Isolation And Identification Of Ralstonia Mannitolilytica Strain From A Patient With Septicaemia And Analysisof Its Drug Resistance Genes

Objective:This study is to isolate and identify the bacterial pathogen in peripheral blood sample from a etiologically-unknown septicemia patient and to determine the drug resistance of isolate and its drug resistance-associated genes.Methods:Two peripheral blood samples were collected from the patient andthen the samples were inoculated on blood agar plates using streak plating. The plates were routinely cultured at 37℃ for 24 h. After incubation, several single colonies on the plates were picked up for bacterial identification by using microscopy after Gram-staining and VITEK 2-compact automatic bacterium detection and analysis system. Moreover, PCR for amplifying segment of 16S rRNA gene and sequencing of the PCR products were performed to further determine the results of bacteriologic identication above. Micro-double dilutiontest was applied to detect the susceptibility of bacterial isolate against antibiotics. Both β-lactamase and broad-spectrumβ-lactamase confirmatorytestsas well as both AmpC enzyme and carbapenemase phenotype tests were used to detect these enzymes produced by the isolate and their types. The 19 genes for encoding common P-lactamases, broad-spectrumβ-lactamases, AmpC enzymes or carbapenemases derived from strains of Enterobacteriaceaeas well as the 21 genes which products annotated as the β-lactamasesof Ralstonia mannitolilytica （GenBank Accession No.:CP010799, CP010800, AJ270257） in the isolate were detected by PCR. The PCR products were sequenced after T-A cloning.Results:Same Gram-negative short bacillus was isolated from the two peripheral blood samples. The bacterial isolates were identified as Ralstonia mannitolilytica according to the detection results of VITEK 2-compact automatic bacterium detection and analysis system plus GN bacterial identification card with an identity= 99% and an identification No.= 4003611303500001. The PCR and sequencing results also demonstrated that the nucleotide sequence of 16S rRNA gene amplification segments from the isolate of Ralstonia mannitolilytica had 100% identity compared to that reported in the GenBank （Accession No.:AJ270257）. The isolate of Ralstonia mannitolilytica was sensitive to ceftriaxone, cefepime, ciprofloxacin, ofloxacin, tigecycline and SMZ-TMP, but resistant to ceftazidime, amoxicillin/clavulanate, ampicillin, ticarcillin/clavulanate, piperacillin/tazobactam, imipenem, meropenem, aztreonam, gentamicin, amikacin and nitrofurantoin. All the PCRs to detect the 19 genes encoding common drug resistance-associated enzymes derived from strains of Enterobacteriaceae were negative, but five β-lactamase encoding genes derived from Ralstonia mannitolilytica, TK49₀9850, TK49₂955, TK49₄470, TK49₄495 and TK49₁8990 genes, could be detectable by PCR. Compared to the corresponding genes of Ralstonia mannitolilytica in GenBank （Accession No.:CP010799, CP010800, AJ270257）, the nucleotide and amino acid sequence identities of TK49₀9850, TK49₂955, TK49₄470, TK49₄495 and TK49₁8990 genes from the isolate of Ralstonia mannitolilytica were 96.2% （737/766） and 96.2% （251/261）,97.3% （838/861） and 99.0% （283/286）,100% （795/795） and 100% （264/264）,97.5% （980/1005） and 98.8% （330/334）, and 97.5% （807/828） and 97.1% （267/275）, respectively. However, these sequences had very lower identities with those of the common β-lactamase or broad-spectrum β-lactamase encoding genes derived from strains of Enterobacteriaceae.Conclusions:The septicemia patient was rarely infected with Ralstonia mannitolilytica. The isolate of Ralstonia mannitolilytica has a high drug resistance with a noticeable diversity against different β-lactam antibiotics, and its β-lactamase. encoding genes completely differ to those from Enterobacteriaceae strains.