RESUMO
Objective@#To analyze the susceptibility and distribution of β-lactamase encoding and efflux pump genes as well as the inhibitory effect of efflux pump inhibitors on the carbapenem resistance of the carbapenem resistant Klebsiella pneumoniae without producing carbapenemase (CRKPPC). @*Methods@#One hundred and eight strains of carbapenem non-susceptible Klebsiella pneumoniae were collected from our hospital during 2012-2014. The strains producing carbapenemase were screened by Mastdiscs combi Carba plus disc system. For CRKPPC strains, the susceptibilities to antimicrobial agents were determined by micro-dilution broth methods. PCR and DNA sequencing technology were used to analyze the prevalence of β-lactamase encoding extended-spectrum β-lactamase (ESBL) and plasmid mediated AmpC genes as well as efflux genes including acrAB-tolC, kexD, kdeA, kpnEF and kpnGH. The inhibitory experiments were implemented by using carbonylcyanide-m-chlorophenylhydrazone (CCCP) and Reserpine to observe the role of efflux pumps on the carbapenem resistance. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed to analyze the expression of outer membrane proteins OmpK35 and OmpK36. @*Results@#Twenty-six strains out of the 108 carbapenem non-susceptible Klebsiella pneumoniae were identified to be CRKPPC strains which displayed quite high resistance to β-lactam and high resistance to amikacin, gentamicin, levofloxacin and ciprofloxacin. Very good sensitivities were observed to tigecycline, polymyxin, ceftazidime/avibatan and aztreonam/avibatan. The high prevalence of bla CTX-M and bla SHV were also displayed with the prevalent rates being 76.9% and 57.7%, respectively. The loss of outer membrane proteins OmpK35 and OmpK36 with varying degrees was observed, among which 57.7% strains lost ompK35 and 19.2% strains lost OmpK36. More than 80% strains contained efflux genes including acrAB-tolC, kexD, kdeA, kpnEF and kpnGH. The results of inhibitory experiments showed that CCCP displayed quite obviously inhibitory effects on the carbepenem resistance whereas no inhibitory effect was observed for Reserpine. @*Conclusion@#Tigecycline and polymyxin could be used as the basis of combined drug for CRKPPC. The prevalence of AmpC, ESBLs and loss of OmpK35 and OmpK36 with varying degrees among these strains were observed, but the over-expression of efflux pumps should be the main mechanism of the carbapenem resistance in the Klebsiella pneumoniae strains, and efflux inhibitors may have potential value in treating the infections caused by these bacteria.
RESUMO
This study was aimed to illustrate the interaction mechanism between Chinese herbal medicines and CASP3 target,and to analyze the structural characteristics of CASP3 inhibitors.Molecular docking,molecular dynamics and binding energy were employed to analyze the interactions and mechanism between CASP3 target and ligands which were screened from a series of nature products.The results showed that the binding forces of tanshinone ⅡA and scutellarin with CASP3 target were stronger than others.And the theoretical stable structures of tanshinone ⅡA and scutellarin combined with CASP3 target were obtained by molecular dynamics method.It also can be found that hydrophobic interaction was crucial for tanshinone ⅡA binding to amino acid residues of CASP3 such as Phe256,Ser205 and Trp206.Meanwhile,one hydrogen bond was formed between ligand and receptor.The main interactions between scutellarin and CASP3 target were found to arise from hydrophobic effect in ligand and nine amino acid residues of receptor (such as Ser249,Trp214,and Trp206),four hydrogen bonds with different stabilities and electrostatic interaction.It was concluded that tanshinone ⅡA and scutellarin can form stable structures with CASP3 target.And their similar structures may be useful to screen effective CASP3 inhibitors.