Visualization of interrelationships among various urogenital conditions involving micro-organisms.

<p>Green colors indicate desirable conditions, and red colors indicate undesirable conditions. In both cases, the darker the color, the more desirable or undesirable the condition, respectively. The size of the circles is relative to the size of the respective epidemics, but only very roughly. The STI circle does not include viral STIs. The circles on the far left and far right appear as if they do not overlap because the image is two dimensional, but they do overlap somewhat. It is important to note that few studies on the associations between urogenital conditions and host responses or adverse outcomes (which determine whether a condition is desirable or undesirable) have been holistic. For example, many studies only employ 16S ribosomal RNA sequencing of the vaginal microbiota, but this does not cover fungi, protozoa, and viruses and does not reliably identify <i>Chlamydia trachomatis</i> and <i>Neisseria gonorrhoeae</i>. Abbreviations: GBS, Group B streptococcus; STI, sexually transmitted infection. * Complications include HIV acquisition, pelvic inflammatory disease, adverse pregnancy outcomes, and maternal and neonatal infections.</p

Box and whisker plot showing DNA yield obtained by each pretreatment lysis method.

<p>Boxes extend from the lower quartiles to the upper quartiles with median values indicated by the line within each box. Whiskers represent maximum and minimum values, excluding any outliers (values indicated by circles which lie outside 1.5 times the interquartile range). Significant differences between methods are starred (* P <0.05; ** P ≤0.01; *** P ≤0.001).</p

Overview of experimental design.

<p>Schematic showing how samples were processed for 16S rRNA amplicon sequencing.</p

Principal coordinate analysis ordination of a Bray-Curtis dissimilarity matrix.

<p>Extracts are coloured by sample of origin. Extracts cluster closely with other extracts originating from the same sample and there is no observable effect of pretreatment lysis method. Extracts from samples that are dominated by <i>Lactobacillus iners</i> with variable proportions of <i>Gardnerella</i> have clustered on the left, extracts from samples that are dominated by <i>L</i>. <i>acidophilus</i> group have clustered on the bottom right and extracts from high diversity samples that contained a mixture of strict and facultative anaerobes cluster towards the top.</p

Heat map showing most abundant operational taxonomic units (OTUs) with sample extracts arranged by hierarchical clustering.

<p>All OTUs that were present at 1% or higher in at least one sample are shown. Extracts are named according to the sample of origin followed by the pretreatment lysis method used and are arranged by Unweighted Pair Group Method with Arithmetic Mean (UPGMA) clustering on the Bray-Curtis dissimilarity matrix. The coloured bar indicates which extracts have clustered most closely with all other extracts from the same sample (green) and those that have not (red). Reads have been assigned to OTUs based on 97% sequence similarity of the V3–V4 region. Note that in some cases this has resulted in multiple OTUs with the same taxonomic species identifier, which is most likely due to a high degree of intraspecies variability in this region of the gene, or incorrect base calling. <i>Lactobacillus</i> species that could not be identified to species level at the 97% cut-off have been assigned to genus subgroups: <i>L</i>. <i>gasseri</i> group (including <i>L</i>. <i>gasseri</i> and <i>L</i>. <i>johnsonii</i>), <i>L</i>. <i>acidophilus</i> group (including <i>L</i>. <i>acidophilus</i>, <i>L</i>. <i>helveticus</i>, <i>L</i>. <i>gallinarum</i>, <i>L</i>. <i>crispatus</i>, <i>L</i>. <i>jensenii</i> and <i>L</i>. <i>delbruekii</i>), <i>L</i>. <i>vaginalis</i> group (including <i>L</i>. <i>vaginalis</i> and <i>L</i>. <i>reuteri</i>) and <i>L</i>. <i>coleohominis</i> group (including <i>L</i>. <i>coleohominis</i> and <i>L</i>. <i>pontis</i>).</p

Vaginal microbiota communities identified by clustering techniques in 17 articles.

<p>qPCR = quantitative polymerase chain reaction.</p>1<p>Includes direct sequencing (next generation sequencing) of DNA extracted from vaginal samples (10), sequencing of culture colonies (2), fingerprinting (3), and qPCR (2); the 5 studies using DNA hybridisation techniques did not employ data clustering and this technique is therefore not represented in this table.</p>2<p>One qPCR study only assessed <i>L. iners</i> and the other qPCR study did not find clusters dominated by just one <i>Lactobacillus</i> species.</p>3<p>Other than <i>Lactobacillus</i> spp., the 25 most abundant taxa in the 10 direct sequencing studies consistently (in at least 50% of studies) include: <u>Phylum Actinobacteria</u>: <i>G. vaginalis</i>, <i>A. vaginae</i>, <i>Eggerthella</i> spp., <i>Mobiluncus</i> spp.; <u>Phylum Fermicutes</u>: <i>Lachnospiraceae</i> (including BVAB1-3), <i>Dialister</i> spp., <i>Megasphaera</i> spp., <i>Parvimonas</i> (formerly <i>Peptostreptococcus</i>) spp., <i>Veillonella</i> spp., <i>Streptococcus</i> spp., <i>Staphylococcus,</i> spp., <i>Gemella</i> spp.; <u>Phylum Sfingobacteria</u>: <i>Prevotella</i> spp., <i>Porphyromonas</i> spp., <i>Bacteroides</i> spp.; <u>Phylum Fusobacteria</u>: <i>Sneathia</i> spp., <i>Leptotrichia</i> spp.; <u>Phylum Tenericutes</u>: <i>Mycoplasma</i> spp., <i>Ureaplasma</i> spp.; <u>Phylum Proteobacteria</u>: <i>Escherichia/Shigella</i> spp.</p>4<p>Includes <i>Streptococcus</i> spp., <i>Staphylococcus</i> spp., <i>Escherichia/Shigella</i> spp., <i>Proteus</i> spp.</p

Characteristics of molecular vaginal microbiota articles published between 1 January 2008 and 15 November 2013.

<p>BV = bacterial vaginosis; DGGE = Denaturing Gradient Gel Electrophoresis; HIV = human immunodeficiency virus; HPV = human papillomavirus; IVF = in-vitro fertilisation; LH-PCR = Length Heterogeneity PCR; NGS = next generation sequencing; qPCR = quantitative polymerase chain reaction; Randomly Amplified Polymorphic DNA (RAPD); STD clinic = sexually transmitted disease clinic; STI = sexually transmitted infection; TRLFP = Terminal Restriction Fragment Length Polymorphism; USA = United States of America; VMB = vaginal microbiota; WSW = women having sex with women.</p>1<p>Until 15 November 20131;</p>2<p>One article could include more than one category;</p>3<p>Europe other: Sweden (4), Greenland (1), Estonia (1), Austria (1);</p>4<p>Asia other: Japan (2), South Korea (1);</p>5<p>East Africa: Kenya (3), Tanzania (1);</p>6<p>West Africa: Burkina Faso (1), Ghana, Togo, Guinea and Mali (1);</p>7<p>Central America: Mexico (2), Costa Rica (1);</p>8<p>5 publications representing only 3 studies;</p>9<p>Genes sequenced: 16S rRNA gene V1–V2 (8), V2–V3 (2), V3 (2), V3–V5 (3), V4–V6 (2), V6 (2), V6–V9 (1), chaperonin gene (1), and not reported (1);</p>10<p>Fingerprinting techniques used: TRFLP (5), DGGE (5), RAPD (2), and LH-PCR (1);</p>11<p>Hybridisation techniques used: DNA microarray (4) and Luminex (oligonucleotides coupled to beads; 1);</p>12<p>Descriptive other includes effects of race/ethnicity, pregnancy, menstrual cycle, menopause, tampon/pad use, vaginal douching, sexual debut and other sexual and contraceptive behaviours;</p>13<p>Cervical or endometrial microbiota (3) and oral and/or rectal reservoirs (3);</p>14<p>Other confirmed infections: Candidiasis (2), cervicitis (1), endometritis (1), and gingivitis (1).</p

Relative abundances of <i>L</i>. <i>iners</i> proteins in cervicovaginal lavages of women with positive <i>L</i>. <i>iners</i> 16S rDNA microarray results (n = 31) among three cervicovaginal microbiota groups.

<p>GAPDH_1, GAPDH_2, ALDO, GPI, and DPS were significantly decreased in women with moderate and severe dysbiosis, compared to women with a <i>L</i>. <i>iners</i>-dominant cervicovaginal microbiota. Box plots represent median (black line), first and third quartiles (box) and range within 1.5 times the interquartile range from the box (whiskers). Outliers are plotted as points. *p-value<0.05; ** p-value<0.01; ***p-value<0.001. Abbreviations: GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ALDO: fructose-bisphosphate aldolase; GPI: glucose-6-phosphate isomerase; DPS: DNA starvation/stationary phase protection protein; EFtu: elongation factor Tu; PK: pyruvate kinase.</p

Cervicovaginal <i>Lactobacillus iners</i> and <i>L</i>. <i>crispatus</i> proteins identified by mass spectrometry.

<p>Cervicovaginal <i>Lactobacillus iners</i> and <i>L</i>. <i>crispatus</i> proteins identified by mass spectrometry.</p

Relative abundances of <i>L</i>. <i>iners</i> proteins in cervicovaginal lavages of women with positive <i>L</i>. <i>iners</i> 16S rDNA microarray results (n = 31) among three vaginal pH categories.

<p>GAPDH_1, GAPDH_2, ALDO, GPI, and DPS were significantly decreased in women with a vaginal pH ≥5, compared to women with a vaginal pH between 4 and 5. Box plots represent median (black line), first and third quartiles (box) and range within 1.5 times the interquartile range from the box (whiskers). Outliers are plotted as points. *p-value<0.05; ** p-value<0.01; ***p-value<0.001. Abbreviations: GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ALDO: fructose-bisphosphate aldolase; GPI: glucose-6-phosphate isomerase; DPS: DNA starvation/stationary phase protection protein; EFtu: elongation factor Tu; PK: pyruvate kinase.</p