Identification of Brucella spp. isolates and discrimination from the vaccine strain Rev.1 by MALDI-TOF mass spectrometry.

Brucellosis' surveillance and control programs require robust laboratory techniques that can reliably identify and biotype Brucella strains and discriminate between vaccine and field infection. In the recent years, Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) has revolutionized the routine identification of several microorganisms in clinical microbiology laboratories. Nevertheless, its application on Brucella spp. identification is limited since there are no reference spectra in the commercial databases, due to the microorganism's potential bioterrorist use. In this study, a custom MALDI-TOF MS reference library was constructed and its performance on identification at species level was evaluated using 75 Brucella spp. isolates. Furthermore, distinct peak biomarkers were detected for biovar assignment and discrimination from vaccine strain Rev.1. Analysis of mass peak profiles allowed Brucella accurate identification at genus and species level (100%) with no misidentifications. Despite the high intrageneric similarity, MALDI-TOF MS database succeeded in classifying at biovar level, 47 out of 62 B. melitensis bv. 3 isolates (75.81%), whereas all B. melitensis strains, except for one, were correctly discriminated from vaccine strain Rev.1. MALDI-TOF MS appeared to be a rapid, cost-effective and reliable method for the routine identification of brucellae which reduces time consumption in pathogen identification and could replace in the near future the current conventional and molecular techniques. Its ability to differentiate vaccine from field infection could facilitate brucellosis' monitoring systems contributing in the effective control of the disease.

Helical and rod-shaped bacteria swim in helical trajectories with little additional propulsion from helical shape.

It has frequently been hypothesized that the helical body shapes of flagellated bacteria may yield some advantage in swimming ability. In particular, the helical-shaped pathogen Helicobacter pylori is often claimed to swim like a corkscrew through its harsh gastric habitat, but there has been no direct confirmation or quantification of such claims. Using fast time-resolution and high-magnification two-dimensional (2D) phase-contrast microscopy to simultaneously image and track individual bacteria in bacterial broth as well as mucin solutions, we show that both helical and rod-shaped H. pylori rotated as they swam, producing a helical trajectory. Cell shape analysis enabled us to determine shape as well as the rotational and translational speed for both forward and reverse motions, thereby inferring flagellar kinematics. Using the method of regularized Stokeslets, we directly compare observed speeds and trajectories to numerical calculations for both helical and rod-shaped bacteria in mucin and broth to validate the numerical model. Although experimental observations are limited to select cases, the model allows quantification of the effects of body helicity, length, and diameter. We find that due to relatively slow body rotation rates, the helical shape makes at most a 15% contribution to propulsive thrust. The effect of body shape on swimming speeds is instead dominated by variations in translational drag required to move the cell body. Because helical cells are one of the strongest candidates for propulsion arising from the cell body, our results imply that quite generally, swimming speeds of flagellated bacteria can only be increased a little by body propulsion.

Spontaneous excision of the O-polysaccharide wbkA glycosyltranferase gene is a cause of dissociation of smooth to rough Brucella colonies.

The brucellae are Gram-negative pathogens that cause brucellosis, a zoonosis of worldwide importance. The genus Brucella includes smooth and rough species that differ in that they carry smooth and rough lipopolysaccharides, respectively. Brucella abortus, B. melitensis, and B. suis are typical smooth species. However, these smooth brucellae dissociate into rough mutants devoid of the lipopolysaccharide O-polysaccharide, a major antigen and a virulence determinant encoded in regions wbo (included in genomic island-2) and wbk. We demonstrate here the occurrence of spontaneous recombination events in those three Brucella species leading to the deletion of a 5.5-kb fragment carrying the wbkA glycosyltranferase gene and to the appearance of rough mutants. Analysis of the recombination intermediates suggested homologous recombination between the ISBm1 insertion sequences flanking wbkA as the mechanism generating the deletion. Excision of wbkA was reduced but not abrogated in a recA-deficient mutant, showing the existence of both RecA-dependent and -independent processes. Although the involvement of the ISBm1 copies flanking wbkA suggested a transpositional event, the predicted transpositional joint could not be detected. This absence of detectable transposition was consistent with the presence of polymorphism in the inverted repeats of one of the ISBm1 copies. The spontaneous excision of wbkA represents a novel dissociation mechanism of smooth brucellae that adds to the previously described excision of genomic island-2. This ISBm1-mediated wbkA excision and the different %GC levels of the excised fragment and of other wbk genes suggest that the Brucella wbk locus is the result of at least two horizontal acquisition events.

Genomic island 2 is an unstable genetic element contributing to Brucella lipopolysaccharide spontaneous smooth-to-rough dissociation.

Brucella is a Gram-negative bacterium that causes a worldwide-distributed zoonosis. The genus includes smooth (S) and rough (R) species that differ in the presence or absence, respectively, of the O-polysaccharide of lipopolysaccharide. In S brucellae, the O-polysaccharide is a critical diagnostic antigen and a virulence determinant. However, S brucellae spontaneously dissociate into R forms, a problem in antigen and S vaccine production. Spontaneous R mutants of Brucella abortus, Brucella melitensis, and Brucella suis carried the chromosomal scar corresponding to genomic island 2 (GI-2) excision, an event causing the loss of the wboA and wboB O-polysaccharide genes, and the predicted excised circular intermediate was identified in B. abortus, B. melitensis, and B. suis cultures. Moreover, disruption of a putative phage integrase gene in B. abortus GI-2 caused a reduction in O-polysaccharide loss rates under conditions promoting S-R dissociation. However, spontaneous R mutants not carrying the GI-2 scar were also detected. These results demonstrate that the phage integrase-related GI-2 excision is a cause of S-R brucella dissociation and that other undescribed mechanisms must also be involved. In the R Brucella species, previous works have shown that Brucella ovis but not Brucella canis lacks GI-2, and a chromosomal scar identical to those in R mutants was observed. These results suggest that the phage integrase-promoted GI-2 excision played a role in B. ovis speciation and are consistent with other evidence, suggesting that this species and B. canis have emerged as two independent lineages.

Immobilization strategies of Brucella particles on optical fibers for use in chemiluminescence immunosensors.

Immunosensors are powerful analytical tools in clinical and veterinary diagnostics. This has led us to design a chemiluminescent immunosensor aimed at identifying anti-Brucella antibodies using optical fibers as the transducer. In order to develop the optimal transducer, to achieve an optimal chemical modification thereby allowing an optimal covalent binding of the protein receptor, several cleaning strategies and silane coupling agents were investigated. Brucella killed organisms were used as a model receptor for quantifying anti-Brucella IgG antibodies in a suspension compared to conventional colorimetric and chemiluminescent ELISA. A silane-benzophenone derivative was selected as the best performing silane coupling agent: the optical fiber immunosensor (OFIS) has showed the lowest limit of detection at 0.207 microg/ml, compared to 0.828 microg/ml and 0.414 microg/ml achieved by colorimetric and chemiluminescent ELISAs, respectively. These results, together with the additional advantages of rapidity, lower reagent volumes and moderate operating conditions, have set the grounds for further study in order to adapt this platform for on-site diagnostics of brucellosis disease markers.

Cellular bioterrorism: how Brucella corrupts macrophage physiology to promote invasion and proliferation.

Brucellosis is a worldwide human zoonosis caused by intracellular bacteria of the genus Brucella. Virulence factors play an important role in allowing Brucella infection and proliferation within macrophages. Brucella enters macrophages through lipid raft microdomains, avoids phagolysosome fusion, and inhibits TNF-alpha secretion and apoptosis. Furthermore, Brucella can perturb bactericidal activity in macrophages by influencing the host cell response to its advantage through its LPS or by activating the cAMP/PKA pathway. To date, small steps have been taken in defining and understanding the virulence factors of Brucella used in macrophage subversion, but further investigation is required to fully explain virulence and persistence.

Brucella intracellular life: from invasion to intracellular replication.

Brucella organisms are pathogens that ultimate goal is to propagate in their preferred niche, the cell. Upon cell contact the bacteria is internalized via receptor molecules by activating small GTPases of the Rho subfamily and by a moderate recruitment of actin filaments. Once inside cells, Brucella localizes in early phagosomes, where it avoids fusion with late endosomes and lysosomes. These early events require the control of Rab small GTPases, and cytokines such as the G-CSF. Then, the bacterium redirects its trafficking to autophagosomes and finally reaches the endoplasmic reticulum, where it extensively replicates. Some of the bacterial molecular determinants involved in the internalization and early events after ingestion are controlled by the BvrS/BvrR two component regulatory system, whereas the intracellular trafficking beyond this early compartments are controlled by the VirB type IV secretion system. Once inside the endoplasmic reticulum, Brucella extensively replicates without restricting basic cellular functions or inducing obvious damage to cells. The integrity of Brucella LPS on the bacterial surface is one of the required factors for Brucella intracellular survival, and therefore for virulence.

Fun stories about Brucella: the "furtive nasty bug".

Although Brucella is responsible for one of the major worldwide zoonosis, our understanding of its pathogenesis remains in its infancy. In this paper, we summarize some of the research in progress in our laboratory that we think could contribute to a better understanding of the Brucella molecular virulence mechanisms and their regulation.

Fluorescent whole-cell hybridization with 16S rRNA-targeted oligonucleotide probes to identify Brucella spp. by flow cytometry.

A whole-cell hybridization assay with fluorescent oligonucleotide probes derived from the 16S rRNA sequence of Brucella abortus in combination with flow cytometry has been developed. With the three fluorescent probes selected, a positive signal was observed with all the representative strains of the species and biovars of Brucella and with a total of nine different Brucella clinical isolates. Using the B9 probe in the hybridization assay, it was possible to discriminate between Brucella suis biovars 2, 3, 4, and 5 and almost all the other Brucella spp. On the basis of differences in fluorescence intensities, no discrimination was established between Brucella spp. and other phylogenetically related microorganisms. No positive fluorescence signals were detected with any of the bacteria showing serological cross-reactions with Brucella spp. and with a total of 17 clinical isolates not belonging to the genus Brucella. These results suggest that the 16S rRNA whole-cell hybridization technique could be a valuable diagnostic tool for the detection and identification of Brucella spp.

Multiplication of Brucella canis in male reproductive organs and detection of autoantibody to spermatozoa in canine brucellosis.

Orchitis, epididymitis and prostatitis have been reported in male dogs infected with Brucella canis (B. canis), but the pathogenesis of infertility in male dogs has not been clarified yet. We examined localization of B. canis in the tissue of infected male reproductive organs and production of autoantibody to spermatozoa in male dogs by immunofluorescence and unlabeled antibody peroxidase-antiperoxidase (PAP) methods and electron microscopy. B. canis were found in the cytoplasm of macrophages and epithelial cells in testis, epididymis and prostate. Particularly in the prostate, B. canis multiplied in the cytoplasm of epithelial cells and emerged in the glandular lumen with destroyed epithelial cells. Head-to-head agglutination of spermatozoa was found in the semen, urine and epididymal duct with varying degrees of intensity among the infected dogs. Appearance of the spermagglutination began following the detection of B. canis in urine and semen, suggesting invasion of the organisms in male reproductive organs. In the sera from the dogs orally inoculated with B. canis, (Ig M), Ig G and Ig A anti-spermantibodies were detected in parallel with the appearance of the serum spermagglutinating activity. The heads of agglutinated spermatozoa in the epididymal duct and semen were coated with Ig A antibody, which is considered to be anti-spermautoantibody locally produced. The target of these circulating and local antibodies was acrosome of the dog spermatozoa and spermatids. It seems probable that multiplication of B. canis in epithelial cells is the direct cause of damage to the infected cells, and the damage acts as a trigger of the production of autoantibody to spermatozoa.

Evolution and taxonomy in the genus Brucella: steroid hormone induction of filterable forms with altered characteristics after reversion.

Several strains of Brucella abortus, Brucella suis, and Brucella melitensis were exposed to physiologic concentrations of testosterone, progesterone, and diethylstilbestrol by incorporating them into the growth medium. The hormones induced Brucella to form cell wall-defective organisms, including filterable forms. The filterable forms from 1 strain of B melitensis had altered characteristics when it reverted to growth as an intact cell. This is the 1st reported instance of induction of filterable forms of Brucella by progesterone, testosterone, and diethylstilbestrol.

Recommendations for the description of species and biotypes of the genus Brucella.

The properties of the genus Brucella are summarized and recommendations made for the definition of species and biotypes within the genus. In the first instance it is recommended that the classification of an organism as a member of the genus should be based on the following criteria: gramnegative coccobacillary morphology, a G + C content for the DNA of 56-58 moles %, a minimum of 90% homology with the DNA of reference strains in hybridization tests, disk electrophoretograms of acid-phenol soluble proteins of identical pattern to reference strains, a cytochrome C absorption spectrum with absorption maxima between 522-530 nm and 552-560 nm, extensive serological cross-reactions of intra-cellular antigens with those of reference strains. Supporting evidence is obtained from cultural, biochemical and pathogenic properties, although variations in these occur within the genus. Subdivision into species is dependent upon principal natural host, oxidative metabolic pattern and phage sensitivity. Classification into biotypes is dependent upon CO2 requirement, H2S production, dye sensitivity and reaction with monospecific antisera to A and M antigens. It is recommended that any organism proposed as a new member of the genus should have characters which approximate the ranges quoted. In all cases comparisons should be made with the established reference/neotype strains.

[Structure and constituents of Brucella. Characterization and biological properties of the fractions]. / Structure et constituants des Brucella. Caractérisation des fractions et propriétés biologiques

The analysis of the components of a bacterium may be envisaged from the biological aspect (fractionation), the ultrastructural aspect (staining of the structures examined electron-microscopically), and the biological aspect (measure of an activity). In this report we attempt to examine the components of brucella from all three aspects simultaneously. The brucella envelopes have the same ultrastructure as that of gramnegative bacteria: outer membrane, thick stratum or peptidoglycane, periplasmic space, cytoplasmic membrane. The outer membrane of brucella in phase S contains many types of polysaccharides: (1) the lipopolysaccharide (LPS) (S) and polysaccharide B are solubilized by the phenol-uater and ether-water methods, by trichloracetic acid (TCA), by heated sodium dodecyl-sulfate (SDS). The exact localization of polysaccharide B is not known; by the phenol-water extraction method, the LPS (S) in its toxic form (endotoxin) passes in solution into the phenol phase, unlike the endotoxin of enterobacteria, which passes into the aqueous phase. In addition to its toxicity, this LPS (S) is responsible for reactions of immediate hypersensitivity as well as serological reactions towards the standard antigen. It presents A + M antigenic sites; (2) one or more of the polysaccharides remains unsolubilized by the ether-water method, but solubilized by heated SDS; (3) a polysaccharide is linked to peptidoglycane. The structure of the outer membrane of the brucella in phase R is analogous to that of LPS, carrying antigen R, characteristic of these strains. This antigen may be utilized for the serological diagnosis of infections due to brucella R (B. ovis) or vaccinations by a vaccine in phase R. The peptidoglycane fraction extracted by the heated SDS has a more complex structure than that of E. coli: it consists of a supplementary outer layer containing amino acids and polysaccharides. This fraction has a vaccinal activity. A soluble protein fraction, without organized structure, no doubt of cytoplasmic origin, may be extracted by a cold saline solution. This fraction, known as "brucelline", reveals delayed hypersensitivity when injected intradermally. The biological activity of the other structures (periplasm, cytoplasmic membrane, ribosomes...) is not known. Biological activities have been attributed to fractions, but since these are badly defined from the structural point of view it is difficult to determine the connection between activities and structures.

[Biological properties and chemical composition of a soluble fraction of Brucella abortus. Monospecific A or M antigens]. / Propriétés biologiques et composition chimique d'une fraction soluble (ABS) de Brucella abortus. mise en évidence des antigènes monospécifiques A ou M

ABS, the supernatant of smooth B. abortus phenol-protected suspensions, is a protein, lipid, sugar and ARN complex containing all amino acids found in phenol-water fractions from B. abortus and B. melitensis. ABS is non-toxic and immunizes mice against B. abortus challenge. Chromium chloride easily binds ABS to sheep erythrocytes (E ABS) for a specific and accurate passive hemagglutination test in brucellosis. E ABS are agglutinated by all antisera to fractions of B. abortus or B. melitensis, but not by the monospecific anti M serum. A study of the antigenic relationships between ABS and phenol-water fractions from smooth brucella strains leads to the isolation of sub-fractions fron the phenol precipitates of B. abortus or of B. melitensis that could contain only A or M antigens which react only with their respective monospecific A or M serum.

[Brucella and modification of the host response to bacterial and viral infection. Effect of the S-R variation]. / Brucella et modifications de la reponse de l'organisme à des infections bacteriennes et à une infection virale. Influence de la variation S-R

Within the framework of a study on the immunostimulant properties of Brucella abortus and of its extracts, we have investigated the protective activity on infection of mice by Klebsiella pneumoniae and on Charlotte Friend's leukemia, of inactivated Brucella abortus and brucella lysates by ultrasound. The injection of inactivated B 19 R preparations and their lysates three days before infection by klebsiella has allowed to protect mice against a large number of DL50 (100 and 1000 in certain cases). To determine the role of the surface antigen in this type of immunostimulant, the activity of the B 19 R inactivated preparations has also been examined. It appears that the protection obtained by the injection on day J + 3, in relation to the infection, does not depend on the presence of the complete surface antigen, while the long term effect seems to be more dependent on this. The development of Charlotte Friend's leukemia has been favourably influenced by the previous injection of B 19 S or inactivated B 19 R. Immunotherapy on days J + 1 or J + 3 gave encouraging results with B 19 R, but facilitation with B 19 S. These results confirm the immunostimulant activity of brucella which has already been reported by ourselves and by other authors.

[Immunogenic properties of the cellular components of Brucella]. / Immunogennye svoistva kletochnykh komponentov brutsell

A study was made of immunogenic properties of the cell wall and other cellular components of brucellae obtained by mechanical disntegration of bacteria. The greatest immunogenic properties proved to be possessed by the cell walls. In using the vaccine consisting o the living vaccine of the Br. abortus strain 19 and the cell walls of brucellae the efficacy of the preparation was not reduced in experiments on guinea pigs and mice.