Leptospira Is an Environmental Bacterium That Grows in Waterlogged Soil

Leptospires survive for an extended time in the environment.

We collected 110 samples (106 soil and 4 water) in the Philippines and 36 samples (31 soil and 5 water) in Japan (Table 1 and Table S1). The presence of leptospires in the soil and water samples was determined by the isolation of bacteria with characteristic helical morphology and PCR amplification of Leptospira-specific rrl or sequencing of the rrs genes. Additionally, the flaB gene associated with the pathogenic (P) clade of Leptospira was detected by PCR. The presence of Leptospira was confirmed in 86 of 110 samples (78%; 83 of 106 soil samples and 3 of 4 water samples) in the Philippines and 15 of 36 samples (42%; 13 of 31 soil samples and 2 of 5 water samples) in Japan. Of 101 culture-positive samples, 21 (21%) were positive for flaB PCR. Environmental samples in the Philippines showed a higher positivity rate of leptospires than those in Japan, which was consistent with previous reports (Table 1) (1, 27). Interestingly, in the collection of leptospires from the environment, leptospires were detected even in the sample collected from harsh environments, such as forest soil at an altitude of more than 1,200 m in Mt. Yatsugatake and in soil (−2°C) under a 40-cm-thick layer of snow in Sapporo, where chances of contamination by leptospires from infected animals are minimal. These results suggest that leptospires survive for an extended time in the soil, as reported in water (11, 12).

To confirm the long-term survival of leptospires in the soil, 12 selected Leptospira-positive soil samples were stored in closed plastic conical tubes at room temperature (Table 2). After a certain period of time, the presence of leptospires was examined by the reisolation of the bacteria. Although the incubation time before reisolation varied among samples, leptospires were reisolated from all tested soil samples for up to 379 days after the incubation regardless of harboring the flaB gene. Therefore, soil is potentially one of the reservoirs for both pathogenic and saprophytic leptospires.

Leptospires multiply in waterlogged soil.

Since postflooding soil conditions might be suitable for growing leptospires, a growth experiment of Leptospira using waterlogged soils, a 1:2.5 mixture of soil and water, was conducted (Fig. 1). In this experiment, four strains were selected from different subclades (Fig. S1). In Korthof’s medium, all strains grew as expected and reached a cell count of more than 10⁸ cells/mL by day 8. In sterile Milli-Q water, the count of leptospires was below the detectable limit (60,000 cells/mL) at all the time points, indicating that leptospires do not multiply in Milli-Q water. When leptospires were inoculated into waterlogged ranch soil, the cell count of the four Leptospira strains used in this experiment increased to more than 10⁶ cells/mL by day 8 (Fig. 1A to D), although the growth rate was lower than that in Korthof’s medium. Comparing growth at day 5 of seeding, which is considered the logarithmic growth phase, Ictero No. 1 increased the count only to 1.56 × 10⁵ cells/mL (Fig. 1A), whereas Lepto 2 reached a cell count of 3.75 × 10⁶ cells/mL (Fig. 1C), suggesting that the growth speed in the waterlogged soil varies among strains. This experiment was repeated multiple times, and the growth of leptospires in waterlogged soil was observed consistently. However, the growth rate of leptospires varied among experiments and sometimes reached a cell count of more than 10⁷ cells/mL in waterlogged soil. We also conducted the growth experiment using autoclaved soils without adding water. Although it might be difficult to make a direct comparison with the waterlogged soil because of the completely different method of collecting the supernatant from soil, we could not detect that these Leptospira strains grew (less than 60,000 cells/mL) in the autoclaved soil without water, as shown in Fig. 1.

Considering the effect of carrying over Korthof’s medium contained in bacterial stock solutions on leptospiral proliferation, a growth experiment of Leptospira strain Lepto 14 was conducted in waterlogged soil after washing with Milli-Q water (Fig. 1D). Although the count of Lepto 14 was under the detection limit (60,000 cells/mL) on day 5 of incubation, it reached 1.7 × 10⁶ cells/mL on day 7. As for the reason for the lower initial growth when seeding the bacteria after washing, we cannot exclude the possibility that the medium in bacterial stock solutions is involved in the growth at an initial phase, but the cells may have been damaged by washing and centrifuging, possibly decreasing their viability.

In order to examine the point without damaging the cells by washing, we conducted growth experiments in which leptospires were repeatedly passed in waterlogged soil until the remaining Korthof’s medium was fully diluted. Ictero No. 1, Lepto 2, and Lepto 14 increased their numbers even after five passages, which gave 6.25 × 10⁶-fold dilution of the original stock solution (Fig. 2), although the number of leptospiral cells in each strain tended to decrease with each passage, probably due to overdilution. The average doubling time for each strain was 29.2, 35.4, and 32.8 h, respectively, while the doubling time of leptospires in the liquid medium was between 8 and 20 h. Although the growth rate in waterlogged soil was lower than that in the growth medium, such as Korthof’s medium, our results indicated that leptospires in both pathogenic and saprophytic clades are capable of multiplying in a postflood environment like waterlogged soil.

Isolation of leptospires from environmental samples.

Isolation of leptospires was attempted from environmental soil and water collected in various locations in the Philippines and Japan during different seasons. The date and location information (city name and latitude/longitude) of the Leptospira strain isolation are shown in Table S1. Soil and water samples (approximately 10 g and 10 mL, respectively) were collected in sterile 15-mL screw-cap tubes. For soil samples, 10 mL of sterile water was added and mixed. Each tube containing 10 g of soil sample soaked in 10 mL of water or 10 mL of water sample alone was incubated in a vertical position for 1 h to allow the sediments to settle. Then, 2.0 mL of supernatant from the sample was added to 2.5 mL of 2-fold concentrated Korthof’s medium (Thomas scientific, Swedesboro, NJ, USA) supplemented with 500 μL of 10-fold concentrated antimicrobial reagent called STAFF (400 μg/mL of sulfamethoxazole, 200 μg/mL of trimethoprim, 50 μg/mL of amphotericin B, 4 mg/mL of fosfomycin, and 1 mg/mL of 5-fluorouracil) (41). All antimicrobial agents were purchased from Sigma-Aldrich (St. Louis, MO, USA). These tubes were incubated at 30°C, and droplet aliquots taken from each tube culture were observed daily by dark-field microscopy for the presence of Leptospira, confirmed by the characteristic thin helical structures with prominent hooked ends and motility. Samples were considered negative and discarded if no Leptospira was detected after 28 days of incubation. When Leptospira was confirmed microscopically, the whole volume of sample was filtered using a 0.2-μm-pore-size membrane filter to remove the contaminants (other microbes such as bacteria and fungi). A half milliliter of the filtrate was then added to 4.5 mL of fresh Korthof’s medium or semisolid medium containing 0.1% (wt/vol) agar without STAFF and cultured at 30°C. The number of the bacteria was counted using a Petroff-Hausser bacterial cell-counting chamber under dark-field microscopy. Single-colony isolation was performed in solid Korthof’s medium containing 1% (wt/vol) agar. Bacteria in the liquid culture medium were diluted to 10⁴ cells/mL and inoculated over the surface of the solid medium with a glass spreader. The inoculated plate was incubated at 30°C and observed daily for the appearance of subsurface colonies to pick a single colony up. The selected strains were purely cultured in Korthof’s medium to prepare bacterial stocks, and their aliquots were stored at −80°C for growth experiments.

DNA extraction.

Bacterial DNA was extracted from 0.5 mL of the primary culture before filtration and 0.1 mL of the pure confluent culture of Leptospira isolates using an Illustra bacteria genomic prep mini spin kit (GE Healthcare, Chalfont St Giles, Buckinghamshire, United Kingdom) following the protocol designated for Gram-negative bacteria. Pure confluent culture of Leptospira strain Ictero No. 1 and Milli-Q water were used as positive and negative controls for the following PCR, respectively.

23S rRNA (rrl) gene PCR and flaB nested PCR.

It has been reported that all Leptospira species can be identified by detecting 23S rRNA (rrl) gene via PCR (42). For the 23S rRNA gene PCR, the primers rrl-F (5′-GACCCGAAGCCTGTCGAG-3′) and rrl-R (5′-GCCATGCTTAGTCCCGATTAC-3′) were used (28). Leptospiral DNA from the samples was amplified in a thermal cycler under the following conditions: 30 cycles of 94°C for 20 s, 54°C for 30 s, and 72°C for 1 min and an extension at 72°C for 6 min using Ex Taq HS DNA polymerase (TaKaRa, Otsu, Shiga, Japan). PCR products were checked by electrophoresis on 1.5% (wt/vol) agarose gel.

The flaB nested PCR for detecting leptospires in a pathogenic clade was performed as reported previously (7). For the first PCR, the primers l-flaB-F1 (5′-CTCACCGTTCTCTAAAGTTCAAC-3′) and l-flaB-R1 (5′-TGAATTCGGTTTCATATTTGCC-3′) were used (29). PCR amplification was performed under the following conditions: an initial cycle of 1 min at 94°C and 25 cycles at 94°C for 10 s, 50°C for 30 s, and 72°C for 1 min using Ex Taq HS DNA polymerase (TaKaRa). Then, 1 μL of the first-round PCR product was added to 19 μL of the second-round PCR mixture. For the second PCR, the primers l-flaB-F2 (5′-TGTGCACAAGACGATGAAAGC-3′) and l-flaB-R2 (5′-AACATTGCCGTACCACTCTG-3′) were used (29). The second PCR amplification consisted of an initial cycle of 1 min at 94°C, 30 cycles for 10 s at 94°C, 30 s at 55°C, and 50 s at 72°C and a final extension for 7 min at 72°C using Ex Taq HS DNA polymerase (TaKaRa). PCR products were checked to confirm the detection of flaB by electrophoresis on 1.5% (wt/vol) agarose gels. The PCR was conducted with reference to the “Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-04-001)” issued by U.S. Environmental Protection Agency.

16S rRNA (rrs) gene sequencing and phylogenetic analysis.

The 16S rRNA gene was amplified with bacterial universal primers P16S-8UA (5′-AGAGTTTGATCMTGGCTCAG-3′) and P16S-1485R (5′-TACGGYTACCTTGTTACGACTT-3′) using Ex Taq HS DNA polymerase (TaKaRa) (29). PCR amplification was performed under the following conditions: 30 cycles at 96°C for 1 min, 55°C for 1 min, and 72°C for 1.5 min. After the amplicons of the 16S rRNA gene by electrophoresis on 1% (wt/vol) agarose gels were confirmed, the PCR products were purified by using a PCR purification kit (Qiagen, Venlo, Netherlands), and the sequence was determined using a 3130 genetic analyzer (Thermo Fisher Scientific, Waltham, MA, USA). The 16S sequences of Leptospira obtained in this study were deposited in GenBank (accession numbers MW727617, MW727618, MW727619, and MW727620). The sequence data were compared with those of representative reference strains collected from public databases (GenBank, NCBI, NIH) and phylogenetically analyzed using GENETYX Network v.13.1.1 (GENETYX). According to the Bayesian Information Criterion (43), the model with the lowest score was used in maximum likelihood analysis to establish the best-fit substitution model. Phylogenetic trees were constructed using MEGA v.7.0 (http://www.megasoftware.net/) under the best-fit model.

Survival test of Leptospira in soil.

The long-term survival test was conducted with 100 g of 12 selected soil samples positive for leptospires. Initially, leptospires were isolated from 10 g of the soil and cultured in Korthof’s medium as described above, followed by the identification of rrl and flaB genes by PCR. Then, the remaining 90 g of the soil samples with confirmed Leptospira isolation was stored in a closed conical tube at room temperature (24 to 26°C) for the indicated period. Subsequently, isolation and identification of Leptospira were attempted from 10 g of soil in the remaining samples as described above.

Growth experiments in waterlogged soil.

To examine whether leptospires multiply in the environmental samples without the animal host, in vitro growth experiments were conducted in water, soil, and waterlogged soil (mimicking the situation after floods). We used soil from a ranch in Hokkaido University Campus after it was autoclaved to sterilize any potential leptospires and other bacteria in the soil, as well as sterile Milli-Q water. The moisture content of the ranch soil was 45% (mean value from two experiments), which was calculated from the weight before and after drying as reported previously (29), and the pH examined by paper pH strips (Thermo Fisher Scientific) was 6.8 (mean value from three experiments; range was 6.5 to 7.0).

For this experiment, the following four strains were selected from the P1, P2, S1, and S2 subclades, respectively: L. interrogans serovar Icterohaemorrhagiae strain Ictero No. 1 kindly provided by Y. Kobayashi, L. wolffii strain YH 112 (sample number 115), L. yanagawae strain Lepto 2 (sample number 130), and Lepto 14 (sample number 122) closely related to L. ryugenii (Table S1 and Fig. S1). The Leptospira stock culture was diluted in Milli-Q water to make the concentration to 1 × 10⁶ cells/mL.

In growth experiments, 5 × 10⁵ cells in 500 μL of the diluted stock were inoculated into 100-mL glass bottles containing 25 mL of sterile Milli-Q water (water bottle) or 10 g of autoclaved soil without or with 25 mL of sterile Milli-Q water (soil bottle or waterlogged soil bottle, respectively). In addition, 25 mL of Korthof’s medium without soil was prepared in the bottle as positive control. The closed bottles were incubated at room temperature (24 to 26°C) for up to 12 days. A small portion (about 100 μL) of the supernatant was aseptically collected continuously from each bottle at different time points. To collect leptospires from the soil bottle at each indicated time point, soil was resuspended in 25 mL of water for 30 min with gentle agitation, and then 100 μL of the supernatant was collected. The number of leptospires in collected supernatants was counted using a Petroff-Hausser bacterial cell-counting chamber under dark-field microscopy. The detection limit of cells was 60,000 cells/mL. The reproducibility was confirmed by at least three repeated experiments.

To exclude the possibility that bacterial growth was supported by culture medium from the stock, Leptospira stock culture of Lepto 14 was centrifuged (4,400 × g, 20 min) and washed twice with Milli-Q water to remove the remaining Korthof’s medium. The washed cells were diluted to 6 log cell counts/mL with Milli-Q water, 500 μL (5 × 10⁵ cells) was inoculated into waterlogged soil, and the cell count was performed as described above.

In addition, transplanting tests were conducted to reduce the effect of Korthof’s medium in the initial inoculation by serial dilution on the leptospiral growth in waterlogged soil. After inoculation of Ictero No.1, Lepto 2, and Lepto 14 in waterlogged soil bottles, 500 μL of the supernatant was transferred into a new waterlogged soil bottle every 4 to 6 days, which means 50-fold dilution per passage. The passage was repeated four times, and finally the medium contained in the initial inoculum was diluted by 6.25 × 10⁶-fold. Six days after the last passage, the cell number in the supernatant was measured as described above.

Data availability.

The 16S sequences of Leptospira obtained in this study were deposited in GenBank (accession numbers MW727617, MW727618, MW727619, and MW727620).