Viability of Leptospira Isolates from a Human Outbreak in Thailand in Various Water Types, pH, and Temperature Conditions

Abstract

Abstract. Leptospira spp. isolated from patients during a multiyear outbreak in Thailand were genotyped using multilocus sequence typing and a majority were identified as ST34, especially in earlier years. We tested whether ST34 isolates were better adapted to survive in various pH levels, temperatures, and water sources. Motility and growth were monitored over a 12-week period. Early year ST34 isolates did not appear to have a significant fitness advantage over non-ST34, however, this may have been because a majority of the isolates survived to the termination of the study, with the exception being at high temperature (37 C) and/or basic pH (8.65). Failure to detect a significant fitness advantage of ST34 may be a result of the length of the study or the small sample size. Lengthening the study and looking at virulence and maintenance in the host could yield additional information about this outbreak.Leptospirosis has a broad range of symptoms, from mild flu-like illness to acute renal failure and pulmonary hemorrhage.1,2 The disease is found worldwide, but is more common in countries with a rainy season and warmer climate that promotes the survival of leptospires in the environment. 3 The outbreak could not be explained by climate or regional flooding; the authors hypothesized that the increased incidence may have been caused by the presence of a biologically successful strain of pathogenic Leptospira.3 In this study, we tested whether early year ST34 isolates (2001 and 2003) were better adapted than later year ST34 and/or non-ST34 isolates (2005 and 2006) from this outbreak for survival in water from different sources and at variable pH and temperature.Isolates representing four of the 11 STs from various years of the outbreak were selected (Table 1). Isolates had been maintained at 25-30 C and had gone through 18-23 passages in Ellinghausen, McCullough, Johnson, and Harris (EMJH) semisoild media. Isolates were inoculated into liquid EMJH, grown at 30 C to the logarithmic phase, and adjusted to a McFarland standard of 0.5. [5][6][7][8][9] Spiking was performed by centrifuging 12 mL of the adjusted culture, discarding the supernatant, and then resuspending with 12 mL of suspending fluid. Rice field water and pond water from the outbreak location in Thailand were autoclaved before use. For each suspending fluid, conditions were tested in duplicate with various temperatures (25 C, 30 C, or 37 C) and pH (unadjusted, 5.65 or 8.65) combinations. The unadjusted pH was found to be pH 6.95 in rice field water and pH 7.79 in pond water. Once the Leptospira were resuspended, tubes were placed in a dark incubator at the appropriate temperature for 12 weeks. For each test combination, growth and viability of the Leptospira isolates were determined by measuring optical density (OD), leptospiral motility, and growth in semisolid EMJH (Figure 1).The OD at 400 nm was measured twice each week as a method of monitoring viable Leptospira ( Figure 1A). 10,11 Repeated measures analysis of variance and Wilks test were performed to...

Rainfall and other meteorological factors as drivers of urban transmission of leptospirosis

“…The 1 to 2 weeks lag between rainfall, temperature, or humidity and leptospirosis identified here, as well as the 2 weeks lag between rainfall and flooding reported in Manila, contrast with results from previous studies that used monthly data and suggested longer temporal association between onset of symptoms of leptospirosis and meteorological data (2 to 10month lags respectively) [18][19][20]46]. Therefore, though leptospires survive for up to one year and remain virulent in culture conditions that mimic the external environment in vitro [45,47,48], L. interrogans survival in spring water decreases to 3 weeks [49].…”

A systematic review of Leptospira in water and soil environments

“…Factors usually reported to influence the survival of Leptospira in water or soils such as pH, salinity, temperature, moisture were not sufficiently reported in the studies selected to allow meta-analysis. However, it should be noted that a number of studies demonstrated Leptospira survival at low pH or low temperature in water or soils [11,[40][41][42][43][44][45].…”

In-vitro antileptospiral activity of Canarium odontophyllum Miq. (Dabai) leaves extract

“…In each row, 50 µL of various concentrations of leaf extract at 0.049 mg/mL, 0.098 mg/mL, 0.195 mg/mL, 0.391 mg/mL, 0.781 mg/mL, 1.563 mg/mL, 3.125 mg/mL, 6.25 mg/mL, 12.5 mg/mL and 25 mg/mL was added horizontally and incubated for three days at 30 °C in the dark condition. After incubation time, the plate was read by spectrophotometer at the wavelength of 400 nm (Stoddard et al, 2014) in triplicate and the average OD of test was calculated. The percentage of inhibition of Leptospira growth by Canarium odontophyllum extract was calculated through the following formula (Wang et al, 2010):…”