Developing Multilocus Sequence Typing And Antibiotic Resistance Of Serratia Marcescens

ObjectiveDeveloping multilocus sequence typing(MLST), studying the antibiotic resistance and the epidemic character of Serratia marcescens to control the nosocomial infection and prevent outbreak. They were evidence for clinical rational use of antimicrobial agents. MaterialsSixty five clinical isolates were collected from unrelated patients in First Affiliated Hospital of Soochow University from September 2013 to December 2015, Collecting source and drug resistance of specimen, distribution of departments, gender and age of patients.26 Serratia marcescens complete gonome sequences date in public databases were compared. Methods1. Sixty five clinical isolates were collected from unrelated patients in First Affiliated hospital of soochow university from September 2013 to December 2015,Collecting source and drug resistance of specimen,distribution of departments,gender and age of patients.26 Serratia marcescens complete gonome sequences date in public databases were compared.2. Bacterial stain purification and K-B to measure drug susceptibility.3. All isolates were cultured and genomic DNA was isolated from all strains with Wizard Genomic DNA purification kit, which were stored at-20°C.4. Seven housekeeping genes(dnaA, gyrB, rec A, infB, pgi, adk and rpoB) were selected as targets for Serratia marcescens, design the primer sequences for PCR amplification.5. Sequence alignment and phylogenetic analysis send the PCR production to Shang hai sheng gong company sequencing Bioedit software was utilized to align and edit the sequencechromatograms.DnaSPv5,Splitstree,CLUSTAL(http://www.genome.jp/tools/clust alw/),eBurstV3 were used to analysis GC%,dN/dS, polymorphism from the sequence alignments,Phylogenetic analysis,clonal complexes(CCs), split decomposition trees and so on in order to detect and discusses the relationship of the evolution between strains. Results1. Serratia marcescens specimens were comprised of sputum(63.1%),urine(12.3%)and wound(9.2%). distribution of departments Serratia marcescens specimens respectively were respiratory medicine(55.4%),haematology department(15.4%) and ICU(9.2%).Serratia marcescens isolated stains were more from men than women(64.6%),75.4% of total patient over the age of 50 patients.2. The results of antibiotics susceptibility testing demonstrated that the drug resistance rate to thirteen antibiotics differed from 10.0% to 53.8%,Piperacillin is one of the highest percentages of drug resistance rate(53.8%), imipenem resistance against 32.5%. aminoglycosides(amikacin) specificity is 86.2%.3. MLST based on seven housekeeping genes indicated 26 Serratia marcescens isolates into 18 STs and 65 Serratia marcescens isolates into 38 STs. The number of sequence type ranged from 25 to 35,rpoB possessed the most STs(35)following gyrB( 33).The number of alleles ranged from 34 to 98, gyrB possessed the most polymorphism sites(98)and adk the fewest polymorphism sites(34).polymorphism loci mutation rate is between7.2% and 13.0%,mutation rate of highest is gyr B(13.0%),minimum value is adk(7.2%).The GC content of the seven housekeeping genes were 58.0%~61.9%, gyrB possessed the most GC( 61.9%).The dN/dS calculated for all loci and ranged from 0 for infB to 0.0494 for adk.The dN/dS ratio of infB was zero, suggesting that infB is under strong selection pressure.4. SplitTree Version was used to draw split decomposition trees of the concatenated sequences of the STs and Serratia marcescens to detect possible recombination-based network structures.Splits tree research indicated that the majority of Serratia marcescens isolates and STs were classified into Cluster1,Cluster 2,Cluster 3,Cluster 4,Cluster 5 and Cluster A,Cluster C,Cluster D,Cluster E.5. CLUSTALW research indicated that all isolates clustered into 56 STs, of which 20 isolates in ST25 and classified into one clonal complex CC25.6. eBurst V3 analysis was done to assess the presence of clonal complexes that share 5group and 39 Singleton, 4 double couplets(DLV).7. Tajima’s D、Fu and Li’s D*and Fu and Li’s F* neutrality tests were powerful at detecting population growth, indicated that only rpoB alleles of the synonymous substitutions have statistical significance. Conclusions1. Different department of vulnerable groups in our hospital is different, mainly men over the age of 50 had caused respiratory and urinary tract infections.2. The drug resistance rate to imipenem is relatively high in Serratia marcescens, and show a trend of pan-drug resistance. The molecular epidemiological trends of Serratia marcescens are relatively slow, and its genome is relatively concentrated. Our key tasks locate on monitoring the prevalence of ST25-CC25 Type in Serratia marcescens and prevent its major outbreak or prevalence in hospital.3. we established MLST classification technology of Serratia marcescens for the first time all over the world,this technology will plan an important role in bacteria strain identification、biological roots and epidemic outbreak investigation.4. We will further improve MLST technology in the future.