Background: Heteroresistance is a phenomenon that occurs in all bacteria and can cause treatment failure. Yet, the exact mechanisms responsible for heteroresistance still remain unknown. The following study investigated the mechanisms of imipenem-heteroresistance and -resistance in Pseudomonas aeruginosa  clinical isolates from Wenzhou, China.
Methods: Imipenem resistance was detected by the agar dilution method; heteroresistance was determined by population analysis profiles. Biofilm formation assay and modified carbapenem inactivation methods were also performed. Polymerase chain reaction (PCR) was conducted to detect oprD , and quantitative real-time PCR was used to determine expression levels of oprD , ampC  and four efflux pump coding genes (mexB, mexD, mexE  and mexY ).
Results: Six imipenem-heteroresistant and -resistant P. aeruginosa  isolates were selected respectively. Deficient oprD  was detected in all resistant isolates and two heteroresistant isolates. No strains produced carbapenemases. Expression levels of oprD  were down-regulated in heteroresistant isolates. Transcription levels of the mexE  and mexY  were significantly increased in all heterogeneous subpopulations compared with their respective native ones. Compared with the susceptible group, increased mean relative expression levels of mexE  and mexY  or the decreased mean relative expression levels of oprD  were observed in the resistant group (P < 0.05), whereas transcription levels of the mexB  and mexD  remained unchanged.
Conclusion: Down-regulation of oprD  contributed to the resistance and heteroresistance of imipenem in our P. aeruginosa  clinical isolates. In addition, the marginal up-regulation of efflux systems may indirectly affect imipenem resistance. Contrarily, defective oprD  was less common in our experimental heteroresistant strains than resistant strains.
Keywords: Pseudomonas aeruginosa , imipenem, resistance, heteroresistance, molecular mechanism, oprD