage through human hosts, mutated back to original spectinornycin sensitive states. It is suggested that continual surveillance for spectinomycin resistant N. gonorrhoeae should be conducted.