Molecular characterisation and antibiotic susceptibility of meningococcal isolates from healthy men who have sex with men.

OBJECTIVES: To evaluate and characterise meningococcal carriage among healthy men who have sex with men (MSM) within a screening programme for Neisseria gonorrhoeae infection at the San Gallicano Dermatological Institute, Italy. METHODS: A total of 441 MSM attending the STI/HIV Centre of the San Gallicano Institute, Rome, Italy, in 2016 were routinely screened for N. gonorrhoeae infection by pharyngeal and rectal swabs. N. meningitidis isolates were evaluated for antibiotic susceptibility and characterised by whole genome sequencing. Genetic relationships among the meningococcal carriage isolates were determined using core genome multilocus sequence typing analysis. The soluble domain of AniA (sAniA) protein expression by western blotting was also evaluated. RESULTS: A total of 62 (14.1%, 95% CI 11.1 to 17.6) carriage meningococci were found among 441 MSM. Forty-three viable N. meningitidis isolates were cultivated (42 from pharyngeal and 1 from rectal swabs). All the viable isolates were susceptible to cefotaxime, ceftriaxone, ciprofloxacin and rifampicin. Four isolates were penicillin G-resistant and 73% of those penicillin G-susceptible showed a minimum inhibitory concentration from 0.064 µg/mL to 0.25 µg/mL. Serogroup B was the most frequent (44.2%), followed by Z (16.3%), E (9.3%), and Y and W (2.3%), respectively. Multilocus sequence typing analysis identified 29 sequence types belonging to 12 clonal complexes. The sAniA protein was expressed in 8 out of 28 (29%) screened meningococcal carriage isolates. CONCLUSIONS: Serogroup B meningococcal carriage identified from oral and anal specimens among healthy MSM was the most frequent serogroup identified in this study. Molecular evaluation revealed a degree of similarity among strains belonging to the same clonal complex.

Reactive vaccination as control strategy for an outbreak of invasive meningococcal disease caused by Neisseria meningitidis C:P1.5-1,10-8:F3-6:ST-11(cc11), Bergamo province, Italy, December 2019 to January 2020.

In Italy, serogroup C meningococci of the clonal complex cc11 (MenC/cc11) have caused several outbreaks of invasive meningococcal disease (IMD) during the past 20 years. Between December 2019 and January 2020, an outbreak of six cases of IMD infected with MenC/cc11 was identified in a limited area in the northern part of Italy. All cases presented a severe clinical picture, and two of them were fatal. This report is focused on the microbiological and molecular analysis of meningococcal isolates with the aim to reconstruct the chain of transmission. It further presents the vaccination strategy adopted to control the outbreak. The phylogenetic evaluation demonstrated the close genetic proximity between the strain involved in this outbreak and a strain responsible for a larger epidemic that had occurred in 2015 and 2016 in the Tuscany Region. The rapid identification and characterisation of IMD cases and an extensive vaccination campaign contributed to the successful control of this outbreak caused by a hyperinvasive meningococcal strain.

Whole genome and phylogenetic analysis of two SARS-CoV-2 strains isolated in Italy in January and February 2020: additional clues on multiple introductions and further circulation in Europe.

Whole genome sequences of SARS-CoV-2 obtained from two patients, a Chinese tourist visiting Rome and an Italian, were compared with sequences from Europe and elsewhere. In a phylogenetic tree, the Italian patient's sequence clustered with sequences from Germany while the tourist's sequence clustered with other European sequences. Some additional European sequences in the tree segregated outside the two clusters containing the patients' sequences. This suggests multiple SARS-CoV-2 introductions in Europe or virus evolution during circulation.

Coronavirus disease (COVID-19) in a paucisymptomatic patient: epidemiological and clinical challenge in settings with limited community transmission, Italy, February 2020.

Data concerning the transmission of the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) in paucisymptomatic patients are lacking. We report an Italian paucisymptomatic case of coronavirus disease 2019 with multiple biological samples positive for SARS-CoV-2. This case was detected using the World Health Organization protocol on cases and contact investigation. Current discharge criteria and the impact of extra-pulmonary SARS-CoV-2 samples are discussed.

Co-existence of virulence factors and antibiotic resistance in new Klebsiella pneumoniae clones emerging in south of Italy.

BACKGROUND: Endemic presence of Klebsiella pneumoniae resistant to carbapenem in Italy has been due principally to the clonal expansion of CC258 isolates; however, recent studies suggest an ongoing epidemiological change in this geographical area. METHODS: 50 K. pneumoniae strains, 25 carbapenem-resistant (CR-Kp) and 25 susceptible (CS-Kp), collected from march 2014 to march 2016 at the Laboratory of Bacteriology of the Paolo Giaccone Polyclinic University hospital of Palermo, Italy, were characterized for antibiotic susceptibility and fully sequenced by next generation sequencing (NGS) for the in silico analysis of resistome, virulome, multi-locus sequence typing (MLST) and core single nucleotide polymorphism (SNP) genotypes RESULTS: MLST in silico analysis of CR-Kp showed that 52% of isolates belonged to CC258, followed by ST395 (12%), ST307 (12%), ST392 (8%), ST348 (8%), ST405 (4%) and ST101 (4%). In the CS-Kp group, the most represented isolate was ST405 (20%), followed by ST392 and ST15 (12%), ST395, ST307 and ST1727 (8%). The in silico ß-lactamase analysis of the CR-Kp group showed that the most detected gene was blaSHV (100%), followed by blaTEM (92%), blaKPC (88%), blaOXA (88%) and blaCTX-M (32%). The virulome analysis detected mrk operon in all studied isolates, and wzi-2 was found in three CR-Kp isolates (12%). Furthermore, the distribution of virulence genes encoding for the yersiniabactin system, its receptor fyuA and the aerobactin system did not show significant distribution differences between CR-Kp and CS-Kp, whereas the Klebsiella ferrous iron uptake system (kfuA, kfuB and kfuC genes), the two-component system kvgAS and the microcin E495 were significantly (p < 0.05) prevalent in the CS-Kp group compared to the CR-Kp group. Core SNP genotyping, correlating with the MLST data, allowed greater strain tracking and discrimination than MLST analysis. CONCLUSIONS: Our data support the idea that an epidemiological change is ongoing in the Palermo area (Sicily, Italy). In addition, our analysis revealed the co-existence of antibiotic resistance and virulence factors in CR-Kp isolates; this characteristic should be considered for future genomic surveillance studies.

Meningococci of Serogroup X Clonal Complex 181 in Refugee Camps, Italy.

Four cases of infection with serogroup X meningococci (MenX) (1 in 2015 and 3 in 2016) occurred in migrants living in refugee camps or reception centers in Italy. All MenX isolates were identified as clonal complex 181. Our report suggests that serogroup X represents an emerging health threat for persons arriving from African countries.

Botulism in Italy, 1986 to 2015.

Botulism is a rare but severe neuroparalytic disease caused by botulinum toxins. Because of its high potential impact on public health, botulism is a closely monitored communicable disease in Europe. In Italy, which has one of the highest incidence rates in Europe (0.03 cases per 100,000 population), botulism is monitored through a case-based passive surveillance system: the front-line physician who diagnoses a suspected case must notify the Local Health Units immediately, and the Ministry of Health's office within 12 hours. From 1986 to 2015, 466 confirmed cases of botulism were recorded in Italy (of 1,257 suspected cases). Of these, 421 were food-borne (the most frequently seen form of botulism due to the consumption of improperly home-canned foods), 36 were infant botulism, which accounts for ca 50% of all these types of cases registered in Europe, six were wound-related and three were due to adult intestinal colonisation. This scenario suggests that stronger efforts should be made towards raising public awareness of the risk of food-borne botulism, especially with respect to home-preserved foods, as well as improving the training of front-line medical personnel, to ensure that a quick and accurate diagnosis of botulism can be made.

Smartphone-assisted paper-based electrochemical immunosensor for SARS-CoV-2 detection in saliva.

Herein, we developed a new waste solution-free paper-based electrochemical immunosensor for SARS-CoV-2 detection in saliva, by combining vertical and lateral flow. In detail, the device was constituted of a reservoir containing all reagents for the construction of the immunological chain onto the magnetic beads and a lateral flow holder which contained a polyester-based electrode, a magnet, and an adsorbent pad. The measurement was carried out by adding the saliva sample into the reservoir, followed by the addition of this solution in the hole present in the lateral flow holder. The successive additions of washing buffer and TMB solution in the lateral flow holder allowed the detection of N protein in saliva in the range of 0.06 to 4 µg/mL with a detection limit equal to 30 ng/mL. The analysis of several saliva samples with the sensing tool and the reference method, demonstrated the effectiveness of this device, being able to identify positive patients with high values of CT e.g. 35. This new configuration paves the way for the realization of any magnetic beads-based immunosystem without waste solution production, enlarging the application of paper-based devices.

FHbp variants among meningococci of serogroup B in Italy: Evolution and selective pressure, 2014-2017.

BACKGROUND: Neisseria meningitidis (meningococcus) is the causative agent of invasive meningococcal disease (IMD). Meningococcus of serogroup B (MenB) is one of the main serogroup causing IMD. MenB strains may be prevented by meningococcal B vaccines. In particular, vaccines with Factor H-binding protein (FHbp), classified into two subfamilies (A or B) or in three variants (v1, v2 or v3), are those available. The objective of the study was to investigate the phylogenetic relationships of FHbp subfamilies A and B (variants v1, v2 or v3) genes and proteins, together with their evolution patterns and selective pressure. MATERIALS AND METHODS: Overall, alignments of FHbp nucleotide and protein sequence from 155 MenB samples collected in different parts of Italy, from 2014 to 2017, were analyzed by ClustalW. JModeltest and the Smart Model Selection software were used for the statistical selection of the best-fit substitution models for nucleotide and protein alignments. Site-specific positive and negative selection were estimated through the HYPHY package. The phylogenetic signal was investigated with the likelihood mapping method. The Maximum Likelihood (ML) phylogenetic reconstructions were performed with Phyml. RESULTS: The phylogenic analysis identified different clusters within the FHbp subfamily A and B variants, confirming sequence diversity. The pattern of selective pressure in our study indicated that subfamily B FHbp sequences are subjected to greater variations and positive selective pressure respect to subfamily A, with 16 positively supported selected sites identified. CONCLUSION: The study pointed out the need for continued genomic surveillance for meningococci to monitor selective pressure and amino acidic changes. Monitoring the genetic diversity and molecular evolution of FHbp variants may be useful to investigate genetic diversity which may emerge over time.

Antimicrobial susceptibility profiles and genotyping of Neisseria meningitidis of serogroup C, Italy, 2000-2020.

Background: In Italy the introduction of meningococcal C conjugate vaccine in 2005 has led to a significant reduction of invasive meningococcal disease (IMD) caused by Neisseria meningitidis of serogroup C (MenC). However, this serogroup is still responsible of sporadic cases, clusters and local outbreaks. The study aims to investigate the genotype and antimicrobial susceptibility profile of MenC isolates collected in Italy from 2000 to 2020. Methods: Bacterial isolates and biological samples (blood or cerebrospinal fluid) from invasive meningococcal cases are collected and characterized at the National Reference Laboratory for IMD of Istituto Superiore di Sanità. Antimicrobial susceptibility was determined by MIC Test Strip Method and interpreted according to the EUCAST breakpoints guideline. Genotypic characteristics, including multi locus sequence typing (MLST), finetype, and antimicrobial resistance target genes were performed and analyzed using the PubMLST database. Genomic comparison of core genome MLST (cgMLST) of MenC genomes was also carried out. Results: From 2000 to 2020, a total of 665 MenC isolates were investigated for antimicrobial susceptibility and 301 for genotyping. Over two decades, almost all MenC isolates resulted susceptible to antimicrobials with few isolates resulting resistant to ciprofloxacin (N = 2), penicillin G (N = 13), and rifampicin (N = 9), respectively. Molecular typing of MenC obtained from isolates or clinical specimens identified mostly the genotype C:P1.5-1,10-8:F3-6:ST-11(cc11). However, phylogenetic analysis, performed on genomes from MenC isolates, identified two sub lineages, 11.1 and 11.2, among cc11, of which the sub lineage 11.2 was the predominant. Conclusion: Wider application of the genomic analysis and monitoring of antimicrobial susceptibility represent key aspects of IMD surveillance and to monitor the continued evolution of these hyperinvasive strains.

Phylogenetic and Evolutionary Genomic Analysis of Listeria monocytogenes Clinical Strains in the Framework of Foodborne Listeriosis Risk Assessment.

Listeria monocytogenes is one of the most important foodborne pathogens responsible for listeriosis, a severe disease with symptoms ranging from septicemia, meningoencephalitis, and abortion. Given the strong impact of listeriosis on human health and the difficulty of controlling L. monocytogenes along the food production chain, listeriosis has become a priority subjected to molecular surveillance in European Union/European Economic Area since 2007. From 2018, surveillance is based on whole-genome sequence using the core genome multilocus sequence type. The complete sequences of 132 clinical strains were used to define the evolutionary relatedness among subtypes of L. monocytogenes isolated in Italy from 2010 to 2016, allowing the identification of clades and/or clusters associated with outbreaks or sporadic cases of listeriosis. All the strains analyzed are clustered in lineages I and II, and the majority of the strains were classified as lineage II. A probable epidemic entrance in different years for every clade and cluster from each different region was defined. The persistence of the same specific clonal complexes of L. monocytogenes has been found over long periods; this may be related to the fact that some strains are able to survive better than others in a food production environment. Phylogenic studies, using whole-genome sequence data, are able to identify the emergence of highly persistent pathogenic variants, contributing to improving the hazard characterization of L. monocytogenes.

Genetic Diversity of Antimicrobial Resistance and Key Virulence Features in Two Extensively Drug-Resistant Acinetobacter baumannii Isolates.

In recent decades, Acinetobacter baumannii emerged as a major infective menace in healthcare settings due to scarce therapeutic options to treat infections. Therefore, undertaking genome comparison analyses of multi-resistant A. baumannii strains could aid the identification of key bacterial determinants to develop innovative anti-virulence approaches. Following genome sequencing, we performed a molecular characterization of key genes and genomic comparison of two A. baumannii strains, #36 and #150, with selected reference genomes. Despite a different antibiotic resistance gene content, the analyzed strains showed a very similar antibiogram profile. Interestingly, the lack of some important virulence determinants (i.e., bap, ata and omp33-36) did not abrogate their adhesive abilities to abiotic and biotic surfaces, as reported before; indeed, strains retained these capacities, although to a different extent, suggesting the presence of distinct vicarious genes. Conversely, secretion systems, lipopolysaccharide (LPS), capsule and iron acquisition systems were highly similar to A. baumannii reference strains. Overall, our analyses increased our knowledge on A. baumannii genomic content and organization as well as the genomic events occurring in nosocomial isolates to better fit into changing healthcare environments.

Paper-based immunoassay based on 96-well wax-printed paper plate combined with magnetic beads and colorimetric smartphone-assisted measure for reliable detection of SARS-CoV-2 in saliva.

Coronavirus disease 2019 (COVID-19) has been recognized as a global pandemic outbreak, opening the most severe socio-economic crisis since World War II. Different scientific activities have been emerged in this global scenario, including the development of innovative analytical tools to measure nucleic acid, antibodies, and antigens in the nasopharyngeal swab, serum, and saliva for prompt identification of COVID-19 patients and to evaluate the immune response to the vaccine. The detection of SARS-CoV-2 in saliva remains a challenge for the lack of sufficient sensitivity. To address this issue, we developed a novel paper-based immunoassay using magnetic beads to support the immunological chain and 96-well wax-printed paper plate as a platform for color visualization by using a smartphone combined with Spotxel free-charge app. To assess the reliability of the measurement of SARS-CoV-2 in saliva, untreated saliva was used as a specimen and the calibration curve demonstrated a dynamic range up to 10 µg/mL, with a detection limit equal to 0.1 µg/mL. The effectiveness of this sustainable analytical tool in saliva was evaluated by comparing the data with the nasopharyngeal swab specimens sampled by the same patients and tested with Real-Time PCR reference method, founding 100% of agreement, even in the case of high Cycle Threshold (CT) numbers (low viral load). Furthermore, the positive saliva samples were characterized by the next-generation sequencing method, demonstrating the capability to detect the Delta variant, which is actually (July 2021) the most relevant variant of concern.

Analysis of Genomic Characteristics of SARS-CoV-2 in Italy, 29 January to 27 March 2020.

We performed next-generation sequencing (NGS), phylogenetic analysis, gene flows, and N- and O-glycosylation prediction on SARS-CoV-2 genomes collected from lab-confirmed cases from different Italian regions. To this end, a total of 111 SARS-CoV-2 genomes collected in Italy between 29 January and 27 March 2020 were investigated. The majority of the genomes belonged to lineage B.1, with some descendant lineages. The gene flow analysis showed that the spread occurred mainly from the north to the center and to the south of Italy, as confirmed by epidemiological data. The mean evolutionary rate estimated here was 8.731 × 10-4 (95% highest posterior density, HPD intervals 5.809 × 10-4 to 1.19 × 10-3), in line with values reported by other authors. The dated phylogeny suggested that SARS-CoV-2 lineage B.1 probably entered Italy between the end of January and early February 2020. Continuous molecular surveillance is needed to trace virus circulation and evolution.

Hydrogen peroxide induced by nerve injury promotes axon regeneration via connective tissue growth factor.

Regeneration of the neuromuscular junction (NMJ) leverages on extensive exchange of factors released from motor axon terminals (MATs), muscle fibers and perisynaptic Schwann cells (PSCs), among which hydrogen peroxide (H2O2) is a major pro-regenerative signal. To identify critical determinants of NMJ remodeling in response to injury, we performed temporal transcriptional profiling of NMJs from 2 month-old mice during MAT degeneration/regeneration, and cross-referenced the differentially expressed genes with those elicited by H2O2 in SCs. We identified an enrichment in extracellular matrix (ECM) transcripts, including Connective Tissue Growth Factor (Ctgf), which is usually expressed during development. We discovered that Ctgf levels are increased in a Yes-associated protein (YAP)-dependent fashion in response to rapid, local H2O2 signaling generated by stressed mitochondria in the injured sciatic nerve, a finding highlighting the importance of signals triggered by mechanical force to motor nerve repair. Through sequestration of Ctgf or inactivation of H2O2, we delayed the recovery of neuromuscular function by impairing SC migration and, in turn, axon-oriented re-growth. These data indicate that H2O2 and its downstream effector Ctgf are pro-regenerative factors that enable axonal growth, and reveal a striking ECM remodeling process during nerve regeneration upon local H2O2 signaling. Our study identifies key transcriptomic changes at the regenerating NMJ, providing a rich source of pro-regenerative factors with potential for alleviating the consequences of peripheral nerve injuries.

Genome analysis of Legionella pneumophila ST23 from various countries reveals highly similar strains.

Legionella pneumophila serogroup 1 (Lp1) sequence type (ST) 23 is one of the most commonly detected STs in Italy where it currently causes all investigated outbreaks. ST23 has caused both epidemic and sporadic cases between 1995 and 2018 and was analysed at genomic level and compared with ST23 isolated in other countries to determine possible similarities and differences. A core genome multi-locus sequence typing (cgMLST), based on a previously described set of 1,521 core genes, and single-nucleotide polymorphisms (SNPs) approaches were applied to an ST23 collection including genomes from Italy, France, Denmark and Scotland. DNAs were automatically extracted, libraries prepared using NextEra library kit and MiSeq sequencing performed. Overall, 63 among clinical and environmental Italian Lp1 isolates and a further seven and 11 ST23 from Denmark and Scotland, respectively, were sequenced, and pangenome analysed. Both cgMLST and SNPs analyses showed very few loci and SNP variations in ST23 genomes. All the ST23 causing outbreaks and sporadic cases in Italy and elsewhere, were phylogenetically related independent of year, town or country of isolation. Distances among the ST23s were further shortened when SNPs due to horizontal gene transfers were removed. The Lp1 ST23 isolated in Italy have kept their monophyletic origin, but they are phylogenetically close also to ST23 from other countries. The ST23 are quite widespread in Italy, and a thorough epidemiological investigation is compelled to determine sources of infection when this ST is identified in both LD sporadic cases and outbreaks.

Clostridium botulinum group I strain genotyping by 15-locus multilocus variable-number tandem-repeat analysis.

Clostridium botulinum is a taxonomic designation that encompasses a broad variety of spore-forming, Gram-positive bacteria producing the botulinum neurotoxin (BoNT). C. botulinum is the etiologic agent of botulism, a rare but severe neuroparalytic disease. Fine-resolution genetic characterization of C. botulinum isolates of any BoNT type is relevant for both epidemiological studies and forensic microbiology. A 10-locus multiple-locus variable-number tandem-repeat analysis (MLVA) was previously applied to isolates of C. botulinum type A. The present study includes five additional loci designed to better address proteolytic B and F serotypes. We investigated 79 C. botulinum group I strains isolated from human and food samples in several European countries, including types A (28), B (36), AB (4), and F (11) strains, and 5 nontoxic Clostridium sporogenes. Additional data were deduced from in silico analysis of 10 available fully sequenced genomes. This 15-locus MLVA (MLVA-15) scheme identified 86 distinct genotypes that clustered consistently with the results of amplified fragment length polymorphism (AFLP) and MLVA genotyping in previous reports. An MLVA-7 scheme, a subset of the MLVA-15, performed on a lab-on-a-chip device using a nonfluorescent subset of primers, is also proposed as a first-line assay. The phylogenetic grouping obtained with the MLVA-7 does not differ significantly from that generated by the MLVA-15. To our knowledge, this report is the first to analyze genetic variability among all of the C. botulinum group I serotypes by MLVA. Our data provide new insights into the genetic variability of group I C. botulinum isolates worldwide and demonstrate that this group is genetically highly diverse.

High throughput MLVA-16 typing for Brucella based on the microfluidics technology.

BACKGROUND: Brucellosis, a zoonosis caused by the genus Brucella, has been eradicated in Northern Europe, Australia, the USA and Canada, but remains endemic in most areas of the world. The strain and biovar typing of Brucella field samples isolated in outbreaks is useful for tracing back source of infection and may be crucial for discriminating naturally occurring outbreaks versus bioterrorist events, being Brucella a potential biological warfare agent. In the last years MLVA-16 has been described for Brucella spp. genotyping. The MLVA band profiles may be resolved by different techniques i.e. the manual agarose gels, the capillary electrophoresis sequencing systems or the microfluidic Lab-on-Chip electrophoresis. In this paper we described a high throughput system of MLVA-16 typing for Brucella spp. by using of the microfluidics technology. RESULTS: The Caliper LabChip 90 equipment was evaluated for MLVA-16 typing of sixty-three Brucella samples. Furthermore, in order to validate the system, DNA samples previously resolved by sequencing system and Agilent technology, were de novo genotyped. The comparison of the MLVA typing data obtained by the Caliper equipment and those previously obtained by the other analysis methods showed a good correlation. However the outputs were not accurate as the Caliper DNA fragment sizes showed discrepancies compared with real data and a conversion table from observed to expected data was created. CONCLUSION: In this paper we described the MLVA-16 using a rapid, sophisticated microfluidics technology for detection of amplification product sizes. The comparison of the MLVA typing data produced by Caliper LabChip 90 system with the data obtained by different techniques showed a general concordance of the results. Furthermore this platform represents a significant improvement in terms of handling, data acquiring, computational efficiency and rapidity, allowing to perform the strain genotyping in a time equal to one sixth respect to other microfluidics systems as e.g. the Agilent 2100 bioanalyzer.Finally, this platform can be considered a valid alternative to standard genotyping techniques, particularly useful dealing with a large number of samples in short time. These data confirmed that this technology represents a significative advancement in high-throughput accurate Brucella genotyping.

Reliable identification at the species level of Brucella isolates with MALDI-TOF-MS.

BACKGROUND: The genus Brucella contains highly infectious species that are classified as biological threat agents. The timely detection and identification of the microorganism involved is essential for an effective response not only to biological warfare attacks but also to natural outbreaks. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a rapid method for the analysis of biological samples. The advantages of this method, compared to conventional techniques, are rapidity, cost-effectiveness, accuracy and suitability for the high-throughput identification of bacteria. Discrepancies between taxonomy and genetic relatedness on the species and biovar level complicate the development of detection and identification assays. RESULTS: In this study, the accurate identification of Brucella species using MALDI-TOF-MS was achieved by constructing a Brucella reference library based on multilocus variable-number tandem repeat analysis (MLVA) data. By comparing MS-spectra from Brucella species against a custom-made MALDI-TOF-MS reference library, MALDI-TOF-MS could be used as a rapid identification method for Brucella species. In this way, 99.3% of the 152 isolates tested were identified at the species level, and B. suis biovar 1 and 2 were identified at the level of their biovar. This result demonstrates that for Brucella, even minimal genomic differences between these serovars translate to specific proteomic differences. CONCLUSIONS: MALDI-TOF-MS can be developed into a fast and reliable identification method for genetically highly related species when potential taxonomic and genetic inconsistencies are taken into consideration during the generation of the reference library.