Genomic analysis of group A Streptococcus isolated during a correctional facility outbreak of MRSA in 2004.

BACKGROUND: In 2004-2005, an outbreak of impetigo occurred at a correctional facility during a sentinel outbreak of methicillin- resistant Staphylococcus aureus (MRSA) in Alberta, Canada. Next-generation sequencing (NGS) was used to characterize the group A Streptococcus (GAS) isolates and evaluate whether genomic biomarkers could distinguish between those recovered alone and those co-isolated with S. aureus. METHODS: Superficial wound swabs collected from all adults with impetigo during this outbreak were cultured using standard methods. NGS was used to characterize and compare all of the GAS and S. aureus genomes. RESULTS: Fifty-three adults were culture positive for GAS, with a subset of specimens also positive for MRSA (n = 5) or methicillin-sensitive S. aureus (n = 3). Seventeen additional MRSA isolates from this facility from the same time frame (no GAS co-isolates) were also included. All 78 bacterial genomes were analyzed for the presence of known virulence factors, plasmids, and antimicrobial resistance (AMR) genes. Among the GAS isolates were 12 emm types, the most common being 41.2 (n = 27; 51%). GAS genomes were phylogenetically compared with local and public datasets of invasive and non-invasive isolates. GAS genomes had diverse profiles for virulence factors, plasmids, and AMR genes. Pangenome analysis did not identify horizontally transferred genes in the co-infection versus single infections. CONCLUSIONS: GAS recovered from invasive and non-invasive sources were not genetically distinguishable. Virulence factors, plasmids, and AMR profiles grouped by emm type, and no genetic changes were identified that predict co-infection or horizontal gene transfer between GAS and S. aureus.

HISTORIQUE: En 2004­2005, une éclosion d'impétigo s'est manifestée dans un établissement correctionnel pendant une éclosion sentinelle de Staphylococcus aureus résistante à la méthicilline (SRAM) en Alberta, au Canada. Le séquençage de prochaine génération (SPG) a été utilisé pour caractériser les isolats du streptocoque du groupe (SGA) et évaluer si les biomarqueurs génomiques peuvent distinguer ceux qui se rétablissent seuls de ceux qui ont co-isolé le S. aureus. MÉTHODOLOGIE: Les chercheurs ont mis en culture des écouvillons de plaies cutanées superficielles prélevés chez tous les adultes atteints d'impétigo pendant cette éclosion. Ils ont utilisé le SNG pour caractériser et comparer tous les génomes du SPG et du S. aureus récupérés. RÉSULTATS: Cinquante-trois adultes étaient positifs au SGA, un sous-groupe d'échantillons était également positif au SRAM (n = 5) ou au S. aureus (n = 3) sensible à la méthicilline. Dix-sept autres isolats de SRAM provenant de cet établissement pendant la même période (pas de co-isolats du SGA) ont été inclus. Ils ont analysé l'ensemble des 78 génomes bactériens pour déceler la présence de facteurs de virulence connus, de plasmides et de gènes de résistance antimicrobienne (RAM). Parmi les isolats du SGA se trouvaient 12 types d'emm, les plus courants étant 41,2 (n = 27, 51 %). Les génomes du SGA ont été comparés sur le plan phylogénétique aux données locales et publiques sur les isolats invasifs et non invasifs. Les génomes du SGA présentaient divers profils de facteurs de virulence, de plasmides et de gènes de RAM. L'analyse du pangénome n'a pas permis de repérer les gènes transférés dans les génomes de co-infection ou les adaptations génétiques associées à une co-infection plutôt par rapport aux infections uniques. CONCLUSIONS: Le SGA prélevé de sources invasives ou non invasives n'était pas reconnaissable sur le plan génétique. Les facteurs de virulence, les plasmides et les profils de résistance antimicrobienne regroupés par type d'emm ne présentaient pas de changements génétiques prédicteurs d'une co- infection ou d'un transfert génétique horizontal entre le SGA et le S. aureus.

High Diversity and Variability in the Vaginal Microbiome in Women following Preterm Premature Rupture of Membranes (PPROM): A Prospective Cohort Study.

OBJECTIVE: To characterize the vaginal microbiota of women following preterm premature rupture of membranes (PPROM), and determine if microbiome composition predicts latency duration and perinatal outcomes. DESIGN: A prospective cohort study. SETTING: Canada. POPULATION: Women with PPROM between 24+0 and 33+6 weeks gestational age (GA). METHODS: Microbiome profiles, based on pyrosequencing of the cpn60 universal target, were generated from vaginal samples at time of presentation with PPROM, weekly thereafter, and at delivery. MAIN OUTCOME MEASURES: Vaginal microbiome composition, latency duration, gestational age at delivery, perinatal outcomes. RESULTS: Microbiome profiles were generated from 70 samples from 36 women. Mean GA at PPROM was 28.8 wk (mean latency 2.7 wk). Microbiome profiles were highly diverse but sequences representing Megasphaera type 1 and Prevotella spp. were detected in all vaginal samples. Only 13/70 samples were dominated by Lactobacillus spp. Microbiome profiles at the time of membrane rupture did not cluster by gestational age at PPROM, latency duration, presence of chorioamnionitis or by infant outcomes. Mycoplasma and/or Ureaplasma were detected by PCR in 81% (29/36) of women, and these women had significantly lower GA at delivery and correspondingly lower birth weight infants than Mycoplasma and/or Ureaplasma negative women. CONCLUSION: Women with PPROM had mixed, abnormal vaginal microbiota but the microbiome profile at PPROM did not correlate with latency duration. Prevotella spp. and Megasphaera type I were ubiquitous. The presence of Mollicutes in the vaginal microbiome was associated with lower GA at delivery. The microbiome was remarkably unstable during the latency period.

Gardnerella vaginalis Subgroups Defined by cpn60 Sequencing and Sialidase Activity in Isolates from Canada, Belgium and Kenya.

Increased abundance of Gardnerella vaginalis and sialidase activity in vaginal fluid is associated with bacterial vaginosis (BV), a common but poorly understood clinical entity associated with poor reproductive health outcomes. Since most women are colonized with G. vaginalis, its status as a normal member of the vaginal microbiota or pathogen causing BV remains controversial, and numerous classification schemes have been described. Since 2005, sequencing of the chaperonin-60 universal target (cpn60 UT) has distinguished four subgroups in isolate collections, clone libraries and deep sequencing datasets. To clarify potential clinical and diagnostic significance of cpn60 subgroups, we undertook phenotypic and molecular characterization of 112 G. vaginalis isolates from three continents. A total of 36 subgroup A, 33 B, 35 C and 8 D isolates were identified through phylogenetic analysis of cpn60 sequences as corresponding to four "clades" identified in a recently published study, based on sequencing 473 genes across 17 isolates. cpn60 subgroups were compared with other previously described molecular methods for classification of Gardnerella subgroups, including amplified ribosomal DNA restriction analysis (ARDRA) and real-time PCR assays designed to quantify subgroups in vaginal samples. Although two ARDRA patterns were observed in isolates, each was observed in three cpn60 subgroups (A/B/D and B/C/D). Real-time PCR assays corroborated cpn60 subgroups overall, but 13 isolates from subgroups A, B and D were negative in all assays. A putative sialidase gene was detected in all subgroup B, C and D isolates, but only in a single subgroup A isolate. In contrast, sialidase activity was observed in all subgroup B isolates, 3 (9%) subgroup C isolates and no subgroup A or D isolates. These observations suggest distinct roles for G. vaginalis subgroups in BV pathogenesis. We conclude that cpn60 UT sequencing is a robust approach for defining G. vaginalis subgroups within the vaginal microbiome.

Characterization of the vaginal microbiota of healthy Canadian women through the menstrual cycle.

BACKGROUND: The vaginal microbial community plays a vital role in maintaining women's health. Understanding the precise bacterial composition is challenging because of the diverse and difficult-to-culture nature of many bacterial constituents, necessitating culture-independent methodology. During a natural menstrual cycle, physiological changes could have an impact on bacterial growth, colonization, and community structure. The objective of this study was to assess the stability of the vaginal microbiome of healthy Canadian women throughout a menstrual cycle by using cpn60-based microbiota analysis. Vaginal swabs from 27 naturally cycling reproductive-age women were collected weekly through a single menstrual cycle. Polymerase chain reaction (PCR) was performed to amplify the universal target region of the cpn60 gene and generate amplicons representative of the microbial community. Amplicons were pyrosequenced, assembled into operational taxonomic units, and analyzed. Samples were also assayed for total 16S rRNA gene content and Gardnerella vaginalis by quantitative PCR and screened for the presence of Mollicutes by using family and genus-specific PCR. RESULTS: Overall, the vaginal microbiome of most women remained relatively stable throughout the menstrual cycle, with little variation in diversity and only modest fluctuations in species richness. Microbiomes between women were more different than were those collected consecutively from individual women. Clustering of microbial profiles revealed the expected groupings dominated by Lactobacillus crispatus, Lactobacillus iners, and Lactobacillus jensenii. Interestingly, two additional clusters were dominated by either Bifidobacterium breve or a heterogeneous mixture of nonlactobacilli. Direct G. vaginalis quantification correlated strongly with its pyrosequencing-read abundance, and Mollicutes, including Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum, were detected in most samples. CONCLUSIONS: Our cpn60-based investigation of the vaginal microbiome demonstrated that in healthy women most vaginal microbiomes remained stable through their menstrual cycle. Of interest in these findings was the presence of Bifidobacteriales beyond just Gardnerella species. Bifidobacteriales are frequently underrepresented in 16S rRNA gene-based studies, and their detection by cpn60-based investigation suggests that their significance in the vaginal community may be underappreciated.

Resolution and characterization of distinct cpn60-based subgroups of Gardnerella vaginalis in the vaginal microbiota.

Bacterial vaginosis (BV), characterized by a shift of the vaginal microbiota from a Lactobacillus-dominated community to a dense biofilm containing a complex mixture of organisms, is an important risk factor in poor reproductive health outcomes. The Nugent score, based on Gram stain, is used to diagnose BV and Gardnerella vaginalis abundance in the sample is one factor determining Nugent score. A high Nugent score is indicative of BV but does not always correspond to the presence of clinical symptoms. G. vaginalis is recognized as a heterogeneous group of organisms, which can also be part of the normal, healthy vaginal microbiome. In addition, asymptomatic BV and non-Gardnerella types of BV are being recognized. In an attempt to resolve the heterogeneous group of G. vaginalis, a phylogenetic tree of cpn60 universal target sequences from G. vaginalis isolates was constructed that indicates the existence of four subgroups of G. vaginalis. This subdivision, supported by whole genome similarity calculation of representative strains using JSpecies, demonstrates that these subgroups may represent different species. The cpn60 subgroupings did not correspond with the Piot biotyping scheme, but did show consistency with ARDRA genotyping and sialidase gene presence. Isolates from all four subgroups produced biofilm in vitro. We also investigated the distribution of G. vaginalis subgroups in vaginal samples from Kenyan women with Nugent scores consistent with BV, Intermediate and Normal microbiota (n = 44). All subgroups of G. vaginalis were detected in these women, with a significant difference (z = -3.372, n = 39, p = 0.001) in frequency of G. vaginalis subgroup B between BV and Normal groups. Establishment of a quantifiable relationship between G. vaginalis subgroup distribution and clinical status could have significant diagnostic implications.

Comparing the similarity of different groups of bacteria to the human proteome.

Numerous aspects of the relationship between bacteria and human have been investigated. One aspect that has recently received attention is sequence overlap at the proteomic level. However, there has not yet been a study that comprehensively characterizes the level of sequence overlap between bacteria and human, especially as it relates to bacterial characteristics like pathogenicity, G-C content, and proteome size. In this study, we began by performing a general characterization of the range of bacteria-human similarity at the proteomic level, and identified characteristics of the most- and least-similar bacterial species. We then examined the relationship between proteomic similarity and numerous other variables. While pathogens and nonpathogens had comparable similarity to the human proteome, pathogens causing chronic infections were found to be more similar to the human proteome than those causing acute infections. Although no general correspondence between a bacterium's proteome size and its similarity to the human proteome was noted, no bacteria with small proteomes had high similarity to the human proteome. Finally, we discovered an interesting relationship between similarity and a bacterium's G-C content. While the relationship between bacteria and human has been studied from many angles, their proteomic similarity still needs to be examined in more detail. This paper sheds further light on this relationship, particularly with respect to immunity and pathogenicity.

Complete genome sequence of the beer spoilage organism Pediococcus claussenii ATCC BAA-344T.

Pediococcus claussenii is a common brewery contaminant. We have sequenced the chromosome and plasmids of the type strain P. claussenii ATCC BAA-344. A ropy variant was chosen for sequencing to obtain genetic information related to growth in beer, as well as exopolysaccharide and possibly biofilm formation by this organism.