Analysis of Efflux Pump System and Other Drug Resistance Related Gene Mutations in Tigecycline-Resistant .

Background: The isolation of tigecycline-resistant in recent years has brought great difficulties to clinical prevention and treatment.

Purpose: To explore the effect of efflux pump system and other resistance related gene mutations on tigecycline resistance in .

Methods: Fluorescence quantitative PCR was used to detect the expression levels of major efflux pump genes (, , and ) in extensive drug-resistant . The minimum inhibitory concentration (MIC) of tigecycline was detected by the broth microdilution testing and efflux pump inhibition experiment to assess the role of efflux pump in tigecycline resistance of . Efflux pump regulatory genes ( and ) and tigecycline resistance related genes (, , and ) were amplified by PCR and sequenced. By sequence alignment, tigecycline sensitive and tigecycline-insensitive were compared with standard strains to analyze the presence of mutations in these genes.

Results: The relative expression of in the tigecycline-insensitive was significantly higher than that in the tigecycline sensitive (114.70 (89.53-157.43) vs 86.12 (27.23-129.34), = 0.025). When efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was added, the percentage of tigecycline-insensitive with tigecycline MIC decreased was significantly higher than that of tigecycline-sensitive (10/13 (76.9%) vs 26/59 (44.1%)), = 0.032); the relative expression of in the MIC decreased group was significantly higher than that in the MIC unchanged group (110.29 (63.62-147.15) vs 50.06 (26.10-122.59), = 0.02); The relative expression levels of efflux pumps and did not increase significantly, and there was no significant difference between these groups. One point mutation (Gly232Ala) and eight point mutations (Ala97Thr, Leu105Phe, Leu172Pro, Arg195Gln, Gln203Leu, Tyr303Phe, Lys315Asn, Gly319Ser) were newly detected. Consistent mutations in and genes were detected in both tigecycline-insensitive and tigecycline-sensitive , but no mutation in gene was detected in them.

Conclusion: Tigecycline-insensitive efflux pump overexpression was an important mechanism for tigecycline resistance, and the mutations of efflux pump regulator genes ( and ) are responsible for overexpression. The effect of , , and gene mutations on the development of tigecycline resistance in remains controversial.