ABSTRACT
Objective To isolate and identify the pathogenic bacteria from peripheral blood of a patient with septicemia of unknown etiology and to analyze their drug resistance genes.Methods Two peripheral blood samples were collected from the patient after having fever.Several assays including culturing bacteria on blood agar plates by using streaking technique,Gram-staining of bacterial colonies and microscopic observation,VITEK 2-compact automatic bacterium detection and analysis system as well as a sequencing analysis of the 16s rRNA gene were performed to identify the bacterial pathogens in blood samples.Microdilution test was performed to detect the drug susceptibilities of isolated bacteria to antibiotics.Confirmatory tests were performed to detect the production of β-lactamase and extended spectrum β-lactamase by the isolated strains and the phenotypes of AmpC enzyme and carbapenemase.PCR was used to identify the β-lactamase-encoding genes in the isolated strains by using the primers of 19 common β-lactamase-,AmpC enzyme-and carbapenemase-encoding genes in Enterobacteriaceae strains and the primers of 21 annotated gene sequences encoding the β-lactamase of a Ralstonia mannitolilytica strain.The PCR products were sequenced and analyzed after T-A cloning.Results Ralstonia mannitolilytica strains were isolated from the two peripheral blood samples.The isolated strains were sensitive to ceftriaxone,cefepime,ciprofloxacin,ofloxacin,tigecycline and compound sulfamethoxazole (SMZ-TMP),but resistant to the other 11 tested antibiotics.Results of PCR amplification by using the primers of common genes encoding β-lactamase of Enterobacteriaceae strains were all negative.Fragments of genes encoding the β-lactamase of the isolated Ralstonia mannitolilytica strain were successfully amplified,which were TK49_09850,TK49_12955,TK49_14470,TK49_14495and TK49_18990.The sequences of the amplified gene fragments were not similar to those of the common β-lactamase-encoding genes in Enterobacteriaceae strains.Conclusion The patient was infected with Ralstonia mannitolilytica.The isolated Ralstonia mannitolilytica strain showed a high-level drug resistance with a noticeable diversity against different β-lactam antibiotics.The genes encoding β-lactamase of the isolated Ralstonia mannitolilytica strain were completely different to those of Enterobacteriaceae strains.
ABSTRACT
Ralstonia mannitolilytica junto con Ralstonia pickettii han sido asociadas con brotes hospitalarios relacionados con la contaminación de algún dispositivo o fluido. El objetivo de este trabajo fue describir un brote por R. mannitolilytica a partir de bacteriemias asociadas a catéteres implantables y semiimplantables ocurrido en un hospital pediátrico de alta complejidad y evaluar la utilidad del empleo de métodos moleculares para su investigación.Se detectó la presencia de bacilos gram negativos no fermentadores, con igual antibiotipo, en hemocultivos y retrocultivos a partir de dos pacientes que tenían catéteres implantables y estaban atendidos en una misma área del hospital. Se realizaron estudios microbiológicos de muestras de frascos de heparina, soluciones de dextrosa y soluciones antisépticas con resultado negativo. Algunos pacientes tuvieron signos y/o síntomas clínicos de bacteriemia al habilitar los catéteres para su uso. Se citaron para su estudio a todos los pacientes que habían tenido un procedimiento de apertura y cierre de catéter durante las fechas cercanas a los hallazgos en hemocultivos (N expuestos = 45). Ocurrieron 17 casos (infectados), a partir de los cuales se analizaron 23 aislamientos, en los que se pudo documentar la presencia de R. mannitolilytica (23 aislamientos). Por métodos moleculares se determinó que los aislamientos provenientes de muestras de pacientes involucrados en el brote se encontraban estrechamente relacionados y podrían representar una misma cepa o clon. Por evidencia circunstancial se consideró a la "solución heparínica de cierre" como fuente posible del brote (AU)
Both Ralstonia mannitolilytica and Ralstonia pickettii have been associated with hospital outbreaks due to device or fluid contamination. The aim of this study was to describe an implantable- or semi-implantable-catheter-related bacteremia outbreak by R. mannitolilytica in a tertiary-care hospital and to assess the usefulness of molecular analysis for the identification of the organism. Non-fermenting gram-negative bacilli, with identical antibiotypes, were detected in hemocultures of two patients with implantable catheters in the same hospital area. Microbiological studies of heparin and dextrose and antiseptic solution vials were negative. Some of the patients had clinical signs and/or symptoms of bacteremia when the catheter was prepared for use. All patients who underwent a procedure of accessing or locking the port of the catheter around the time of the positive hemoculture findings were contacted (N exposed = 45). Seventeen infections were detected, of which 23 isolates were analyzed. The presence of R. mannitolilytica was recorded in 23 isolates. Molecular analysis showed that the isolates from the samples of the patients involved in the outbreak were closely related and might represent the same strain or clone. Circumstantial evidence suggested that the heparin-lock solution may have been the source of the outbreak (AU)