The Detection, Localization And Genetic Environment Of The Tet(A) Variant In Gram-negative Bacteria

The horizontal transmission and vertical transmission of drug resistance genes had brought great troubles to clinical treatment,so the problem of bacterial resistance had became the biggest problem in in medical science and veterinarian clinical infection.And the gram-negative bacteria not only had been identified the main pathogens of nosocomial infections,but also became bunker for various resistance genes.With the use of antibiotics,more and more gram-negative bacteria became multi-resistant pathogen and extensively resistant bacteria,the number of drugs can effectively treat gram-negative bacterial infections is decreasing.Tigecycline is considered to be the “last line of defense” to against multi-resistant gram-negative bacteria because of its good antibacterial activity and clinical safety.However,Klebsiella pneumoniae and Escherichia coli have emerged "superbugs" wich can resist tigecycline nowadays,the existing research can not explain thire resistance mechanism of Tigecycline.So,regular monitoring the sensitivity of Klebsiella pneumoniae and Escherichia coli to tigecycline,exploring in a great depth of the main mechanisms howt to mediate the resistance,have a very important public health significance.In this study,203 strains of clinical K.pneumoniae and 251 strains of clinical E.coli were used to screen the tigecycline resistant,using PCR amplification,electroporation assays,functional assays,drug sensitivity tests,whole-genome sequencing,and other technical tools explored the resistance mechanisms of tigecycline in Klebsiella pneumoniae and Escherichia coli,confirmed that the new resistance gene tet(A)mutants can lead to the Gram-negative bacteria resist to tigecycline in the end.The results showed that 1 strain Klebsiella pneumoniae from human origin and 1 strain E.coli from animal origin were resisted to tigecycline.Electrotransformation experiment of this tigecycline-resistant Klebsiella pneumoniae obtained a single plasmid carry Tetracycline resistance;Functional analysis showed that tet(A)gene happened a double frameshift mutation in the electrorotator plasmid;drug susceptibility testing confirmed that this mutant could independently cause the MIC of tigecycline,minocycline and tetracycline increased 8 times,128 time and 128 times;full-plasmid sequencing and circular intermediate detection demonstrate that the tet(A)mutant is located on a transposon similar to Tn1721 and can be successfully cyclized between plasmid to chromosomes.PCR amplification of the tet(A)mutant sequence revealed the presence of a double frameshift mutation at the same site in the tigecycline-resistant E.coli isolated from animal sources.Electrotransformation assays,drug susceptibility testing and whole genome sequencing confirmed the tet(A)Mutants can mediate tigecycline resistance to E.coli.These data suggest that the tet(A)mutant reduces the sensitivity of Klebsiella pneumoniae and Escherichia coli to tigecycline and that it is spread horizontally within the bacterial species or between species using transposons or plasmids,accelerated the clinical epidemic of tigecycline-resistant strains.In summary,transposons or plasmid-mediated tet(A)mutants are important mechanisms leading to the resistance of gram-negative bacteria to tigecycline,which explains the molecular mechanism of bacteria against tigecycline.The reason for the rapid increase in drug resistance has broadened the understanding of the mechanism of resistance to gefitinobactam against tigecycline,and provided a basis for the diffusion control of tet(A)mutants in gram-negative bacteria.