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The impact of peripheral artery disease (PAD) in patients undergoing chronic total occlusion (CTO) percutaneous coronary intervention (PCI) has received limited study. We reviewed 3999 CTO PCIs performed in 3914 patients between 2012 and 2018 at 25 centers, 14% of whom had a history of PAD. We compared the clinical and angiographic characteristics and procedural outcomes of patients with versus without history of PAD. Patients with PAD were older (67 ± 9 vs 64 ± 10 years, P < .001) and had a higher prevalence of cardiovascular risk factors. They also had more complex lesions as illustrated by higher Japanese CTO score (2.7 ± 1.2 vs 2.4 ± 1.3, P < .001). In patients with PAD, the final crossing technique was less often antegrade wire escalation (40% vs 51%, P < .001) and more often the retrograde approach (23 vs 20%, P < .001) and antegrade dissection/reentry (20% vs 16%, P < .001). Technical success was similar between the 2 study groups (84% vs 87%, P = .127), but procedural success was lower for patients with PAD (81% vs 85%, P = .015). The incidence of in-hospital major adverse cardiac events was higher among patients with PAD (3% vs 2%, P = .046). In conclusion, patients with PAD undergoing CTO PCI have more comorbidities, more complex lesions, and lower procedural success.

Bacterial lipases play important roles during infection. The Staphylococcus aureus genome contains several genes that encode well-characterized lipases and several genes predicted to encode lipases or esterases for which the function has not yet been established. In this study, we sought to define the function of an uncharacterized S. aureus protein, and we propose the annotation S. aureus lipase 3 (SAL3) (SAUSA300_0641). We confirmed that SAL3 is a lipase and that it is surface associated and secreted through an unknown mechanism. We determined that SAL3 specifically hydrolyzes short chain (4-carbon and fewer) fatty acids and specifically binds negatively charged lipids including phosphatidic acid, phosphatidylinositol phosphate, and phosphatidylglycerol, which is the most abundant lipid in the staphylococcal cell membrane. Mutating the catalytic triad S66-A, D167-A, S168-A, and H301-A in the recombinant protein abolished lipase activity without altering binding to host lipid substrates. Taken together we report the discovery of a novel lipase from S. aureus specific to short chain fatty acids with yet to be determined roles in host pathogen interactions.

Background When crossing into the distal true lumen fails during chronic total occlusion (CTO) percutaneous coronary intervention (PCI), subintimal plaque modification (SPM) is often performed to restore antegrade flow and facilitate subsequent lesion recanalization. Methods Between January 2012 and May 4, 2019, 4,659 CTO PCIs were included in the PROGRESS-CTO registry, of which 935 (20%) had a prior unsuccessful attempt. Of those 935 patients, 119 (13%) had prior SPM. We analyzed the outcomes of the 58 SPM procedures for which data were available, as well as the outcomes of the 60 subsequent CTO PCI attempts. Results Mean patient age was 67 ± 9 years and 86% were men. Patients had high prevalence of cardiovascular risk factors such as dyslipidemia (91%), hypertension (93%) diabetes (48%), prior PCI (61%), and prior coronary artery bypass graft surgery (47%). The target CTO lesions often had proximal cap ambiguity (54%), moderate/severe calcification (73%), moderate/severe tortuosity (63%), and high J-CTO score (mean 3.2 ± 1.1). The technical and procedural success of subsequent CTO PCI were high (83% for both) with an acceptable rate of in-hospital major adverse cardiovascular events (3.3%). Technical and procedural success were higher for repeat attempts that were performed ≥60 days after the index CTO PCI (94% vs. 69%, p = .015). Median (interquartile range) subsequent procedure time was 147 (100, 215) min, contrast volume was 185 (150, 260) ml, and air kerma radiation dose was 2.5 (1.4, 4.2) Gray. Conclusion Repeat CTO PCI attempts after SPM are associated with high likelihood for successful revascularization with acceptable risks.

The microbiome of the female reproductive tract has implications for women’s reproductive health. We examined the vaginal microbiome in two cohorts of women who experienced normal term births: a cross-sectionally sampled cohort of 613 pregnant and 1,969 non-pregnant women, focusing on 300 pregnant and 300 non-pregnant women of African, Hispanic or European ancestry case-matched for race, gestational age and household income; and a longitudinally sampled cohort of 90 pregnant women of African or non-African ancestry. In these women, the vaginal microbiome shifted during pregnancy toward Lactobacillus-dominated profiles at the expense of taxa often associated with vaginal dysbiosis. The shifts occurred early in pregnancy, followed predictable patterns, were associated with simplification of the metabolic capacity of the microbiome and were significant only in women of African or Hispanic ancestry. Both genomic and environmental factors are likely contributors to these trends, with socioeconomic status as a likely environmental influence. Ancestry and socioeconomic factors influence predictable changes in the vaginal microbiome that occur early in pregnancy in women who experience normal term birth.

Background The human placenta has been traditionally viewed as sterile, and microbial invasion of this organ has been associated with adverse pregnancy outcomes. Yet, recent studies that utilized sequencing techniques reported that the human placenta at term contains a unique microbiota. These conclusions are largely based on the results derived from the sequencing of placental samples. However, such an approach carries the risk of capturing background-contaminating DNA (from DNA extraction kits, polymerase chain reaction reagents, and laboratory environments) when low microbial biomass samples are studied. Objective To determine whether the human placenta delivered at term in patients without labor who undergo cesarean delivery harbors a resident microbiota (“the assemblage of microorganisms present in a defined niche or environment”). Study Design This cross-sectional study included placentas from 29 women who had a cesarean delivery without labor at term. The study also included technical controls to account for potential background-contaminating DNA, inclusive in DNA extraction kits, polymerase chain reaction reagents, and laboratory environments. Bacterial profiles of placental tissues and background technical controls were characterized and compared with the use of bacterial culture, quantitative real-time polymerase chain reaction, 16S ribosomal RNA gene sequencing, and metagenomic surveys. Results (1) Twenty-eight of 29 placental tissues had a negative culture for microorganisms. The microorganisms retrieved by culture from the remaining sample were likely contaminants because corresponding 16S ribosomal RNA genes were not detected in the same sample. (2) Quantitative real-time polymerase chain reaction did not indicate greater abundances of bacterial 16S ribosomal RNA genes in placental tissues than in technical controls. Therefore, there was no evidence of the presence of microorganisms above background contamination from reagents in the placentas. (3) 16S ribosomal RNA gene sequencing did not reveal consistent differences in the composition or structure of bacterial profiles between placental samples and background technical controls. (4) Most of the bacterial sequences obtained from metagenomic surveys of placental tissues were from cyanobacteria, aquatic bacteria, or plant pathogens, which are microbes unlikely to populate the human placenta. Coprobacillus, which constituted 30.5% of the bacterial sequences obtained through metagenomic sequencing of placental samples, was not identified in any of the 16S ribosomal RNA gene surveys of these samples. These observations cast doubt as to whether this organism is really present in the placenta of patients at term not in labor. Conclusion With the use of multiple modes of microbiologic inquiry, a resident microbiota could not be identified in human placentas delivered at term from women without labor. A consistently significant difference in the abundance and/or presence of a microbiota between placental tissue and background technical controls could not be found. All cultures of placental tissue, except 1, did not yield bacteria. Incorporating technical controls for potential sources of background-contaminating DNA for studies of low microbial biomass samples, such as the placenta, is necessary to derive reliable conclusions.

During chronic total occlusion (CTO) percutaneous coronary intervention (PCI), sometimes non-CTO lesions are also treated.We compared the clinical and procedural characteristics and outcomes of CTO PCIs with and without concomitant treatment of a non-CTO lesion in a contemporary multicenter CTO registry.Of the 3598 CTO PCIs performed at 21 centers between 2012 and 2018, 814 (23%) also included PCI of at least one non-CTO lesion. Patients in whom non-CTO lesions were treated were older (65 ± 10 vs. 64 ± 10 years, p = 0.03), more likely to present with an acute coronary syndrome (32% vs. 23%, p 0.01), and less likely to undergo PCI of a right coronary artery (RCA) CTO (46% vs. 58%, p 0.01). The most common non-CTO lesion location was the left anterior descending artery (31%), followed by the circumflex (29%) and the RCA (25%).Combined non-CTO and CTO-PCI procedures had similar technical (88% vs. 87%, p = 0.33) and procedural (85% vs. 85%, p = 0.74) success and major in-hospital complication rates (3.4% vs. 2.7%, p = 0.23), but had longer procedure duration (131 [88, 201] vs. 117 [75, 179] minutes, p 0.01), higher patient air kerma radiation dose (3.0 [1.9, 4.8] vs. 2.8 [1.5, 4.6] Gray, p 0.01) and larger contrast volume (300 [220, 380] vs. 250 [180, 350] ml, p 0.01).Combined CTO PCI with PCI of non-CTO lesions is associated with similar success and major in-hospital complication rates compared with cases in which only CTOs were treated, but requires longer procedure duration and higher radiation dose and contrast volume.

The incidence of preterm birth exceeds 10% worldwide. There are significant disparities in the frequency of preterm birth among populations within countries, and women of African ancestry disproportionately bear the burden of risk in the United States. In the present study, we report a community resource that includes 'omics' data from approximately 12,000 samples as part of the integrative Human Microbiome Project. Longitudinal analyses of 16S ribosomal RNA, metagenomic, metatranscriptomic and cytokine profiles from 45 preterm and 90 term birth controls identified harbingers of preterm birth in this cohort of women predominantly of African ancestry. Women who delivered preterm exhibited significantly lower vaginal levels of Lactobacillus crispatus and higher levels of BVAB1, Sneathia amnii, TM7-H1, a group of Prevotella species and nine additional taxa. The first representative genomes of BVAB1 and TM7-H1 are described. Preterm-birth-associated taxa were correlated with proinflammatory cytokines in vaginal fluid. These findings highlight new opportunities for assessment of the risk of preterm birth.

The impact of intravascular ultrasound (IVUS) for stent optimization on long-term outcomes in Chronic Total Occlusion (CTO) Percutaneous Coronary Intervention (PCI) has received limited study. We examined the outcomes of CTO PCI with and without IVUS use for stent optimization in 942 CTO PCIs

Mucosal surfaces of the female reproductive tract contain a variety of antimicrobial components that provide the first line of defense against bacteria involved in the development of bacterial vaginosis (BV). Microbiological analysis of BV has shown Gardnerella vaginalis to be a prominent species in BV development. However, G. vaginalis colonization does not always lead to BV. Over the last decade, phenotypic and genotypic studies have demonstrated the existence of strain variants. Therefore, this study aimed to investigate if the major components of the vaginal immune response, specifically lysozyme, lactoferrin and β-defensin 2, differently affected virulence traits of G. vaginalis strains isolated from healthy women or from women with BV. Gardnerella vaginalis strains were first genotyped by the clade classification system and then phenotypically characterized. Our results revealed that key differences in initial adhesion existed among the isolates but that these differences could not be predicted using the clade-genotyping approach. Importantly, we found that growth, initial adhesion and biofilm formation were strongly affected by lysozymes, but at similar levels in both groups, suggesting that the response to host immune components is not a distinguishing characteristic of isolates from women with BV versus those from healthy women., This study was supported by the project PTDC/BIA-MIC/ 28271/2017 funded by Portuguese Foundation for Science and Technology (FCT), under the scope of COMPETE 2020 (POCI01–0145-FEDER-028271) and by the strategic funding of unit UID/BIO/04469/2013., info:eu-repo/semantics/publishedVersion

Studies have implicated Gardnerella vaginalis as an important etiological agent in bacterial vaginosis (BV). It produces a cholesterol-dependent cytolysin, vaginolysin (VLY). In this study, we sought to characterize the interaction between vaginal epithelium, G. vaginalis, and VLY using EpiVaginal tissues from MatTek. These tissues are three-dimensional and have distinct apical and basolateral sides, enabling comparison of the effects of G. vaginalis and VLY following exposure to either side. We measured cytotoxicity, cytokine production, and bacterial growth, following apical versus basolateral exposure. G. vaginalis exhibited more-rapid growth in coculture with the tissue model when it was exposed to the apical side. VLY permeabilized cells on the basolateral side of the tissues but failed to permeabilize apical epithelial cells. Cytokine secretion in response to VLY and G. vaginalis also depended on the polarity of exposure. VLY did not cause significant changes in cytokine levels when exposed apically. Apical tissue challenge by G. vaginalis appeared to dampen the inflammatory response, as decreases in granulocyte-macrophage colony-stimulating factor (GM-CSF) (6.6-fold), RANTES (14.8-fold), and interferon gamma inducible protein 10 kDa (IP-10) (53-fold) and an increase in interleukin-1 receptor antagonist (IL-1ra) (5-fold) were observed. In vivo, G. vaginalis normally colonizes the apical face of the vaginal epithelium. Results from this study suggest that while G. vaginalis may grow on the apical face of the vaginal epithelium, its VLY toxin does not target these cells in this model. This phenomenon could have important implications regarding colonization of the vagina by G. vaginalis and may suggest an explanation for the lack of an overt immune response to this organism.

Objective: Evidence supports an inverse association between vitamin D and bacterial vaginosis (BV) during pregnancy. Furthermore, both the vaginal microbiome and vitamin D status correlate with pregnancy outcome. Women of African ancestry are more likely to experience BV, to be vitamin D deficient, and to have certain pregnancy complications. We investigated the association between vitamin D status and the vaginal microbiome. Study Design: Subjects were assigned to a treatment (4 400 IU), or a control group (400 IU vitamin D daily), sampled 3 times during pregnancy, and vaginal 16S rRNA profiles and plasma 25-hydroxyvitamin D [25(OH)D] concentrations were examined. Result: Gestational age and ethnicity were significantly associated with the microbiome. Megasphaera correlated negatively (p = 0.0187) with 25(OH)D among women of African ancestry. Among controls, women of European ancestry exhibited a positive correlation between plasma 25(OH)D and L. crispatus abundance. Conclusion: Certain vaginal bacteria are associated with plasma 25(OH)D concentration.

Background Frequent laboratory testing may be necessary at times for critically ill patients. However, the practice of indiscriminate laboratory test ordering is common. Purpose The purpose of this quality improvement project was to assess the effectiveness of the acute care nurse practitioner (ACNP) in reducing the number of unwarranted laboratory tests ordered for ICU patients. To determine whether the presence of an ACNP would make a difference, an ACNP was present on daily ICU multidisciplinary rounds to facilitate the discussion of the laboratory testing needs for each patient for the following 24-hour period. Conclusions Eighty-one patients were enrolled in the project, 41 in the comparison and 40 in the intervention group. No significant differences were noted between the two groups. The project demonstrated that although there was an increase in tests ordered for the intervention group, the increase was brought about by an increase in specific individual tests rather than an increase in panels of laboratory tests. A reduction in patient cost was observed for the number of tests ordered. No increase in adverse events was noted. Implications for practice Acute care nurse practitioner presence on multidisciplinary rounds may be an effective method to change the practice toward the ordering of tests based on clinical indication.

Modification of the host lipidome via secreted enzymes is an integral, but often overlooked aspect of bacterial pathogenesis. In the current era of prevalent antibiotic resistance, knowledge regarding critical host pathogen lipid interactions has the potential for use in developing novel antibacterial agents. While most studies to date on this matter have focused on specific lipids, or select lipid classes, this provides an incomplete picture. Modern methods of untargeted lipidomics have the capacity to overcome these gaps in knowledge and provide a comprehensive understanding of the role of lipid metabolism in the pathogenesis of infections. In an attempt to determine the role of lipid modifying enzymes produced by staphylococci, we exposed bovine heart lipids, a standardized model for the mammalian lipidome, to spent medium from staphylococcal cultures, and analyzed lipid molecular changes by MS/MSALL shotgun lipidomics. We elucidate distinct effects of different staphylococcal isolates, including 4 clinical isolates of the pathogenic species Staphylococcus aureus, a clinical isolate of the normally commensal species S. epidermidis, and the non-pathogenic species S. carnosus. Two highly virulent strains of S. aureus had a more profound effect on mammalian lipids and modified more lipid classes than the other staphylococcal strains. Our studies demonstrate the utility of the applied untargeted lipidomics methodology to profile lipid changes induced by different bacterial secretomes. Finally, we demonstrate the promise of this lipidomics approach in assessing the specificity of bacterial enzymes for mammalian lipid classes. Our data suggests that there may be a correlation between the bacterial expression of lipid-modifying enzymes and virulence, and could facilitate the guided discovery of lipid pathways required for bacterial infections caused by S. aureus and thereby provide insights into the generation of novel antibacterial agents.

The microbiome influences humans in many still underappreciated respects, including but not limited to development and growth, immunity, metabolism and even behavior1,2. Most bacterial communities exist in mutualistic relationships with the healthy human host, and it is clear that our microbiota evolved in concert with our genome, the product of which is a true human-microbial symbiosis. However, it is also clear that microbial dysbiosis can result in disease, and the outgrowth of opportunistic pathogens can threaten the health and life of the human host. Fueled in part by the Human Microbiome Project (HMP) of the National Institutes of Health (NIH), and similar efforts by other groups worldwide3–5, large-scale efforts have been made to define the “normal” microbiome of healthy individuals across multiple body sites. Facilitated by the advent of next-generation sequencing, a major success of the first phase of these efforts has been the wealth of data generated, which collectively has revealed the previously poorly recognized complexity and dynamic nature of the human microbiome and its stunning impacts on human health and well-being. To further explore the functional role of the microbiome in human health and disease, the NIH has launched HMP2, now termed the integrative HMP or iHMP, a second phase of study that mandates a more in depth ‘multi-omic’ approach to explore host-bacterial interactions and community dynamics in the context of human health and disease. The Vaginal Microbiome Consortium (vmc.vcu.edu) at Virginia Commonwealth University (VCU) has a two-stage project funded by the NIH HMP1 and iHMP programs. The first stage, the Vaginal Human Microbiome Project, is a cross-sectional community based study on over 6,000 visitors to multiple women's clinics in Central Virginia, with the goal of investigating the roles of the vaginal microbiome in women's urogenital health. Vaginal and buccal samples were collected from women volunteers over the age of 18, with the exception of women who were incarcerated, independent of their state of health. Embedded within this study is the collection and analysis of samples from approximately 250 monozygotic and dizygotic twin pairs from VCU's Mid Atlantic Twin Registry6. The microbial populations in each sample were defined by high-throughput metagenomic 16S rRNA gene sequencing, whole metagenome shotgun analysis, and by microbiologically culturing, cloning by single colony isolation and sequencing of the genomes of target bacterial species or taxa. In the Multi-Omic Microbiome Study-Pregnancy Initiative, the second stage of this program, samples from over 2,000 pregnant women and their infants are being collected longitudinally at multiple prenatal visits during their pregnancies, at delivery and at early post natal visits. Samples are collected from the vagina, rectum, nares, mouth and skin from each participant over the age of 15 who is not incarcerated and who is not a surrogate. Samples from these participants are subjected to six ‘omics’ technologies: i) targeted 16S rRNA gene surveys to generate species-level microbiome profiles; ii) whole genome sequencing of relevant taxa that we are able to culture and bacteriologically clone; iii) whole metagenomic shotgun sequencing (WMGS) to generate ‘gene-centric’ and ‘taxonomy-centric’ profiles of the metabolic and pathogenic potential, and to generate genome sequences of abundant taxa that we are unable to cultivate; iv) metatranscriptomic analysis to assess expression levels of relevant prokaryotic and host genes; v) metabolomic/lipidomic analyses to provide insight into the signaling and regulatory pathways controlling the environment in the vagina; and vi) immunoproteomic analyses to measure cytokines and immune factors impacting vaginal function during pregnancy. The objective of the latter study is to elucidate the role(s) of the vaginal microbiome in the etiology or prevention of adverse outcomes of pregnancy, with a specific focus on preterm birth and stillbirth.

Arthropods transmit diverse infectious agents; however, the ways microbes influence their vector to enhance colonization are poorly understood. Ixodes scapularis ticks harbor numerous human pathogens, including Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis. We now demonstrate that A. phagocytophilum modifies the I. scapularis microbiota to more efficiently infect the tick. A. phagocytophilum induces ticks to express Ixodes scapularis antifreeze glycoprotein (iafgp), which encodes a protein with several properties, including the ability to alter bacterial biofilm formation. IAFGP thereby perturbs the tick gut microbiota, which influences the integrity of the peritrophic matrix and gut barrier-critical obstacles for Anaplasma colonization. Mechanistically, IAFGP binds the terminal d-alanine residue of the pentapeptide chain of bacterial peptidoglycan, resulting in altered permeability and the capacity of bacteria to form biofilms. These data elucidate the molecular mechanisms by which a human pathogen appropriates an arthropod antibacterial protein to alter the gut microbiota and more effectively colonize the vector.

Background Bacterial vaginosis (BV) is the leading dysbiosis of the vaginal microbiome. The pathways leading towards the development of BV are not well understood. Gardnerella vaginalis is frequently associated with BV. G. vaginalis produces the cholesterol-dependent cytolysin (CDC), vaginolysin, which can lyse a variety of human cells and is thought to play a role in pathogenesis. Because membrane cholesterol is required for vaginolysin to function, and because HMG-CoA reductase inhibitors (statins) affect not only serum levels of cholesterol but membrane levels as well, we hypothesized that statins might affect the vaginal microbiome. Methods To investigate the relationship between use of the statins and the vaginal microbiome, we analyzed 16S rRNA gene taxonomic surveys performed on vaginal samples from 133 women who participated in the Vaginal Human Microbiome Project and who were taking statins at the time of sampling, 152 women who reported high cholesterol levels but were not taking statins, and 316 women who did not report high cholesterol. To examine the effect of statins on the cytolytic effect of vaginolysin, the cholesterol-dependent cytolysin (CDC) produced by Gardnerella vaginalis, we assessed the effect of simvastatin pretreatment of VK2E6/E7 vaginal epithelial cells on vaginolysin-mediated cytotoxicity. Results The mean proportion of G. vaginalis among women taking statins was significantly lower relative to women not using statins. Women using statins had higher mean proportions of Lactobacillus crispatus relative to women with normal cholesterol levels, and higher levels of Lactobacillus jensenii relative to women with high cholesterol but not taking statins. In vitro, vaginal epithelial cells pretreated with simvastatin were relatively resistant to vaginolysin and this effect was inhibited by cholesterol. Conclusions In this cross-sectional study, statin use was associated with reduced proportions of G. vaginalis and greater proportions of beneficial lactobacilli within the vaginal microbiome. The negative association between statin use and G. vaginalis may be related to inhibition of vaginolysin function.

The current investigation serves to assess the usefulness, lessons learned, and leader development of eight male students in a senior level university leadership class based on reading and reflection. Thematic analysis of participants’ responses revealed three major themes to explain students’ lessons learned in leader development through journaling: (a) Relationship Development, (b) Technical Skill Development, and (c) Awareness Development. Support exists for the effectiveness of reading and reflection as a pedagogical tool for leader lesson development with a college-age sample.

The use of saphenous vein grafts (SVGs) for retrograde crossing during chronic total occlusion (CTO) percutaneous coronary intervention (PCI) has received limited study. We examined 1,615 retrograde CTO PCIs performed between 2012 and 2019 at 25 centers. We compared the clinical, angiographic, and

Bacterial vaginosis (BV) is the most common vaginal disorder in women of child-bearing age. It is widely accepted that the microbial switch from normal microflora to the flora commonly associated with BV is characterized by a decrease in vaginal colonization by specific Lactobacillus species together with an increase of G. vaginalis and other anaerobes. However, the order of events leading to the development of BV remains poorly characterized and it is unclear whether the decrease in lactobacilli is a cause or a consequence of the increase in the population density of anaerobes. Our goal was to characterize the interaction between two Gardnerella vaginalis strains, one of which was isolated from a healthy woman (strain 5-1) and the other from a woman diagnosed with BV (strain 101), and vaginal lactobacilli on the adherence to cervical epithelial cells. In order to simulate the transition from vaginal health to BV, the lactobacilli were cultured with the epithelial cells first, and then the G. vaginalis strain was introduced. We quantified the inhibition of G. vaginalis adherence by the lactobacilli and displacement of adherent lactobacilli by G. vaginalis. Our results confirmed that pathogenic G vaginalis 101 had a higher capacity for adhesion to the cervical epithelial cells than strain 5-1. Interestingly, strain 101 displaced L. crispatus but not L. iners whereas strain 5-1 had less of an effect and did not affect the two species differently. Furthermore, L. iners actually enhanced adhesion of strain 101 but not of strain 5-1. These results suggest that BV-causing G. vaginalis and L. iners do not interfere with one another, which may help to explain previous reports that women who are colonized with L. iners are more likely to develop BV., This work was supported by European Union funds (FEDER/COMPETE) and by national funds (FCT) under the project with reference FCOMP-01-0124-FEDER-008991 (PTDC/BIA-MIC/098228/2008) and in part by funds from the National Institutes of Health (P60-MD002256). AM acknowledges the FCT individual fellowship SFRH/BD/62375/2009).

Bacterial vaginosis is the most common gynecological disorder affecting women of reproductive age. Bacterial vaginosis is frequently associated with the development of a Gardnerella vaginalis biofilm. Recent data indicates that G. vaginalis biofilms are more tolerant to antibiotics and are able to incorporate other bacterial vaginosis -associated species, yielding a multi-species biofilm. However, despite its apparent role in bacterial vaginosis, little is known regarding the molecular determinants involved in biofilm formation by G. vaginalis. To gain insight into the role of G. vaginalis in the pathogenesis of bacterial vaginosis, we carried out comparative transcriptomic analysis between planktonic and biofilm phenotypes, using RNA-sequencing. Significant differences were found in the expression levels of 815 genes. A detailed analysis of the results obtained was performed based on direct and functional gene interactions. Similar to other bacterial species, expression of genes involved in antimicrobial resistance were elevated in biofilm cells. In addition, our data indicate that G. vaginalis biofilms assume a characteristic response to stress and starvation conditions. The abundance of transcripts encoding proteins involved in glucose and carbon metabolism was reduced in biofilms. Surprisingly, transcript levels of vaginolysin were reduced in biofilms relative to planktonic cultures. Overall, our data revealed that gene-regulated processes in G. vaginalis biofilms resulted in a protected form of bacterial growth, characterized by low metabolic activity. This phenotype may contribute towards the chronic and recurrent nature of bacterial vaginosis. This suggests that G. vaginalis is capable of drastically adjusting its phenotype through an extensive change of gene expression., Vaginal infection: Gene activity protects biofilms Altered gene activity may promote chronic and recurrent infections in the most common gynecological disorder in women of reproductive age. Bacterial vaginosis (BV) frequently involves the formation of biofilms by Gardnerella vaginalis bacteria. Nuno Cerca and colleagues at the University of Minho in Portugal, with co-workers elsewhere in Portugal and in the USA, studied changes in gene activity in BV biofilms. Their results linked differences in the activity of 815 genes to an ability of the bacteria to enter a protected form of growth when producing biofilms. The differences included increased activity of specific genes associated with antibiotic resistance. This insight into the bacterial ability to undergo drastic changes in gene activity could guide work to understand chronic and recurrent BV. It may also lead to new ways to treat this common and troublesome condition.

Vaginal lactobacilli can inhibit colonization by and growth of other bacteria, thereby preventing development of bacterial vaginosis (BV). Amongst the lactobacilli, Lactobacillus crispatus appears to be particularly effective at inhibiting growth of BV-associated bacteria. Nonetheless, some women who are colonized with this species can still develop clinical BV. Therefore, we sought to determine whether strains of L. crispatus that colonize women with lactobacilli-dominated vaginal microbiomes are distinct from strains that colonize women who develop BV. The genomes of L. crispatus isolates from four women with lactobacilli-dominated vaginal microbiomes ( 12 % 16S rRNA reads from bacterial taxa associated with BV) were sequenced and compared. Lactic acid production by the different strains was quantified. Phage induction in the strains was also analysed. There was considerable genetic diversity between strains, and several genes were exclusive to either the strains from Lactobacillus-dominated microbiomes or those containing BV-associated bacteria. Overall, strains from microbiomes dominated by lactobacilli did not differ from strains from microbiomes containing BV-associated bacteria with respect to lactic acid production. All of the strains contained multiple phage, but there was no clear distinction between the presence or absence of BV-associated bacteria with respect to phage-induced lysis. Genes found to be exclusive to the Lactobacillus-dominated versus BV-associated bacteria-containing microbiomes could play a role in the maintenance of vaginal health and the development of BV, respectively.

Staphylococcus aureus is a leading cause of catheter infections, and biofilm formation plays a key role in the pathogenesis. Metal ion chelators inhibit bacterial biofilm formation and viability, making them attractive candidates as components in catheter lock solutions. The goal of this study was to characterize further the effect of chelators on biofilm formation. The effect of the calcium chelators ethylene glycol tetraacetic acid (EGTA) and trisodium citrate (TSC) on biofilm formation by 30 S. aureus strains was tested. The response to subinhibitory doses of EGTA and TSC varied dramatically depending on strain variation. In some strains, the chelators prevented biofilm formation, in others they had no effect, and they actually enhanced biofilm formation in others. The molecular basis for this phenotypic variability was investigated using two related strains: Newman, in which biofilm formation was inhibited by chelators, and 10833, which formed strong biofilms in the presence of chelators. It was found that deletion of the gene encoding the surface adhesin clumping factor B (clfB) completely eliminated chelator-induced biofilm formation in strain 10833. The role of ClfB in biofilm formation activity in chelators was confirmed in additional strains. It was concluded that biofilm-forming ability varies strikingly depending on strain background, and that ClfB is involved in biofilm formation in the presence EGTA and citrate. These results suggest that subinhibitory doses of chelating agents in catheter lock solutions may actually augment biofilm formation in certain strains of S. aureus, and emphasize the importance of using these agents appropriately so that inhibitory doses are achieved consistently.

Staphylococcus aureus is the leading cause of nosocomial infections and a major cause of community-acquired infections. Biofilm formation is a key virulence determinant in certain types of S. aureus infection, especially those involving inserted medical devices. We found in a previous study that the calcium chelators sodium citrate and EGTA inhibit biofilm formation in certain strains of S. aureus but actually augment biofilm formation in other strains. Even two closely related strains, Newman and 10833, exhibited strikingly different biofilm phenotypes in the presence of calcium chelators, in that biofilm formation was inhibited in Newman but augmented in 10833. We also found that the surface protein clumping factor B (ClfB) plays a role in this phenomenon. In this study, we confirm that ClfB is required for biofilm formation under calcium-depleted conditions. We investigated the post-translational regulation of ClfB-mediated biofilm formation and found evidence that both calcium and the protease aureolysin disrupt established ClfB-dependent biofilms. Finally, we investigated the genetic basis for the biofilm-negative phenotype in strain Newman versus the biofilm-positive phenotype in strain 10833 under calcium-depleted conditions and found that strain 10833 contains a deletion that results in a stop codon within the aureolysin gene (aur). When 10833 expressed Newman aur, surface-associated ClfB and the ability to form a biofilm in chelating conditions was lost. Thus, the positive effect of chelating agents on biofilm formation in certain strains can be explained by increased ClfB activity in the absence of calcium and the discrepancy in the response of strains 10833 and Newman can be explained by point mutations in aur. This study reveals a previously unknown, to our knowledge, role for ClfB in biofilm formation and underscores the potential for striking phenotypic variability resulting from minor differences in strain background.

Staphylococcus aureus has a variety of genes that can influence the process of biofilm formation. The ability to establish a biofilm is an important virulence factor for this pathogen, and yet, the regulation of this process in vivo is not well understood. S. aureus can form biofilms on intravenous catheters and this process plays a key role in the pathogenesis of catheter infections. In order to investigate whether or not serum is conducive to the process of biofilm formation, we grew S. aureus in serum and analyzed biofilm thickness and expression of biofilm-related genes. Whereas serum supported planktonic bacterial growth, it was a potent inhibitor of biofilm formation. The inhibitory serum component had a molecular weight less than 3000 kDa. The component was protease-resistant and heat stable. The serum component induced a significant increase in the transcription of the intercellular adhesin gene icaA and the fibronectin binding protein gene fnbA. Transcription of other biofilm-related genes was affected in a strain-dependent manner. These results reveal that serum inhibits biofilm formation despite the fact that biofilms form on intravenous catheters. This may suggest that in vivo, biofilm formation is “selected for” by the force of blood flow and/or immune pressure rather than “induced” by serum.

Purpose To determine the degree of overlap between foods considered part of African American (AA) culture and those considered to be healthy. Methods A total of 44 AA men and women were recruited from the Birmingham, AL area, 25 years of age and older to participate in four Nominal Group Technique (NGT) meetings. Participants from the first two groups generated 90 unique food items in response to the question “What are the foods you associate with being African American?” Participants individually ranked their top three most unhealthy foods. The next two groups generated 116 unique food items in response to the question “What foods do you consider to be healthy?” Participants individually ranked their top three foods that were considered most associated with AA. Results The top five foods associated with AA were chitterlings, fried chicken, pig parts, greens prepared with ham hock, and pork ribs. Of the foods associated with AA, chitterlings, pig parts, fatback, fried chicken, and greens prepared with ham hocks were ranked as the unhealthiest. The top five healthy foods were broccoli, boiled greens, baked fish, grapefruit, and broiled fish. From these top five healthy foods, only boiled greens were considered to be associated with AA. Conclusions Many of the foods AA consider as traditional foods are also perceived as unhealthy. On the contrary, foods perceived to have the most health value may not be a routine part of AA food patterns. Understanding AA perceptions of the healthfulness of foods can be informative for culturally appropriate nutrition intervention development.

Context: The workplace may be an ideal venue for engaging African American women in behavioral interventions for weight reduction. Objective: To examine the effectiveness of a culturally enhanced EatRight dietary intervention among a group of predominately African American women in a workplace setting. Design: Crossover design study. Setting: Workplace. Participants: A total of 39 women volunteered for this study, of whom 27 completed it. Intervention: The control period involved observation of participants for 22 weeks after receiving standard counseling on lifestyle methods to achieve a healthy weight; following the control period, participants crossed over to the 22-week intervention period. The intervention was culturally enhanced using feedback derived from formative assessment and delivered as 15 group sessions. Main Outcome Measures: The primary outcome measure was the difference in weight change between the control and intervention periods; changes in waist circumference and quality of life were secondary outcomes. Results: Most participants were obese, with a mean baseline body mass index of 36 kg/m 2 , weight of 97.9 kg, and waist circumference of 111 cm. Weight increased during the control period by 0.7 kg but decreased by 2.6 kg during the intervention (net difference =− 3.4 kg, P

Escherichia coli contains a four-gene operon, pgaABCD, which encodes the proteins necessary for the synthesis of polymeric N-acetylglucosamine, or PGA. Poly-N-acetyl-glucosamine was first described in Staphylococcus aureus and Staphylococcus epidermidis and was found to have important roles in biofilm formation and immune evasion. PGA also plays a role in biofilm formation in E. coli, but its role in immune evasion has not been thoroughly studied. We previously reported that E. coli PGA cross-reacts with an opsonic-antibody raised against S. aureus PNAG and this is the basis for an ongoing investigation regarding the development of a vaccine against both pathogens. In this paper we investigated pga expression in wild type and csrA or nhaR deletion mutant strains during different growth phases and temperatures, and in response to chemical stimuli using a pga promoter-reporter fusion construct, real-time reverse transcriptase-PCR, immunoblotting, and biofilm assays. Expression of pga and polysaccharide synthesis were induced by glucose, NaCl, and ethanol, but only glucose augmented biofilm formation. The regulatory factor NhaR was required for NaCl-induced pga expression, whereas the effects of glucose and ethanol were independent of CsrA and NhaR.

Background Characterizing microbial communities via next-generation sequencing is subject to a number of pitfalls involving sample processing. The observed community composition can be a severe distortion of the quantities of bacteria actually present in the microbiome, hampering analysis and threatening the validity of conclusions from metagenomic studies. We introduce an experimental protocol using mock communities for quantifying and characterizing bias introduced in the sample processing pipeline. We used 80 bacterial mock communities comprised of prescribed proportions of cells from seven vaginally-relevant bacterial strains to assess the bias introduced in the sample processing pipeline. We created two additional sets of 80 mock communities by mixing prescribed quantities of DNA and PCR product to quantify the relative contribution to bias of (1) DNA extraction, (2) PCR amplification, and (3) sequencing and taxonomic classification for particular choices of protocols for each step. We developed models to predict the “true” composition of environmental samples based on the observed proportions, and applied them to a set of clinical vaginal samples from a single subject during four visits. Results We observed that using different DNA extraction kits can produce dramatically different results but bias is introduced regardless of the choice of kit. We observed error rates from bias of over 85% in some samples, while technical variation was very low at less than 5% for most bacteria. The effects of DNA extraction and PCR amplification for our protocols were much larger than those due to sequencing and classification. The processing steps affected different bacteria in different ways, resulting in amplified and suppressed observed proportions of a community. When predictive models were applied to clinical samples from a subject, the predicted microbiome profiles were better reflections of the physiology and diagnosis of the subject at the visits than the observed community compositions. Conclusions Bias in 16S studies due to DNA extraction and PCR amplification will continue to require attention despite further advances in sequencing technology. Analysis of mock communities can help assess bias and facilitate the interpretation of results from environmental samples. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0351-6) contains supplementary material, which is available to authorized users.

Objective Microbial invasion of the amniotic cavity is associated with spontaneous preterm labor and adverse pregnancy outcome, and Mycoplasma hominis often is present. However, the pathogenic process by which M hominis invades the amniotic cavity and gestational tissues, often resulting in chorioamnionitis and preterm birth, remains unknown. We hypothesized that strains of M hominis vary genetically with regards to their potential to invade and colonize the amniotic cavity and placenta. Study Design We sequenced the entire genomes of 2 amniotic fluid isolates and a placental isolate of M hominis from pregnancies that resulted in preterm births and compared them with the previously sequenced genome of the type strain PG21. We identified genes that were specific to the amniotic fluid/placental isolates. We then determined the microbial burden and the presence of these genes in another set of subjects from whom samples of amniotic fluid had been collected and were positive for M hominis. Results We identified 2 genes that encode surface-located membrane proteins (Lmp1 and Lmp-like) in the sequenced amniotic fluid/placental isolates that were truncated severely in PG21. We also identified, for the first time, a microbial gene of unknown function that is referred to in this study as gene of interest C that was associated significantly with bacterial burden in amniotic fluid and the risk of preterm delivery in patients with preterm labor. Conclusion A gene in M hominis was identified that is associated significantly with colonization and/or infection of the upper reproductive tract during pregnancy and with preterm birth.

Despite an increased knowledge of risk factors for atherosclerotic heart disease, it remains nearly endemic in Western society. Despite the high penetrance, only a fraction of those with the disease progress to develop a frank myocardial infarction (MI). Over the past decade, it has become clear that inflammation plays an important role in the pathogenesis of MI. Inflammatory arterial disease therefore may be a better term for the subset of patients that develop the serious adverse consequences related to the rupture of the intracoronary plaque. Using newer molecular techniques such as high-throughput SNP analysis, genome-wide scanning, and enriched pedigree analysis, many of the specific mechanisms underlying the inflammatory milieu involved in this transition have been elucidated and may help identify those at risk for the adverse events associated with atherosclerotic disease.

When bacteria assume the biofilm mode of growth, they can tolerate levels of antimicrobial agents 10 to 1,000 times higher than the MICs of genetically equivalent planktonic bacteria. The properties of biofilms that give rise to antibiotic resistance are only partially understood. Inhibition of antibiotic penetration into the biofilm may play a role, but this has not been proven directly. In this report, penetration of the glycopeptide antibiotic vancomycin into viable Staphylococcus aureus biofilms was analyzed by confocal scanning laser microscopy using a fluorescently labeled derivative of the drug. We found that while vancomycin bound to free-floating bacteria in water within 5 min, it took more than 1 h to bind to cells within the deepest layers of a biofilm. These results indicate that the antibiotic is transported through the depth of the biofilm but that the rate is significantly reduced with respect to its transport through flowing water. This suggests that, whereas planktonic bacteria were rapidly exposed to a full bolus of vancomycin, the bacteria in the deeper layers of the biofilm were exposed to a gradually increasing dose of the drug due to its reduced rate of penetration. This gradual exposure may allow the biofilm bacteria to undergo stress-induced metabolic or transcriptional changes that increase resistance to the antibiotic. We also investigated the role of poly- N -acetylglucosamine, an important component of the S. aureus biofilm matrix, and found that its production was not involved in the observed decrease in the rate of vancomycin penetration.

This study examined the association of single nucleotide polymorphisms in 4 candidate genes in a cohort of subjects with aspirin resistance. Aspirin resistance was significantly associated with genetic variation in the platelet surface adenosine 5-diphosphate receptor gene P2Y1.

Biofilm formation is an important aspect of the pathogenesis of staphylococcal infections. A beta-1,6-linked N-acetyl glucosamine polysaccharide is critical to biofilm elaboration and is synthesized by proteins encoded by the intercellular adhesion (ica) locus. These studies were undertaken to characterize the mechanism by which transcription of the ica locus in S. aureus is regulated using isogenic S. aureus MN8 and MN8 mucoid (MN8m) strains, the latter of which constitutively overproduces biofilm. Transformation of the ica locus from MN8m to the ica knock-out mutants of two strains, MN8 and NCTC 10833, conferred a strong biofilm-producing phenotype. Sequence analysis revealed a 5-nucleotide deletion within the promoter region of the ica locus in MN8m compared with the sequence in the wild-type locus. Deletion or substitution of these 5 nucleotides within the wild-type ica locus augmented transcription of the ica locus and induced the strong biofilm-producing phenotype. Gel shift analysis demonstrated that a protein(s) within cell-free lysates from strain MN8 bind(s) specifically to oligonucleotides representative of the wild-type ica promoter sequence and that this binding is greatly diminished by the deletion or substitution of the 5 nucleotides. DNase I footprint analysis revealed that purified IcaR, thought to be a regulator of ica transcription, also binds to the ica promoter sequence just upstream of the ica start codon, but its affinity for the ica promoter is unaffected by deletion of the 5-nucleotide motif. These findings identify a 5-nucleotide motif within the ica promoter region that has a functional role in transcriptional regulation of the ica locus that is independent of IcaR, and also show that IcaR binds to the promoter region of the S. aureus ica locus.

Mitral valve endocarditis typically results in mitral regurgitation. However, endocarditis leading to functional mitral stenosis is uncommon and, when present, fungal organisms are typically implicated. Thus, obstructive-type bacterial endocarditis due to large vegetations blocking the mitral valve orifice is a rare occurrence, with approximately 20 reported cases in the literature. We report on two patients with bacterial endocarditis and severe functional mitral stenosis requiring emergent surgery. Additionally, this is the first report of vancomycin-resistant enterococcus causing endocarditis and functional mitral stenosis. The discussion emphasizes the hemodynamic instability of these patients and need for early surgical intervention.

Gardnerella vaginalis is the most common species found in bacterial vaginosis (BV). However, it is also present in a significant proportion of healthy women and G. vaginalis vaginal colonization does not always lead to BV. In an effort to better understand the differences between G. vaginalis isolated from women with a positive (BV) versus a negative (non-BV) diagnosis of BV, we compared the virulence potential of 7 BV and 7 non-BV G. vaginalis isolates and assessed the virulence factors related to biofilm formation, namely: initial adhesion and cytotoxic effect, biofilm accumulation, susceptibility to antibiotics, and transcript levels of the known vaginolysin, and sialidase genes. Furthermore, we also determined the ability of G. vaginalis to displace lactobacilli previously adhered to HeLa cells. Our results showed that non-BV strains were less virulent than BV strains, as suggested by the lower cytotoxicity and initial adhesion to Hela cells. Significant differences in expression of known virulence genes were also detected, further suggesting a higher virulence potential of the BV associated G. vaginalis. Importantly, we demonstrated that BV associated G. vaginalis were able to displace pre-coated vaginal protective lactobacilli and we hypothesize this to be a trigger for BV development., European Union funds (FEDER/COMPETE) and by national funds (FCT) under the project with reference FCOMP-01-0124-FEDER-008991 (PTDC/BIA-MIC/098228/2008). FCT Strategic Project of UID/BIO/04469/2013 unit the project NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte(ON.2 – O Novo Norte), QREN, FEDER, and the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462). FCT individual fellowship SFRH/BD/93963/2013.

We qualitatively assessed how life stressors affected African American women's participation in a weight reduction program. A sample of 9 women, who completed a behavioral lifestyle intervention, participated in individual, structured, in-depth interviews. Life stressors, ranging from personal illness to changes in employment status, had varied effects on participation. Some women coped with life stressors by using them as a motivational tool to improve their own health, while others reported limited ability to devote time to attend meetings or engage in the prescribed lifestyle changes due to life stressors. A critical key to improving weight loss outcomes for African American women may be using intervention strategies that teach positive coping skills to alter maladaptive responses to life stressors.