The Analysis Of Bacterial Identification, Antibiotic Susceptibility And Genotyping Of Ralstonia Pickettii

1. Objective:(1)To provide reference for clinical accurate identification of Ralstonia pickettii, a specific PCR experiment was operated. (2) To understand the antibiotics resistant phenotypes of Ralstonia pickettii and provide reference for reasonable application of drugs in clinical work, antibiotics sensitivity of the strains was determined.(3) To investigate the genotypic features of Ralstonia pickettii, a preliminary MLST system was established. (4) An infection caused by Ralstonia insidiosa in the domestic was firstly reported.2. Methods:(1) 48 nonrepetitive Ralstonia pickettii strains identified by VITEK 2 system were collected from January 2008 to December 2013 and these strains were identified again through a specific PCR experiment and 16S rDNA experiment to compare the identification accuracy. (2) The antibiotic sensitivity of these strains were determined With K-B method and MIC method and the Carba NP experiment was operated to detect the B-lactamase in these strains. (3) 6 house keeping genes was chosen and the nucleotide sequences was measured, these sequences were compared to that of the different alleles of these genes to determine the sequence types.(4) The clinical data of the patient infection caused by R. insidiosa was retrospectively analyzed.3. Results:(1) By VITEK bacterial identification system,all the 48 strains were R.pickettii; By the specific PCR experiment,1 strain was R. insidiosa,8 strains were R. mannitolilytica,30 strains were R. pickettii; By 16S rDNA experiment,1 strain was R. insidiosa,7 strains were R. mannitolilytica,34 strains were R. pickettii,1 strain was Achromobacter xylosoxidans,2 strains were Burkholderia cenocepacia,3 strains were Stenotrophomonas maltophilia. (2) R. mannitolilytica was resistant to Aztreonam, aminoglycosides and Polymyxin B and was sensitive to ciprofloxacin, levofloxacin and trimethoprim/sulfamethoxazole. R. pickettii was sensitive to β-lactamase/β lactamase inhibitor, levofloxacin, minocycline, trimethoprim/Sulfamethoxazole and was resistant to aztreonam, meropenem, aminoglycosides, polymyxin B and nitrofurantoin. The Carpa NP experiment was negative in all the strains.(3) The MLST analysis showed that the 42 strains gained 18 sequence types (STs), ST9 was the most,followed be ST2 and ST4. UPGMA analysis showed that the 42 strains were divided into 2 groups (A and B) and 18 genotypes, with A group containing 8 genotypes and B group 10 genotypes. (4) An R. insidiosa strain was found in the sputum of a 93-year-old female patient, the strain was sensitive to β-lactamase/β-lactamase inhibitor, quinolones and trimethoprim/Sulfamethoxazole and was resisitant to Aztreonam, aminoglycosides, nitrofurantoin and Polymyxin B.The patient was improved through the treatment of ceftazidime and Ciprofloxacin.4. Conclusions:(1) In the clinical work, the identification results of R. picketti by VITEK 2 system are not accurate. The PCR experiment is more simple, fast and specific. (2) The drug susceptibility results by K-B method and MIC method are not consistent in some strains.The Ralstonia strains may exist other mechanisms to mediate the drug resistance to Carbapenems. (3)Through analysis of atpD, gltB, gyrB, lepA, phaC and recA, a MLST analysis system was preliminarily established. (4)The infections caused by R. insidiosa was rare in clinical work and the infection is lack of characteristic clinical symptoms. The diagnosis is dependent on the accurate microbiology identification and the antibiotics can be given according to the drug susceptibility results.