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Antimicrobial Disk Susceptibility Testing of Leptospira spp. Using Leptospira Vanaporn Wuthiekanun (LVW) Agar.

Wuthiekanun V, Amornchai P, Langla S, White NJ, Day NP, Limmathurotsakul D, Peacock SJ - Am. J. Trop. Med. Hyg. (2015)

Bottom Line: Ten pathogenic Leptospira isolates were tested, all of which were susceptible to 17 antimicrobial agents (amoxicillin/clavulanic acid, amoxicillin, azithromycin, cefoxitin, ceftazidime, ceftriaxone, chloramphenicol, ciprofloxacin, clindamycin, doripenem, doxycycline, gentamicin, linezolid, nitrofurantoin, penicillin, piperacillin/tazobactam, and tetracycline).All 10 isolates had no zone of growth inhibition for four antimicrobials (fosfomycin, nalidixic acid, rifampicin, and trimethoprim/sulfamethoxazole).Of the ten Leptospira, seven had a growth inhibition zone of ≤ 21 mm for aztreonam, the zone diameter susceptibility break point for Enterobacteriaceae.

View Article: PubMed Central - PubMed

Affiliation: Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Nuffield Department of Medicine, Centre for Tropical Medicine and Global Health, University of Oxford, Headington, Oxford, United Kingdom; Department of Tropical Hygiene, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge, United Kingdom lek@tropmedres.ac direk@tropmedres.ac.

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Zone of inhibition (50 mm) for penicillin G disk diffusion method on Leptospira Vanaporn Wuthiekanun (LVW) agar for Leptospira interrogans serovar Autumnalis strain NR-20161. The plate was prepared by spreading 300 μL of 108 CFU/mL and preincubating at 30°C in 5% CO2 for 2 days followed by application of the disk and further incubation at 30°C in air for a total of 7 days.
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Figure 1: Zone of inhibition (50 mm) for penicillin G disk diffusion method on Leptospira Vanaporn Wuthiekanun (LVW) agar for Leptospira interrogans serovar Autumnalis strain NR-20161. The plate was prepared by spreading 300 μL of 108 CFU/mL and preincubating at 30°C in 5% CO2 for 2 days followed by application of the disk and further incubation at 30°C in air for a total of 7 days.

Mentions: The antimicrobial agents selected for testing (N = 22) represent the spectrum of drugs used in tropical settings for the treatment of suspected bacterial sepsis. Disk susceptibility testing was performed by spread plating 300 μL of each isolate (108 CFU/mL) across the surface of a LVW agar plate. These were preincubated at 30°C in 5% CO2 for 2 days (the established optimal incubation conditions for LVW agar), after which a standard disk was applied in the center of a single plate for the following antimicrobials (disk content): amoxicillin/clavulanic acid (20/10 μg), amoxicillin (10 μg), azithromycin (15 μg), aztreonam (30 μg), cefoxitin (30 μg), ceftazidime (30 μg), ceftriaxone (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), clindamycin (2 μg), doripenem (10 μg), doxycycline (30 μg), fosfomycin (50 μg), gentamicin (10 μg), linezolid (30 μg), nalidixic acid (30 μg), nitrofurantoin (300 μg), penicillin (10 units), piperacillin/tazobactam (100/10 μg), rifampicin (5 μg), tetracycline (30 μg), and trimethoprim/sulfamethoxazole (1.25/23.75 μg) (Oxoid Ltd, Basingstoke, United Kingdom). An additional plate (without discs) was used as a growth control. Plates were then incubated at 30°C in air and observed every day for 7 days. The growth inhibition zone sizes were measured at the point at which a bacterial lawn was clearly discernible by the naked eye (usually at day 7) (Figure 1). As disk diffusion testing has not been performed previously for Leptospira, we used Clinical and Laboratory Standards Institute (CLSI) performance standards (M100-S25) for threshold zone sizes primarily for Enterobacteriaceae, extending to Pseudomonas aeruginosa or Staphylococcus spp. where zone sizes were not available for the drug being tested (Supplemental Table 1). The results for four antimicrobials (penicillin, doxycycline, ceftriaxone, and chloramphenicol) were also compared with susceptibility testing using a published minimum inhibitory concentration (MIC) method (Etest),4 which was performed in parallel with disk testing.


Antimicrobial Disk Susceptibility Testing of Leptospira spp. Using Leptospira Vanaporn Wuthiekanun (LVW) Agar.

Wuthiekanun V, Amornchai P, Langla S, White NJ, Day NP, Limmathurotsakul D, Peacock SJ - Am. J. Trop. Med. Hyg. (2015)

Zone of inhibition (50 mm) for penicillin G disk diffusion method on Leptospira Vanaporn Wuthiekanun (LVW) agar for Leptospira interrogans serovar Autumnalis strain NR-20161. The plate was prepared by spreading 300 μL of 108 CFU/mL and preincubating at 30°C in 5% CO2 for 2 days followed by application of the disk and further incubation at 30°C in air for a total of 7 days.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4530741&req=5

Figure 1: Zone of inhibition (50 mm) for penicillin G disk diffusion method on Leptospira Vanaporn Wuthiekanun (LVW) agar for Leptospira interrogans serovar Autumnalis strain NR-20161. The plate was prepared by spreading 300 μL of 108 CFU/mL and preincubating at 30°C in 5% CO2 for 2 days followed by application of the disk and further incubation at 30°C in air for a total of 7 days.
Mentions: The antimicrobial agents selected for testing (N = 22) represent the spectrum of drugs used in tropical settings for the treatment of suspected bacterial sepsis. Disk susceptibility testing was performed by spread plating 300 μL of each isolate (108 CFU/mL) across the surface of a LVW agar plate. These were preincubated at 30°C in 5% CO2 for 2 days (the established optimal incubation conditions for LVW agar), after which a standard disk was applied in the center of a single plate for the following antimicrobials (disk content): amoxicillin/clavulanic acid (20/10 μg), amoxicillin (10 μg), azithromycin (15 μg), aztreonam (30 μg), cefoxitin (30 μg), ceftazidime (30 μg), ceftriaxone (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), clindamycin (2 μg), doripenem (10 μg), doxycycline (30 μg), fosfomycin (50 μg), gentamicin (10 μg), linezolid (30 μg), nalidixic acid (30 μg), nitrofurantoin (300 μg), penicillin (10 units), piperacillin/tazobactam (100/10 μg), rifampicin (5 μg), tetracycline (30 μg), and trimethoprim/sulfamethoxazole (1.25/23.75 μg) (Oxoid Ltd, Basingstoke, United Kingdom). An additional plate (without discs) was used as a growth control. Plates were then incubated at 30°C in air and observed every day for 7 days. The growth inhibition zone sizes were measured at the point at which a bacterial lawn was clearly discernible by the naked eye (usually at day 7) (Figure 1). As disk diffusion testing has not been performed previously for Leptospira, we used Clinical and Laboratory Standards Institute (CLSI) performance standards (M100-S25) for threshold zone sizes primarily for Enterobacteriaceae, extending to Pseudomonas aeruginosa or Staphylococcus spp. where zone sizes were not available for the drug being tested (Supplemental Table 1). The results for four antimicrobials (penicillin, doxycycline, ceftriaxone, and chloramphenicol) were also compared with susceptibility testing using a published minimum inhibitory concentration (MIC) method (Etest),4 which was performed in parallel with disk testing.

Bottom Line: Ten pathogenic Leptospira isolates were tested, all of which were susceptible to 17 antimicrobial agents (amoxicillin/clavulanic acid, amoxicillin, azithromycin, cefoxitin, ceftazidime, ceftriaxone, chloramphenicol, ciprofloxacin, clindamycin, doripenem, doxycycline, gentamicin, linezolid, nitrofurantoin, penicillin, piperacillin/tazobactam, and tetracycline).All 10 isolates had no zone of growth inhibition for four antimicrobials (fosfomycin, nalidixic acid, rifampicin, and trimethoprim/sulfamethoxazole).Of the ten Leptospira, seven had a growth inhibition zone of ≤ 21 mm for aztreonam, the zone diameter susceptibility break point for Enterobacteriaceae.

View Article: PubMed Central - PubMed

Affiliation: Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Nuffield Department of Medicine, Centre for Tropical Medicine and Global Health, University of Oxford, Headington, Oxford, United Kingdom; Department of Tropical Hygiene, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge, United Kingdom lek@tropmedres.ac direk@tropmedres.ac.

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Related in: MedlinePlus