Abstract
.Leptospirosis is a global zoonotic disease caused by pathogenic bacteria of the Leptospira genus, which are fastidious, slow-growing organisms. Antimicrobial susceptibility data are limited; traditionally, the organisms have not been culturable on solid media. The recent development of Leptospira Vanaporn Wuthiekanun (LVW) agar, which facilitates rapid growth of Leptospira spp., provides the opportunity for antimicrobial susceptibility testing. Eighty-three Leptospira spp. clinical isolates originating from patients in Laos between 2006 and 2016 were tested against six antimicrobials (azithromycin, ceftriaxone, ciprofloxacin, doxycycline, gentamicin, and penicillin G) using disk diffusion on LVW agar. Quality control was undertaken using American Type Culture Collection (ATCC) reference strains with known susceptibilities on both standard media and LVW agar. All Leptospira spp. isolates produced large zones of inhibition around each of the six antimicrobials. All zones were greater than 25 mm: gentamicin produced the smallest zones (median 35 mm; interquartile range 30 mm–37 mm) and azithromycin produced the largest zones (median 85 mm; interquartile range 85 mm–85 mm). Zones produced by non-leptospiral ATCC reference strains on LVW agar were within 2 mm of accepted strain-specific quality control range on standard media. Antimicrobial activity on LVW agar appears to be similar to that on standard media. As there are no published susceptibility guidelines for the Leptospira genus, zone interpretation was subjective. Leptospira Vanaporn Wuthiekanun agar enabled antimicrobial susceptibility testing of multiple Leptospira isolates on solid media; the large zone sizes observed suggest that resistance has not emerged to these six antimicrobials in Lao Leptospira spp.
Highlights
Leptospirosis is a widespread zoonosis caused by spirochete bacteria of the genus Leptospira
The aim of our study was to expand on this initial study and use disk diffusion assays on Leptospira Vanaporn Wuthiekanun (LVW) agar to assess the antimicrobial susceptibility of 83 Lao People’s Democratic Republic (Laos) human Leptospira spp. isolates to six antimicrobials, which are commonly used to empirically treat fever in Laos.[25,26,27]
All zones produced by E. coli American Type Culture Collection (ATCC) 25922, S. pneumoniae ATCC 49619, S. aureus ATCC 25923, and P. aeruginosa ATCC 27853 on Clinical and Laboratory Standards Institute (CLSI) standard media were within the strain-specific quality control ranges published in CLSI guidelines (Table 2).[26]
Summary
Leptospirosis is a widespread zoonosis caused by spirochete bacteria of the genus Leptospira. The highest disease burden is in tropical and subtropical areas, including Southeast Asian countries such as the Lao People’s Democratic Republic (Laos), where it is commonly under-recognized.[1] The infection is spread to humans through environmental contamination by the urine of infected animals and causes a spectrum of clinical presentations ranging from mild febrile illness to severe disease that can result in organ failure and death.[2] First-line therapy for severe leptospirosis is intravenous penicillin with oral doxycycline or azithromycin indicated in less-severe disease.[3] Because of limited diagnostic capability and nonspecific clinical presentation, leptospirosis patients are commonly treated empirically for undifferentiated fever usually with betalactams, doxycycline, gentamicin, or ciprofloxacin.[4,5,6,7,8]. Susceptibility testing of Leptospira spp. has been limited as leptospires are fastidious, slow-growing organisms unable to grow on standard laboratory media.[10,11] There has been no robust method of culturing leptospires on solid media and no standard method for in vitro testing of antimicrobial agents against Leptospira spp.[10,11,12] Culture using special liquid or semiliquid media can be performed but is labor intensive, requiring expertise and may take weeks to months to achieve growth.[10,13] Both macro- and micro-dilution methods have been used to test the susceptibility of isolates from humans and animals, but they are time consuming and difficult to control.[10,11] Both clinical evidence and in vitro studies suggest that leptospires are susceptible to beta-lactams, macrolides, tetracyclines, and fluoroquinolones, but the lack of large-scale studies means there is little information about the prevalence of antimicrobial resistance within the genus.[14,15,16,17,18,19,20,21,22,23]
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