Identification Of Crucial Defense Antibiotic Resistance Gene TetX By Multipiex PCR And Preliminary Study Of Resistance Mechanism Of Nmcr-2 Gene

Tigecycline and colistin are known as the "last line of defense" in the treatment of complex infections caused by multi-drug resistant Gram-negative and Gram-positive bacteria.Plasmid-mediated tigecycline resistance gene tet X and colistin resistance gene mcr have been continuously reported,making this line of defense seriously threatened.A growing number of tet X-type tigecycline resistance determinants(tet X1 to tet X5)constitutes an expanding family of tetracycline-inactivating enzymes,posing a potential risk to global public health.Here,we report the development of an efficient multiplex PCR method to detect the family of tet X variants.This method is successfully applied in the screen and validation of tet X genes in the field and clinic bacterial samples.The emergence and dissemination of mobile colistin resistance(MCR)determinants in Gram-negative Enterobacteriaceae is a global concern in public health and food security.MCR-8 is a new member of this expanding MCR family,the origin and mechanism of it is poorly understood.Here we report a non-mobile colistin resistance enzyme,termed NMCR-2,originating from the plant pathogen Kosakonia pseudosacchari.Physiological consequences of both nmcr-2 or mcr-8 in Escherichia coli,are addressed.Genetic study reveals that nmcr-2 is comparable to mcr-1 and mcr-8 in the ability of producing phenotypic colistin resistance.This membrane proteins NMCR-2 is prepared,and purified to homogeneity.Biochemical and biophysical characteristics of NMCR-2 is analogous to that of other members of the MCR family.Combined with MS identity of lipid A pools,that these two enzymes catalyze the transfer of PEA from the donor PE lipid substrate to the recipient lipid A molecule by a putative “ping-pong” trade-off.The resistance mechanism of nmcr-2 gene is similar to other members of mcr family.NMCR-2(551aa)gives 67.3% identity to MCR-8(565aa).Therefore,our characterization of NMCR-2 suggests that it might be a progenitor of MCR-8.The research results of this experiment can quickly and accurately screen the line of antibiotics-tigecycline resistance gene tet X1～5,which helps humans to quickly identify the gene family and monitor the direction of transmission in real time.The study of the evolutionary relationship and resistance mechanism of another line of antibiotics-colistin resistance gene nmcr-2 and other members of the MCR protein family,can help future generations to better study the mcr gene resistance mechanism and evolution of mcr gene.