The Activities Of Antimicrobial Agents Against Intracellular Legionella And Quantification Of Viable Legionella In Hospital Water Distribution Systems

Part oneActivity of macrolides and fluoroquinolones against intracellular Legionella pneumophilaObjective:Evaluate the activity of macrolides and fluoroquinolones against legionella pneumophila by intracellular suspceptibility testing.Methods:Minimum inhibitory concentration (MIC) was determined by standard agar dilution test according to the CLSI. For intracellular assays, legionella pneumonia was used to infect human monocytic cell line THP-1. Erythromycin, Azithromycin, Levofloxacin and Moxifloxacin at 1×MIC,4×MIC,8×MIC were added following phagocytosis. Number of viable bacteria was enumerated at 24h on BCYE (Buffered charcoal yeast extract) agar in duplicate using standard plate count method. Results:Percentage inhibition at 24h were:Erythromycin 1×MIC (50.18±27.29)%,4XMIC (79.48±20.08)%,8×MIC (91.46±8.70)%; Azithromycin 1 XMIC (66.77±26.18)%,4XMIC (91.73±8.72)%,8XMIC (97.10±3.37)%; Levofloxacin 1 XMIC (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). The fluoroquinolones showed greater inhititory activity than macrolides against legionella pneumophila(U=1.0,2.0,5.0, P<0.05). The levofloxacin and moxifloxacin had the same intracellular activity against legionella pneumophila (U=190,183,217, P>0.05). Azithromycin was more effective than erythromycin in inhibiting intracellular legionella pneumophila (U=132,128,125, P<0.05). Conclusions:The fluoroquinolones were markedly more active than macrolides against legionella pneumophila. The intracellular activity of levofloxacin against legionella pneumophila appeard to be similar to moxifloxacin. Azithromycin was demonstrated to have superior activity against legionella pneumophila compared with erythromycin.Part 2Quantification of viable legionellae in hospital water distribution systems by combining EMA and Real time PCRObjective:To investigate the contamination of viable legionella in hospital water distribution systems by combining EMA and real time PCR(EMA-qPCR). Compare the results of EMA-qPCR with those of real time PCR(qPCR) and culture. Methods:EMA and qPCR were applied to heated and unheated legionella pneumophila to quantify viable cells, which were also simultaneously determined by BacLight Bacterial Viability kit with Fluorescence microplate reader. Apply EMA-qPCR, qPCR and culture to quantify the legionella after treatment by heating(70℃, 1h) and chlorine (active chlorine 10g/L,1h). And then,50 water samples from the water distribution systems of 2 hospitals in Shanghai were collected. The number of legionella species and legionella pneumophila was quantified by EMA-qPCR, qPCR and culture. Results:EMA at 2.5ug/mL was determined as the optimal EMA-qPCR assay. The number (Log(Lp-l/mL) of legionella quantified by EMA-qPCR, qPCR and culture is:thermal disinfection 4.0, 7.87,0; chlorine treatment 3.8,7.61,1.34. Legionella was detected in 48 samples of hospital water distribution systems by EMA-qPCR. The concentration of 40 samples (80.0%) was equal to or more than 103GU/L. More than 104GU/L legionella was detected in 7 samples (14.0%), while the concentration of 1 sample (2.0%) is more than≥105GU/L. The concentration of legionella by EMA-qPCR, qPCR and culture is respectively (7.75±22.4)x103GU/L, (2.37±6.71) x104GU/L, (3.80±25.50)×102 CFU/L, while the concentration of legionella pneumophila is respectively (8.40±2.47)×102GU/L, (1.11±2.65)×103 GU/L, (2.15±43.87)×102 CFU/L. Conclusions:EMA-qPCR can evaluate the disinfection efficacy more accurately. The two investigated hospitals have high concentration of viable legionella and a substantial proportion of legionella was in Viable but nonculturable state. Disinfection treatment should be considered to eliminate legionella of water distribution system.