Determination And Study Of Genes Involved In The Susceptibility Of Pseudomonas Aeruginosa To Carbapenems

Pseudomonas aeruginosa is an important opportunistic pathogen and a leading cause of nosocomial infections. Infections are usually among compromised patients such as organ transplant patients, burn victims and cystic fibrosis (CF) patients. The infection is difficult to eradicate due to both high intrinsic and acquired resistance to all antimicrobial drugs available on the market.Carbapenems is currently one of the most important antimicrobial against P. aeruginosa. The characteristic of this kind is stronger activity against almost pathogen and lower virulence. The emergence and spread of resistance to carbapenems, in this species, have challenged the success of therapeutic. Investigation of the molecular mechanisms leading to carbapenems resistance is extremely urgent.A P.aeruginosa transposon insertion library containing30,000mutant clones was constructed and screened for changes in the MICs of imipenem, meropenem and biapenem compared with PAO1.48genes involved in intrinsic carbapenems resistance were identified in this study by arbitrary primed PCR、sequencing and alignment. Among these genes,30genes is class IV and unreported in any case of antimicrobial resistance.Two of the mutants had transposon insertion at PA0011. The susceptibility of which to meropenem and biapenem was increased approximately twofold compared with the wild type PAO1. This gene encode a probable2-OH-lauroytransferase that control the synthesis of LPS. In this study, we constructed a PA0011knockout mutant and investigated the function of the PA0011gene in terms of resistance and virulence in PAO1.PA0011is responsible for the resistance to many kinds of antibiotics, including β-lactams, aminoglycosides, erythromycin, carbenicillin, tetracycline, ciprofloxacin and polymyxins. PA0011mutant showed a significant decrease in outer membrane stability and integrity compared to the wild-type PAO1. The result suggested that PA0011plays an important role in antibiotic resistance by affecting outer membrane integrity and stability. The expression level of the acyltransferase is temperature regulated, The expression of PA0011in PAO1was apparently higher at21℃than at37℃. the change of susceptibility to antibiotics was affected by temperature, PAOI(△0011) were more susceptible to carbapenems at21℃than37℃.In several pathogens, the lipid A acylation status were important for virulence, we demonstrated that the PA0011mutant decreased virulence of P. aeruginosa in vitro or vivo. The endotoxin bioactivity of the lipopolysaccharide from the PA0011mutant were markedly attenuated, which accounted for the reduced virulence at least partialy. Decreased expression of some virulence factors such as flagellar motility leaded to attenuated virulence. However, PA0011mutant had no effect on susceptibility of the human serum and murine macrophages.The expression levels of TTSS virulence factors were affected by the LPS status in Shigella and P. aeruginosa. Our date indicate that modification of lipid A acylation would change the expression and secretion of TTSS cytotoxins.