Atopobium vaginae and Prevotella bivia are able to incorporate and influence gene expression in a pre-formed Gardnerella vaginalis biofilm

Bacterial vaginosis (BV) is associated with a highly structured polymicrobial biofilm on the vaginal epithelium where Gardnerella species presumably play a pivotal role. Gardnerella vaginalis, Atopobium vaginae, and Prevotella bivia are vaginal pathogens detected during the early stages of incident BV. Herein, we aimed to analyze the impact of A. vaginae and P. bivia on a pre-established G. vaginalis biofilm using a novel in vitro triple-species biofilm model. Total biofilm biomass was determined by the crystal violet method. We also discriminated the bacterial populations in the biofilm and in its planktonic fraction by using PNA FISH. We further analyzed the influence of A. vaginae and P. bivia on the expression of key virulence genes of G. vaginalis by quantitative PCR. In our tested conditions, A. vaginae and P. bivia were able to incorporate into pre-established G. vaginalis biofilms but did not induce an increase in total biofilm biomass, when compared with 48-h G. vaginalis biofilms. However, they were able to significantly influence the expression of HMPREF0424_0821, a gene suggested to be associated with biofilm maintenance in G. vaginalis. This study suggests that microbial relationships between co-infecting bacteria can deeply affect the G. vaginalis biofilm, a crucial marker of BV.This research was partially funded by the National Institute of Allergy and Infectious Diseases (R01AI146065-01A1). It was also partially funded by the Portuguese Foundation for Science and Technology (FCT), by the research project (PTDC/BIA-MIC/28271/2017), under the scope of COMPETE 2020 (POCI-01-0145-FEDER-028271), and by the strategic funding of unit (UIDB/04469/2020).info:eu-repo/semantics/publishedVersio

Assessing recovery rates of distinct exogenous controls for gDNA extraction efficiency using phenol-chloroform or silica-column based extractions

Assessment of genomic DNA (gDNA) extraction efficiency is required for accurate bacterial quantification by qPCR. Exogenous DNA molecules are often added after bacterial cultures are lysed, but before DNA purification steps, to determine extraction efficiency. Herein we found that different exogenous DNA controls have different recovery rates, suggesting distinct DNA extraction efficiencies. Recovery rates are also affected by the gDNA extraction method being more affected in silica-based columns than in phenol-chloroform extraction. Overall, we determined that the use of long DNA fragments, such as gDNA, as exogenous controls have a higher recovery rate than use of smaller size DNA molecules.This work was partially funded by the Portuguese Foundation for Science and Technology (FCT), with the strategic funding of the unit (UIDB/04469/2020). It was also partially funded by the National Institute of Allergy and Infectious Diseases (R01AI146065-01A1 to CAM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation.info:eu-repo/semantics/publishedVersio

Bacterial vaginosis multi-species biofilms: can standard quantification methods accurately quantify in vitro biofilms?

Background. While it is well established that Bacterial Vaginosis (BV), the most common cause of vaginal discharge, involves the presence of a multi-species biofilm adhered to vaginal epithelial cells, in-depth study has been limited due to the complexity of the bacterial community comprising the biofilm. Assessing bacterial interactions between bacterial species that inhabit the BV biofilm can provide key information regarding synergism or antagonism between these species and provide insights into the pathogenesis of BV. Thus, proper biofilm quantification approaches are essential to further this body of research. Objectives. To evaluate BV biofilm formation by several key individual BV-associated bacteria (Gardnerella vaginalis, Fannyhessea vaginae, and Prevotella bivia) and compare with a multispecies biofilm formed simultaneously by all three bacterial species. Methods. Single- or multi-species biofilms were quantified by the crystal violet (CV) staining method, total cell counts by epifluorescence microscopy, and the plate counting technique (CFU); individual traits were assessed by bacterial species. Results. Each individual species had a unique signature assessed by the distinct relationship between the total number of cells, CFUs, and total biofilm biomass. Conclusions & Significance: The assessment of multi-species BV biofilm quantification results in significant bias, mainly since individual species quantification signatures cant be related to the multi-species consortia. To minimize this bias, a multiple-technical approach should be considered when quantifying multi-species BV biofilms, to circumvent the caveats of individual techniques alone, tailoring a more complete picture of the biofilm-forming capacity of key bacterial species and furthering the field of BV pathogenesis research.This work was partially supported by the Portuguese Foundation for Science and Technology (FCT) by the research project [PTDC/BIA-MIC/28271/2017] under the scope of COMPETE 2020 [POCI-01-0145-FEDER 028271] and by the strategic funding of unit [UID/BIO/04469/2020]. It was also partially funded by the National Institute of Allergy and Infectious Diseases (R01AI146065-01A1)info:eu-repo/semantics/publishedVersio

A novel Gardnerella, Prevotella, and Lactobacillus standard that improves accuracy in quantifying bacterial burden in vaginal microbial communities

Bacterial vaginosis (BV) is the most common vaginal dysbiosis. In this condition, a polymicrobial biofilm develops on vaginal epithelial cells. Accurately quantifying the bacterial burden of the BV biofilm is necessary to further our understanding of BV pathogenesis. Historically, the standard for calculating total bacterial burden of the BV biofilm has been based on quantifying Escherichia coli 16S rRNA gene copy number. However, E. coli is improper for measuring the bacterial burden of this unique micro-environment. Here, we propose a novel qPCR standard to quantify bacterial burden in vaginal microbial communities, from an optimal state to a mature BV biofilm. These standards consist of different combinations of vaginal bacteria including three common BV-associated bacteria (BVAB) Gardnerella spp. (G), Prevotella spp. (P), and Fannyhessea spp. (F) and commensal Lactobacillus spp. (L) using the 16S rRNA gene (G:P:F:L, G:P:F, G:P:L and 1G:9L). We compared these standards to the traditional E. coli (E) reference standard using known quantities of mock vaginal communities and 16 vaginal samples from women. The E standard significantly underestimated the copy numbers of the mock communities, and this underestimation was significantly greater at lower copy numbers of these communities. The G:P:L standard was the most accurate across all mock communities and when compared to other mixed vaginal standards. Mixed vaginal standards were further validated with vaginal samples. This new G:P:L standard can be used in BV pathogenesis research to enhance reproducibility and reliability in quantitative measurements of BVAB, spanning from the optimal to non-optimal (including BV) vaginal microbiota

Persistent Chlamydia trachomatis, Neisseria gonorrhoeae or Trichomonas vaginalis positivity after treatment among human immunodeficiency virus-infected pregnant women, South Africa

The objective of this study is to assess the predictors and frequency of persistent sexually transmitted infection (STI) positivity in human immunodeficiency virus (HIV)-infected pregnant women treated for Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) or Trichomonas vaginalis (TV) infection. We enrolled HIV-infected pregnant women attending their first antenatal care visit and tested them for urogenital CT, NG and TV infection using Xpert® CT/NG and TV assays (Cepheid, Sunnyvale, CA). Those testing positive were treated. Participants either notified partners to seek treatment or were given extra medication to deliver to partners for treatment. Repeat testing was conducted approximately 21 days post-treatment or treatment initiation. Among 427 participants, 172 (40.3%) tested positive for any STI. Of the 136 (79.1%) that returned for repeat testing, 36 (26.5%) tested positive for the same organism: CT = 27 (26.5%), NG = 1 (6.3%), TV = 11 (16.7%). Persistent CT positivity was independently associated with having more than one sex partner in the preceding 12 months (adjusted-prevalence ratio [aPR] = 3.03, 95% CI: 1.44–6.37) and being newly diagnosed with HIV infection during the first antenatal care visit compared to those currently on antiretroviral therapy (aPR = 3.97, 95% CI: 1.09–14.43). Persistent TV positivity was associated with not knowing if a partner sought treatment following STI disclosure (aPR = 12.6, 95% CI: 2.16–73.5) and prior diagnosis of HIV but not currently on antiretroviral therapy. (aPR = 4.14; 95% CI: 1.25–13.79). We identified a high proportion of HIV-infected pregnant women with persistent CT or TV positivity after treatment. To decrease the risk of re-infection, enhanced strategies for partner treatment programmes are needed to improve the effectiveness of STI screening and treatment in pregnancy. The relationship between not being on antiretroviral therapy and persistent STI positivity needs further study.The Eunice Kennedy Shriver Institute of Child Health and Human Development, National Institutes of Health (NIH), award R21HD084274 and the President’s Emergency Plan for AIDS Relief through the United States Agency of the Cooperative Agreement AID 674-A-12-00017 funded this study. Noah Kojima was supported by the U.S. NIH Fogarty International Center (award number D43TW009343) and the University of California Global Health Institute. Christina A Muzny was supported by K23AI106957-01A1 from the National Institute of Allergy and Infectious Diseases.https://journals.sagepub.com/home/stdhj2021Medical MicrobiologyPaediatrics and Child Healt

Sexually transmitted infection screening to prevent adverse birth and newborn outcomes: study protocol for a randomized-controlled hybrid-effectiveness trial

Background Sexually transmitted infections (STIs) during pregnancy are associated with adverse birth outcomes, including preterm birth, low birth weight, perinatal death, and congenital infections such as increased mother-to-child HIV transmission. Prevalence of STIs among pregnant women in South Africa remains high, with most women being asymptomatic for their infection(s). Unfortunately, most STIs remain undetected and untreated due to standard practice syndromic management in accordance with World Health Organization (WHO) guidelines. Although lab-based and point-of-care molecular tests are available, optimal screening strategies during pregnancy, their health impact, and cost-effectiveness are unknown. Methods We will implement a 3-arm (1:1:1) type-1 hybrid effectiveness-implementation randomized-controlled trial (RCT). We will enroll 2500 pregnant women attending their first antenatal care (ANC) visit for their current pregnancy at participating health facilities in Buffalo City Metro District, Eastern Cape Province, South Africa. Participants allocated to arms 1 and 2 (intervention) will receive GeneXpert® point-of-care diagnostic testing for Neisseria gonorrhoeae, Chlamydia trachomatis, and Trichomonas vaginalis, with same-day treatment for detected infection(s). Arm 1 will additionally receive a test-of-cure 3 weeks post-treatment, while Arm 2 will receive a repeat test at 30–34 weeks’ gestation. Those allocated to Arm 3 will receive syndromic management (standard-of-care). The RE-AIM framework will be used to guide collection of implementation indicators to inform potential future scale up. Primary outcome measures include (1) frequency of adverse birth outcomes among study arms, defined by a composite measure of low birth weight and pre-term delivery, and (2) change in STI prevalence between baseline and birth outcome among intervention arms and compared to standard-of-care. Estimates and comparative costs of the different screening strategies relative to standard-of-care and the costs of managing adverse birth outcomes will be calculated. Cost-effectiveness will be assessed per STI and disability-adjusted life year averted. Discussion This trial is the first RCT designed to identify optimal, cost-effective screening strategies that decrease the burden of STIs during pregnancy and reduce adverse birth outcomes. Demonstrating the impact of diagnostic screening and treatment, compared to syndromic management, on birth outcomes will provide critical evidence to inform changes to WHO guidelines for syndromic management of STIs during pregnancy. Trial registration ClinicalTrials.gov NCT04446611 . Registered on 25 June 2020

A common allele in RPGRIP1L is a modifier of retinal degeneration in ciliopathies

Despite rapid advances in the identification of genes involved in disease, the predictive power of the genotype remains limited, in part owing to poorly understood effects of second-site modifiers. Here we demonstrate that a polymorphic coding variant of RPGRIP1L (retinitis pigmentosa GTPase regulator-interacting protein-1 like), a ciliary gene mutated in Meckel-Gruber (MKS) and Joubert (JBTS) syndromes, is associated with the development of retinal degeneration in individuals with ciliopathies caused by mutations in other genes. As part of our resequencing efforts of the ciliary proteome, we identified several putative loss-of-function RPGRIP1L mutations, including one common variant, A229T. Multiple genetic lines of evidence showed this allele to be associated with photoreceptor loss in ciliopathies. Moreover, we show that RPGRIP1L interacts biochemically with RPGR, loss of which causes retinal degeneration, and that the Thr229-encoded protein significantly compromises this interaction. Our data represent an example of modification of a discrete phenotype of syndromic disease and highlight the importance of a multifaceted approach for the discovery of modifier alleles of intermediate frequency and effect.This work was supported by grants R01EY007961 from the National Eye Institute (H.K. and A.S.), R01HD04260 from the National Institute of Child Health and Development (N.K.), R01DK072301, R01DK075972 (N.K.), R01DK068306, R01DK064614, R01DK069274 (F.H.), NRSA fellowship F32 DK079541 (E.E.D.) from the National Institute of Diabetes, Digestive and Kidney disorders, Intramural program of NEI (A.S.), the Macular Vision Research Foundation (N.K.), the Foundation for Fighting Blindness (H.K., S.S.B., A.S. and N.K.), the Foundation for Fighting Blindness Canada (R.K.K.), Le Fonds de la recherche en sante du Québec (FRSQ) (R.K.K.), Research to Prevent Blindness (A.S.), Harold Falls Collegiate Professorship (A.S.), the Midwest Eye Banks and Transplantation Center (H.K.), the Searle Scholars Program (M.A.B.), the Deutsche Forschungsgemeinschaft (DFG grant BE 3910/4-1; C.B.) the UK Medical Research Council (grant number G0700073; C.A.J.), NIHR Biomedical Research Centre for Ophthalmology (S.S.B.) and EU-GENORET Grant LSHG-CT-2005-512036 (S.S.B.). F.H. is an investigator of the Howard Hughes Medical Institute (HHMI) and a Doris Duke Distinguished Clinical Scientist (DDCF)

The genomes of two key bumblebee species with primitive eusocial organization

Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats. Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits. Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation