A Study of the Infant Nasal Microbiome Development over the First Year of Life and in Relation to Their Primary Adult Caregivers Using cpn60 Universal Target (UT) as a Phylogenetic Marker.

Whereas the infant gut microbiome is the subject of intense study, relatively little is known regarding the nares microbiome in newborns and during early life. This study aimed to survey the typical composition and diversity of human anterior nare microflora for developing infants over time, and to explore how these correlate to their primary caregivers. Single nare swabs were collected at five time points over a one-year period for each subject from infant-caregiver pairs. Our study comprised of 50 infants (recruited at 2 weeks, post delivery) and their 50 primary caregivers. Applying the chaperonin-60 (cpn60) universal target (UT) amplicon as our molecular barcoding marker to census survey the microbial communities, we longitudinally surveyed infant nares microbiota at 5 time points over the course of the first year of life. The inter- and intra-subject diversity was catalogued and compared, both longitudinally and relative to their adult primary caregivers. Although within-subject variability over time and inter-subject variability were both observed, the assessment detected only one or two predominant genera for individual infant samples, belonging mainly to phyla Actinobacteria, Firmicutes, and Proteobacteria. Consistent with previously observed microbial population dynamics in other body sites, the diversity of nares microflora increased over the first year of life and infants showed differential operational taxonomic units (OTUs) relative to their matched primary caregiver. The collected evidence also support that both temporal and seasonal changes occur with respect to carriage of potentially pathogenic bacteria (PPBs), which may influence host predisposition to infection. This pilot study surveying paired infant/caregiver nare microbiomes provides novel longitudinal diversity information that is pertinent to better understanding nare microbiome development in infants.

Phylogenetic relationship and virulence inference of Streptococcus Anginosus Group: curated annotation and whole-genome comparative analysis support distinct species designation.

BACKGROUND: The Streptococcus Anginosus Group (SAG) represents three closely related species of the viridans group streptococci recognized as commensal bacteria of the oral, gastrointestinal and urogenital tracts. The SAG also cause severe invasive infections, and are pathogens during cystic fibrosis (CF) pulmonary exacerbation. Little genomic information or description of virulence mechanisms is currently available for SAG. We conducted intra and inter species whole-genome comparative analyses with 59 publically available Streptococcus genomes and seven in-house closed high quality finished SAG genomes; S. constellatus (3), S. intermedius (2), and S. anginosus (2). For each SAG species, we sequenced at least one numerically dominant strain from CF airways recovered during acute exacerbation and an invasive, non-lung isolate. We also evaluated microevolution that occurred within two isolates that were cultured from one individual one year apart. RESULTS: The SAG genomes were most closely related to S. gordonii and S. sanguinis, based on shared orthologs and harbor a similar number of proteins within each COG category as other Streptococcus species. Numerous characterized streptococcus virulence factor homologs were identified within the SAG genomes including; adherence, invasion, spreading factors, LPxTG cell wall proteins, and two component histidine kinases known to be involved in virulence gene regulation. Mobile elements, primarily integrative conjugative elements and bacteriophage, account for greater than 10% of the SAG genomes. S. anginosus was the most variable species sequenced in this study, yielding both the smallest and the largest SAG genomes containing multiple genomic rearrangements, insertions and deletions. In contrast, within the S. constellatus and S. intermedius species, there was extensive continuous synteny, with only slight differences in genome size between strains. Within S. constellatus we were able to determine important SNPs and changes in VNTR numbers that occurred over the course of one year. CONCLUSIONS: The comparative genomic analysis of the SAG clarifies the phylogenetics of these bacteria and supports the distinct species classification. Numerous potential virulence determinants were identified and provide a foundation for further studies into SAG pathogenesis. Furthermore, the data may be used to enable the development of rapid diagnostic assays and therapeutics for these pathogens.

Genome sequence of a chimpanzee herpesvirus and its relation to other primate alphaherpesviruses.

This study reports the complete genome sequence of chimpanzee herpesvirus (ChHV), an alphaherpesvirus isolated from a chimpanzee. Although closely related to human herpes simplex virus type 2 (HSV2), the level of sequence diversity confirms that ChHV is sufficiently distinct to be considered a member of a different virus species rather than a variant strain of HSV2. Phylogenetic comparison with other simplexviruses at several levels supports the hypothesis that HSV2 and ChHV co-evolved with their respective human and chimpanzee hosts and raises questions regarding the evolutionary origins of HSV1.

The attenuated genotype of varicella-zoster virus includes an ORF0 transitional stop codon mutation.

Varicella-zoster virus (VZV) is the first of the human herpesviruses to be attenuated and subsequently approved as a live vaccine to prevent varicella and herpes zoster. Both the attenuated VZV vaccine, called vaccine Oka or vOka, and the parental strain pOka have been completely sequenced. Yet the specific determinants of attenuation are uncertain. The open reading frame (ORF) with the most single nucleotide polymorphisms (SNPs), ORF62, encodes the regulatory protein IE62, but IE62 studies have failed to define a specific SNP associated with attenuation. We have completed next-generation sequencing of the VZV Ellen genome, a strain known to be highly attenuated by its very limited replication in human skin xenografts in the SCID mouse model of VZV pathogenesis. A comparative analysis of the Ellen sequence with all other complete VZV sequences was extremely informative. In particular, an unexpected finding was a stop codon mutation in Ellen ORF0 (herpes simplex virus UL56 homolog) identical to one found in vOka, combined with the absence of polymorphisms in most Ellen ORFs that were known to be mutated in vOka. The mutated ORF0 protein was also imaged in both two dimensions and three dimensions by confocal microscopy. The probability of two VZV strains not connected by a recent common ancestor having an identical ORF0 SNP by chance would be 1 × 10(-8), in other words, extremely unlikely. Taken together, these bioinformatics analyses strongly suggest that the stop codon ORF0 SNP is one of the determinants of the attenuation genotype of live VZV vaccines.

Genetic and antigenic analysis of invasive serogroup Y Neisseria meningitidis isolates collected from 1999 to 2003 in Canada.

One hundred forty serogroup Y Neisseria meningitidis isolates recovered from patients with invasive meningococcal disease (IMD) in Canada from 1999 to 2003 were analyzed by genetic and serological methods. Seventy-four isolates (52.9%) belonged to serotype 2c, and most have serosubtype antigen P1.5,2 (37 isolates, 26%) or P1.5 (31 isolates, 22%). Forty-eight isolates (34.3%) belonged to serotype 14 and have serosubtype antigen P1.5,2 (13 isolates, 9%) or P1.5 (7 isolates, 5%) or were nonserosubtypeable (27 isolates, 19%). Thirteen isolates (9.3%) were nonserotypeable. Multilocus sequence typing identified two unrelated clonal populations of serogroup Y meningococci causing invasive disease in Canada: ST-23 and ST-167 clonal complexes. Almost all ST-167-related isolates were typed as 2c:P1.5, while strains of the ST-23 clonal complex were either serotype 14 or 2c but with the serosubtype antigen P1.5,2. In contrast to previous reports that patients with serogroup Y disease are usually older, 26% of the Canadian serogroup Y cases were found in the 10-to-19-year-old age group and another 11% were in the 20-to-39-year-old age group.

Temperate bacteriophages affect pulsed-field gel electrophoresis patterns of Campylobacter jejuni.

The recently sequenced genome of Campylobacter jejuni RM1221 revealed the presence of three integrated bacteriophage-like elements. In this study, genes from the first element, a Mu-like bacteriophage, were amplified by PCR and used to probe pulsed-field gels of clinical C. jejuni strains obtained from a waterborne outbreak (Ontario, Canada, 2000). These highly similar strains differed only by their pulsed-field gel electrophoresis (PFGE) patterns due to an apparent insertion or deletion of a 40-kb fragment. Bacteriophage probes hybridized to these different bands in Southern blot analysis, indicating that homologues of bacteriophage genes were present in the outbreak strains. Investigation of the bacteriophage insertion sites in these isolates suggested that bacteriophage acquisition, loss, or transposition was responsible for the PFGE pattern variation. The bacteriophage gene sequences were similar, but not identical, in the outbreak strains and RM1221, indicating that differences may exist between the bacteriophages.

A full-genome phylogenetic analysis of varicella-zoster virus reveals a novel origin of replication-based genotyping scheme and evidence of recombination between major circulating clades.

Varicella-zoster virus (VZV) is a remarkably stable virus that until recently was thought to exhibit near-universal genetic homogeneity among circulating wild-type strains. In recent years, the expanding knowledge of VZV genetics has led to a number of groups proposing sequence-based typing schemes, but no study has yet examined the relationships between VZV genotypes at a full-genome level. A central hypothesis of this study is that VZV has coevolved with humankind. In this study, 11 additional full VZV genomic sequences are presented, bringing the current number of complete genomic sequences publicly available to 18. The full-genome alignment contained strains representing four distinct clades, but the possibility exists that a fifth clade comprised of African and Asian-like isolates was not represented. A consolidated VZV genotyping scheme employing the origin-associated region between reiteration region R4 and open reading frames (ORFs) 63 and 70 is described, one which accurately categorizes strains into one of four clades related to the geographic origin of the isolates. The full-genome alignment also provided evidence for recombination having occurred between the major circulating VZV clades. One Canadian clinical isolate was primarily Asian-like in origin, with most of the genome showing strong sequence identity to the Japanese-like clade B, with the exceptions being two putative recombination regions, located in ORFs 14 to 17 and ORFs 22 to 26, which showed clear similarity to the European/North American clade A. The very low rate of single-nucleotide polymorphisms scattered across the genome made full-genome sequencing the only definitive method for identifying specific VZV recombination events.

The complete genome sequence of herpesvirus papio 2 (Cercopithecine herpesvirus 16) shows evidence of recombination events among various progenitor herpesviruses.

We have sequenced the entire genome of herpesvirus papio 2 (HVP-2; Cercopithecine herpesvirus 16) strain X313, a baboon herpesvirus with close homology to other primate alphaherpesviruses, such as SA8, monkey B virus, and herpes simplex virus (HSV) type 1 and type 2. The genome of HVP-2 is 156,487 bp in length, with an overall GC content of 76.5%. The genome organization is identical to that of the other members of the genus Simplexvirus, with a long and a short unique region, each bordered by inverted repeats which end with an "a" sequence. All of the open reading frames detected in this genome were homologous and colinear with those of SA8 and B virus. The HSV gene RL1 (gamma(1)34.5; neurovirulence factor) is not present in HVP-2, as is the case for SA8 and B virus. The HVP-2 genome is 85% homologous to its closest relative, SA8. However, segment-by-segment bootstrap analysis of the genome revealed at least two regions that display closer homology to the corresponding sequences of B virus. The first region comprises the UL41 to UL44 genes, and the second region is located within the UL36 gene. We hypothesize that this localized and defined shift in homology is due to recombination events between an SA8-like progenitor of HVP-2 and a herpesvirus species more closely related to the B virus. Since some of the genes involved in these putative recombination events are determinants of virulence, a comparative analysis of their function may provide insight into the pathogenic mechanism of simplexviruses.

Complete genome sequence of cercopithecine herpesvirus 2 (SA8) and comparison with other simplexviruses.

We have obtained the complete sequence of the herpesvirus simian agent 8 (SA8; cercopithecine herpesvirus 2) a baboon simplexvirus closely related to the monkey B virus and herpes simplex virus types 1 and 2. The genome of SA8 is 150,715 bp long, with an overall G/C content of 76%, the highest among the simplexviruses sequenced so far. The sequencing has confirmed that the genomic arrangement of SA8 is similar to that of other simplexviruses: unique long and unique short regions bordered by two sets of inverted repeats. All genes identified in SA8 are homologous and collinear with those of the monkey B virus, including the lack of the RL1 open reading frame, a gene responsible for neurovirulence in human herpes simplex viruses. This latter finding supports the hypothesis that a different pathogenetic mechanism may have developed in human simplexviruses, after their divergence from monkey simplexviruses.

Potential capsule switching from serogroup Y to B: The characterization of three such Neisseria meningitidis isolates causing invasive meningococcal disease in Canada.

Three group B Neisseria meningitidis isolates, recovered from meningococcal disease cases in Canada and typed as B:2c:P1.5, were characterized. Multilocus sequence typing showed that all three isolates were related because of an identical sequence type (ST) 573. Isolates typed as 2c:P1.5 are common in serogroup Y meningococci but rare in isolates from serogroups B or C. Although no serogroup Y isolates have been typed as ST-573, eight isolates showed five to six housekeeping gene alleles that were identical to that of ST-573. This suggested that the B:2c:P1.5 isolates may have originated from serogroup Y organisms, possibly by capsule switching.