A novel combination of selective agents for isolation of Leptospira species

Abstract

A novel combination of antimicrobial agents (sulfamethoxazole, 40 μg/mL; trimethoprim, 20 μg/mL; amphotericin B, 5 μg/mL; fosfomycin, 400 μg/mL; and 5-fluorouracil, 100 μg/mL) was developed for selective isolation of leptospires from contaminated samples. The growth of 16 microorganisms considered as possible contaminants during isolation of Leptospira were inhibited by this antimicrobial cocktail. In contrast, the growth of a smaller inoculum (10 1 cells per mL) of 25 Leptospira strains (representing 18 serovars/serogroups of 5 species) was not suppressed by this antimicrobial combination. This cocktail, after being incorporated into Leptospira growth medium (Korthof 's), successfully detected leptospires in environmental soil and water. Based on the results, this selective medium has the potential to meet the existing need for an effective selective medium for the isolation of Leptospira.

PCR and Culture Identification of Pathogenic Leptospira spp. from Coastal Soil in Leyte, Philippines, after a Storm Surge during Super Typhoon Haiyan (Yolanda)

“…All the tubes were kept in a vertical position for 1 h to allow the sample to settle. After checking the pH of the supernatant using a urine pH test kit (Nippon Chemiphar, Tokyo, Japan), 2 ml was transferred to 2.5 ml 2ϫ-concentrated liquid and semisolid Korthof's media supplemented with 500 l of 10ϫ-concentrated STAFF (sulfamethoxazole, 400 g/ml; trimethoprim, 200 g/ml; amphotericin B, 50 g/ml; fosfomycin, 4 mg/ml; 5-fluorouracil, 1 mg/ml) (8). These tubes were incubated at 30°C for 28 days and checked daily by dark-field microscopy for the presence of Leptospira, which was confirmed by observing the characteristic thin helical structures with prominent hooked ends and motility.…”

“…However, isolation from environmental samples is usually unsuccessful due to the slow growth of leptospires and the overgrowth of coexisting microorganisms. We previously reported a novel combination of antimicrobial agents (sulfamethoxazole, trimethoprim, amphotericin B, fosfomycin, and 5-fluorouracil [STAFF]) for the selective isolation of leptospires from contaminated samples (8). After this cocktail was incorporated into Leptospira growth medium, it inhibited the growth of contaminants and successfully isolated pathogenic leptospires as well as saprophytic species from environmental samples (9).…”

Comparative Analysis of Leptospira Strains Isolated from Environmental Soil and Water in the Philippines and Japan

“…However, isolation from environmental samples is challenging due to the slow growth of leptospires and the overgrowth of coexisting microorganisms. We previously reported a novel combination of antimicrobial agents (sulfamethoxazole, trimethoprim, amphotericin B, fosfomycin, and 5-fluorouracil [STAFF]) for selective isolation of leptospires from contaminated samples (4). This cocktail, after being incorporated into Leptospira growth medium, inhibited the growth of contaminants and successfully isolated leptospires in environmental samples.…”

Molecular and phenotypic characterization of Leptospira johnsonii sp. nov., Leptospira ellinghausenii sp. nov. and Leptospira ryugenii sp. nov. isolated from soil and water in Japan

“…The genus Leptospira was previously classified into 23 species that included ten pathogenic, six intermediate and seven saprophytic species. However, since the discovery of a novel antibiotic combination (STAFF) for Leptospira culture, various Leptospira have been isolated from soil and water. In this study, the molecular and phenotypic characteristics of three strains isolated from soil in Japan were compared with those of Leptospira species and 12 novel Leptospira species isolated from soil in New Caledonia …”