Molecular Evolutionary Study Of CTX-M Extended Spectrum Beta-Lactamases Of Klebsiella Pneumoniae

BackgroundKlebsiella pneumoniae has emerged as one of the most epidemic clinical bacterium, essentially because of its ability to persist in clinical environments and its ability to infect patients. K. pneumoniae isolates can persist on environmental surface and human skin, respiratory and urinary tract and easily transfer among patients via clinical operations and examinations. K. pneumoniae has become one of the most frequent causes of outbreaks in intensive care units and respiratory unites. Multidrug resistant K. pneumoniae isolates, especially those ESBLs-producing or carbapenem-producing strains increased in the past decades because of the abuse of antibiotic drugs. Novel antibiotic-resistant enzymes appeared and transmitted in clinical unites under antibiotic selections, and this has lead to the high cost of spends both patients and society. The research about evolutionary mechanisms of antibiotic resistant genes has become one of hotspots in global research.CTX-M ESBLs producing isolates has replaced SHV and TEM as the most epidemiology isolates. A prevalent beta-lactam resistance mechanism consists of the CTX-M type extended spectrum beta-lactamase(ESBL) enzymes. CTX-M ESBLs persist high hydrolytic of cephalosporin anbiotics, and it could be transmitted by plasmids. And the CTX-M ESBLs producing isolates may persist kinds of resistance mechanisms, so they could evolve rapidly under the selection of anbiotics. The research of novel resistance genes and evolutionary mechanisms of CTX-M ESBLs has become an important spot of global research.Based on the rapid development of bioinformatics, the computational bacterium evolutionary study developed in recent decades. Bioinformatic software helps the building of resistance gene bank with enormous gene sequences. The evolutionary map of SHV and TEM ESBLs, and then the evolutionary origin and evolutionary trajectory of CTX-M ESBLs were found by bioinformatic methods. However, the precise evolutionary relationship cannot be detected simply based on resistance gene sequences. Only with the consideration of clinical data, can the precise evolutionary trajectory be detected.On other way, the experimental evolutionary researches are hotspot globally. Angela Novais had illustrated the CTX-M-1 ESBLs evolutionary map by antibiotic selections. But this research cannot help guide the usage of antibiotic drugs.The purpose of this study was to investigate the antibiotic resistance mechanism of multidrug resistant K. pneumoniae in Central China and to examine the epidemiologic and evolutionary properties of these isolates isolated in Henan. Methods and materials(1) Isolates. We performed a retrospective study of K. pneumoniae infected patients from August 2014 to November 2014, 72 isolates were collected and detected by Vitek ? Compact.(2) Antimicrobial susceptibility testing. The antimicrobial susceptibility of all isolates was tested by Vitek? GNI+ card, and interpreted according to the Clinical and Laboratory Standards Institute(CLSI) 2014 guidelines.(3) 5.PCR detection and sequencing of ESBL and carbapenemase genes. ESBL and carbapenemase genes were detected by PCR methods and sequenced.(4) Homology analysis of antibiotic resistance isolates. RAPD-PCR was used to analyze the homology of antibiotic resistance isolates.(5) Evolutionary analysis of CTX-M ESBLs. Sequence alignment, phylogenetic analysis, positive selection and protein structure analysis were used to analyze the evolutionary trajectory of CTX-M ESBLs.Results 1. Detection of ESBLs producing Klebsiella pneumoniae Total 60 CTX-M ESBLs producing isolates were detected, including CTX-M-3, CTX-M-15, CTX-M-55, CTX-M-9, CTX-M-14, CTX-M-27 and CTX-M-65. SHV, TEM and OXA-10 were also detected in CTX-M producing isolates, and 21 producing isolates were detected to produce KPC-2 or NDM-1 carbapenemases. 2. CTX-M ESBLs evolutionary trajectory Evolutionary trajectory: CTX-M-3?CTX-M-15? CTX-M-55 was detected. The 80 th and 242 th amino acid positions were positive selection positions. However no evolutionary trajectory was detected in CTX-M-14 ESBLs. 3. CTX-M-65 and KPC-2 producing isolates CTX-M-65 ESBLs and KPC-2 carbapenemases were detected in one Klebsiella pneumoniae isolates, and this is the first detection of CTX-M-65 in Henan province.Conclusions 1. CTX-M ESBLs producing Klebsiella pneumoniae isolates also produce other ESBLs and carbapenemases. Antibiotic drugs should be used reasonably in Klebsiella pneumoniae infections. And the combination of antibiotic drugs should be considered. 2. The evolutionary trajectory CTX-M-3?CX-M-15?CTX-M-55 has indicated the possibility of novel CTX-M ESBLs. The detection of CTX-M resistance genes should be improved to prevent the evolution and epidemiology of resistance genes. 3. The first detection of CTX-M-65 implies the origin of novel CTX-M ESBLs. The detection of novel CTX-M ESBLs should be emphasized to prevent the evolution and epidemiology of resistance genes in the local area.