First Argentine database for the accurate identification of Brucella to species level by MALDI-TOF MS.

MALDI-TOF mass spectrometry (MS) has proven to be a fast and reliable method for the identification of a large number of taxonomic groups. It offers the advantage of being able to incorporate protein spectra of microorganisms that are absent or poorly represented in commercial databases, such as the genus Brucella. The aim of the study was to build the first database of protein spectra of local biological variants of Brucella in Argentina and of standard strains. First, the identification performance of a panel of 135 strains was evaluated with the Swedish database ¨Folkhälsomyndigheten¨ (containing protein spectra of several international standards of the genus Brucella) imported from the open access site https://spectra.folkhalsomyndigheten.se/spectra/. With this library 100 % of the strains were correctly identified by mass spectrometry to genus level, but not to species level. Due to the limitation found, an in-house database was designed with local Brucella isolates from Argentina and standard strains used in routine bacteriological diagnosis. For its validation, a panel of strains, different from those used to develop the extended local database (n: 177), was used to, simultaneously, challenge both libraries. The samples were processed by triplicate and the results obtained were: 177 strains correctly identified to genus and species level compared to the gold standard method (phenotypic typing), meeting the criteria accepted by the literature and the manufacturer as reliable identification. Only 2 of these isolates had score values lower than 2 (1.862) and were therefore not included in the calculation of results. According to these results, MALDI-TOF MS is a fast and reliable method for the routine identification of the different Brucella species, and even has the advantage of reducing the time of exposure to pathogenic microorganisms for laboratorians. It could be considered a valuable technique to replace, in the near future, the current conventional techniques due to the ease of transferring protein spectra, avoiding the use of reference strains that are difficult to find commercially available and commonly used in phenotypic typing.

Higher diversity of Brucella canis in Latin America, according to an MLVA_13 Bc analysis.

Brucella canis is the main causative agent of canine brucellosis, which affects domestic and wild canids and leads to clinical signs and symptoms of the reproductive and locomotor systems. Owing to the scarce information on this pathogen, here we addressed the genetic diversity of the circulating strains of this species in Argentina by following an MVLA_13 Bc scheme. The analyzed sample set consisted of 101 strains of B. canis isolates collected between 2006 and 2020 from canines of the Autonomous City of Buenos Aires (CABA) and other regions of Argentina, as well as 235 isolates from North America. The analysis yielded 336 variants (Hunter-Gaston Diversity Index, HGDI equal to 1.0) showing high diversity on a global scale. The analysis of the six most variable markers also reveled high diversity and allowed further analysis regarding variant relationships. Although the diversity obtained using both schemes (all or the 6 most variable markers) was higher for the Latin American than for the North American strains, we cannot discard that this was due to biases in the sampling methodology or to the different health policies employed in these regions regarding the management of infected individuals. Altogether, the Argentine circulating strains are genetically diverse, but with no apparent geographical association. The markers used in the MLVA_13 Bc are variable and highly useful for the evaluation of outbreaks. Furthermore, the reduced panel of 6 markers (MLVA_6 Bc) proposed in this study is convenient for the study of B. canis strain diversity.