Prevalence Of Plasmid-encoded 16S RRNA Methylase Genes In Association With β-lactamases Among Clinical Isolates Of Serratia Marcescens

ObjectiveTo investigate the prevalence of plasmid-mediated 16 S r RNA methylase genes and their association with β-lactamases genes in clinical isolates of Serratia marcesceas in Anhui.Materials and MethodsIsolatesA total of 201 consecutive non-duplicate isolates of Serratia marcesceas were collected from 2005 to 2012 at 34 hospitals in Anhui, China. In this study, Escherichia coli ATCC 25922 and sodium azide-resistant Escherichia coli J53 were used as a quality control strain for antimicrobial susceptibility testing and the recipient for conjugation experiments respectively.Method1. Minimal inhibitory concentrations(MICs) of different antimicrobial agents were determined for each isolate by broth microdilution method as described by the Clinical and Laboratory Standards Institute(CLSI, 2013)2. Bacterial plasmid DNA was extracted from the AMK-resistant isolates with Plasmid DNA Extraction Kit. The 16 S r RNA methylase genes were screened by polymerase chain reaction(PCR) in 58 AMK-resistant clinical isolates of S. marcescens. The PCR fragmentation was examined by agarose gel electrophoresis. The positive isolates were subsequently confirmed by sequencing and the sequence was compared with the Gen Bank nucleotide database.3. Conjugation experiment was conducted for the 16 S r RNA methylase gene positive isolates using Escherichia coli J53 AZR as the recipient strain. The MIC of S. marcescens isolates, recipient strains and conjugants were tested as above.4. The β-lactamases genes were analyzed by PCR with the methods described previously for the 16 S r RNA methylase gene positive isolates and conjugants. All the purified PCR products were sequenced and the sequence was compared with the Gen Bank nucleotide database.5. Plasmid DNAs were extracted from the conjugants and the β-lactamases genes were analyzed by PCR. All the purified PCR products were sequenced and the sequence was compared with the Gen Bank nucleotide database.Results1. Among the total of 201 isolates, the results of antimicrobial susceptibility test showed that the IMP and MEM were the most susceptible drug and rates of susceptibility can be up to 98.51%. Compared to other 3nd generation cephalosporins, the 4th generation cephalosporins and antibiotic compound preparation showed more effective antibacterial ability. Rates of susceptibility to GAT and LVX in relation to total isolates were 77.11% and 75.12%. 58 isolates showed resistant to AMK and GEN simultaneously. The 58 isolates was the frequently isolated from sputum, followed by urine and secretion.2. 7 strains of the 58 AMK-resistant S. marcescens isolates harboured 16 S r RNA methylase genes. With regard to the genotypes, the most prevalent genes were arm A(5), followed by rmt B(2), whereas no rmt B,rmt C,rmt D,and npm A-positive isolate was detected. the prevalence of 16 S r RNA methylase genes was confirmed to be 3.5%(7/201) overall and 12.1%(7/58) among AMK-resistant S. marcescens isolates.3. Conjugative transfer of 16 S r RNA methylase gene to E. coli J53 AZ R was positive for 6 of the 7 selected isolates, including 5 isolates carrying arm A and 1 isolate positive for rmt B. All transconjugants exhibited an increase in the MICs of aminoglycosides compared to the recipient strains.4. 7 strains of the armA and rmt B-positive strains were screened by PCR. A total of 6 isolates were found to carry at least one of these β-lactamase genes. The dominant β-lactamase genes in this study were CTX-M-14 and TEM-1 genes, followed by SHV-5 and OXA-1.5. All the transconjugants were detected β-lactamase genes, including 3 strains harbouring bla CTX-M-14, 2 strains harbouring bla TEM-1 and 1 strains harbouring bla SHV-5.Conclusion1. According to the MICs, multi-drug resistant S. Marcescens have been often identified from different specimen. The dissemination of these isolates is very worrisome and continuous monitoring should be reinforced.2. Plasmid-mediated 16 S r RNA methylase genes were first discovered among S. Marcescens in our country.3. All transconjugants exhibited an increase in the MICs of aminoglycosides compared to the recipient strains.4. It was observed that the significant relationship of 16 S rRNA methylase with β-lactamse amongst S. Marcescens. These linked genes can be disseminated by horizontal transfer simultaneously.