The genus Brucella contains highly infectious species that have been found to cause infections in a wide variety of mammals. Most Brucella species have a narrow host range. Infection in humans arises from direct or indirect contact with infected animals or through consumption of contaminated meat or dairy products . Diagnostic laboratory workers are also at risk; 2% of all cases of brucellosis are laboratory acquired. Person-to-person transmission is extremely rare [1–3]. Characteristically, Brucella species have a low infectious dose and are capable of transmission via aerosols, and the treatment of infections is lengthy with a risk of complications. For these reasons, Brucella is classified as a potential warfare threat agent, and Brucella suis has been weaponized in the past by the United States, the former Soviet Union, and China .
Brucella species belong to the family Brucellaceae in the order Rhizobiales of the class Alphaproteobacteria and are small, non-motile Gram-negative rods. Until recently, six species, some of which may be subdivided into biovars, were assigned to the Brucella genus. These species are Brucella abortus (seven biovars), Brucella melitensis (three biovars), Brucella suis (five biovars), Brucella ovis, Brucella canis, and Brucella neotomae. However, four new species have recently been described. Three of these species were isolated from sea mammals and 'wild' mammals: Brucella ceti, Brucella pinnipedialis, and Brucella microti [5–10]. Finally, a new species, Brucella inopinata, was isolated from a breast implant (strain BO1) and from a lung biopsy (strain BO2) [11, 12].
The Brucella species primarily considered to be pathogenic for humans are B. melitensis, B. suis (biovars 1, 3, and 4), B. abortus, and sporadically B. canis [1, 2, 13]. B. suis biovars 2 and 5 are considered not to be human pathogens because no human cases have been documented for these agents .
The DNA-DNA hybridization results suggest that the classification system used for Brucella is open to debate. Among the different Brucella species, the DNA-DNA hybridization relatedness varies from 87% to 99%, indicating that the Brucella species may actually be considered a single species [13–15]. However, the traditional nomenclature was maintained because the specific host range and pathogenicity differ among the Brucella species . The conventional methods used to identify Brucella isolates are complex, labor-intensive, and time consuming. In addition, Brucella is a potential health hazard to laboratory personnel. Traditionally, the identification of Brucella species is mainly based on host specificity, pathogenicity, and minor phenotypic differences that are determined using several separate tests, which include tests for the oxidation of carbohydrate and amino acid substrates, phage sensitivity, CO2 requirement, H2S production, serum agglutination, and growth in the presence of thionine and basic fuchsine . The scheme to discriminate to the level of biovars is inconclusive because the biological differences between the biovars described are limited, and the interpretation of the results can be subjective . In addition, some Brucella isolates appear unable to be typed .
DNA-based approaches have been widely introduced to identify microorganisms, including Brucella species. A relatively rapid approach is the 'Bruce-ladder', a multiplex PCR that is able to distinguish the six classical species [13, 16]. To complement the 'Bruce-ladder', a single PCR was added to distinguish the marine mammal-derived Brucellae as well. This method, called bp26 PCR, is based on the IS711 [13, 16].
Another method, mainly developed for the epidemiological investigation of outbreaks, is multilocus variable-number tandem repeat analysis (MLVA). MLVA is based on the differences in the number of tandem repeats in several loci of the bacterial chromosome . The MLVA developed for Brucella has been proven to be a reliable, reproducible, and highly discriminatory method that is able to classify all of the Brucella strains [13, 18–20]. In this study, we the previously described MLVA-16 assay to identify Brucella species was used [13, 18–20].
Genomic studies have shown that the nomenclature for several Brucella species is not consistent if the genetic relationships among species are considered to be the gold standard for discriminating between species . For example, B. ceti is divided into two separate groups, one of which is more closely related to B. pinnipedialis than to the other group of B. ceti . Additionally, B. suis biovar 5 is more related to B. ceti, B. neotomae, B. pinnipedialis and B. ovis than to the other B. suis biovars .
The timely detection and rapid identification of the microorganisms involved are essential for the most-effective response to an infectious disease outbreak, regardless of whether the outbreak is natural or deliberate. This rapid identification is necessary not only to treat patients effectively but also to establish outbreak management, source tracing, and threat analyses.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a rapid method used to analyze biological differences in microorganisms. MALDI-TOF-MS emerged as a new diagnostic tool in established microbiological laboratories . The advantages of MALDI-TOF-MS over conventional techniques are that it is a fast, cost-effective, accurate method, which is suitable for the high-throughput identification of bacteria by less-skilled laboratory personnel because preliminary identification steps are unnecessary [21–24]. The bacteria are identified by comparing the obtained MS spectra to the MS spectra or profiles of MS spectra from a reference library. Hence, the reliability of the identification is based on the content and quality of this library, among other factors. Recently, a reference library to identify Brucella species was constructed using 12 Brucella strains, but using this 'Brucella library', the discrimination was insufficient for identification at the species level . In contrast, reliable identification at the species level was shown for other genetically closely related species, such as Fransicella species, Bacillus species, and species from the Burkholderia cepacia complex [26–28].
The aim of this study was to improve identification using MALDI-TOF-MS at the species level of Brucella. Therefore, a custom reference library was constructed with strains that represent the known genetic variation of Brucella at the species and biovar level according to MLVA. Subsequently, this custom reference library was evaluated using 152 Brucella isolates that were identified using MLVA.