Mechanism of CCCP-induced non-canonical autophagy / 中国生化药物杂志

Objective To identify the factors those regulate CCCP-induced non-canonical autophagy.Methods Different cells expressing GFP-LC3 were treated with or without CCCP (30μM) for 6 h.Fluorescent images were taken and cell lysates were analyzed by western blot assay.Real-time PCR was used to measure the mRNA levels of LC3B.FIP200-KO MEF cells were cultured and treated by 30 μM CCCP with or without water channel inhibitors, for 6 h.Cell lysates were analyzed by Western blot assay.Results CCCP could not induced autophagy in Atg5-KO MEF cells.CCCP could induce non-canonical autophagy in ULK1-KO MEF, FIP200-KO MEF, and Beclin1-KD U251.CCCP treatment in FIP200-KO MEF cells had no effect on the expression level of LC3B mRNA.We also found two distinct aquaporin water channel inhibitors could inhibit the generation of LC3 which was induced by CCCP.Conclusion CCCP induced non-canonical autophagy was Atg5-dependent, but Beclin1-, ULK1-and FIP200-independent.Osmotic imbalance could regulate CCCP-induce non-canonical autophagy.

Relationship of AcrAB-TolC efflux pump and its regulatory gene mutation with fluoroquinolones resistance by Shigella.spp / 天津医药

Objective To investigate the role of AcrAB-TolC efflux pump in fluoroquinolones resistance by Shigella. spp and to explore the significance of AcrAB-TolC efflux pump on mutation of acrR, soxS and marOR as well as on drug re?sistence. Methods Drug resistant bacteria were selected by Kirby-Bauer disk diffusion test. After addition of efflux pump inhibitor carbonylcyanide-m-chlorophenylhydrazone (CCCP), change of minimal inhibitory concentration (MIC)s of nilidixic acid, Levofloxacin, ofloxacin, ciprofloxacin and Norfloxacin were examined. The DNA binding region of acrA, acrB, soxS, acrR and marOR gene in these mutants were amplified by PCR and sequenced. Results Among the 159 clinical isolates of Shigella,11 strains are resistant to fluoroquinolone. After the addition of CCCP, MICs of 2 fluoroquinolone resistant strains decreased; the MICs of 7 fluoroquinolone resistant strains did not change; MICs of 2 fluoroquinolone resistant strains in?creased. The corresponding nucleotides C, A, T, T on the 36th to 39th of marOR gene were missing, showing by sequencing, in fluoroquinolone resistent strains which might be regulated by the efflux pump gene AcrAB-TolC. Conclusion Efflux pump inhibitor could restrain the activity of efflux partially. The mutations of marOR might play an important role in fluoroquino?lone resistent by shigella.

Epidemiology and antimicrobial resistance of staphylococci isolated from different infectious diseases

A total of 187 isolates from 470 clinical specimens were collected from three hospitals in El-Minia governorate and identified as 132 Staphylococcus aureus strains and 55 coagulase-negative staphylococci (CoNS) strains. Susceptibility of isolates to antimicrobial agents was tested by the agar dilution method. The isolated S. aureus strains showed low resistance to vancomycin (1.5 percent), amikacin (2.3 percent) and gatifloxacin (3.8 percent). Vancomycin was the most effective antibiotic against CoNS. The ampicillin-resistant isolates were tested for â-lactamase production where, 61.7 percent of S. aureus and 42.9 percent of CoNS were positive for â-lactamase enzyme. Beta-lactamase producing strains were screened for their plasmid profile using alkaline lysis method. Some of these strains carried at least one plasmid suggesting plasmid-mediated antibiotic resistance. When cells of these strains were exposed to curing agent ethidium bromide, the production of the â-lactamase was lost. Resistance by efflux was studied by a modified fluorometric assay. Addition of uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) increased norfloxacin accumulation in quinolone resistant S. aureus strains, suggesting endogenous energy-dependent efflux. Combinations of ciprofloxacin with four antimicrobial agents against methicillin resistant S.aureus (MRSA) strains were investigated using decimal assay for additivity (DAA) technique. Synergistic interaction was observed between ciprofloxacin and oxacillin. ciprofloxacin plus cefepime and gentamicin appeared to be additive, while ciprofloxacin plus erythromycin was antagonistic.

Relationship Between Efflux Pumps and Klebsiella pneumoniae Resistance Mechanism / 中华医院感染学杂志

OBJECTIVE To investigate whether efflux mechanism is involved in fluoroquinolone-resistant Klebsiella pneumoniae strains isolated in China. METHODS We compared the ciprofloxacin accumulation in clinically isolated K. pneumoniae with or without CCCP by fluorospectrophotometry. Use reverse transcriptase-polymerase chain reaction (RT-PCR) to measure the mRNA expression level of acrAB-tolC efflux gene. RESULTS The accumulation of ciprofloxacin in resistant strains was lower than that in susceptible ones, and it could increase to a high level nearly to the susceptible strains. The mRNA level of efflux gene acrA was higher in resistant strains than in susceptible ones. CONCLUSIONS Efflux mechanism is associated with the resistance to fluroquinolones in K. pneumoniae strains isolated in China and CCCP can inhibit its active efflux mechanism , which provides a sensitive method to detect the active efflux system of K. pneumoniae.