Mechanisms Of Resistance And Molecular Epidemiology Of Imipenem-resistant Pseudomonas Aeruginosa

Pseudomonas aeruginosa（PA）is a common non-fermenting Gram-negative bacillus with mutable and multi-drug resistant characteristics,which often leads to an increased chance of infection when the body is immunocompromised or treated with invasive operations.Imipenem,as a representative of carbapenems,has an important role in the treatment of PA-associated infections,but with the widespread use of this drug,Imipenem-resistant Pseudomonas aeruginosa（IRPA）has been detected.It has been shown that IRPA may also be a multi-drug resistant Pseudomonas aeruginosa（MDRPA）that is not susceptible to three or more antimicrobial drugs and is associated with many high-risk clones at the international level.The massive spread of these clones coupled with highly resistant strains has caused increasing concern for the monitoring and treatment of these pathogens.The aim of this study was to investigate IRPA resistance,resistance mechanisms,molecular epidemiology and potential combination treatment options in a tertiary care hospital in Hebei Province,and to provide a theoretical basis for the rational application of antimicrobial drugs by clinicians in this hospital to control the production and spread of IRPA.In this study,49 strains of IRPA isolated from clinical specimens in a tertiary hospital in Hebei Province from June 2021 to June 2022 were collected,and the susceptibility of IRPA to common and novel antimicrobial drugs such as amikacin,meropenem,ceftazidime/avibactam and imipenem/relebactam was determined by micro-broth dilution method;the modified carbapenem inactivation test(Modified carbapenem inactivation method（m CIM）and colloidal gold immunochromatography were used to detect carbapenemase phenotypes;DNA was extracted by boiling method and polymerase chain reaction（PCR）was used to amplify KPC,IMP,NDM and VIM.The main carbapenemase genes,OXA-10 gene and outer membrane pore protein Opr D₂ gene were amplified by PCR and the positive products were sequenced;Multilocus Sequence Typing（MLST）was used to analyze the homology of the strains;the whole genome of IRPA was sequenced by second generation sequencing technology;the paper diffusion method was used to combine the genomes of the strains.The results showed that 49 strains of IRPA were tested for their genomic identity.The results showed that 49 strains of IRPA had high resistance rates to meropenem and aminotransimide,75.51%（37/49）and 65.31%（32/49）,respectively,and relatively low resistance rates to amikacin and imipenem/relebactam,both 28.57%（14/49）,and the lowest resistance rate to polymyxin B,6.12%（3/49）.In the m CIM and colloidal gold immunochromatography tests,three and two strains were detected as carbapenemase-positive strains,respectively;PCR test results showed that15 strains produced OXA-10-type enzymes,13 strains had membrane pore protein Opr D₂ deletion,and two strains carried VIM-type metalloenzymes,both of which were sequenced as VIM-2.ST2651,ST3134,ST207,and the novel STn3 as the major typing,accounting for 28.57%（14/49）,10.20%（5/49）,10.20%（5/49）,4.08%（2/49）,and 4.08%（2/49）,respectively,and there was clonal transmission.Whole-genome sequencing results showed that each IRPA strain hadβ-lactamase genes,aminoglycoside resistance genes,phosphomycin resistance genes,and chloramphenicol acetyltransferase genes,and fluoroquinolone resistance genes were also detected in most strains,and the detection of resistance genes was highly consistent with the resistance results,and it also showed that some strains were closely related to each other.The results of the combined drug sensitivity test showed that ceftazidime/avibactam and imipenem,ceftazidime/avibactam and amineptine,and ceftazidime/avibactam and meropenem were synergistic,with the synergy rates of 53.06%（26/49）,44.90%（22/49）and 26.53%（13/49）,respectively.In conclusion,IRPA in this study showed high resistance to a variety of drugs,with low resistance rates only to polymyxin B,amikacin and imipenem/relebactam;and there were complex resistance mechanisms and clonal transmission,with OXA-10-producing enzyme and membrane pore protein deficiency as the main resistance mechanisms;m CIM and immunogold standard method can be used as a screening method for carbapenemase-producing strains in this hospital;ceftazidime/avibactam combined with imipenem,aminotrans and meropenem,respectively,are potential combination drug regimens in this hospital,and we still need to apply antimicrobial drugs rationally and further strengthen the prevention and control measures for nosocomial infections.