Genetic element Φ1207.3 (formerly Tn1207.3) is a prophage of Streptococcus pyogenes which carries the macrolide eﬄux resistance genes mef(A)/msr(D) and is capable of conjugal transfer among streptococci. Complete nucleotide sequence showed that Φ1207.3 is 52,491 bp in length and contained 58 open reading frames (ORFs). A manual homology-based annotation with functional prediction of the hypothetical gene product was possible only for 34 out of 58 ORFs. Φ1207.3 codes for two different C-methylation systems, several phage structural genes, a lysis cassette (composed by a holin and a peptidoglycan hydrolase), and three site-specific resolvases of the serine recombinase family.
prophage; ICE; Φ1207.3; Tn1207.3; Tn1207.1; S. pyogenes; macrolide resistance
Conjugative transposon Tn5253, an integrative conjugative element (ICE) of Streptococcus pneumoniae carrying the cat and tet(M) genes, was shown to be 64,528 bp in size and to contain 79 open reading frames, of which only 38 could be annotated. Two distinct genetic elements were found integrated into Tn5253: Tn5251 (18,033 bp), of the Tn916-Tn1545 family of ICEs, and Ωcat(pC194) (7,627 bp), which could not conjugate but was capable of intracellular mobility by excision, circularization, and integration by homologous recombination. The highest conjugation frequency of Tn5253 was observed when Streptococcus pyogenes was the donor (6.7 × 10−3 transconjugants/donor).
Approximately 7% of survivors from meningococcal meningitis (MM) suffer from neurological sequelae due to brain damage in the course of meningitis. The present study focuses on the role of matrix metalloproteinases (MMPs) in a novel mouse model of MM-induced brain damage.
The model is based on intracisternal infection of BALB/c mice with a serogroup C Neisseria meningitidis strain. Mice were infected with meningococci and randomised for treatment with the MMP inhibitor batimastat (BB-94) or vehicle. Animal survival, brain injury and host-response biomarkers were assessed 48 h after meningococcal challenge.
Mice that received BB-94 presented significantly diminished MMP-9 levels (p < 0.01), intracerebral bleeding (p < 0.01), and blood-brain barrier (BBB) breakdown (p < 0.05) in comparison with untreated animals. In mice suffering from MM, the amount of MMP-9 measured by zymography significantly correlated with both intracerebral haemorrhage (p < 0.01) and BBB disruption (p < 0.05).
MMPs significantly contribute to brain damage associated with experimental MM. Inhibition of MMPs reduces intracranial complications in mice suffering from MM, representing a potential adjuvant strategy in MM post-infection sequelae.
Electronic supplementary material
The online version of this article (doi:10.1186/s12879-014-0726-6) contains supplementary material, which is available to authorized users.
Neisseria meningitidis; Meningococcal meningitis; Mouse model; Brain damage; Matrix metalloproteinases; Metalloproteinase inhibitors
Mycobacterium tuberculosis is successfully evolving antibiotic resistance, threatening attempts at tuberculosis epidemic control. Mechanisms of resistance, including the genetic changes favored by selection in resistant isolates, are incompletely understood. Using 116 newly and 7 previously sequenced M. tuberculosis genomes, we identified genomewide signatures of positive selection specific to the 47 resistant genomes. By searching for convergent evolution, the independent fixation of mutations at the same nucleotide site or gene, we recovered 100% of a set of known resistance markers. We also found evidence of positive selection in an additional 39 genomic regions in resistant isolates. These regions encode pathways of cell wall biosynthesis, transcriptional regulation and DNA repair. Mutations in these regions could directly confer resistance or compensate for fitness costs associated with resistance. Functional genetic analysis of mutations in one gene, ponA1, demonstrated an in vitro growth advantage in the presence of the drug rifampicin.
The pathogenesis of bacteraemia after challenge with one million pneumococci of three isogenic variants was investigated. Sequential analyses of blood samples indicated that most episodes of bacteraemia were monoclonal events providing compelling evidence for a single bacterial cell bottleneck at the origin of invasive disease. With respect to host determinants, results identified novel properties of splenic macrophages and a role for neutrophils in early clearance of pneumococci. Concerning microbial factors, whole genome sequencing provided genetic evidence for the clonal origin of the bacteraemia and identified SNPs in distinct sub-units of F0/F1 ATPase in the majority of the ex vivo isolates. When compared to parental organisms of the inoculum, ex-vivo pneumococci with mutant alleles of the F0/F1 ATPase had acquired the capacity to grow at low pH at the cost of the capacity to grow at high pH. Although founded by a single cell, the genotypes of pneumococci in septicaemic mice indicate strong selective pressure for fitness, emphasising the within-host complexity of the pathogenesis of invasive disease.
Decades of research on bacterial sepsis have been devoted to analysing the steps that lead from a local event, either carriage or a localised infection, to systemic disease. Our work analyses in depth the events determining systemic infection by one of the main human pathogens, Streptococcus pneumoniae. Consistent with similar findings on the pathogenesis of bacteraemia due to other commensal pathogens, our results show that after an intravenous inoculum of a million pneumococci, the resulting septicaemia is often founded by a single bacterial cell. Investigation into the nature of this monoclonal infection identified strong within-host selective pressure for metabolic fitness during outgrowth of the bacterial population.
Multidrug-resistant (MDR) Mycobacterium tuberculosis complex (MTBC) strains represent a major threat for tuberculosis (TB) control. Treatment of MDR-TB patients is long and less effective, resulting in a significant number of treatment failures. The development of further resistances leads to extensively drug-resistant (XDR) variants. However, data on the individual reasons for treatment failure, e.g. an induced mutational burst, and on the evolution of bacteria in the patient are only sparsely available. To address this question, we investigated the intra-patient evolution of serial MTBC isolates obtained from three MDR-TB patients undergoing longitudinal treatment, finally leading to XDR-TB. Sequential isolates displayed identical IS6110 fingerprint patterns, suggesting the absence of exogenous re-infection. We utilized whole genome sequencing (WGS) to screen for variations in three isolates from Patient A and four isolates from Patient B and C, respectively. Acquired polymorphisms were subsequently validated in up to 15 serial isolates by Sanger sequencing. We determined eight (Patient A) and nine (Patient B) polymorphisms, which occurred in a stepwise manner during the course of the therapy and were linked to resistance or a potential compensatory mechanism. For both patients, our analysis revealed the long-term co-existence of clonal subpopulations that displayed different drug resistance allele combinations. Out of these, the most resistant clone was fixed in the population. In contrast, baseline and follow-up isolates of Patient C were distinguished each by eleven unique polymorphisms, indicating an exogenous re-infection with an XDR strain not detected by IS6110 RFLP typing. Our study demonstrates that intra-patient microevolution of MDR-MTBC strains under longitudinal treatment is more complex than previously anticipated. However, a mutator phenotype was not detected. The presence of different subpopulations might confound phenotypic and molecular drug resistance tests. Furthermore, high resolution WGS analysis is necessary to accurately detect exogenous re-infection as classical genotyping lacks discriminatory power in high incidence settings.
The ability of fluorescence resonance energy transfer, molecular-beacon, and TaqMan probes to detect single nucleotide polymorphism (SNP) in the presence of a wild-type allele was evaluated using drug resistance-conferring SNPs in mixed Mycobacterium tuberculosis DNA. It was found that both the absolute quantity and the ratio of alleles determine the detection sensitivity of the probe systems.
A PCR-microarray assay was developed in which PCR primers and hybridization probes were designed for the 16S rRNA genes of 16 clinically relevant mycobacteria and for IS6110 of Mycobacterium tuberculosis. The assay, based on a multiplex PCR followed by hybridization with oligonucleotide probes, was tested against 16 Mycobacterium species and 70 clinical samples.
When grown on glucose and beta-glucosides, S. pneumoniae shows sequential use of sugars resulting in diauxic growth with variable time extent of the lag phase separating the biphasic growth curve. The pneumococcal beta-glucoside uptake locus containing the PTS transporter spr0276-82, is regulated by a multi-domain transcriptional regulator CelR. In this work, we address the contribution of phosphorylation of the phosphorylable cysteine in the EIIB domain of CelR to diauxic lag. Utilising site-directed mutagenesis of the phosphorylable amino acids in the EIIB and EIIA domains of CelR, we show that the EIIB domain activation is linked to the duration of the lag phase. Analysis of mutants for other PTS systems indicates that a second beta-glucoside PTS (spr0505), not able to support growth on cellobiose, is responsible for the lag during diauxic growth. A mathematical model of the process is devised together with a nonlinear identification procedure which provides model parameter estimates characterizing the single phases of bacterial growth. Parameter identification performed on data recorded in appropriate experiments on mutants allows for establishing a relationship between a specific model parameter, the EIIB domain and the time extent of the diauxic lag. The experimental results and the related insights provided by the mathematical model provide evidence that the conflicting activation of the CelR regulator is at the origin of the lag phase during sequential growth on glucose and cellobiose. This data is the first description of diauxic lag regulation involving two PTS and a multidomain regulator and could serve as a promising approach for studying the S. pneumoniae growth process on complex carbon sources as possibly encountered in the human host.
Streptococcus pneumoniae expresses three distinct sialidases, NanA, NanB, and NanC, that are believed to be key virulence factors and thus, potential important drug targets. We previously reported that the three enzymes release different products from sialosides, but could share a common catalytic mechanism before the final step of product formation. However, the kinetic investigations of the three sialidases have not been systematically done thus far, due to the lack of an easy and steady measurement of sialidase reaction rate.
In this work, we present further kinetic characterization of pneumococcal sialidases by using a direct spectrophotometric method with the chromogenic substrate p-nitrophenyl-N-acetylneuraminic acid (p-NP-Neu5Ac). Using our assay, the measured kinetic parameters of the three purified pneumococcal sialidase, NanA, NanB and NanC, were obtained and were in perfect agreement with the previously published data. The major advantage of this alternative method resides in the direct measurement of the released product, allowing to readily determine of initial reaction rates and record complete hydrolysis time courses.
We developed an accurate, fast and sensitive spectrophotometric method to investigate the kinetics of sialidase-catalyzed reactions. This fast, sensitive, inexpensive and accurate method could benefit the study of the kinetics and inhibition of sialidases in general.
Sialidase; Neuraminidase; Chromogenic sialic acids; Kinetic assay; Streptococcus pneumoniae
During infection, Streptococcus pneumoniae exists mainly in sessile biofilms rather than in planktonic form, except during sepsis. The capacity to form biofilms is believed to be important for nasopharyngeal colonization as well as disease pathogenesis, but relatively little is known about the regulation of this process. Here, we investigated the effect of exogenous iron [Fe(III)] as well as the role of luxS (encoding S-ribosylhomocysteine lyase) on biofilm formation by S. pneumoniae D39. Fe(III) strongly enhanced biofilm formation at concentrations of ≥50 μM, while Fe(III) chelation with deferoxamine was inhibitory. Importantly, Fe(III) also upregulated the expression of luxS in wild-type D39. A luxS-deficient mutant (D39luxS) failed to form a biofilm, even with Fe(III) supplementation, whereas a derivative overexpressing luxS (D39luxS+) exhibited enhanced biofilm formation capacity and could form a biofilm without added Fe(III). D39luxS exhibited reduced expression of the major Fe(III) transporter PiuA, and the cellular [Fe(III)] was significantly lower than that in D39; in contrast, D39luxS+ had a significantly higher cellular [Fe(III)] than the wild type. The release of extracellular DNA, which is an important component of the biofilm matrix, also was directly related to luxS expression. Similarly, genetic competence, as measured by transformation frequency as well as the expression of competence genes comD, comX, comW, cglA, and dltA and the murein hydrolase cbpD, which is associated with fratricide-dependent DNA release, all were directly related to luxS expression levels and were further upregulated by Fe(III). Moreover, mutagenesis of cbpD blocked biofilm formation. We propose that competence, fratricide, and biofilm formation are closely linked in pneumococci, and that luxS is a central regulator of these processes. We also propose that the stimulatory effects of Fe(III) on all of these parameters are due to the upregulation of luxS expression, and that LuxS provides for a positive Fe(III)-dependent amplification loop by increasing iron uptake.
During infection, pneumococci exist mainly in sessile biofilms rather than in planktonic form, except during sepsis. However, relatively little is known about how biofilms contribute to pneumococcal pathogenesis. Here, we carried out a biofilm assay on opaque and transparent variants of a clinical serotype 19F strain WCH159. After 4 days incubation, scanning electron microscopy revealed that opaque biofilm bacteria produced an extracellular matrix, whereas the transparent variant did not. The opaque biofilm-derived bacteria translocated from the nasopharynx to the lungs and brain of mice, and showed 100-fold greater in vitro adherence to A549 cells than transparent bacteria. Microarray analysis of planktonic and sessile bacteria from transparent and opaque variants showed differential gene expression in two operons: the lic operon, which is involved in choline uptake, and in the two-component system, ciaRH. Mutants of these genes did not form an extracellular matrix, could not translocate from the nasopharynx to the lungs or the brain, and adhered poorly to A549 cells. We conclude that only the opaque phenotype is able to form extracellular matrix, and that the lic operon and ciaRH contribute to this process. We propose that during infection, extracellular matrix formation enhances the ability of pneumococci to cause invasive disease.
Different models for biofilm in Streptococcus pneumoniae have been described in literature. To permit comparison of experimental data, we characterised the impact of the pneumococcal quorum-sensing competence system on biofilm formation in three models. For this scope, we used two microtiter and one continuous culture biofilm system.
In both microtiter models the competence system influences stability and structure of biofilm in the late attachment phase and synthetic competence stimulating peptide (CSP) restored wild type phenotypes in the comC mutants unable to produce the peptide. Early attachment of single cells to well bottoms was found for both systems to be competence independent, while later phases, including microcolony formation correlated to an intact competence system. The continuous culture biofilm model was not affected by mutations in the competence locus, but deletion of capsule had a significant impact in this model.
Since biofilm remains a largely uncharacterised multi-parameter phenotype it appears to be advisable to exploit more than one model in order to draw conclusion of possible relevance of specific genotypes on pneumococcal physiology.
Streptococcus pneumoniae is an important human pathogen representing a major cause of morbidity and mortality worldwide. We sequenced the genome of a serotype 11A, ST62 S. pneumoniae invasive isolate (AP200), that was erythromycin-resistant due to the presence of the erm(TR) determinant, and carried out analysis of the genome organization and comparison with other pneumococcal genomes.
The genome sequence of S. pneumoniae AP200 is 2,130,580 base pair in length. The genome carries 2216 coding sequences (CDS), 56 tRNA, and 12 rRNA genes. Of the CDSs, 72.9% have a predicted biological known function. AP200 contains the pilus islet 2 and, although its phenotype corresponds to serotype 11A, it contains an 11D capsular locus. Chromosomal rearrangements resulting from a large inversion across the replication axis, and horizontal gene transfer events were observed. The chromosomal inversion is likely implicated in the rebalance of the chromosomal architecture affected by the insertions of two large exogenous elements, the erm(TR)-carrying Tn1806 and a functional prophage designated ϕSpn_200. Tn1806 is 52,457 bp in size and comprises 49 ORFs. Comparative analysis of Tn1806 revealed the presence of a similar genetic element or part of it in related species such as Streptococcus pyogenes and also in the anaerobic species Finegoldia magna, Anaerococcus prevotii and Clostridium difficile. The genome of ϕSpn_200 is 35,989 bp in size and is organized in 47 ORFs grouped into five functional modules. Prophages similar to ϕSpn_200 were found in pneumococci and in other streptococcal species, showing a high degree of exchange of functional modules. ϕSpn_200 viral particles have morphologic characteristics typical of the Siphoviridae family and are capable of infecting a pneumococcal recipient strain.
The sequence of S. pneumoniae AP200 chromosome revealed a dynamic genome, characterized by chromosomal rearrangements and horizontal gene transfers. The overall diversity of AP200 is driven mainly by the presence of the exogenous elements Tn1806 and ϕSpn_200 that show large gene exchanges with other genetic elements of different bacterial species. These genetic elements likely provide AP200 with additional genes, such as those conferring antibiotic-resistance, promoting its adaptation to the environment.
The IgA1 protease of Streptococcus pneumoniae is a proteolytic enzyme that specifically cleaves the hinge regions of human IgA1, which dominates most mucosal surfaces and is the major IgA isotype in serum. This protease is expressed in all of the known pneumococcal strains and plays a major role in pathogen's resistance to the host immune response. The present work was focused at identifying the immunodominant regions of pneumococcal IgA1 protease recognized by the human antibody response.
An antigenic sequence corresponding to amino acids 420–457 (epiA) of the iga gene product was identified by screening a pneumococcal phage display library with patients' sera. The epiA peptide is conserved in all pneumococci and in two out of three S. mitis strains, while it is not present in other oral streptococci so far sequenced. This epitope was specifically recognized by antibodies present in sera from 90% of healthy adults, thus representing an important target of the humoral response to S. pneumoniae and S. mitis infection. Moreover, sera from 68% of children less than 4 years old reacted with the epiA peptide, indicating that the human immune response against streptococcal antigens occurs during childhood.
The broad and specific recognition of the epiA polypeptide by human sera demonstrate that the pneumococcal IgA1 protease contains an immunodominant B-cell epitope. The use of phage display libraries to identify microbe or disease-specific antigens recognized by human sera is a valuable approach to epitope discovery.
Pneumococcal surface protein C (PspC) is a virulence factor of Streptococcus pneumoniae previously shown to play a role in bacterial adherence, invasion, and evasion of complement. We investigated the role of this protein in our murine models of pneumococcal pneumonia with different pneumococcal strains. The deletion of pspC in strains of serotypes 2, 3, and 19F did not significantly alter host survival times in the pneumonia model. In contrast, pspC deletion significantly reduced the virulence of the serotype 4 strain, TIGR4, in both the pneumonia and bacteremia models. Therefore, pspC is a systemic and pulmonary virulence determinant for S. pneumoniae, but its effects are influenced by the pneumococcal strain. Finally, pneumonia infection of complement-deficient (C3−/−) mice enhanced pspC virulence, illustrating that PspC-mediated complement evasion contributes to virulence. However, other functions of PspC also contribute to virulence, as demonstrated by the finding that the pspC-deficient TIGR4 mutant was still attenuated relative to the wild-type parent, even in the absence of C3.
A DNA microarray was developed to detect bacterial genes conferring resistance to macrolides and related antibiotics. A database containing 65 nonredundant genes selected from publicly available DNA sequences was constructed and used to design 100 oligonucleotide probes that could specifically detect and discriminate all 65 genes. Probes were spotted on a glass slide, and the array was reacted with DNA templates extracted from 20 reference strains of eight different bacterial species (Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus haemolyticus, Escherichia coli, and Bacteroides fragilis) known to harbor 29 different macrolide resistance genes. Hybridization results showed that probes reacted with, and only with, the expected DNA templates and allowed discovery of three unexpected genes, including msr(SA) in B. fragilis, an efflux gene that has not yet been described for gram-negative bacteria.
Two main patterns of gene expression of Streptococcus pneumoniae were observed during infection in the host by quantitative real time RT-PCR; one was characteristic of bacteria in blood and one of bacteria in tissue, such as brain and lung. Gene expression in blood was characterized by increased expression of pneumolysin, pspA and hrcA, while pneumococci in tissue infection showed increased expression of neuraminidases, metalloproteinases, oxidative stress and competence genes. In vitro situations with similar expression patterns were detected in liquid culture and in a newly developed pneumococcal model of biofilm respectively. The biofilm model was dependent on addition of synthetic competence stimulating peptide (CSP) and no biofilm was formed by CSP receptor mutants. As one of the differentially expressed gene sets in vivo were the competence genes, we exploited competence-specific tools to intervene on pneumococcal virulence during infection. Induction of the competence system by the quorum-sensing peptide, CSP, not only induced biofilm formation in vitro, but also increased virulence in pneumonia in vivo. In contrast, a mutant for the ComD receptor, which did not form biofilm, also showed reduced virulence in pneumonia. These results were opposite to those found in a bacteraemic sepsis model of infection, where the competence system was downregulated. When pneumococci in the different physiological states were used directly for challenge, sessile cells grown in a biofilm were more effective in inducing meningitis and pneumonia, while planktonic cells from liquid culture were more effective in inducing sepsis. Our data enable us, using in vivo gene expression and in vivo modulation of virulence, to postulate the distinction – from the pneumococcal point of view – between two main types of disease. During bacteraemic sepsis pneumococci resemble planktonic growth, while during tissue infection, such as pneumonia or meningitis, pneumococci are in a biofilm-like state.
The emergence and spread of multidrug-resistant (MDR) Mycobacterium tuberculosis (MT) represents a worldwide health care problem because of the difficulty in treating these infections. Development of drug resistance in MT arises mainly by mutation of chromosomal genes. To investigate the evolution of a MT population during a long-lasting infection, the phenotypic and genotypic changes in the drug resistance of 10 sequential MT isolates from a noncompliant chronically infected patient were investigated. During more than 12 years of active disease, a MDR population developed; molecular typing showed one single parental strain that infected the patient and persisted throughout the disease. Molecular analysis of the drug resistance-related genes revealed that discrete subpopulations evolved over time from the parental strain by acquiring and accumulating resistance-conferring mutations to isoniazid, rifampin, and streptomycin. Overall, these observations indicate that during a chronic infection, several subpopulations may coexist in the same patient with different drug susceptibility profiles.
In the past ten years there has been a growing interest in engineering Gram-positive bacteria for biotechnological applications, including vaccine delivery and production of recombinant proteins. Usually, bacteria are manipulated using plasmid expression vectors. The major limitation of this approach is due to the fact that recombinant plasmids are often lost from the bacterial culture upon removal of antibiotic selection. We have developed a genetic system based on suicide vectors on conjugative transposons allowing stable integration of recombinant DNA into the chromosome of transformable and non-transformable Gram-positive bacteria.
The aim of this work was to select a strong chromosomal promoter from Streptococcus gordonii to improve this genetic system making it suitable for expression of single-copy recombinant genes. To achieve this task, a promoterless gene encoding a chloramphenicol acetyltransferase (cat), was randomly integrated into the S. gordonii chromosome and transformants were selected for chloramphenicol resistance. Three out of eighteen chloramphenicol resistant transformants selected exhibited 100% stability of the phenotype and only one of them, GP215, carried the cat gene integrated as a single copy. A DNA fragment of 600 base pairs exhibiting promoter activity was isolated from GP215 and sequenced. The 5' end of its corresponding mRNA was determined by primer extention analysis and the putative -10 and a -35 regions were identified. To study the possibility of using this promoter (PP) for single copy heterologous gene expression, we created transcriptional fusions of PP with genes encoding surface recombinant proteins in a vector capable of integrating into the conjugative transposon Tn916. Surface recombinant proteins whose expression was controlled by the PP promoter were detected in Tn916-containing strains of S. gordonii and Bacillus subtilis after single copy chromosomal integration of the recombinant insertion vectors into the resident Tn916. The surface recombinant protein synthesized under the control of PP was also detected in Enterococcus faecalis after conjugal transfer of a recombinant Tn916 containing the transcriptional fusion.
We isolated and characterized a S. gordonii chromosomal promoter. We demonstrated that this promoter can be used to direct expression of heterologous genes in different Gram-positive bacteria, when integrated in a single copy into the chromosome.
Streptococcus pneumoniae, a major cause of human disease, produces a 17-mer autoinducer peptide pheromone (competence-stimulating peptide [CSP]) for the control of competence for genetic transformation. Due to previous work linking CSP to stress phenotypes, we set up an in vivo sepsis model to assay its effect on virulence. Our data demonstrate a significant increase in the rates of survival of mice, reductions of blood S. pneumoniae counts, and prolonged times to death for mice treated with CSP. In vitro the dose of CSP used in the animal model produced a transitory inhibition of growth. When a mutant with a mutation in the CSP sensor histidine kinase was assayed, no bacteriostatic phenotype was detected in vitro and no change in disease outcome was observed in vivo. The data demonstrate that CSP, which induces in vitro a temporary growth arrest through stimulation of its cognate histidine kinase receptor, is able to block systemic disease in mice. This therapeutic effect is novel, in that the drug-like effect is obtained by stimulation, rather than inhibition, of a bacterial drug target.
Streptococcus pneumoniae is the leading cause of bacterial meningitis. Pneumococcal meningitis is associated with the highest mortality among bacterial meningitis and it may also lead to neurological sequelae despite the use of antibiotic therapy. Experimental animal models of pneumococcal meningitis are important to study the pathogenesis of meningitis, the host immune response induced after infection, and the efficacy of novel drugs and vaccines.
In the present work, we describe in detail a simple, reproducible and efficient method to induce pneumococcal meningitis in outbred mice by using the intracranial subarachnoidal route of infection. Bacteria were injected into the subarachnoid space through a soft point located 3.5 mm rostral from the bregma. The model was tested with several doses of pneumococci of three capsular serotypes (2, 3 and 4), and mice survival was recorded. Lethal doses killing 50 % of animals infected with type 2, 3 and 4 S. pneumoniae were 3.2 × 10, 2.9 × 10 and 1.9 × 102 colony forming units, respectively. Characterisation of the disease caused by the type 4 strain showed that in moribund mice systemic dissemination of pneumococci to blood and spleen occurred. Histological analysis of the brain of animals infected with type 4 S. pneumoniae proved the induction of meningitis closely resembling the disease in humans.
The proposed method for inducing pneumococcal meningitis in outbred mice is easy-to-perform, fast, cost-effective, and reproducible, irrespective of the serotype of pneumococci used.
Streptococcus pneumoniae possesses large zinc metalloproteinases on its surface. To analyse the importance in virulence of three of these metalloproteinases, intranasal challenge of MF1 outbred mice was carried out using a range of infecting doses of wild type and knock-out pneumococcal mutant strains, in order to compare mice survival.
Observation of survival percentages over time and detection of LD50s of knock out mutants in the proteinase genes in comparison to the type 4 TIGR4 wild type strain revealed two major aspects: i) Iga and ZmpB, present in all strains of S. pneumoniae, strongly contribute to virulence in mice; (ii) ZmpC, only present in about 25% of pneumococcal strains, has a lower influence on virulence in mice.
These data suggest Iga, ZmpB and ZmpC as candidate surface proteins responsible for pneumococcal infection and potentially involved in distinct stages of pneumococcal disease.
A mass-screening protocol for the diagnosis of anthrax from nasal swabs based on an enrichment step in liquid medium was devised. Incubation for growth was performed in autoclavable vials and racks which allow real-time PCR analysis of sterilized cultures. A dual-color PCR was set up with primers and probes for the chromosomal marker rpoB and the plasmid marker lef. Specific primer and probe sets were designed for the differentiation of Bacillus anthracis from B. cereus and for the differentiation of the Sterne vaccine strain from field isolates and the Ames strain, which was used in the recent anthrax bioterrorist attack. The present protocol thus combines the high specificity and sensitivity of real-time PCR with excellent biosafety and the low hands-on time necessary for the processing of large numbers of samples, which is extremely important during control programs involving the processing of large numbers of samples.
We developed a novel surface display system based on the use of bacterial spores. A protein of the Bacillus subtilis spore coat, CotB, was found to be located on the spore surface and used as fusion partner to express the 459-amino-acid C-terminal fragment of the tetanus toxin (TTFC). Western, dot blot and fluorescent-activated cell sorting analyses were used to monitor TTFC surface expression on purified spores. We estimated that more than 1.5 × 103 TTFC molecules were exposed on the surface of each spore and recognized by TTFC-specific antibodies. The efficient surface presentation of the heterologous protein, together with the simple purification procedure and the high stability and safety record of B. subtilis spores, makes this spore-based display system a potentially powerful approach for surface expression of bioactive molecules.