Analysis of Multilocus Sequence Typing for Identification of Leptospira Isolates in Brazil

Abstract

A collection of 101 Leptospira isolates was tested by multilocus sequence typing (MLST) and by traditional serotyping. MLST divided the isolates into 4 sequence types (STs), while serotyping classified them into 6 serogroups. Two isolates failed to generate products for some genes by MLST. MLST was less discriminatory than serotyping for uncommonly occurring isolates from humans in Brazil.Leptospirosis, caused by pathogenic leptospires, is one of the most widespread zoonotic diseases known. The genus Leptospira is divided into 20 species with more than 200 pathogenic serovars organized into 24 serogroups on the basis of antigenic relatedness (2,9,13,30). The disease occurs in wild and domesticated animals, both of which can be a source of human infection. Exposures that occur during flooding events are the main risk factors of human leptospirosis in Brazil (1,3,14,22). In São Paulo State, Brazil, 13,620 cases have been reported in the last 20 years (ftp://ftp.cve.saude.sp.gov.br/doc_tec/zoo /lepto09_perfil.pdf). However, information about circulating isolates of Leptospira spp. in Brazil is limited. Identification of isolates to the serovar level is essential for understanding the epidemiology of the disease in both humans and animals.Since certain serovars are often associated with specific mammalian hosts and with the symptoms and severity of the disease, the identification of the serovar usually permits the prediction of sources of infection, thereby enabling control of the spread of the disease (17). Monitoring the serovars and genetic profiles of strains collected over time and from different regions is also important to better understand the current circulating Leptospira population worldwide. Isolate discrimination can be performed by a microagglutination test (MAT) at the serogroup level and by a cross-agglutinin absorption test (CAAT) at the serovar level (5). However, performing those methods is tedious, as live cultures of collection strains must be maintained for use as antigens and rabbit hyperimmune sera are required. Although identification by serotyping is valuable, molecular methods with higher reproducibility and discriminatory power may be more useful in epidemiological investigations.New molecular methods such as multilocus sequence typing (MLST) have been recently developed and applied to the study of many bacterial species (4,6,7,15,18). MLST is a simple PCR-based technique that makes use of automated DNA sequencers to assign and characterize the alleles present in different target genes. The method allows one to generate sequence data on a low-to high-throughput scale that is unambiguous and suitable for epidemiological and population studies. The selected loci are generally housekeeping genes, which evolve very slowly over an evolutionary time scale (8).Our goal was to evaluate the discriminatory power of MLST compared to serotyping using a set of Brazilian human isolates. A total of 101 Leptospira clinical strains (95 from blood, 5 from cerebrospinal fluid, and 1 from urine) isolated fro...

High-Resolution Typing of Leptospira interrogans Strains by Multispacer Sequence Typing

“…MLST produced nonambiguous, specific results with a good resolution. However, MLST demonstrated an inability to distinguish certain isolates at the serovar level, such as for Icterohaemorrhagiae and Copenhageni (23).…”

Characterization of Leptospira interrogans serovar Pomona isolated from swine in Brazil

“…As MLST does not always specify Leptospira serovars [9], the same ST 37 also encompasses serovar Canicola when using the scheme previously proposed [19,9,11]. This is also observed with the new MLST protocol [25] in which serovar Pomona can be assigned to STs 38 and 140 together with serovars Gem and Guaratuba, respectively.…”

“…While PFGE distinguished closely related serovars, the MLST technique grouped all of the isolates into one ST. ST 37 was expected for the Pomona isolates because it had already been assigned this serovar, independently of its origin [19,9,11]. As MLST does not always specify Leptospira serovars [9], the same ST 37 also encompasses serovar Canicola when using the scheme previously proposed [19,9,11].…”

“…The increasing number of sequenced genomes enables the use of sequencebased approaches such as multilocus sequence typing (MLST) for concrete serovar identification [11]. A microdilution technique was developed for antibiotic selection and leptospirosis treatment [12].…”

Fifteen years of human leptospirosis in São Paulo, Brazil

“…The identification of isolated Leptospira was performed by serogrouping, pulsedfield gel electrophoresis (PFGE) [17] and multilocus sequence typing (MLST). [18] …”