Isolation and identification of Ralstonia mannitolilytica strains from a patient with septicaemia and analysis of their drug resistance genes / 中华微生物学和免疫学杂志

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.