Study On Aminoglycosides Resistance Mechanism And Pathogenicity Of Multidrug-resistant Pseudomonas Aeruginosa In Critically Ill Patients Sputum Secretions Of The Hospital Respiratory Unit

Objective: To understand the aminoglycoside resistance mechanism and pathogenicity of clinical multidrug-resistant Pseudomonas aeruginosa(MDR-PA),in order to provide a basis for rational application of antibiotics and effective control of the spread of drug-resistant strains.Methods: Twenty-five clinical isolates strains of Pseudomonas aeruginosa were collected from the respiratory department of our hospital,and 15 strains of multidrug-resistant Pseudomonas aeruginosa were screened by drug sensitivity tests using micro-broth dilution and K-B agar diffusion methods as well as typed by ERIC-PCR.The aminoglycoside-modifying enzyme genes(ant(2’’)-Ⅰ,aac(3)-Ⅰ,ant(3’’)-Ⅰ,aac(6’)-Ⅰ,aac(3)-Ⅱ,aac(6’)-Ⅱ),16 S r RNA methylesterase genes(arm A,rmt A,rmt B,rmt C and rmt D)and virulence genes(apr E,exo S,exo U,exo Y,plc H,las B,prp L,exo T and alg D)were detected by PCR and sequenced for validation,the carrying status of aminoglycoside-resistance genes and virulence factors in clinical isolates of P.aeruginosa in the hospital were obtained.A ST292 multidrug-resistant strain of PA172 was selected for framework map sequencing and whole genome resequencing.Finally,detection of biofilm formation ability of PA172 by crystalline violet staining,the phenotype screening of PA172 aminoglycoside efflux pump was detected in 96-well plates.Results:(1)The proportion of multidrug-resistant strains among the 25 Pseudomonas aeruginosa strains isolated in the in-hospital respiratory unit was 60%;the rate of resistance to aminoglycoside antibiotics was 88%.(2)Most of the strains were endogenously infected and have high genetic polymorphism.(3)The PCR results showed that multiple aminoglycoside resistance genes could co-exist in 15 MDR-PA strains,most of which were aminoglycoside modifying enzyme genes,among which the proportion of aac(6’)-Ⅰ was100%,the proportion of ant(3’’)-Ⅰ was 80%,the proportion of both genes was 80%,the proportion of aac(3)-II and arm A were both 13.3% and the proportion of rmt B was 6.6%.(4)PCR results showed that the genotypes of all 15 strains of MDR-PA virulence genes were a combination of apr E,exo Y,plc H,las B,prp L,exo T and alg D.(5)Framework map sequencing revealed that PA172 was an ST292-type clone that carried multiple aminoglycoside antibiotic resistance genes and highly pathogenic virulence factors,and comparative genomics revealed that PA172-specific SNPs tend to evolved to a large extent in the direction of adaptation to external stressful environments.(6)Resequencing revealed a high degree of PA172 variability,and comparison with the PAO1 reference genome predicted6347 nonsynonymous mutations containing multiple plasmid or integron-mediated aminoglycoside resistance genes,pro-aminoglycoside antibiotic efflux pump genes and pathogenicity associated virulence genes.(7)Strongly positive for biofilm formation ability of PA172.(8)Positive for aminoglycoside antibiotic efflux-pump of PA172.Conclusion: Clinical isolates of MDR-PA aminoglycoside resistance mechanisms are diverse and have a pathogenic basis.Traditional DNA striping techniques combined with genome sequencing can provide a theoretical basis for the aminoglycosides resistance mechanisms and pathogenesis of the prevalent clonal strain ST292 multidrug-resistant Pseudomonas aeruginosa in Kunming.