Multilocus sequence typing reveals diverse known and novel genotypes of Leptospira spp. circulating in Sri Lanka

Background Leptospirosis has gained much attention in Sri Lanka since its large outbreak in 2008. However, most of the cases were clinically diagnosed and information on Leptospira genotypes and serotypes currently prevailing in the country is lacking. Methodology/Principal findings We retrospectively analyzed 24 Leptospira strains from human patients as well as isolated and characterized three Leptospira strains from black rats using the microscopic agglutination test with antisera for 19 serovars and multilocus sequence typing. The isolates were identified as Leptospira borgpetersenii sequence types (STs) 143 and 144; L. interrogans STs 30, 34, 43, 44, 74, 75, 80, 308, 313, 314, 316, and 317; and L. kirschneri ST318. Six of the 15 STs were identified for the first time in this study. Five serogroups such as Autumnalis, Grippotyphosa, Hebdomadis, Javanica, and Pyrogenes were detected among the isolates. Contrary to previous studies, various genotypes including novel STs were isolated during an outbreak in Southern Province. L. borgpetersenii serogroup Javanica ST143 was isolated both from a human and black rat. Conclusions/Significance This study revealed that genetically diverse Leptospira strains currently circulate in Sri Lanka: some genotypes have been circulating and others have emerged recently, which may explain the recent surge of leptospirosis patients with varying clinical manifestations and frequent outbreaks of leptospirosis. Black rats were identified as the source of infection for humans, but reservoir animals for other genotypes remain unknown.


Methodology/Principal findings
We retrospectively analyzed 24 Leptospira strains from human patients as well as isolated and characterized three Leptospira strains from black rats using the microscopic agglutination test with antisera for 19 serovars and multilocus sequence typing. The isolates were identified as Leptospira borgpetersenii sequence types (STs) 143 and 144; L. interrogans STs 30,34,43,44,74,75,80,308,313,314,316, and 317; and L. kirschneri ST318. Six of the 15 STs were identified for the first time in this study. Five serogroups such as Autumnalis, Grippotyphosa, Hebdomadis, Javanica, and Pyrogenes were detected among the isolates. Contrary to previous studies, various genotypes including novel STs were isolated during an outbreak in Southern Province. L. borgpetersenii serogroup Javanica ST143 was isolated both from a human and black rat.

Conclusions/Significance
This study revealed that genetically diverse Leptospira strains currently circulate in Sri Lanka: some genotypes have been circulating and others have emerged recently, which may explain the recent surge of leptospirosis patients with varying clinical manifestations

Introduction
Leptospirosis is a zoonotic disease caused by pathogenic spirochaetes of Leptospira spp. belonging to subclades P1 and P2 [1][2][3][4]. Human leptospirosis is an acute febrile illness with an extremely broad clinical spectrum ranging from mild influenza-like illness to severe disease forms characterized by jaundice, bleeding, renal failure, and death [1,3]. Approximately one million cases and 58,900 deaths of leptospirosis are estimated to occur worldwide each year, more than 70% of which are reported from the tropical, poorest regions of the world [5]. Pathogenic Leptospira spp. colonize the proximal renal tubules and are excreted in the urine of reservoir animals [1,3]. Human leptospirosis is primarily transmitted by exposure to water or soil contaminated with the urine of infected animals [1,3]. Leptospirosis is a re-emerging disease in Sri Lanka and has gained much attention since the large outbreak in 2008 [6]. A recent systematic review indicated that the cumulative annual incidence of leptospirosis that required hospitalization from 2008 to 2015 was 52.1 per 100,000 people, with an estimated case fatality ratio of 7.0% [7]. A wide array of new clinical entities such as pulmonary hemorrhage, pancreatitis, and myocarditis with high case fatality were seen in recent outbreaks [8][9][10][11]. Most of the cases, however, were clinically diagnosed, and some were diagnosed serologically by the microscopic agglutination test (MAT) using a non-pathogenic Leptospira strain Patoc I [12]. Recent studies performed MAT using a panel of Leptospira serovars and detected antibodies to various leptospiral serovars in human patients, rodents, and cattle [11,[13][14][15]. In addition, recent molecular investigations detected L. borgpetersenii, L. interrogans, L. kirschneri, and L. weilii in clinical samples of febrile human patients and regional differences of predominant Leptospira species in the country [13,[15][16][17][18]. Leptospira DNAs belonging to L. borgpetersenii, L. interrogans, and L. kirschneri have been recently detected in cattle kidney or urine samples collected in distinct areas [14,19]. Although Leptospira strains were isolated from humans, rodents, shrews, and dogs in 1960s and early 1970s [20,21], only one report of culture isolation of L. interrogans unidentified serogroups has been published in Sri Lanka during the past 40 years [22]. Therefore, Leptospira genotypes and serotypes currently prevailing in the country remain largely unknown.
In the present study, we characterized Leptospira isolates from clinically-suspected leptospirosis patients and rats in various Provinces in Sri Lanka using the microscopic aggulutination test with antisera for 19 serovars and multilocus sequence typing (MLST).

Maintenance of Leptospira isolates from clinically-suspected leptospirosis patients
We retrospectively analyzed 24 Leptospira strains stored at National Reference Laboratory for Leptospirosis, Medical Research Institute, Sri Lanka. These strains were isolated from clinically-suspected leptospirosis patients defined by the National Guideline on Management of Leptospirosis 2016, Ministry of Health, Sri Lanka (www.epid.gov.lk) between 2012 to January 2019 by bedside inoculation of two drops of fresh blood into liquid Ellinghausen-McCullough-Johnson-Harris (EMJH) medium that comprised Difco Leptospira Medium Base EMJH and Difco Leptospira Enrichment EMJH (Becton, Dickinson and Company, Franklin Lakes, NJ) with no antibiotics. During the period, 401 blood cultures were collected and 28 Leptospira strains were isolated (isolation rate: 7.0%), of which four cultures were contaminated and not included in this study. The isolates were stored in semisolid EMJH as described above at ambient temperature and inoculated into liquid medium before serological and molecular characterization and cultured at 30˚C. This study was approved by the Ethics Review Committee of Medical Research Institute.

Isolation of Leptospira spp. from small mammals
For the survey of reservoir animals, we used live traps and captured 108 rodents and shrews in Kandy, Central Province in 2015 and 116 rodents in Giradurukotte, Uva Province in 2017. Animal dissections were performed according to the American Veterinary Medical Association guidelines under the ethical approval of the Institutional Review Board of the University of Peradeniya. The species of animals captured was identified by DNA sequencing of the mitochondrial cytochrome b gene, cytb [23]. DNA was extracted from animal kidney tissues using DNAZOL Reagent (Invitrogen, Carlsbad, CA). cytb was amplified with AmpliTaq Gold 360 Mater Mix (Applied Biosystems, Foster City, CA) using primer pairs H15300 (5 0 -GTTTACAA GACCAGAGTAAT-3 0 ) and L497A (5 0 -CCTAGTAGAATGAATCTGAGG-3 0 ) and H655A (5 0 -TGTGTAGTATGGGTGGAATGG-3 0 ) and L14115 (5 0 -GACATGAAAAATCATCGTTG-3 0 ) [23] or L14274 (5 0 -CGAAGCTTGATATGAAAAACCATCGTTG-3 0 )and H15149 (5 0 -AAA CTGCAGCCCCTCAGAATGATATTTGTCCTCA-3 0 ) [24]. After an initial denaturation step at 95˚C for 10 min, the reaction mixture was subjected to 35 cycles of denaturation at 95˚C for 30 s, annealing at 50˚C for 30 s, and extension at 72˚C for 60 s, followed by a final extension at 72˚C for 5 min. DNA sequencing of the amplicons was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) with the PCR primers and the resulting sequences were subjected to phylogenetic analyses as previously described [23]. All black rats captured in this study belonged to Rattus rattus complex lineage 1 [25]. For the isolation of Leptospira spp., kidney tissues were inoculated into 5 ml liquid EMJH medium that composed Difco Leptospira Medium Base EMJH and Difco Leptospira Enrichment EMJH (Becton, Dickinson and Company, Franklin Lakes, NJ) and supplemented with 100 μg/ mL of 5-fluorouracil and cultured at 30˚C for 3 months.

Molecular characterization of Leptospira isolates
DNA was extracted from 0.5 ml cultures of the isolates using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). Partial flaB sequence was amplified with TaKaRa Ex Taq Hot Start Version (Takara Bio, Japan) using primers L-flaB-F1 (5 0 -CTCACCGTTCTCTAAAGTT CAAC-3 0 ) and L-flaB-R1 (5 0 -TGAATTCGGTTTCATATTTGCC-3 0 ) for species classification [26]. After an initial denaturation step at 95˚C for 1 min, the reaction mixture was subjected to 30 cycles of denaturation at 98˚C for 10 s, annealing at 50˚C for 30 s, and extension at 72˚C for 50 s. DNA sequencing of the amplicons was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) with the PCR primers, and the resulting sequences were compared with those of reference strains through BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The flaB sequences determined have been deposited in a public database (DDBJ accession numbers LC565816-LC565842). MLST was performed using seven housekeeping genes, glmU, pntA, sucA, tpiA, pfkB, mreA, and caiB, as previously described by Boonsilp et al. [27]. Sequence types (STs) were assigned through the MLST database (https://pubmlst.org/leptospira/). New STs obtained in this study have been deposited in the database (ST308, ST313, ST314, ST316, ST317, and ST318). A minimum spanning tree (MST) based on the allelic profiles of MLST of the isolates was created using BioNumerics Software (version 7.6, Applied-Maths, Sint Maartens-Latem, Belgium) with categorical efficiency.

Information on culture-positive population
A total of 24 Leptospira strains isolated from 401 clinically-suspected leptospirosis patients (positive rate including four contaminated cultures: isolation rate, 7.0%) were analyzed. The median (range) age of the culture-positive population was 39.5 (12-72) years, with 95.8% male predominance (n = 23). Majority (67%, n = 16) were from Western Province, and others were from Southern Province (n = 6), North-Western Province (n = 1) and Uva Province (n = 1) (Fig 1). Among 21 cases with clinical information available (Western Province; 14, Southern Province; 6, North-Western Province; 1), severe symptoms were seen in eight cases, of which six were from the Elpitiya Medical Officer of Health region in Southern Province (S1 Table). All patients with severe disease survived.

Molecular and serological characterization of Leptospira isolates from human patients
By partial flaB gene sequencing and MAT using a panel of 19 antisera, species and serogoup of the isolates were identified as Leptospira interrogans serogroup Pyrogenes (14 strains), L. interrogans serogroup Autumnalis (4 strains), L. kirschneri serogroup Grippotyphosa (2 strains), L. borgpetersenii serogroup Javanica (1 strain), and L. interrogans serogroup Hebdomadis (1 strain) ( Table 1). Serogroups of two L. interrogans isolates could not be identified: one of the isolates reacted weakly with anti-serovar Rachmati antiserum at 1:100, and the other reacted with none of the 19 antisera.
MST indicated genetic diversity in the same serogroups (Fig 2 and S1-S3 Figs): for example, in L. interrogans serogroup Pyrogenes, ST74, ST75, and ST308 are of the same group (>5/7 alleles were identical), whereas ST30 and ST316 showed more than four allele differences (Fig  2). Regarding novel STs identified in this study, ST318 is the same group with ST68 (5/7 loci) that was isolated from humans in Thailand (S3 Fig). ST313 is the same group with ST44 (5/7 loci) found in Sri Lanka, but their serogroups are different. On the other hand, there are no known STs that are the same group with STs 314, 316, and ST317, although ST314 and ST317 form the same group (Fig 2 and S2 Fig).

PLOS NEGLECTED TROPICAL DISEASES
Genetic diversity of Leptospira spp. in Sri Lanka

Discussion
This study revealed that genetically diverse Leptospira strains currently cause human leptospi-  (Table 1 and Fig 1). This finding is in contrast to the previous studies based on nucleotide sequences of a single gene (16S rRNA gene) that indicated that single genotype was predominant in certain regions [13,16]. This discrepancy may be related to their restricted sampling areas and difference in discriminatory power of MLST and 16S rRNA gene sequencing analysis. The digits on the lines between two circles represent the number of different alleles between two STs. One isolate of ST144 and two isolates of ST143 were isolated from rats, and others were isolated from human patients. ST308, ST313, ST314, ST316, ST317, and ST318 were newly assigned in this study. https://doi.org/10.1371/journal.pntd.0008573.g002 Leptospira interrogans serogroup Pyrogenes ST75 has been found only in Sri Lanka since 2006 [MLST database (https://pubmlst.org/leptospira/)] and six of the seven loci are identical between ST75 and ST308 (Fig 2 and S2 Fig), indicating that new ST308 emerged from ST75. However, the way of emergence of other new STs remains unclear (Fig 2 and S2 and S3 Figs). Although they may have existed in the country for some time, the importation of livestock or unintentional introduction of rodent reservoirs via merchant ships may be the reason for the emergence of new Leptospira genotypes. Recent whole genome sequencing study (WGS) suggested that L. interrogans serovar Copenhageni may have spread globally via reservoir rats, R. norvegicus [28]. L. interrogans ST34 found in this study was isolated from bandicoot rats (Bandicota indica and B. savilei) in Thailand [29], which may have been introduced to and then spread to the indigenous bandicoot rats in Sri Lanka, but more comprehensive analysis like WGS of Sri Lankan and Thai isolates of ST34 needs to verify this assumption.
In this study, we found that L. interrogans serogroup Pyrogenes ST75 is the dominant clone in Sri Lanka (8/24). This ST strain was isolated from four mild and three severe patients (and one patient without clinical records; S1 Table). In addition, its genetically related ST308 was also isolated from severe patients, suggesting that these clonal complex strains can potentially cause severe leptospirosis in humans. These results suggest that the expansion of these clonal complex strains may be related to recent frequent outbreaks of leptospirosis. Besides ST308, novel STs, ST313 and ST314 were also isolated from severe patients during the outbreak from 2015 to 2017 in Elpitiya, Southern Province [9] (S1 Table). The relationship between Leptospira genotypes and their virulence has been suggested: a specific clone belonging to L. interrogans serovars Copenhageni and Autumnalis has been shown to be associated with severe pulmonary hemorrhage syndrome in Brazil and outbreaks of leptospirosis in Thailand, respectively [29,30]. Besides bacterial virulence, the association between serum levels of cytokines and clinical outcome in patients with leptospirosis has been demonstrated [31]. Although the number of patients infected with the clonal complex strains is limited and more cases are needed, comprehensive proteomic, transcriptomic, and metabolomic analysis of blood or immune cells in mild and severe patients infected with this L. interrogans serogroup Pyrogenes clonal complex would give insights into the pathogenesis and disease severity of leptospirosis.
Although the isolates were from different provinces, L. borgpetersenii ST144 was isolated both from a human patient and a black rat, indicating that the black rat is likely to be the infection source of this strain for humans. However, there is no information on reservoir animals for other STs. As mentioned above, bandicoot rats are likely to be reservoir animals for ST34. DNA of L. borgpetersenii, L. interrogans, and L. kirschneri has recently been detected in cattle, suggesting its importance as a reservoir animal in Sri Lanka [14,19]. In addition, antibodies against serogroup Pyrogenes were detected in buffaloes and buffalo farmers in Homagama area, Western Province [32]. Six L. interrogans serogroup Pyrogenes ST30 (2) and ST75 (4) were isolated from patients in the Base Hospital Homagama, implying buffaloes as an important reservoir in Homagama and suburbs. L. interrogans DNA and DNA of unknown Leptospira species have also been detected in elephant and buffalo urine samples, respectively [33,34]. Furthermore, Leptospira strains belonging to serogroups Autumnalis, Hebdomadis, and Grippotyphosa without species identification were isolated from Rattus sp, dogs, or Bandicota bengalensis (Gunomys gracilis) [21]. In this study, the isolation rate of Leptospira spp. from small mammals was less than that previously described: Leptospira spp. were isolated from 49 of 592 (8.3%) rodents of five species about 50 years ago [21]. Leptospira DNA was detected from 11% rodents by real-time PCR [34]. Low carriage rate in this study may be related to environmental differences in the areas and/or season investigated. L. interrogans was linked to humid habitats, whereas L. borgpetersenii was abundant in both humid and dry habitats [35]. A higher prevalence during the dry season than rainy season has been observed in some rodent species [36]. Further investigations on cattle and other animals as well as the importance of rodents as a reservoir animal are necessary to identify reservoir animals and the transmission route(s) of leptospirosis in Sri Lanka.
In summary, genetically diverse Leptospira strains, both known and novel genotypes, currently cause human leptospirosis in Sri Lanka. The number of reported cases of leptospirosis with new clinical entities has increased in recent times. Three new genotypes, ST308, ST313, and ST314, were isolated from severe patients during the outbreak from 2015 to 2017 in Elpitiya, Southern Province [9]. Further studies are required to verify the relationship between the emergence of new Leptospira genotypes and the recent surge of leptospirosis patients and frequent outbreaks of leptospirosis in Sri Lanka.