Is there an association between the vaginal microbiome and first trimester miscarriage? A prospective observational study.

AIM: To examine whether there are differences in the vaginal microbiome of women who miscarry compared to those who have normal pregnancy outcomes. METHODS: Prospective observational study conducted at the Canberra Hospital, Australia, with 24 participant women in the first trimester of pregnancy. The vaginal microbiomes of the 24 women were characterized using sequencing analysis of the V4 region of the 16S rRNA gene employing an Illumina MiSeq instrument with QIAGEN reagents. Vaginal microbiome data were correlated with pregnancy clinical metadata. RESULTS: Ordination plots showed differences in the composition of microbiomes of women who miscarried and controls. In nulliparous women, Lactobacillus crispatus was the dominant bacterium in 50% of women. Lactobacillus iners was the dominant bacterium in 50% of women with a history of prior miscarriage and a miscarriage in the study compared to 15% (p = 0.011) in those with no history of miscarriage and no miscarriage in the study. There were significant differences in the number of operational taxonomic units and the richness of the microbiomes of women who miscarried compared to those who delivered at term. Eight taxa were found in different relative abundances in both groups of women. CONCLUSIONS: The study indicated that the composition of the vaginal microbiome varies with pregnancy history. Also, there was a significant difference in the vaginal microbiomes between women who suffered miscarriage and those who continued to term delivery both in the overall microbiome populations and in the abundances of individual taxa.

Is It Time for Australia to Reassess Its Legislation on Human Embryo Experimentation?

The United Kingdom Warnock Committee (1984) was a landmark contributor to the ethics and law governing human embryo experimentation. It recommended a time limit up to 14 days of development after fertilisation within which such experimentation may take place, which mirrors the late 1970s' proposal of the United States Department of Health, Education, and Welfare Ethics Advisory Board (EAB). This study analyses the EAB's and the Warnock Committee's reasoning and conclusions regarding what constitutes ethical behaviour towards the human embryo. Current embryology and recently created embryo-like structures are considered. After the Warnock Report, several Australian Federal and State committees in Australia investigated the ethics which should guide experimentation on human embryos. The reports of these Australian committees are reviewed and the potential influence of both earlier committees on their deliberations is discussed. The rationale informing current Australian law governing human embryo experimentation is examined. Considering current more advanced knowledge of embryology, it is concluded that this legislation should be reassessed.

Insights into the Genital Microbiota of Women Who Experienced Fetal Death in Utero.

The aim of this work was to achieve a better understanding of the bacterial pathogens associated with stillbirths that would serve to inform clinical interventions directed at reducing this adverse pregnancy outcome. A prospective observational study was conducted with the participation of 22 women from northern Peru, of whom 11 experienced fetal death in utero and 11 delivered preterm births. Swabs were taken from the vagina, placenta, amniotic fluid and axilla of the infant at birth by Caesarean section. The bacterial populations in the vagina and the amniotic space of each participant were determined by employing the amplicon sequencing of the V4 region of the 16S rRNA genes. The sequence data were analysed using bioinformatics tools. The work showed differences in the composition of the genital microbiomes of women who experienced preterm birth or fetal death in utero. There were no differences in the alpha diversity between the genital microbiotas of both groups of women, but there were more different taxa in the vagina and amniotic space of the preterm participants. Lactobacillus spp. was less abundant in the stillbirth cases. E. coli/Shigella, Staphylococcus, Gardnerella, Listeria and Bacteroides taxa were associated with the stillbirths. In each woman, there was a minimal concordance between the bacterial populations in the vagina and amniotic space.

Effects of human and porcine bile on the proteome of Helicobacter hepaticus.

BACKGROUND: Helicobacter hepaticus colonizes the intestine and liver of mice causing hepatobiliary disorders such as hepatitis and hepatocellular carcinoma, and has also been associated with inflammatory bowel disease in children. In its habitat, H. hepaticus must encounter bile which has potent antibacterial properties. To elucidate virulence and host-specific adaptation mechanisms of H. hepaticus modulated by human or porcine bile, a proteomic study of its response to the two types of bile was performed employing two-dimensional gel electrophoresis (2-DE) and mass spectrometry. RESULTS: The 2-DE and mass spectrometry analyses of the proteome revealed that 46 proteins of H. hepaticus were differentially expressed in human bile, 18 up-regulated and 28 down-regulated. In the case of porcine bile, 32 proteins were differentially expressed of which 19 were up-regulated, and 13 were down-regulated. Functional classifications revealed that identified proteins participated in various biological functions including stress response, energy metabolism, membrane stability, motility, virulence and colonization. Selected genes were analyzed by RT-PCR to provide internal validation for the proteomic data as well as provide insight into specific expressions of motility, colonization and virulence genes of H. hepaticus in response to human or porcine bile. CONCLUSIONS: Overall, the data suggested that bile is an important factor that determines virulence, host adaptation, localization and colonization of specific niches within host environment.

Vulnerability and Resilience: Phenomenological Analysis of Cancer Patients Value Directives.

CONTEXT: Personal values are individual conceptions of the desirable appraisals and actions that guide our attitudes and behaviour. Advance care planning (ACP) now emphasises the consideration of personal life goals and values expressed as a Values Directive (VD) to guide discussions concerning medical treatment. OBJECTIVE: To investigate the diversity of values, experiences and adaptations expressed in cancer patients VDs. METHODS: Contents of the VDs of ACPs of cancer patients who participated in a randomised control trial comparing a video intervention showing values communication between cancer patient-caregivers with usual care were analysed. Qualitative phenomenological content analysis was used to understand how participants made meaning of their lived experiences. RESULTS: Forty-two participants completed an ACP (37.2% response rate), with 97.6% of these completing a VD (57.1% female, mean age 72 years, 30.1% gastrointestinal cancer). Participants described diverse adjustments to frailty and adaptive coping with deteriorating functionality. Emotional and financial concerns were eased through experiencing benevolence and trust established through family and friendship bonds and reciprocation of care. Death anxiety and ambivalence were expressed concurrently with the experiential acceptance of dying. Secular and sacred rituals featured as an affirmation of their faith or beliefs. CONCLUSION: Cancer patients seek to make meaning of their experiences, concurrently posturing vulnerability and resilience, despite conflicting emotions and experiences. Given that the choices people make as they approach dying relate to their most deeply held values, ACP conversations should explore how patients draw from their values and life goals to optimise their adaptations to illness.

Posthumanism: Creation of 'New Men' Through Technological Innovation.

The posthumanist project proposes directing the evolution of human beings by promoting their improvement through technological means to create a variety of entities that will have few or no common characteristics with current humans. Its agenda is extremely broad and this study mostly addresses enhancement of the human organism through genetic modification techniques. An overview of posthumanist values and a brief discussion of its philosophical background provide a framework to understand its ideals. Genetics and ethics are employed to assess some claims of the posthumanist program of creating evolved humans; in particular, the capabilities and limitations of techniques for somatic and germline genome editing. Consequences of the creation of posthumans are discussed in relation to accepted current human beings and values. It is concluded that the posthumanist program rests on a large number of hypotheses without sufficient evidence and with little or no consideration of the consequences of its implementation.

Transhumanist Genetic Enhancement: Creation of a 'New Man' Through Technological Innovation.

The transhumanist project of reshaping human beings by promoting their improvement through technological innovations has a broad agenda. This study focuses on the enhancement of the human organism through genetic modification techniques. Transhumanism values and a discussion of their philosophical background provide a framework to understand its ideals. Genetics and ethics are employed to assess the claims of the transhumanist program of human enhancement. A succinct description of central concepts in genetics and an explanation of current techniques to edit the human genome serve to assess the capabilities and limitations of editing techniques. Potential benefits and liabilities of human enhancement through genome editing are discussed to appraise its feasibility. Ethical considerations of genome editing inform a reflection on the implications of introducing heritable changes in the genome of individuals. It is concluded that the transhumanist program is underpinned by a large number of hypotheses rather than by sufficient evidence.

Diagnosing death 50 years after the Harvard brain death report.

More than 50 years after the publication of the Harvard Committee Report that sought to define death according to whole-brain function criteria, this document continues to generate a diversity of opinions regarding how death should be defined. The various perspectives show that doubts linger regarding when brain death should be diagnosed, the criteria to pinpoint the occurrence of death, and the alignment of medical practices seeking to establish human death with these criteria. This study reviews and assesses three perspectives that have made significant contributions to the debate. Attention is also given to definitions of death that depart from the recommendations of the Harvard Report. Appraisals of various arguments lead to the conclusion that changes in the definition of death have resulted from advances in knowledge of human biology, medical technology and diagnostic techniques. A commentary is included on expediting the time of death with the view of organ donation.

Response of Helicobacter hepaticus to bovine bile.

Helicobacter hepaticus is an enterohepatic bacterium associated with inflammatory bowel disease in children and causes severe hepatobiliary disorders in mice. To elucidate the molecular response of H. hepaticus to bovine bile, a proteomic investigation was conducted. Bacteria were grown for 48 h in liquid media supplemented with different concentrations of bovine bile to determine its effects on bacterial growth and morphology. Protein expression profiles of bacteria grown at a bile concentration of 0.1% and in the absence of bile were obtained using two-dimensional gel electrophoresis. Gel spots with differences in intensities greater than 2-fold between both conditions were determined, and 55 differentially expressed proteins were identified using tandem mass spectrometry. Identified proteins participate in various biological functions including cell envelope biosynthesis, cell response to stress, iron homeostasis and transport, motility, primary and secondary metabolism, and virulence. Changes in the expression of H. hepaticus genes related to proteins involved in virulence and oxidative stress that were differentially expressed in the presence of bile were investigated using real-time reverse transcriptase PCR. The results indicated that the effects of bile on H. hepaticus included a strong response to oxidative stress and an expression of factors that can promote host colonization.

Phosphonate metabolism in Helicobacter pylori.

Helicobacter pylori has been shown to degrade two phosphonates, N-phosphonoacetyl-L: -aspartate and phosphonoacetate; however, the bacterium does not have any genes homologous to those of the known phosphonate metabolism pathways suggesting that H. pylori may have a novel phosphonate metabolism pathway. Growth of H. pylori on phosphonates was studied and the catabolism of these compounds was measured employing (1)H-nuclear magnetic resonance spectroscopy. The specificity of the catabolic enzymes was elucidated by assaying the degradation of several phosphonates and through substrate competition studies. H. pylori was able to utilise phenylphosphonate as a sole source of phosphate for growth. Three strains of H. pylori showed sigmoidal enzyme kinetics of phenylphosphonate catabolism. Allosteric kinetics were removed when lysates were fractionated into cytosolic and membrane fractions. Catabolic rates increased with the addition of DTT, Mg(2+) and phosphate and decreased with the addition of EDTA. The physiological properties of H. pylori phosphonate metabolism were characterised and the presence of at least two novel phosphonate catabolism pathways that do not require phosphate starvation growth conditions for activity has been established.

Agency, Autonomy and Euthanasia.

Agency is the human capacity to freely choose one's thoughts, motivations and actions without undue internal or external influences; it is distinguished from decisional capacity. Four well-known conditions that can deeply affect agency are depression, demoralization, existential distress, and family dysfunction. The study reviews how they may diminish agency in persons whose circumstances may lead them to consider or request euthanasia or assisted suicide. Since agency has been a relatively neglected dimension of autonomous choice at the end of life, it is argued that to respect the autonomy of individuals, it is essential to establish their agency.

Comparison of the Genital Microbiomes of Pregnant Aboriginal and Non-aboriginal Women.

The genital microbiomes of women varies with racial background. Preterm birth and early-onset neonatal sepsis are two outcomes associated with genital infections during pregnancy. The rate of preterm birth in Aboriginal Australian mothers is high, as is the rate of early-onset sepsis in their infants. To date, no studies have been conducted to investigate genital microbiome taxa associated infection in this group of women. A prospective cohort study to characterize the vaginal and placental microbiomes of a group of these women from the Pilbara region was conducted at the Hedland Health Campus in Western Australia. Included in the study were gravidae Aboriginal (n = 23) and Non-aboriginal (n = 27) women in labor or for planned lower uterine segment Caesarean section. Employing sterile swabs, vaginal samples were obtained under sterile conditions immediately prior to vaginal delivery or planned Caesarean section; and placental samples were obtained under the same conditions during labor. Taxa present in the samples were identified by 16S rRNA amplicon sequencing (V4 region, 515F-806R). Taxon identity and abundance were established from Operational Taxonomic Unit (OTU) counts. Statistical analyses combining clinical metadata and sequencing results were employed to determine associations of taxa with racial background. The findings of this work served to enhance the current understanding of microbiota associated with health and disease in Aboriginal and Non-Aboriginal women. Differences were found between the vaginal and placental microbiomes of Aboriginal and Non-aboriginal women during pregnancy, as well as notable differences between the abundance of specific taxa in each racial group. The relative abundances of specific taxa were significantly different between participants with clinical signs of infection and those with healthy pregnancies. This work will contribute to understanding the causes of differences in rates of infection-driven preterm birth in various racial populations.

Helicobacter spp. other than Helicobacter pylori.

Non-H. pylori Helicobacter species (NHPHS) are associated with several important human and animal diseases. In the past year research into this group of bacteria has continued to gain attention, and novel species have been described in new niches owing to improvements in detection methods. Polymerase chain reaction and/or sequencing remain the gold standard for the detection of this genus. New insights into the pathogenesis of the NHPHS in hepatobiliary, gastric, and intestinal diseases were gained. In particular, data revealed interaction between hepatic steatosis and infectious hepatitis in the development of hepatocellular carcinoma. Evidence of an association between hepatitis C virus and Helicobacter spp. in hepatocarcinoma development was also provided; and male sex hormone signaling appeared to influence infectious hepatitis induced by Helicobacter hepaticus. More findings support an association between Helicobacter heilmannii and gastric adenocarcinoma; and in mice, mucins MUC4 and MUC5 but not MUC1 influence the colonization and pathogenesis of Helicobacter felis. Data indicated that the roles of the adaptive immune system in H. hepaticus-induced intestinal tumorigenesis are different in the small and large intestines, and environmental factors, such as bile acids may modulate H. hepaticus carcinogenic potential. New reports in the prevention and eradication of NHPHS showed a protective response against Helicobacter suis induced by vaccine administration, and a successful cross-foster rederivation method successfully eradicated Helicobacter spp. from contaminated mice litters. Overall, the studies provided insights into the pathophysiology of Helicobacter species other than Helicobacter pylori.

Insights into the molecular basis of the microaerophily of three Campylobacterales: a comparative study.

The concentration of oxygen in the atmosphere is a common environmental factor which can also be a source of stress for microorganisms. Comparative analyses of the responses of the epsilon-proteobacteria Campylobacter jejuni, Helicobacter pylori and Wolinella succinogenes to elevated oxygen concentrations were carried out using transcriptomics. Microarray data were analysed to determine genes differentially expressed under elevated oxygen concentrations. The results indicated 158, 58 and 82 genes were upregulated and 46, 40 and 65 were downregulated in C. jejuni, H. pylori and W. succinogenes, respectively. The gene encoding the enzyme alkyl hydroperoxide reductase was the only one upregulated at higher oxygen tensions in all three bacterial species. No genes were found to be downregulated in all three species. Functional classification analyses were performed on the genes whose expression was modulated in order to identify common pathways and functional categories which were differentially expressed in the three organisms. Processes upregulated at higher oxygen tensions included translation, oxidative phosphorylation, antioxidation, and nucleic acid metabolism. ABC and ion-coupled transport proteins were generally downregulated at higher oxygen tensions. Finally, insights into the preferred environment were gained from the analyses of the bacterial responses, specifically motility and chemotaxis proteins. W. succinogenes preferred anaerobic conditions as opposed to C. jejuni and H. pylori preference for microaerobic conditions. These comparative studies provide a better understanding of bacterial adaptation to and interaction with their environment.

Roles of alpha and beta carbonic anhydrases of Helicobacter pylori in the urease-dependent response to acidity and in colonization of the murine gastric mucosa.

Carbon dioxide occupies a central position in the physiology of Helicobacter pylori owing to its capnophilic nature, the large amounts of carbon dioxide produced by urease-mediated urea hydrolysis, and the constant bicarbonate supply in the stomach. Carbonic anhydrases (CA) catalyze the interconversion of carbon dioxide and bicarbonate and are involved in functions such as CO(2) transport or trapping and pH homeostasis. H. pylori encodes a periplasmic alpha-CA (alpha-CA-HP) and a cytoplasmic beta-CA (beta-CA-HP). Single CA inactivation and double CA inactivation were obtained for five genetic backgrounds, indicating that H. pylori CA are not essential for growth in vitro. Bicarbonate-carbon dioxide exchange rates were measured by nuclear magnetic resonance spectroscopy using lysates of parental strains and CA mutants. Only the mutants defective in the alpha-CA-HP enzyme showed strongly reduced exchange rates. In H. pylori, urease activity is essential for acid resistance in the gastric environment. Urease activity measured using crude cell extracts was not modified by the absence of CA. With intact CA mutant cells incubated in acidic conditions (pH 2.2) in the presence of urea there was a delay in the increase in the pH of the incubation medium, a phenotype most pronounced in the absence of H. pylori alpha-CA. This correlated with a delay in acid activation of the urease as measured by slower ammonia production in whole cells. The role of CA in vivo was examined using the mouse model of infection with two mouse-adapted H. pylori strains, SS1 and X47-2AL. Compared to colonization by the wild-type strain, colonization by X47-2AL single and double CA mutants was strongly reduced. Colonization by SS1 CA mutants was not significantly different from colonization by wild-type strain SS1. However, when mice were infected by SS1 Delta(beta-CA-HP) or by a SS1 double CA mutant, the inflammation scores of the mouse gastric mucosa were strongly reduced. In conclusion, CA contribute to the urease-dependent response to acidity of H. pylori and are required for high-grade inflammation and efficient colonization by some strains.

Campylobacter jejuni response to human mucin MUC2: modulation of colonization and pathogenicity determinants.

Campylobacter jejuni is the main cause of bacterial acute gastroenteritis worldwide. In its colonization of the host intestinal tract, it encounters secreted mucins in the mucus layer and surface mucins in the epithelial cells. Mucins are complex glycoproteins that comprise the major component of mucus and give mucus its viscous consistency. MUC2 is the most abundant secreted mucin in the human intestine; it is a major chemoattractant for C. jejuni, and the bacterium binds to it. There are no studies on the transcriptional response of the bacterium to this mucin. Here, cell-culture techniques and quantitative RT-PCR were used to characterize in vitro the effects of MUC2 on C. jejuni growth and the changes in expression of 20 C. jejuni genes related to various functions. The genes encoding cytolethal distending toxin protein (cdtABC), vacuolating cytotoxin (vacB), C. jejuni lipoprotein (jlpA), Campylobacter invasion antigen (ciaB), the multidrug efflux system (cmeAB), putative mucin-degrading enzymes (cj1344c, cj0843c, cj0256 and cj1055c), flagellin A (flaA) and putative rod-shape-determining proteins (mreB and mreC) were upregulated, whereas those encoding Campylobacter adhesion fibronectin-binding protein (cadF) and sialic acid synthase (neuB1) were downregulated. These results showed that C. jejuni utilizes MUC2 as an environmental cue for the modulation of expression of genes with various functions including colonization and pathogenicity.

Genetic microheterogeneity and phenotypic variation of Helicobacter pylori arginase in clinical isolates.

BACKGROUND: Clinical isolates of the gastric pathogen Helicobacter pylori display a high level of genetic macro- and microheterogeneity, featuring a panmictic, rather than clonal structure. The ability of H. pylori to survive the stomach acid is due, in part, to the arginase-urease enzyme system. Arginase (RocF) hydrolyzes L-arginine to L-ornithine and urea, and urease hydrolyzes urea to carbon dioxide and ammonium, which can neutralize acid. RESULTS: The degree of variation in arginase was explored at the DNA sequence, enzyme activity and protein expression levels. To this end, arginase activity was measured from 73 minimally-passaged clinical isolates and six laboratory-adapted strains of H. pylori. The rocF gene from 21 of the strains was cloned into genetically stable E. coli and the enzyme activities measured. Arginase activity was found to substantially vary (>100-fold) in both different H. pylori strains and in the E. coli model. Western blot analysis revealed a positive correlation between activity and amount of protein expressed in most H. pylori strains. Several H. pylori strains featured altered arginase activity upon in vitro passage. Pairwise alignments of the 21 rocF genes plus strain J99 revealed extensive microheterogeneity in the promoter region and 3' end of the rocF coding region. Amino acid S232, which was I232 in the arginase-negative clinical strain A2, was critical for arginase activity. CONCLUSION: These studies demonstrated that H. pylori arginase exhibits extensive genotypic and phenotypic variation which may be used to understand mechanisms of microheterogeneity in H. pylori.

Oxygen requirement and tolerance of Campylobacter jejuni.

The pathogen Campylobacter jejuni is considered a microaerophile yet has been shown to grow in vitro in atmospheres with partial oxygen tension of 21%. To achieve a better understanding of its microaerophily, the oxygen requirement and tolerance of four C. jejuni strains were investigated by measuring their growth under different conditions, by performing bioinformatic analyses and by determining their metronidazole resistance. At high cell densities, C. jejuni showed similar growth under microaerobic and fully aerobic oxygen tensions, but did not grow under oxygen-depleted conditions. At low cell densities, the bacteria grew only under microaerobic conditions. Eighteen genes were identified bioinformatically as potential contributors to the differences in oxygen tolerance between strains. Among them, cj0203, cj0264c, cj0415, cj0425, cj0628, cj0629 and cj0864 were considered the top potential contributors. The oxygen tolerance of the four C. jejuni strains was different, and this tolerance positively correlated with their resistance to metronidazole. This study provided evidence that C. jejuni was an obligate microaerophile. The data indicated that the strains had different oxygen tolerances; it suggested that they could result in phenotypic and physiological differences between strains grown under the same conditions. These differences could modulate the outcome of experiments, and may explain discrepancies in the results between strains.

MiniReview: bioinformatic study of bile responses in Campylobacterales.

Campylobacter, Helicobacter and Wolinella are genera of the order Campylobacterales, belonging to the class Epsilonproteobacteria. Their habitats are various niches in the gastrointestinal tract of higher animals, where they may come into contact with bile. Microorganisms in these environments require mechanisms of resistance to the surface-active amphipathic molecules with potent antimicrobial activities present in bile. This review summarizes current knowledge on the molecular responses to bile by Campylobacterales and other bacterial species that inhabit the intestinal tract and belong to the phyla Proteobacteria, Bacteriodetes, Firmicutes and Actinobacteria. To date, 125 specific genes have been implicated in bile responses, of which 10 are found in Campylobacterales. Genome database searches, analyses of protein sequence and domain similarities, and gene ontology data integration were performed to compare the responses to bile of these bacteria. The results showed that 33 proteins of bacteria belonging to the four phyla had similarities equal to or greater than 50-46% proteins of Campylobacterales. Domain architecture analyses revealed that 151 Campylobacterales proteins had similar domain composition and organization to 60 proteins known to participate in the tolerance to bile in other bacteria. The proteins CmeB, CmeF and CbrR of Campylobacter jejuni involved in bile tolerance were homologous to 42 proteins identified in the Proteobacteria, Bacteriodetes and Firmicutes. On the other hand, the proteins CiaB, CmeA, CmeC, CmeD, CmeE and FlaAsigma(28) also involved in the response to bile of C. jejuni, did not have homologues in other bacteria. Among the bacteria inhabiting the gastrointestinal tract, the Campylobacterales seem to have evolved some mechanisms of bile resistance similar to those of other bacteria, as well as other mechanisms that appear to be characteristic of this order.

Potential role of thiol:disulfide oxidoreductases in the pathogenesis of Helicobacter pylori.

Helicobacter pylori infections are responsible for a sequence of molecular events which ultimately result in the development of gastric diseases. The pathogenesis of H. pylori has been studied extensively with strong focus on the identification of virulence factors. In contrast, the involvement of thiol:disulfide oxidoreductases in bacterial pathogenesis is less well understood. This paper provides a review of the current knowledge of H. pylori putative thiol:disulfide oxidoreductases, and their potential role in promoting virulence and colonization. Several bioinformatic analyses served to complete the information on these oxidoreductases of H. pylori.