Molecular Identification Of Klebsiella Pneumonia And Its β-Lactamases

Klebsiella pneumonia,belonging to genus of Klebsiella,is one of the major pathogenic bacteria for nosocomial infections.Actually,some serious infection diseases,such as pneumonia,urinary tract infection,and sepsis,were caused by K.pneumonia.In recent years,with the extensive use of antibiotics,especially cephalosporins,carbapenems and so on,K.pneumonia is quickly obtaining resistance to all kinds of antibiotics,and even has beenmultiplely resistant or widely resistant to many antibiotics,which lead a grim situation of drug selecting difficultly and even no drug availably.The β-lactamhydrolase has become the main drug resistance mechanism of Klebsiella pneumoniae.Hence,an efficient method to identify K.pneumonia and β-lactamases contained by it plays a particularly important role for guiding the clinical use of drugs and timely treating patients.Inclinicalmicrobiology laboratory,bacterial cultureand succedent drug sensitive test are main methods usedto detect K.pneumonia and its antibiotics resistance,and regarded as golden standard.These methods are easy to operate,low cost,but time-consuming and imprecise.Recently,some automated drug sensitivity identification systems,such as VITEK? 2 COMPACT and ARIS 2X,were introduced in clinical microbiology laboratory,but they were merely used in minormedical institution.In this study,molecular biology methods,including PCR,multiplex PCR,and Loop-mediated isothermal amplification（LAMP）,were developed toidentify K.pneumoniae and β-lactam resistance genes carried by it.Two genes,ureR₁（GenBankID:11847803）and CelB（GenBankID:11847805）,were predicted to be specific genes of K.pneumonia by local and on-line BLAST.Plasmids with ureR₁ and CelB gene and blood mixed with K.pneumonia were used for specific and sensitive assays.The results showed that ureR₁ and CelB genes were conservative and specific in K.pneumonia by using LAMP and PCR in this study,so these genes could be used to differ K.pneumonia from other common pathogenic bacteria.Both PCR and LAMP showed high sensitivity when only one copy plasmids with ureR₁ and CelB or K.pneumonia existed in DNA or mixed blood samples.16 kinds of β-lactam resistance gene of K.pneumoniae were found through searching in online resistance gene database（ARDB）and all subtype genes of 16 kinds of resistance genes were downloaded from NCBI.After multiple sequence alignment,16 pairs of primers targeting to conserved sequence of each kind of resistance gene were designed.A total of 69 multi-drug resistant K.pneumonia were used to validate our PCR assay method.11 kinds of β-lactam resistance genes were found in 69 clinical isolates by PCR.Moreover,the presence of resistance genes was consistent with the resistance phenotype.Meanswhile,PCR assay showed high sensitivity,it can be positive when only one copy plasmids with ndm,cmy,ctx-m,tem,shv,act,oxa,okp,kpc,imp and ten copy plasmids with dha were used as templates.The multiplex PCR assay was established based on above-mentioned two specific genes and 11 drug resistance genes.All the 13 genes were amplified in two reactions,one for ureR₁,dha,ndm,cmy,ctx-m,tem,and shv,another one for CelB,act,oxa,okp,kpc,and imp.The positive plasmids,genome and bacterial lysate were used as templates for multiplex PCR assay.The results showed that multiplex PCR assay could detect 13 genes in the mixed positive plasmid,genome and bacterial lysate,and the detection limit could reach 102 copies per reaction.In conclusion,the methods to rapidly identify K.pneumonia and its β-lactam resistance genesby PCR,multiplex PCR and LAMP assays were established.Compared with bacterial cultureand succedent drug sensitive test,our methods are faster,cheaper,and more sensitive.