Adrian M. Whatmore

Genetic relationships between clinical isolates of Streptococcus pneumoniae, Streptococcus oralis, and Streptococcus mitis: characterization of "Atypical" pneumococci and organisms allied to S. mitis harboring S. pneumoniae virulence factor-encoding genes.

ABSTRACT The oral streptococcal group (mitis phylogenetic group) currently consists of nine recognized species, although the group has been traditionally difficult to classify, with frequent changes in nomenclature over the years. The pneumococcus (Streptococcus pneumoniae), an important human pathogen, is traditionally distinguished from the most closely related oral streptococcal speciesStreptococcus mitis and Streptococcus oralis on the basis of three differentiating characteristics: optochin susceptibility, bile solubility, and agglutination with antipneumococcal polysaccharide capsule antibodies. However, there are many reports in the literature of pneumococci lacking one or more of these defining characteristics. Sometimes called “atypical” pneumococci, these isolates can be the source of considerable confusion in the clinical laboratory. Little is known to date about the genetic relationships of such organisms with classical S. pneumoniae isolates. Here we describe these relationships based on sequence analysis of housekeeping genes in comparison with previously characterized isolates of S. pneumoniae,S. mitis, and S. oralis. While most pneumococci were found to represent a closely related group these studies identified a subgroup of atypical pneumococcal isolates (bile insoluble and/or “acapsular”) distinct from, though most closely related to, the “typical” pneumococcal isolates. However, a large proportion of isolates, found to be atypical on the basis of capsule reaction alone, did group with typical pneumococci, suggesting that they have either lost capsule production or represent as-yet-unrecognized capsular types. In contrast to typical S. pneumoniae, isolates phenotypically identified as S. mitis and S. oralis, which included isolates previously characterized in taxonomic studies, were genetically diverse. While most of the S. oralis isolates did fall into a well-separated group, S. mitis isolates did not cluster into a well-separated group. During the course of these studies we also identified a number of potentially important pathogenic isolates, which were frequently associated with respiratory disease, that phenotypically and genetically are most closely related to S. mitis but which harbor genes encoding the virulence determinants pneumolysin and autolysin classically associated with S. pneumoniae.

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.

The effects of osmotic upshock on the intracellular solute pools of Bacillus subtilis.

The effects of hypersaline treatment (osmotic upshock) on solute accumulation have been studied in the Gram-positive bacterium Bacillus subtilis. Natural abundance 13C NMR spectroscopy studies revealed only proline as a major organic osmoticum in cells grown in defined medium (no exogenous organic solutes) and this finding was confirmed by amino acid analysis. Intracellular concentrations of both K+ and proline rose markedly after osmotic upshock. K+ influx from the medium was rapid (less than 1 h) but proline synthesis was a slower process (5-9 h). Proline synthesis appeared to be dependent on the prior accumulation of K+ and it is possible that K+ serves in some manner as the signal for increased proline synthesis. In cells upshocked in medium enriched in glycine betaine the endogenous synthesis of proline was repressed and glycine betaine served as the sole organic osmoticum. K+ was also accumulated under these conditions.

Development of a Multilocus Sequence Typing Scheme for the Pig Pathogen Streptococcus suis: Identification of Virulent Clones and Potential Capsular Serotype Exchange

ABSTRACT Streptococcus suis is an important pathogen of pigs and occasionally causes serious human disease. However, little is known about the S. suis population structure, the clonal relationships between strains, the potential of particular clones to cause disease, and the relevance of serotype as a marker for epidemiology. Here we describe a multilocus sequence typing (MLST) scheme for S. suis developed in order to begin to address these issues. Seven housekeeping gene fragments from each of 294 S. suis isolates obtained from various S. suis diseases and from asymptomatic carriage representing 28 serotypes and nine distinct countries of origin were sequenced. Between 32 and 46 alleles per locus were identified, giving the ability to distinguish >1.6 × 1011 sequence types (STs). However only 92 STs were identified in this study. Of the 92 STs 18 contained multiple isolates, the most common of which, ST1, was identified on 141 occasions from six countries. Assignment of the STs to lineages resulted in 37 being identified as unique and unrelated STs while the remaining 55 were assigned to 10 complexes. ST complexes ST1, ST27, and ST87 dominate the population; while the ST1 complex was strongly associated with isolates from septicemia, meningitis, and arthritis, the ST87 and ST27 complexes were found to contain significantly higher numbers of lung isolates. In agreement with the observed distribution of disease-causing isolates of S. suis, most isolates previously characterized as of high virulence in porcine infection models belong to ST1, while isolates belonging to other STs appear to be less virulent in general. Finally nine STs were found to contain isolates of multiple serotypes, and many isolates belonging to the same serotypes were found to have very disparate genetic backgrounds. As well as highlighting that the serotype can often be a poor indicator of genetic relatedness between S. suis isolates, these findings suggest that capsular genes may be moving horizontally through the S. suis population.

Rapid Evolution of Virulence and Drug Resistance in the Emerging Zoonotic Pathogen Streptococcus suis

Background Streptococcus suis is a zoonotic pathogen that infects pigs and can occasionally cause serious infections in humans. S. suis infections occur sporadically in human Europe and North America, but a recent major outbreak has been described in China with high levels of mortality. The mechanisms of S. suis pathogenesis in humans and pigs are poorly understood. Methodology/Principal Findings The sequencing of whole genomes of S. suis isolates provides opportunities to investigate the genetic basis of infection. Here we describe whole genome sequences of three S. suis strains from the same lineage: one from European pigs, and two from human cases from China and Vietnam. Comparative genomic analysis was used to investigate the variability of these strains. S. suis is phylogenetically distinct from other Streptococcus species for which genome sequences are currently available. Accordingly, ∼40% of the ∼2 Mb genome is unique in comparison to other Streptococcus species. Finer genomic comparisons within the species showed a high level of sequence conservation; virtually all of the genome is common to the S. suis strains. The only exceptions are three ∼90 kb regions, present in the two isolates from humans, composed of integrative conjugative elements and transposons. Carried in these regions are coding sequences associated with drug resistance. In addition, small-scale sequence variation has generated pseudogenes in putative virulence and colonization factors. Conclusions/Significance The genomic inventories of genetically related S. suis strains, isolated from distinct hosts and diseases, exhibit high levels of conservation. However, the genomes provide evidence that horizontal gene transfer has contributed to the evolution of drug resistance.

Current understanding of the genetic diversity of Brucella, an expanding genus of zoonotic pathogens.

Brucella species are responsible for brucellosis, one of the worlds most widespread zoonotic diseases causing abortion in domestic animals and a potentially debilitating infection of man. Despite the identification of a number of distinct species within the genus with differing host preferences and pathogenicity it has been known for many years that members of the genus are genetically homogeneous. However, since the start of the millennium the application of new technologies to the group has resulted in rapid advances in the understanding of Brucella diversity and, after many years of inactivity, a process of expansion of the genus is underway. This review summarises the current state of knowledge in this area and outlines how this informs understanding of the taxonomy and evolution of the group and is enabling the development of increasingly sophisticated molecular typing tools.

Characterisation of the genetic diversity of Brucella by multilocus sequencing

BackgroundBrucella species include economically important zoonotic pathogens that can infect a wide range of animals. There are currently six classically recognised species of Brucella although, as yet unnamed, isolates from various marine mammal species have been reported. In order to investigate genetic relationships within the group and identify potential diagnostic markers we have sequenced multiple genetic loci from a large sample of Brucella isolates representing the known diversity of the genus.ResultsNine discrete genomic loci corresponding to 4,396 bp of sequence were examined from 160 Brucella isolates. By assigning each distinct allele at a locus an arbitrary numerical designation the population was found to represent 27 distinct sequence types (STs). Diversity at each locus ranged from 1.03–2.45% while overall genetic diversity equated to 1.5%. Most loci examined represent housekeeping gene loci and, in all but one case, the ratio of non-synonymous to synonymous change was substantially <1. Analysis of linkage equilibrium between loci indicated a strongly clonal overall population structure. Concatenated sequence data were used to construct an unrooted neighbour-joining tree representing the relationships between STs. This shows that four previously characterized classical Brucella species, B. abortus, B. melitensis, B. ovis and B. neotomae correspond to well-separated clusters. With the exception of biovar 5, B. suis isolates cluster together, although they form a more diverse group than other classical species with a number of distinct STs corresponding to the remaining four biovars. B. canis isolates are located on the same branch very closely related to, but distinguishable from, B. suis biovar 3 and 4 isolates. Marine mammal isolates represent a distinct, though rather weakly supported, cluster within which individual STs display one of three clear host preferences.ConclusionThe sequence database provides a powerful dataset for addressing ongoing controversies in Brucella taxonomy and a tool for unambiguously placing atypical, phenotypically discordant or newly emerging Brucella isolates. Furthermore, by using the phylogenetic backbone described here, robust and rationally selected markers for use in diagnostic assay development can be identified.

Brucellosis – new aspects of an old disease

Clinical conditions synonymous with brucellosis have been described since the time of Hippocrates. Indeed, it has been suggested that organisms resembling brucellae have been detected in carbonized cheese from the Roman era (Capasso 2002). These observations highlight how both man and animals have lived with this disease since the days of early record keeping. Despite the numerous biotechnological improvements we now have, brucellosis remains a major worldwide zoonosis. Some countries have been successful in eradication of disease, while others, especially in the developing world still live with a huge disease burden. This review outlines some of the early significant historical milestones then brings the reader to the current challenges that face us in detection, microbial typing, taxonomy, vaccine development, understanding the host-microbial interactions and virulence mechanisms of these enigmatic bacteria.

Identification and Characterization of Variable-Number Tandem-Repeat Markers for Typing of Brucella spp.

ABSTRACT Members of the genus Brucella infect many domesticated and wild animals and cause serious zoonotic infection in humans. The availability of discriminatory molecular typing tools to inform and assist conventional epidemiological approaches would be invaluable in controlling these infections, but efforts have been hampered by the genetic homogeneity of the genus. We report here on a molecular subtyping system based on 21 variable-number tandem-repeat (VNTR) loci consisting of 13 previously unreported loci and 8 loci previously reported elsewhere. This approach was applied to a collection of 121 Brucella isolates obtained worldwide and representing all six classically recognized Brucella species. The size of repeats selected for inclusion varied from 5 to 40 bp giving VNTR loci with a range of diversities. The number of alleles detected ranged from 2 to 21, and Simpsons diversity index values ranged from 0.31 to 0.92. This assay divides the 121 isolates into 119 genotypes, and clustering analysis results in groups that, with minor exceptions, correspond to conventional species designations. Reflecting this, the use of six loci in isolation was shown to be sufficient to determine species designation. On the basis of the more variable loci, the assay could also discriminate isolates originating from restricted geographical sources, indicating its potential as an epidemiological tool. Stability studies carried out in vivo and in vitro showed that VNTR profiles were sufficiently stable such that recovered strains could readily be identified as the input strain. The method described here shows great potential for further development and application to both epidemiological tracing of Brucella transmissions and in determining relationships between isolates worldwide.

Streptococcus pyogenes sclB encodes a putative hypervariable surface protein with a collagen-like repetitive structure

Streptococcus pyogenes is the causative agent in a wide range of diseases of humans of varying severity. During a study scanning the genome sequence of a serotype M1 invasive isolate SF370 for novel surface proteins, an ORF, designated sclB, was identified. The putative protein encoded by sclB contains both a signal peptide and classic Gram-positive wall-associated sequences. Comparison of the sequences of this ORF with those from a number of unrelated isolates demonstrated that sclB encodes a putative surface protein with a variable N-terminal sequence followed by a variable length tract of collagen-like GXY(n) repeats. A further feature of sclB is the presence of CAAAA repeat tracts immediately downstream of the putative start codon. The number of these pentameric repeats varies from 4 to 15 between strains and variation in repeat number results in the predicted SclB protein being either in or out of frame relative to the start codon. These observations suggest that expression of this protein may be regulated at the translational level as a result of gain or loss of CAAAA repeats. While the function of SclB remains to be elucidated, an sclB-specific transcript was detected by RT-PCR during in vitro culture. Finally, it is shown that a second gene, sclA, potentially encoding a protein with a similar extensive collagen-like structure and variable N-terminal sequence, is present in all isolates of S. pyogenes tested to date. Thus S. pyogenes harbours a novel family of structurally related and surface-exposed proteins of potential importance in the pathogenic process.