Girlich Delphine, Bonnin Rémy A, Jousset Agnes, Naas Thierry
EA7361, Université Paris-Sud, Université Paris-Saclay, Associated French National Reference Center for Antibiotic Resistance 'Carbapenemase-producing Enterobacteriaceae', LabEx Lermit, Bacteriology-Hygiene Unit, APHP, Hôpital Bicêtre, Le Kremlin-Bicêtre, France.
EERA 'Evolution and Ecology of Resistance to Antibiotics' Unit, Institut Pasteur-APHP-Université Paris-Sud, Paris, France.
J Antimicrob Chemother. 2017 Jun 1;72(6):1597-1601. doi: 10.1093/jac/dkx044.
KPC-producing pathogens exhibit variable carbapenem susceptibility levels, which is probably the result of the genetic environment of the bla KPC genes. Here we determined the transcriptional start sites (TSSs) and the expression of the bla KPC-2 gene in various genetic contexts and in different hosts ( Escherichia coli , Pseudomonas aeruginosa and Acinetobacter baumannii ).
The bla KPC-2 genes along with the upstream sequences derived from Tn 4401b (structure A), Tn 4401b interrupted by Tn 3 /IS 26 (structure B) and Tn 4401b interrupted by Tn 5563 (structure C) were cloned in two E. coli shuttle vectors (pBBR1MCS.3 for expression studies in P. aeruginosa and pIM-arr2 for expression studies in A. baumannii ). MICs were determined by Etests. 5' RACE (where RACE stands for rapid amplification of cDNA ends) and quantitative RT-PCR experiments were performed to determine TSSs and transcription levels, respectively.
Depending on the bacterial host, different promoters were used for bla KPC-2 gene expression. The highest transcriptional level was obtained in P. aeruginosa with structure C, described only in P. aeruginosa . Tn 4401b (structure A), harbouring two promoters (P1 and P2), was the most efficient in E. coli and A. baumannii . This structure was also efficient in P. aeruginosa , although the same deduced promoter was not used (P1, instead of P2 used by E. coli and A. baumannii ). Two novel TSSs and putative promoters (P2b and P3b) were identified in structure B. In this structure, P2b and P3b were preferably used in E. coli and in P. aeruginosa , respectively, whereas P1 was used in A. baumannii .
We determined the preferred TSSs of the bla KPC gene in each species and described two novel deduced promoters in structure B.
产KPC的病原体对碳青霉烯类药物的敏感性水平各异，这可能是bla KPC基因遗传环境所致。在此，我们确定了bla KPC - 2基因在不同遗传背景及不同宿主（大肠杆菌、铜绿假单胞菌和鲍曼不动杆菌）中的转录起始位点（TSS）及表达情况。
将源自Tn4401b（结构A）、被Tn3/IS26中断的Tn4401b（结构B）以及被Tn5563中断的Tn4401b（结构C）的bla KPC - 2基因及其上游序列克隆至两种大肠杆菌穿梭载体中（用于在铜绿假单胞菌中进行表达研究的pBBR1MCS.3和用于在鲍曼不动杆菌中进行表达研究的pIM - arr2）。通过Etest法测定最低抑菌浓度（MIC）。分别进行5' RACE（其中RACE代表cDNA末端快速扩增）和定量逆转录聚合酶链反应（RT - PCR）实验以确定TSS和转录水平。
根据细菌宿主的不同，bla KPC - 2基因表达使用不同的启动子。在仅在铜绿假单胞菌中描述的结构C的情况下，在铜绿假单胞菌中获得了最高转录水平。含有两个启动子（P1和P2）的Tn4401b（结构A）在大肠杆菌和鲍曼不动杆菌中效率最高。该结构在铜绿假单胞菌中也有效，尽管未使用相同推导的启动子（P1，而非大肠杆菌和鲍曼不动杆菌使用的P2）。在结构B中鉴定出两个新的TSS和推定启动子（P2b和P3b）。在该结构中，P2b和P3b分别优选用于大肠杆菌和铜绿假单胞菌，而P1用于鲍曼不动杆菌。
我们确定了每种细菌中bla KPC基因的优选TSS，并在结构B中描述了两个新的推导启动子。
