铜绿假单胞菌blaKPC基因传播机制研究

Since the first report of KPC carbapenemase in 2001, KPC-producing Enterobacteriaceae have now spread worldwide. Detection of blaKPC in Pseudomonas aeruginosa is increasing recently. P. aeruginosa is an important pathogen causing nosocomial infections, the emergence of KPC-producing P. aeruginosa brings great challenge for anti-infection treatment. Our study showed that KPC-producing P. aeruginosa were initially isolated in 2009 in our hospital, and the prevalence of KPC carbapenemase in carbapenem-resistant P. aeruginosa was even up to 40% in some hospital in 2013. Previous studies showed that blaKPC gene spread among Enterobacteriaceae by transposons, plasmids and other mobile genetic elements. However, the dissemination mechanism of blaKPC gene in P. aeruginosa remains unclear. This study intends to determinate the location of blaKPC gene by southern blot, to characterize the blaKPC-carrying plasmid by conjugation, transformation, and plasmid typing, to analyze the genetic environment of blaKPC gene and the mobile genetic elements involving in the transfer of blaKPC gene by whole-plasmid sequencing or whole-genome sequencing. The aim of the project is to reveal the mechanism of rapid dissemination of blaKPC gene in P. aeruginosa, and to provide theoretical basis for anti-infection treatment and prevention of nosocomial infection.

自2001年KPC酶首次报道以来，产KPC酶肠杆菌科细菌在全球范围广泛流行，近年来迅速蔓延到铜绿假单胞菌。铜绿假单胞菌是引起院内感染的重要病原菌，产KPC菌株的出现给临床治疗带来很大挑战。课题组前期研究发现我院产KPC酶铜绿假单胞菌于2009年开始出现，随后KPC酶的检出率逐年递增，至2013年杭州部分医院产KPC酶铜绿假单胞菌的检出率甚至超过40%。以往研究表明blaKPC基因可通过转座子、质粒等可移动元件在不同肠杆菌科细菌之间传播，而铜绿假单胞菌中blaKPC基因的传播机制尚未明确。本研究拟通过Southern杂交明确铜绿假单胞菌blaKPC基因定位；通过质粒接合和转化试验、质粒分型等研究携带blaKPC基因的质粒特性；通过全质粒测序研究blaKPC基因所在遗传背景及介导blaKPC基因转移的可移动元件，揭示blaKPC基因在铜绿假单胞菌中快速传播的机制，为临床抗感染治疗提供理论依据。

铜绿假单胞菌是引起院内感染的重要病原菌之一，而碳青霉烯类耐药铜绿假单胞菌（carbapenem-resistant Pseudomonas aeruginosa, CRPA）的分离率近几年一直居高不下。课题组前期发现的产KPC铜绿假单胞菌，给临床治疗带来严峻挑战。因此，blaKPC基因在铜绿假单胞菌中的流行情况及传播机制亟需明确，防止院内外产KPC-CRPA菌株的广泛流行。课题组利用全质粒测序对几株代表性菌株进行测序，发现铜绿假单胞菌中流行的KPC质粒为新的质粒，但是其blaKPC基因的周围核心序列与我国流行的肺炎克雷伯菌pkp048一致。同时利用全基因组测序对100余株临床分离株测序发现，ST463一直是近几年我院流行的ST型，blakpc基因的传播主要以可移动元件的形式在同一菌种内进行传播。接合转化试验说明，部分流行株的质粒可以在不同菌种间进行传播，其传播效率受质粒大小及克隆株影响。以上结果揭示了blaKPC基因在铜绿假单胞菌中快速传播的机制，为临床抗感染治疗提供理论依据。同时，我们还通过全基因组测序，发现KPC阳性的铜绿假单胞菌中主要毒力基因exoU和pldA的检出率明显高于KPC阴性的铜绿假单胞菌。这一现象，为以后高毒力高耐药铜绿假单胞菌的研究奠定了方向。