Herbert Tomaso

Brucella microti sp. nov., isolated from the common vole Microtus arvalis

Two Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains CCM 4915(T) and CCM 4916), isolated from clinical specimens of the common vole Microtus arvalis during an epizootic in the Czech Republic in 2001, were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA (rrs) and recA gene sequence similarities, both isolates were allocated to the genus Brucella. Affiliation to Brucella was confirmed by DNA-DNA hybridization studies. Both strains reacted equally with Brucella M-monospecific antiserum and were lysed by the bacteriophages Tb, Wb, F1 and F25. Biochemical profiling revealed a high degree of enzyme activity and metabolic capabilities not observed in other Brucella species. The omp2a and omp2b genes of isolates CCM 4915(T) and CCM 4916 were indistinguishable. Whereas omp2a was identical to omp2a of brucellae from certain pinniped marine mammals, omp2b clustered with omp2b of terrestrial brucellae. Analysis of the bp26 gene downstream region identified strains CCM 4915(T) and CCM 4916 as Brucella of terrestrial origin. Both strains harboured five to six copies of the insertion element IS711, displaying a unique banding pattern as determined by Southern blotting. In comparative multilocus VNTR (variable-number tandem-repeat) analysis (MLVA) with 296 different genotypes, the two isolates grouped together, but formed a separate cluster within the genus Brucella. Multilocus sequence typing (MLST) analysis using nine different loci also placed the two isolates separately from other brucellae. In the IS711-based AMOS PCR, a 1900 bp fragment was generated with the Brucella ovis-specific primers, revealing that the insertion element had integrated between a putative membrane protein and cboL, encoding a methyltransferase, an integration site not observed in other brucellae. Isolates CCM 4915(T) and CCM 4916 could be clearly distinguished from all known Brucella species and their biovars by means of both their phenotypic and molecular properties, and therefore represent a novel species within the genus Brucella, for which the name Brucella microti sp. nov. with the type strain CCM 4915(T) (=BCCN 07-01(T)=CAPM 6434(T)) is proposed.

Recombinase Polymerase Amplification Assay for Rapid Detection of Francisella tularensis

ABSTRACT Several real-time PCR approaches to develop field detection for Francisella tularensis, the infectious agent causing tularemia, have been explored. We report the development of a novel qualitative real-time isothermal recombinase polymerase amplification (RPA) assay for use on a small ESEQuant Tube Scanner device. The analytical sensitivity and specificity were tested using a plasmid standard and DNA extracts from infected rabbit tissues. The assay showed a performance comparable to real-time PCR but reduced the assay time to 10 min. The rapid RPA method has great application potential for field use or point-of-care diagnostics.

Isolation of Brucella microti from Mandibular Lymph Nodes of Red Foxes, Vulpes vulpes, in Lower Austria

From the mandibular lymph nodes of wild red foxes (Vulpes vulpes) hunted in the region of Gmünd, Lower Austria, two gram-negative, oxidase- and urease-positive, coccoid rod-shaped bacteria (strains 257 and 284) were isolated. Cells were fast growing, nonmotile, and agglutinated with monospecific anti-Brucella (M) serum. Both strains were biochemically identified as Ochrobactrum anthropi by using the API 20NE test. However, sequencing of the 16S rRNA and recA genes clearly identified strains 257 and 284 as Brucella spp. Further molecular analysis by omp2a/b gene sequencing, multilocus sequence typing and multilocus variable number tandem repeats analysis revealed Brucella microti, a recently described Brucella species that has originally been isolated from diseased common voles (Microtus arvalis) in South Moravia, Czech Republic in 2000. Our findings demonstrate that B. microti is prevalent in a larger geographic area covering the region of South Moravia and parts of Lower Austria. Foxes could have become infected by ingestion of infected common voles.

Coexistence of Pathogens in Host-Seeking and Feeding Ticks within a Single Natural Habitat in Central Germany

ABSTRACT The importance of established and emerging tick-borne pathogens in Central and Northern Europe is steadily increasing. In 2007, we collected Ixodes ricinus ticks feeding on birds (n = 211) and rodents (n = 273), as well as host-seeking stages (n = 196), in a habitat in central Germany. In order to find out more about their natural transmission cycles, the ticks were tested for the presence of Lyme disease borreliae, Anaplasma phagocytophilum, spotted fever group (SFG) rickettsiae, Francisella tularensis, and babesiae. Altogether, 20.1% of the 680 ticks examined carried at least one pathogen. Bird-feeding ticks were more frequently infected with Borrelia spp. (15.2%) and A. phagocytophilum (3.2%) than rodent-feeding ticks (2.6%; 1.1%) or questing ticks (5.1%; 0%). Babesia spp. showed higher prevalence rates in ticks parasitizing birds (13.2%) and host-seeking ticks (10.7%), whereas ticks from small mammals were less frequently infected (6.6%). SFG rickettsiae and F. tularensis were also found in ticks collected off birds (2.1%; 1.2%), rodents (1.8%; 1.5%), and vegetation (4.1%; 1.6%). Various combinations of coinfections occurred in 10.9% of all positive ticks, indicating interaction of transmission cycles. Our results suggest that birds not only are important reservoirs for several pathogens but also act as vehicles for infected ticks and might therefore play a key role in the dispersal of tick-borne diseases.

Genetic diversity and phylogenetic relationships of bacteria belonging to the Ochrobactrum–Brucella group by recA and 16S rRNA gene-based comparative sequence analysis

The genetic diversity and phylogenetic interrelationships among 106 Ochrobactrum strains (O. anthropi: 72, O. intermedium: 22, O. tritici: 5, O. oryzae: 2, O. grignonense: 2, O. gallinifaecis: 1, O. lupini: 2), the type strains of the eight Brucella species and other closely related taxa were studied by recA and rrs gene (16S rRNA) comparative sequence analysis. Both markers correctly delineated the various Ochrobactrum species; however, resolution at the subspecies level was considerably higher in the recA gene-based approach. Phylogenetic analyses using neighbor-joining, parsimony, and maximum likelihood algorithms generated trees with similar topologies but the overall branching order, and also the order of the subclades, were not stable in either assay, which could be explained by generally high recA and rrs sequence similarities. Ochrobactrum and Pseudochrobactrum formed separate clades distinct from other Alphaproteobacteria with Bartonella, Agrobacterium, and Rhizobium as the closest relatives. O. gallinifaecis was the most distinct member, when compared to the type species O. anthropi, with rrs and recA similarities of 96.2% and 81.4%. Brucella species were indistinguishable, exhibiting high rrs and recA gene similarities of 98.6% and 85.5% compared with Ochrobactrum intermedium. At the protein level, all RecA sequences among the various Ochrobactrum species and between Ochrobactrum and Brucella were highly similar with only a few amino acid substitutions. O. anthropi and O. tritici were indistinguishable by means of their RecA proteins. A set of initially biochemically classified strains did not cluster within their assigned species and they either grouped within other known species or grouped as potential novel Ochrobactrum species. In further investigations, these strains were reclassified and described as novel species. In summary, Ochrobactrum is a highly diverse genus comprising several novel species. We recommend recA- in addition to rrs gene-analysis for correct species allocation and subtyping of novel Ochrobactrum isolates.

Isolation of Potentially Novel Brucella spp. from Frogs

ABSTRACT Bacterial isolates from frogs were phenotypically identified as Ochrobactrum anthropi, but 16S rRNA sequencing showed up to 100% identity with Brucella inopinata. Further analysis of recA, omp2a, omp2b, bcsp31, and IS711 and multilocus sequence analysis (MLSA) verified a close relationship with Brucella, suggesting the isolates may actually represent novel members of this growing genus of zoonotic pathogens.

Rapid identification of Burkholderia mallei and Burkholderia pseudomallei by intact cell Matrix-assisted Laser Desorption/Ionisation mass spectrometric typing

BackgroundBurkholderia (B.) pseudomallei and B. mallei are genetically closely related species. B. pseudomallei causes melioidosis in humans and animals, whereas B. mallei is the causative agent of glanders in equines and rarely also in humans. Both agents have been classified by the CDC as priority category B biological agents. Rapid identification is crucial, because both agents are intrinsically resistant to many antibiotics. Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-TOF MS) has the potential of rapid and reliable identification of pathogens, but is limited by the availability of a database containing validated reference spectra. The aim of this study was to evaluate the use of MALDI-TOF MS for the rapid and reliable identification and differentiation of B. pseudomallei and B. mallei and to build up a reliable reference database for both organisms.ResultsA collection of ten B. pseudomallei and seventeen B. mallei strains was used to generate a library of reference spectra. Samples of both species could be identified by MALDI-TOF MS, if a dedicated subset of the reference spectra library was used. In comparison with samples representing B. mallei, higher genetic diversity among B. pseudomallei was reflected in the higher average Eucledian distances between the mass spectra and a broader range of identification score values obtained with commercial software for the identification of microorganisms. The type strain of B. pseudomallei (ATCC 23343) was isolated decades ago and is outstanding in the spectrum-based dendrograms probably due to massive methylations as indicated by two intensive series of mass increments of 14 Da specifically and reproducibly found in the spectra of this strain.ConclusionsHandling of pathogens under BSL 3 conditions is dangerous and cumbersome but can be minimized by inactivation of bacteria with ethanol, subsequent protein extraction under BSL 1 conditions and MALDI-TOF MS analysis being faster than nucleic amplification methods. Our spectra demonstrated a higher homogeneity in B. mallei than in B. pseudomallei isolates. As expected for closely related species, the identification process with MALDI Biotyper software (Bruker Daltonik GmbH, Bremen, Germany) requires the careful selection of spectra from reference strains. When a dedicated reference set is used and spectra of high quality are acquired, it is possible to distinguish both species unambiguously. The need for a careful curation of reference spectra databases is stressed.

Quantitative analysis of the intramacrophagic Brucella suis proteome reveals metabolic adaptation to late stage of cellular infection

A 2‐D DIGE approach allowed the characterization of the intramacrophagic proteome of the intracellular pathogen Brucella suis at the late stage of in vitro infection by efficient discrimination between bacterial and host cell proteins. Using a subtraction model, a total of 168 proteins showing altered concentrations in comparison with extracellularly grown, stationary‐phase bacteria were identified. The majority of the 44 proteins significantly regulated at this stage of infection were involved in bacterial metabolism and 40% were present in lowered concentrations, supporting the hypothesis of an adaptive response by quantitative reduction of processes participating in energy, protein, and nucleic acid metabolism. In the future, the 2‐D DIGE‐based approach will permit to decipher specifically and quantitatively the intracellular proteomes of various pathogens during adaptation to their specific host cell environments.

Rapid field detection assays for Bacillus anthracis, Brucella spp., Francisella tularensis and Yersinia pestis

Rapid detection is essential for timely initiation of medical post-exposure prophylactic measures in the event of intentional release of biological threat agents. We compared real-time PCR assay performance between the Applied Biosystems 7300/7500 and the RAZOR instruments for specific detection of the causative agents of anthrax, brucellosis, tularemia and plague. Furthermore, an assay detecting Bacillus thuringiensis, a Bacillus anthracis surrogate, was developed for field-training purposes. Assay sensitivities for B. anthracis, Brucella spp., Francisella tularensis and Yersinia pestis were 10-100 fg of target DNA per reaction, and no significant difference in assay performance was observed between the instrument platforms. Specificity testing of the diagnostic panels with both instrument platforms did not reveal any cross-reactivity with other closely related bacteria. The duration of thermocycling with the RAZOR instrument was shorter, i.e. 40 min as compared with 100 min for the Applied Biosystems 7300/7500 instruments. These assays provide rapid tools for the specific detection of four biological threat agents. The detection assays, as well as the training assay for B. thuringiensis powder preparation analysis, may be utilized under field conditions and for field training, respectively.

IDENTIFICATION OF BRUCELLA SPECIES AND BIOTYPES USING POLYMERASE CHAIN REACTION-RESTRICTION FRAGMENT LENGTH POLYMORPHISM (PCR-RFLP)

Brucellosis is a worldwide zoonosis causing reproductive failures in livestock and a severe multi-organ disease in humans. The genus Brucella is divided into seven species and various biotypes differing in pathogenicity and host specificity. Although Brucella spp. represent a highly homogenous group of bacteria, RFLPs of selected genes display sufficient polymorphism to distinguish Brucella species and biovars. PCR-RFLP analysis shows excellent typeability, reproducibility, stability, and epidemiological concordance. Consequently, PCR-RFLP assays of specific gene loci can serve as tools for diagnostic, epidemiological, taxonomic, and evolutionary studies. Various PCR-RFLPs used for the identification of Brucella species and biotypes are reviewed.