High virulence in hamsters of four dominant Leptospira serovars isolated from rats in the Philippines.

Leptospirosis is caused by pathogenic species of Leptospira. The aim of this study was to determine and characterize the pathogenicity of four dominant Leptospira isolates prevailing among rats in the Philippines. The isolates were Leptospira interrogans serovar Manilae strain K64, L. interrogans serovar Losbanos strain K37, L. interrogans serovar Ratnapura strain K5 and Leptospira borgpetersenii serovar Javanica strain K6. Pathogenicities were studied using hamsters, which reproduce severe human leptospirosis. The minimum lethal doses were 10(0) ( = 1) leptospires for K64, K37 and K5, and 10(1) leptospires for K6. Weight loss amongst the Leptospira-infected hamsters was observed from 1 day before death (K64-, K37- and K5-infected hamsters) to as much as 1 week before death for K6-infected hamsters. Similar and varied gross and microscopic lesions were observed amongst infected hamsters, even for strains belonging to the same species (i.e. L. interrogans). The most significant and common histopathological findings were congestion of the glomerulus, disarrangement of hepatic cords and erythrophagocytosis. Other findings were foamy splenic macrophages for K6, severe petechial pulmonary haemorrhage for K64, and hematuria and severe pulmonary congestion for K37. Immunostaining and culture revealed the presence of leptospires in different organs of the infected hamsters. Based on these results, Leptospira isolates from rats in the Philippines were shown to be highly virulent, causing pulmonary haemorrhage, severe hepato-renal damage and death in hamsters even at lower doses. The present findings on experimental leptospirosis support clinical data showing that patients with severe manifestations of leptospirosis, such as pulmonary haemorrhage, are increasing in the Philippines. These findings may serve as a basis to strengthen the early diagnosis and treatment of human leptospirosis.

Comparative analysis of Leptospira strains isolated from environmental soil and water in the Philippines and Japan.

There have been few reports on the epidemiological analysis of environmental Leptospira isolates. This is probably because the isolation of leptospires from the environment was usually unsuccessful due to the overgrowth of contaminants and the slow growth of Leptospira. In this study, we collected a total of 88 samples of soil and water from three sites: Metro Manila and Nueva Ecija, Philippines (an area where Leptospira is now endemic), and Fukuoka, Japan (an area where Leptospira was once endemic). We succeeded in isolating Leptospira from 37 samples by using the novel combination of five antimicrobial agents reported in 2011. The frequencies of positive isolation of Leptospira in the Philippines and Japan were 40 and 46%, respectively. For Leptospira-positive samples, five colonies from each sample were isolated and analyzed by pulsed-field gel electrophoresis (PFGE). The isolates from each area showed their respective characteristics in phylogenetic trees based on the PFGE patterns. Some isolates were closely related to each other across borders. Based on 16S rRNA gene-based phylogenetic analysis, four isolates in Fukuoka were identified as a pathogenic species, L. alstonii; however, its virulence had been lost. One isolate from Nueva Ecija was identified as the intermediate pathogenic species Leptospira licerasiae. Most of the isolates from the environment belonged to nonpathogenic Leptospira species. We also investigated the strain variation among the isolates in a puddle over 5 months. We demonstrated, using PFGE analysis, that Leptospira survived in the wet soil on dry days and appeared in the surface water on rainy days. These results showed that the soil could be a reservoir of leptospires in the environment.

Electron microscopic study of the effects of antimicrobial agents on the cellular architecture of Leptospira.

Morphological changes of Leptospira induced by ampicillin, streptomycin, or ciprofloxacin were studied by transmission electron microscopy after staining with uranyl acetate. Irregularities in coiling, loss of hooks, spherical swellings, membrane vesicle formation, disruption of outer membrane, exposure and detachment of flagella, penetration of dye, and lysis of cells were observed. These changes appeared in a time-dependent manner and appeared to be associated with the sites of action of antimicrobial agents. This is the first report delineating the action of aminoglycoside and new quinolone on the morphology of Leptospira cells.

A novel combination of selective agents for isolation of Leptospira species.

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.

In vitro sensitivity and resistance of 46 Leptospira strains isolated from rats in the Philippines to 14 antimicrobial agents.

The in vitro susceptibilities of 46 Leptospira isolates from rats to 14 antimicrobial agents were tested. All of the strains were found to be sensitive to ampicillin, cefotaxime, ciprofloxacin, norfloxacin, doxycycline, erythromycin, and streptomycin. In contrast, the tested isolates showed resistance to amphotericin B, 5-fluorouracil, fosfomycin, trimethoprim, sulfamethoxazole, neomycin, and vancomycin. These findings will help in selecting effective and ineffective antimicrobials for treatment of leptospirosis and for the development of new selective media, respectively.