paul levett

Antimicrobial Susceptibility Testing of Leptospira species: 

Antimicrobial Susceptibility Testing of Leptospira species Carol U. Whittington Paul N. Levett University of the West Indies and Leptospira Laboratory, Barbados

Treatment of Leptospirosis : 

Treatment of Leptospirosis Penicillin 8 MU/day IV Used for severe cases Two controlled trials gave conflicting results Doxycycline 100 mg orally, twice daily for 7 days Used for anicteric cases Contraindicated in pregnant and lactating women, and in children Other Antibiotics Ciprofloxacin, cefotaxime May be effective, but no clinical trials

Prophylaxis of leptospirosis: 

Prophylaxis of leptospirosis Doxycycline 200 mg orally once weekly Effectively prevents disease in healthy subjects (soldiers)

Susceptibility of Leptospires : 

Susceptibility of Leptospires Numerous studies in the literature Different methods used, difficult to compare variable results Leptospires are susceptible to β-lactams, macrolides, tetracyclines, fluoroquinolones and streptomycin Minimal bactericidal concentrations are several orders of magnitude higher than MIC

Why do we need AST?: 

Why do we need AST? Standardized method Reproducible testing of large numbers of isolates Screening of potential antimicrobial agents for treatment NOT for testing of individual patient isolates

Obstacles to Susceptibility Testing : 

Obstacles to Susceptibility Testing Serum required for growth Protein binding of antibiotics Slow growth Degradation of antibiotics Detection of growth Dark-field microscopy Difficult to determine end-point No interpretative criteria

Objective: 

Objective Develop a standardized, reproducible method for antimicrobial susceptibility testing of leptospires Use of microtiter trays to enable testing of large numbers of replicates Use of a redox indicator to allow detection of growth

Methods 1: 

Methods 1 Reference strains of 12 serovars of Leptospira 10 isolates from humans and animals L.interrogans serovar Copenhageni (2) L.kirschneri serovar Bim (4) L.borgpetersenii serovar Arborea (4) Cultures were maintained in semi-solid medium (PLM-5)

Methods 2: 

Methods 2 Medium for susceptibility testing contained PLM-5 (Intergen Co) 2,6-dicholoroindophenol DCIP (Sigma) Antibiotics studied ciprofloxacin (Bayer) doxycycline (Sigma) penicillin

Methods 3: 

Methods 3 Ten-fold dilutions of antibiotics were made in 190 ml PLM-5 medium in the wells of a microtiter tray Inoculated with 10 ml of a 1:5 dilution of a 7-day culture in PLM-5 Microtiter trays were sealed with plastic film Incubated for 3 days at 29oC

Methods 4: 

Methods 4 Minimal inhibitory concentration (MIC) determined by inspecting antibiotic wells for growth 10 ml medium transferred from each well to 190 ml antibiotic-free medium in a fresh microtiter tray Examined for growth after 3 days’ incubation to determine minimal bactericidal concentration (MBC)

Results 1: 

Results 1 Growth of leptospires was detected in microtiter trays by dark-field microscopy by DCIP colour change from blue to colourless

Results 2: 

Results 2 Penicillin MIC range 0.016 – 1.0 mg/ml MBC range 0.016 – 1.0 mg/ml

Results 3: 

Results 3 Ciprofloxacin MIC range 0.5 – 2.0 mg/ml Doxycycline MIC range 2.0 mg/ml

Conclusions 1: 

Conclusions 1 Developed a microtiter-plate method for MIC determination Using redox indicator, reduced the incubation time to 48hr Comparable to other bacterial susceptibility testing methods

Conclusions 2: 

Conclusions 2 Demonstrated that leptospires are highly susceptible to penicillin Penicillin MBC is close to MIC Penicillin is extremely bactericidal against leptospires All strains tested were susceptible to ciprofloxacin and doxycycline

Limitations of This Study: 

Limitations of This Study Studied only three antibiotics Relatively small number of isolates studied One of the largest studies of Leptospira MBC was determined only for penicillin Develop MBC method for other agents Medium contained serum Evaluate this method with protein-free medium Plastic film was impermeable Condensation prevented direct microscopic reading

Recent Publication: 

Recent Publication

Murray & Hospenthal, 2004: 

Murray & Hospenthal, 2004 Variable penicillin MICs: very sensitive L.interrogans sv Copenhageni L.borgpetersenii L.noguchii Some strains much less sensitive Emphasises need for interpretative criteria