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The Activities of Antimicrobial Agents Against Intracellular Legionella and Quantification of Viable Legionella in Hospital Water Distribution Systems

Author: YuLingLing
Tutor: HuBiJie
School: Fudan University
Course: Internal Medicine
Keywords: Legionella pneumophila Drug sensitivity Fluoroquinolone Macrolide Intracellular Legionella Water supply system Real time PCR EMA Quantitative
CLC: R563.1
Type: Master's thesis
Year: 2011
Downloads: 29
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Macrolides and fluoroquinolones first part of the role of intracellular Legionella purpose: to assess the role of macrolides and fluoroquinolones against Legionella pneumophila within macrophages. Methods: The agar dilution method measuring serum macrolides and fluoroquinolones against 20 Legionella pneumophila type 1 (L. pneumophila serogroup 1, Lp-1), the minimum inhibitory concentration (Minimal inhibitory concentration, MIC) . Jiarufobo ester (Phorbol 12-myristate 13-acetate, PMA) human monocytic cell line THP-1 induced to differentiate into macrophage-like cells. Lp-1 with differentiation of THP-1 cells co-cultured cells engulf bacteria, erythromycin, azithromycin, levofloxacin, moxifloxacin four antimicrobial agents in 1 × MIC, 4 × MIC, 8 × MIC concentration culture 24h, plate count double count the the Legionella number of the cells alive. Results are expressed in bacterial inhibition rate, the difference of the different antimicrobial effect using the Mann-Whitney U-test analysis. Results: four antimicrobial agents in the 1 × MIC, 4 × MIC, 8 × MIC concentrations 24h after bacterial inhibition rate were: erythromycin: 1 × MIC (50.18 ± 27.29)%, 4 × MIC (79.48 ± 20.08 Azithromycin)%, 8 × MIC (91.46 ± 8.70)%;: 1 × the MIC (66.77 ± 26.18)%, 4 × the MIC (91.73 ± 8.72)%, 8 × the MIC (97.10 ± 3.37)%; levofloxacin: 1 × the MIC (99.84 ± 0.25)%, 4 × MIC (99.99 ± 0.02)%, 8 × MIC (99.99 ± 0.01)%; moxifloxacin: 1 × MIC (99.90 ± 0.10)%, 4 × MIC (99.99 ± 0.03)% , 8 × MIC (99.99 ± 0.03)%. Fluoroquinolones in 1 × MIC, 4 × MIC and 8 × MIC of Lp-1 cells inhibition rate was significantly higher than macrolides (U-value of 1.0, 2.0 and 5.0, respectively, K0.05 ), levofloxacin and moxifloxacin in the three concentrations of Lp-1 inhibition rate was no significant difference (U-value of 190,183 and 217, respectively, P gt; 0.05), azithromycin intracellular inhibition of Lp-1 rate higher than erythromycin (U-value, respectively, 132,128 and 125, P LT; 0.05). Conclusions: fluoroquinolones cells, anti-Legionella role better than macrolides levofloxacin and moxifloxacin intracellular anti-the Legionella role was no significant difference, the anti legionella role in the intracellular azithromycin better than Erythromycylamine prime. The second part of the EMA-qPCR method for quantitative detection the purpose of survival of Legionella in hospital water systems: the EMA (Ethidium monoazide) and Real time PCR (qPCR) combined (EMA-qPCR) research hospital water supply system in Shanghai survival Legionella pollution, and compare the results with qPCR and culture. Method: application of different concentrations of EMA qPCR combined with quantitative heat-treated and non-heat-treated Legionella, while using Baclight Live / Dead bacterial survival kit analysis as a standard, to determine EMA-qPCR best EMA concentration. EMA-qPCR, qPCR and culture assay for quantification of heat treatment (70 ° C, 1h) and chlorine disinfectants (free chlorine concentration of 10mg / L, 1h) the number of Legionella treatment. Followed by the three methods for quantitative detection of Legionella in the 50 wards of the two hospitals in Shanghai in the water supply system. Results: EMA-qPCR EMA concentration 2.5ug/mL. EMA-qPCR, qPCR, culture method to detect the number of Legionella disinfection treatment (the Log (Lp-1/mL)), respectively: the heat treatment 4.0,7.87,0; chlorine treatment 3.8,7.61,1.34. EMA-qPCR detection hospital water supply system water samples Legionella positive specimens 48, where concentrations ≥ 103GU / L water sample, 40 (80.0%), concentration ≥ 104Gu / L water sample (14.0% %), the concentration of ≥ 105GU / L water samples and 1 (2.0%). EMA-qPCR, qPCR and culture of three methods of determination of the number of Legionella spp were (7.75 ± 22.4) × 103GU / L, (2.37 ± 6.71) x104GU / L, (3.80 ± 25.50) x102CFU / L; Legionella pneumophila number of bacteria were (8.40 ± 2.47) × 102GU / L, (1.11 ± 2.65) × 103Gu / L (2.15 ± 43.87) x102CFU. / L, respectively. Conclusion: EMA-qPCR can more quickly and accurately assess the effect of disinfection. Higher concentration of viable Legionella in hospital water systems, and a large part of the Legionella is alive but can not be cultured, \

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CLC: > Medicine, health > Internal Medicine > Respiratory system and chest diseases > Pulmonary disease > Pneumonia
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