Species L. licerasiae

Varillal VAR 010 2008

Leptospira licerasiae, serovar Varillal, with type strain VAR 010 has been described as a new species and serovar in 2008 (abstract below) and has been recogized by the Subcommittee on the Taxonomy of Leptospiroceae (TSC) as per minutes of the closed meeting, 18 September 2007, Quito, Ecuador (published in 2008).
However, in 2012, L. licerasiae has been implicated as a contamination of cell culture medium. Please note that L. inadai, strain 'Difco' represents an isolate that was frequently encoutered in various studies that later on appeared to be a contamination of culture medium.


As part of a prospective study of leptospirosis and biodiversity of Leptospira in the Peruvian Amazon, a new Leptospira species was isolated from humans with acute febrile illness. Field trapping identified this leptospire in peridomestic rats (Rattus norvegicus, six isolates; R. rattus, two isolates) obtained in urban, peri-urban, and rural areas of the Iquitos region. Novelty of this species was proven by serological typing, 16S ribosomal RNA gene sequencing, pulsed-field gel electrophoresis, and DNA-DNA hybridization analysis. We have named this species ”Leptospira licerasiae” serovar Varillal, and have determined that it is phylogenetically related to, but genetically distinct from, other intermediate Leptospira such as L. fainei and L. inadai. The type strain is serovar Varillal strain VAR 010(T), which has been deposited into internationally accessible culture collections. By microscopic agglutination test, ”Leptospira licerasiae” serovar Varillal was antigenically distinct from all known serogroups of Leptospira except for low level cross-reaction with rabbit anti-L. fainei serovar Hurstbridge at a reciprocal titer of 100. lipL32, although not detectable by PCR, was detectable in ”Leptospira licerasiae” serovar Varillal by both Southern blot hybridization and Western immunoblot, although on immunoblot, the predicted protein was significantly smaller (27 kDa) than that of L. interrogans and L. kirschneri (32 kDa). Isolation was rare from humans (2/45 Leptospira isolates from 881 febrile patients sampled), but high titers of MAT antibodies against ”Leptospira licerasiae” serovar Varillal were common (30%) among patients fulfilling serological criteria for acute leptospirosis in the Iquitos region, and uncommon (7%) elsewhere in Peru. This new leptospiral species reflects Amazonian biodiversity and has evolved to become an important cause of leptospirosis in the Peruvian Amazon.

* Matthias MA, Ricaldi JN, Cespedes M, Diaz MM, Galloway RL, Saito M, Steigerwalt AG, Patra KP, Ore CV, Gotuzzo E, Gilman RH, Levett PN, Vinetz JM. Human leptospirosis caused by a new, antigenically unique leptospira associated with a rattus species reservoir in the peruvian Amazon. PLoS Negl Trop Dis 2 (4), 2008, pp. e213 10.1371/journal.pntd.0000213

* International Committee on Systemeatics of Prokaryotes; Subcommittee on the Taxonomy of Leptospiraceae, Int J Syst Evol Microbiol 2008;58,1049-1050.



Leptospira licerasiae, a novel bacterial contaminant found in Genentech cell culture manufacturing operations, poses a challenge to current microbial control strategies in upstream cell culture processes, as this microorganism is fully capable of passing through 0.1 µm sterilizing-grade filtration and is not detectable by standard microbiological methods described in major pharmaceutical compendia for microbial screening and quantification required for release of raw materials, in-process intermediates, and finished products in biopharmaceutical production. The root cause investigation was greatly aided by the genetic identification of the contaminant and subsequent confirmation by cultural method and real-time polymerase chain reaction assay from the affected product batches. The purpose of this case study is to share knowledge on the novel contaminant, L. licerasiae, and potential routes of contamination in the cell culture manufacturing environment from a series of investigations involving root cause analysis, impact assessments, risk assessment, and global corrective and preventative action, as well as to provide guidance on the detection and prevention of Leptospira contamination with the intent to aid the industry to continually improve microbial control strategies for the benefit of patients.

*Chen J, Bergevin J, Kiss R, Walker G, Battistoni T, Lufburrow P, Lam H, Vinther A. Case Study: A Novel Bacterial Contamination in Cell Culture Production--Leptospira licerasiae. PDA Journal of Pharmaceutical Science and Technology 2012,66 (6), 580-91.

Conventionally, Leptospira were separated into two valid species, Leptospira interrogans for pathogenic leptospires and Leptospira biflexa for saprophytic ones (Brenner et al., 1999).
Current Leptospira speciation is based on DNA heterogeneity as determined by hybridization experiments.
The first of such studies was by Yasuda et al., 1987 followed by Ramadass et al., 1992.
The most complete study, also summarizing the observation of the previous two studies is by Brenner et al., 1999. Brenner and co-workers described 17 DNA hybridization groups (species), representing the 12 previously describes species and five new ‘genomospecies’, including Leptospira alexanderi.
Smythe et al., 2012 re-named the four remaining ‘genomospecies’ 1, 3, 4 and 5 later on to Leptospira alstonii, L. vanthielii , L. terpstrae and L. yanagawae, respectively.
To date, speciation is increasingly based on other molecular techniques, mainly DNA sequences of the rrs gene or multilocus sequence genotyping, which likely will completely replace the DNA hybridization method.

* Yasuda PH, Steigerwalt AG, Sulzer KR, Kaufmann AF, Rogers F, Brenner DJ. Deoxyribonucleic acid relatedness between serogroups and serovars in the family Leptospiroceae with proposals for seven Leptospira species. Int J Syst Bacteriol 1987, 37, 407-415

* Ramadass P, Jarvis BDW, Corner RJ, Penny D, Marshall RB. Genetic characterization of pathogenic Leptospira species by DNA hybridization. Int J Syst Bacteriol 1992, 42, 215-219

* Brenner DJ, Kaufmann AF, Sulzer KR, Steigerwalt AG, Rogers F, Weyant RS. Further determination of DNA relatedness between serogroups and serovars in the family Leptospiroceae with a proposal for Leptospira alexanderi sp. nov. and four new Leptospira genomospecies. Int J Syst Bacteriol 1999, 49, 839-858

* Smythe L, Adler B, Hartskeerl RA, Galloway RL, Turenne CY, Levett PN. Classification of Leptospira genomospecies 1, genomospecies 3, genomospecies 4 and genomospecies 5 as Leptospira alstonii sp. nov., Leptospira vanthielii sp. nov., Leptospira terpstrae sp. nov., Leptospira yanagawae sp. nov., respectively. Int J Syst Evol Microbiol 2012;

Recommended literature

* Ahmed A, Grobusch MP, Klatser P, Hartskeerl RA. Molecular Approaches in the Detection and Characterization of Leptospira. J Bacteriol Parasitol 2012, 3:2 http://dx.doi.org/10.4172/2155-9597.1000133

* Adler, B., & de la Peña Moctezuma, A. (2010). Leptospira and leptospirosis. Veterinary microbiology 140, 287-296.

* Cerqueira, G.M. & Picardeau, M. (2009). A century of Leptospira strain typing. Infection, Genetics and Evolution 9, 760-768.

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