Silent Tears of Midwives: 'I Want Every Mother Who Gives Birth to Have Her Baby Alive'-A Narrative Inquiry of Midwives Experiences of Very Early Neonatal Death from Tanzania.

BACKGROUND: Midwives working in settings with limited clinical resources experience high rates of very early neonatal deaths. Midwives manage the impact of this grief and trauma almost daily, which may affect patient care and their own well-being. RESEARCH AIMS: To explore how midwives are impacted by and cope with high rates of very early neonatal deaths. To document midwives' insights and local solutions that may reduce very early neonatal deaths in limited resource settings. To document the stories of midwives in order to create awareness and garner support for midwives and their critical work in low resource settings. METHODS: Narrative inquiry utilizing semi structured interviews. Twenty-one midwives with at least six months experience who had experienced or witnessed very early neonatal death were interviewed. Data were audio recorded and transcribed, and reflexive thematic analysis of transcripts was conducted. RESULTS AND DISCUSSION: Three themes were identified: (1) deep sadness resulting from very early neonatal deaths leading to internal struggles; (2) use of spirituality, including prayer and occasional beliefs that unexplainable deaths were 'God's plan'; and (3) development of resilience by seeking solutions, educating themselves, taking accountability and guiding mothers. Participating midwives noted that inadequate staff and high caseloads with limited basic supplies hindered their clinical practice. Participants articulated that they concentrated on active solutions to save babies during labour, such as vigilant foetal rate heart monitoring and partogram. Further, reduction and prevention of very early neonatal death is a complex problem requiring multidisciplinary teams and woman-centred care approaches to address issues contributing to the health of mothers and their new-borns. CONCLUSIONS: Midwives' narratives highlighted ways of coping with grief and deep sadness, through prayer, and further education of both mothers and fellow colleagues to achieve better antenatal and intrapartum care and outcomes. This study gave midwives an opportunity for their voices to be heard and to generate solutions or insights that can be shared with colleagues in similar low-resource settings.

Acquisition of Gonococcal AniA-NorB Pathway by the Neisseria meningitidis Urethritis Clade Confers Denitrifying and Microaerobic Respiration Advantages for Urogenital Adaptation.

Neisseria meningitidis historically has been an infrequent and sporadic cause of urethritis and other urogenital infections. However, a nonencapsulated meningococcal clade belonging to the hyperinvasive clonal complex 11.2 lineage has recently emerged and caused clusters of urethritis cases in the United States and other countries. One of the genetic signatures of the emerging N. meningitidis urethritis clade (NmUC) is a chromosomal gene conversion event resulting in the acquisition of the Neisseria gonorrhoeae denitrification apparatus-the N. gonorrhoeae alleles encoding the nitrite reductase AniA, the nitric oxide (NO) reductase NorB, and the intergenic promoter region. The biological importance of the N. gonorrhoeae AniA-NorB for adaptation of the NmUC to a new environmental niche is investigated herein. We found that oxygen consumption, nitrite utilization, and NO production were significantly altered by the conversion event, resulting in different denitrifying aerobic and microaerobic growth of the clade. Further, transcription of aniA and norB in NmUC isolates differed from canonical N. meningitidis, and important polymorphisms within the intergenic region, which influenced aniA promoter activity of the NmUC, were identified. The contributions of three known meningococcal regulators (NsrR, FNR, and NarQP) in controlling the denitrification pathway and endogenous NO metabolism were distinct. Overall, transcription of aniA was dampened relative to canonical N. meningitidis, and this correlated with the lower NO accumulation in the clade. Denitrification and microaerobic respiration were bolstered, and protection against host-derived NO was likely enhanced. The acquisition of the N. gonorrhoeae denitrification pathway by the NmUC supports the clade's adaptation and survival in a microaerobic urogenital environment.

Dissemination of metaldehyde catabolic pathways is driven by mobile genetic elements in Proteobacteria.

Bioremediation of metaldehyde from drinking water using metaldehyde-degrading strains has recently emerged as a promising alternative. Whole-genome sequencing was used to obtain full genomes for metaldehyde degraders Acinetobacter calcoaceticus E1 and Sphingobium CMET-H. For the former, the genetic context of the metaldehyde-degrading genes had not been explored, while for the latter, none of the degrading genes themselves had been identified. In A. calcoaceticus E1, IS91 and IS6-family insertion sequences (ISs) were found surrounding the metaldehyde-degrading gene cluster located in plasmid pAME76. This cluster was located in closely-related plasmids and associated to identical ISs in most metaldehyde-degrading ß- and γ-Proteobacteria, indicating horizontal gene transfer (HGT). For Sphingobium CMET-H, sequence analysis suggested a phytanoyl-CoA family oxygenase as a metaldehyde-degrading gene candidate due to its close homology to a previously identified metaldehyde-degrading gene known as mahX. Heterologous gene expression in Escherichia coli alongside degradation tests verified its functional significance and the degrading gene homolog was henceforth called mahS. It was found that mahS is hosted within the conjugative plasmid pSM1 and its genetic context suggested a crossover between the metaldehyde and acetoin degradation pathways. Here, specific replicons and ISs responsible for maintaining and dispersing metaldehyde-degrading genes in α, ß and γ-Proteobacteria through HGT were identified and described. In addition, a homologous gene implicated in the first step of metaldehyde utilisation in an α-Proteobacteria was uncovered. Insights into specific steps of this possible degradation pathway are provided.

Probe-based qPCR assay enables the rapid and specific detection of bacterial degrading genes for the pesticide metaldehyde in soil.

Metaldehyde, a molluscicide pesticide, has been identified as a pollutant of concern due to its repeated detection in drinking water, thereby generating numerous compliance failures for water utilities. Biological degradation potential for metaldehyde is widespread in soils, occurring at different rates, but to date, no molecular methods for its assessment have been reported. Here, three genes belonging to a shared metaldehyde-degrading gene cluster present in bacteria were used as candidates for development of a quantitative PCR (qPCR) assay for assessing the metaldehyde-degrading potential in soil. Screening of gene targets, primer pairs and optimization of reaction conditions led to the development of a sensitive and specific probe-based qPCR method for quantifying the mahY metaldehyde-degrading gene from soil. The technique was tested across 8 soils with different compositions and origins. The degrading pathway was detected in 4/8 soils, in which a higher number of gene copies correlated with periods of greater metaldehyde removal. Additionally, swift elimination of the pesticide was observed in soils with an elevated initial number of mahY gene copies. The gene cluster was not detected in other soils, even though metaldehyde removal occurred, indicating that other biological degrading pathways are also important in nature. The method described here is the first one available to estimate the microbial metaldehyde degradation potential and activity in soils, and can also be used to detect degrading microorganisms in systems such as sand filters for water purification or to monitor degrading strains in engineered processes.

Silent voices of the midwives: factors that influence midwives' achievement of successful neonatal resuscitation in sub-Saharan Africa: a narrative inquiry.

BACKGROUND: In Tanzania, birth asphyxia is a leading cause of neonatal death. The aim of this study was to identify factors that influence successful neonatal resuscitation to inform clinical practice and reduce the incidence of very early neonatal death (death within 24 h of delivery). METHODS: This was a qualitative narrative inquiry study utilizing the 32 consolidated criteria for reporting qualitative research (COREQ). Audio-recorded, semistructured, individual interviews with midwives were conducted. Thematic analysis was applied to identify themes. RESULTS: Thematic analysis of the midwives' responses revealed three factors that influence successful resuscitation: 1. Hands-on training ("HOT") with clinical support during live emergency neonatal resuscitation events, which decreases fear and enables the transfer of clinical skills; 2. Unequivocal commitment to the Golden Minute® and the mindset of the midwife; and. 3. Strategies that reduce barriers. Immediately after birth, live resuscitation can commence at the mother's bedside, with actively guided clinical instruction. Confidence and mastery of resuscitation competencies are reinforced as the physiological changes in neonates are immediately visible with bag and mask ventilation. The proclivity to perform suction initially delays ventilation, and suction is rarely clinically indicated. Keeping skilled midwives in labor wards is important and impacts clinical practice. The midwives interviewed articulated a mindset of unequivocal commitment to the baby for one Golden Minute®. Heavy workload, frequent staff rotation and lack of clean working equipment were other barriers identified that are worthy of future research. CONCLUSIONS: Training in resuscitation skills in a simulated environment alone is not enough to change clinical practice. Active guidance of "HOT" real-life emergency resuscitation events builds confidence, as the visible signs of successful resuscitation impact the midwife's beliefs and behaviors. Furthermore, a focused commitment by midwives working together to reduce birth asphyxia-related deaths builds hope and collective self-efficacy.

Air, Air, Air: a champion midwife programme in Tanzania using HOT neonatal resuscitation-lessons learned.

BACKGROUND: Tanzania has approximately 40 000 newborn deaths per year, with >25% of these linked to intrapartum-related hypoxia. The Helping Babies Breathe© and Golden minute© (HBB©) programme was developed to teach skilled intervention for non-breathing neonates at birth. While Helping Babies Breathe© and Golden minute©, providing training in simulated bag and mask ventilation, is theoretically successful in the classroom, it often fails to transfer to clinical practice without further support. Furthermore, the proclivity of midwives to suction excessively as a first-line intervention is an ingrained behaviour that delays ventilation, contributing to very early neonatal deaths. METHODS: The 'champion' programme provided guided instruction during a real-life resuscitation. The site was Amana Hospital, Tanzania. The labour ward conducts 13 500 deliveries annually, most of which are managed by midwives. Brief mannikin simulation practice was held two to three times a week followed by bedside hands-on training (HOT) of bag and mask skills and problem solving while reinforcing the mantra of 'air, air, air' as the first-line intervention during a real-life emergency. RESULTS: Champion midwives (trainers) guided instructions given during a real emergency at the bedside caused learners beliefs to change. Trainees observed changes in baby skin colour and the onset of spontaneous breathing after effective ventilation. CONCLUSIONS: Visible success during an actual real-life emergency created confidence, mastery and collective self-efficacy.

Long-term fertilization and tillage regimes have limited effects on structuring bacterial and denitrifier communities in a sandy loam UK soil.

Denitrification causes loss of available nitrogen from soil systems, thereby reducing crop productivity and increasing reliance on agrochemicals. The dynamics of denitrification and denitrifying communities are thought to be altered by land management practices, which affect the physicochemical properties of the soil. In this study, we look at the effects of long-term tillage and fertilization regimes on arable soils following 16 years of treatment in a factorial field trial. By studying the bacterial community composition based on 16S rRNA amplicons, absolute bacterial abundance and diversity of denitrification functional genes (nirK, nirS and nosZ), under conditions of minimum/conventional tillage and organic/synthetic mineral fertilizer, we tested how specific land management histories affect the diversity and distribution of both bacteria and denitrification genes. Bacterial and denitrifier communities were largely unaffected by land management history and clustered predominantly by spatial location, indicating that the variability in bacterial community composition in these arable soils is governed by innate environmental differences and Euclidean distance rather than agricultural management intervention.

Periscope Proteins are variable-length regulators of bacterial cell surface interactions.

Changes at the cell surface enable bacteria to survive in dynamic environments, such as diverse niches of the human host. Here, we reveal "Periscope Proteins" as a widespread mechanism of bacterial surface alteration mediated through protein length variation. Tandem arrays of highly similar folded domains can form an elongated rod-like structure; thus, variation in the number of domains determines how far an N-terminal host ligand binding domain projects from the cell surface. Supported by newly available long-read genome sequencing data, we propose that this class could contain over 50 distinct proteins, including those implicated in host colonization and biofilm formation by human pathogens. In large multidomain proteins, sequence divergence between adjacent domains appears to reduce interdomain misfolding. Periscope Proteins break this "rule," suggesting that their length variability plays an important role in regulating bacterial interactions with host surfaces, other bacteria, and the immune system.

The basis for non-canonical ROK family function in the N-acetylmannosamine kinase from the pathogen Staphylococcus aureus.

In environments where glucose is limited, some pathogenic bacteria metabolize host-derived sialic acid as a nutrient source. N-Acetylmannosamine kinase (NanK) is the second enzyme of the bacterial sialic acid import and degradation pathway and adds phosphate to N-acetylmannosamine using ATP to prime the molecule for future pathway reactions. Sequence alignments reveal that Gram-positive NanK enzymes belong to the Repressor, ORF, Kinase (ROK) family, but many lack the canonical Zn-binding motif expected for this function, and the sugar-binding EXGH motif is altered to EXGY. As a result, it is unclear how they perform this important reaction. Here, we study the Staphylococcus aureus NanK (SaNanK), which is the first characterization of a Gram-positive NanK. We report the kinetic activity of SaNanK along with the ligand-free, N-acetylmannosamine-bound and substrate analog GlcNAc-bound crystal structures (2.33, 2.20, and 2.20 Å resolution, respectively). These demonstrate, in combination with small-angle X-ray scattering, that SaNanK is a dimer that adopts a closed conformation upon substrate binding. Analysis of the EXGY motif reveals that the tyrosine binds to the N-acetyl group to select for the "boat" conformation of N-acetylmannosamine. Moreover, SaNanK has a stacked arginine pair coordinated by negative residues critical for thermal stability and catalysis. These combined elements serve to constrain the active site and orient the substrate in lieu of Zn binding, representing a significant departure from canonical NanK binding. This characterization provides insight into differences in the ROK family and highlights a novel area for antimicrobial discovery to fight Gram-positive and S. aureus infections.

The fitness challenge of studying molecular adaptation.

Advances in bioinformatics and high-throughput genetic analysis increasingly allow us to predict the genetic basis of adaptive traits. These predictions can be tested and confirmed, but the molecular-level changes - i.e. the molecular adaptation - that link genetic differences to organism fitness remain generally unknown. In recent years, a series of studies have started to unpick the mechanisms of adaptation at the molecular level. In particular, this work has examined how changes in protein function, activity, and regulation cause improved organismal fitness. Key to addressing molecular adaptations is identifying systems and designing experiments that integrate changes in the genome, protein chemistry (molecular phenotype), and fitness. Knowledge of the molecular changes underpinning adaptations allow new insight into the constraints on, and repeatability of adaptations, and of the basis of non-additive interactions between adaptive mutations. Here we critically discuss a series of studies that examine the molecular-level adaptations that connect genetic changes and fitness.

A biotin-conjugated photo-activated CO-releasing molecule (biotinCORM): efficient CO-release from an avidin-biotinCORM protein adduct.

Biotinylated pharmaceuticals are of great interest due to the strong interactions between biotinyl-functionality and streptavidin/avidin, which opens up avenues for efficient targeting and localisation. Three new carbon monoxide-releasing molecules (CO-RMs) have been synthesised and characterised using chemical and biological analysis. An alkyne-containing CO-RM 2 was found to be toxic to RAW 264.7 murine macrophages; and thus therapeutically viable CO-RM 1 was employed as the alkyne precursor for [3 + 2] cycloaddition chemistry enabling a new acid-containing CO-RM 4 and biotin-bioconugate-CO-RM (BiotinCORM 5) to be prepared. CO-RM 4 showed significantly improved solubility and BiotinCORM 5 acts as a photo-CO-RM. We have found that an avidin-CORM adduct of 5 is a CO-releasing protein, releasing CO on irradiation with light (400 nm). The avidin-biotinCORM adduct of 5 was found to have a binding energy of 10 kcal mol-1.

Conserved Histidine Adjacent to the Proximal Cluster Tunes the Anaerobic Reductive Activation of Escherichia coli Membrane-Bound [NiFe] Hydrogenase-1.

[NiFe] hydrogenases are electrocatalysts that oxidize H2 at a rapid rate without the need for precious metals. All membrane-bound [NiFe] hydrogenases (MBH) possess a histidine residue that points to the electron-transfer iron sulfur cluster closest ("proximal") to the [NiFe] H2-binding active site. Replacement of this amino acid with alanine induces O2 sensitivity, and this has been attributed to the role of the histidine in enabling the reversible O2-induced over-oxidation of the [Fe4S3Cys2] proximal cluster possessed by all O2-tolerant MBH. We have created an Escherichia coli Hyd-1 His-to-Ala variant and report O2-free electrochemical measurements at high potential that indicate the histidine-mediated [Fe4S3Cys2] cluster-opening/closing mechanism also underpins anaerobic reactivation. We validate these experiments by comparing them to the impact of an analogous His-to-Ala replacement in Escherichia coli Hyd-2, a [NiFe]-MBH that contains a [Fe4S4] center.

Isolation and characterization of metaldehyde-degrading bacteria from domestic soils.

Metaldehyde is a common molluscicide, used to control slugs in agriculture and horticulture. It is resistant to breakdown by current water treatment processes, and its accumulation in drinking water sources leads to regular regulatory failures in drinking water quality. To address this problem, we isolated metaldehyde-degrading microbes from domestic soils. Two distinct bacterial isolates were cultured, that were able to grow prototrophically using metaldehyde as sole carbon and energy source. One isolate belonged to the genus Acinetobacter (strain designation E1) and the other isolate belonged to the genus Variovorax (strain designation E3). Acinetobacter E1 was able to degrade metaldehyde to a residual concentration < 1 nM, whereas closely related Acinetobacter strains were completely unable to degrade metaldehyde. Variovorax E3 grew and degraded metaldehyde more slowly than Acinetobacter E1, and residual metaldehyde remained at the end of growth of the Variovorax E3 strain. Biological degradation of metaldehyde using these bacterial strains or approaches that allow in situ amplification of metaldehyde-degrading bacteria may represent a way forward for dealing with metaldehyde contamination in soils and water.

Plasma-Generated Poly(allyl alcohol) Antifouling Coatings for Cellular Attachment.

Conformal poly(allyl alcohol) (PAA) coatings were grown on a biomedical grade polyurethane scaffold using pulsed plasma polymerization of the allyl alcohol monomer. The creation of a continuous wave polymer primer layer increases the interfacial adhesion and stability of a subsequent pulsed plasma deposited PAA film. The resulting PAA coatings are strongly hydrophilic and stable following 7 days incubation in biological media. Films prepared through this energy-efficient, two-step process promote human dermal fibroblast cell culture, while resisting E. coli biofilm formation.

Toxicity of tryptophan manganese(i) carbonyl (Trypto-CORM), against Neisseria gonorrhoeae.

The potential for carbon monoxide-releasing molecules (CO-RMs) as antimicrobials represents an exciting prospective in the fight against antibiotic resistance. Trypto-CORM, a tryptophan-containing manganese(i) carbonyl, is toxic against E. coli following photo-activation. Here, we demonstrate that Trypto-CORM is toxic against Neisseria gonorrhoeae in the absence of photoactivation. Trypto-CORM toxicity was reversed by the high CO affinity globin leg-haemoglobin (Leg-Hb), indicating that the toxicity is due to CO release. Release of CO from Trypto-CORM in the dark was also detected with Leg-Hb (but not myoglobin) in vitro. N. gonorrhoeae is more sensitive to CO-based toxicity than other model bacterial pathogens, and may serve as a viable candidate for antimicrobial therapy using CO-RMs.

Re-engineering a NiFe hydrogenase to increase the H2 production bias while maintaining native levels of O2 tolerance.

Naturally occurring oxygen tolerant NiFe membrane bound hydrogenases have a conserved catalytic bias towards hydrogen oxidation which limits their technological value. We present an Escherichia coli Hyd-1 amino acid exchange that apparently causes the catalytic rate of H2 production to double but does not impact the O2 tolerance.

Confirmation of co-denitrification in grazed grassland.

Pasture-based livestock systems are often associated with losses of reactive forms of nitrogen (N) to the environment. Research has focused on losses to air and water due to the health, economic and environmental impacts of reactive N. Di-nitrogen (N2) emissions are still poorly characterized, both in terms of the processes involved and their magnitude, due to financial and methodological constraints. Relatively few studies have focused on quantifying N2 losses in vivo and fewer still have examined the relative contribution of the different N2 emission processes, particularly in grazed pastures. We used a combination of a high (15)N isotopic enrichment of applied N with a high precision of determination of (15)N isotopic enrichment by isotope-ratio mass spectrometry to measure N2 emissions in the field. We report that 55.8 g N m(-2) (95%, CI 38 to 77 g m(-2)) was emitted as N2 by the process of co-denitrification in pastoral soils over 123 days following urine deposition (100 g N m(-2)), compared to only 1.1 g N m(-2) (0.4 to 2.8 g m(-2)) from denitrification. This study provides strong evidence for co-denitrification as a major N2 production pathway, which has significant implications for understanding the N budgets of pastoral ecosystems.

Meningitis in adolescents: the role of commensal microbiota.

The pathogen Neisseria meningitidis causes disease amongst infants and adolescents/young adults. Here we argue that disease amongst adolescents is due largely to interaction between N. meningitidis and other members of the upper respiratory tract microbiota, through a metabolic interaction involving exchange of propionic acid.

Lipid-modified azurin of Neisseria meningitidis is a copper protein localized on the outer membrane surface and not regulated by FNR.

The laz gene of Neisseria meningitidis is predicted to encode a lipid-modified azurin (Laz). Laz is very similar to azurin, a periplasmic protein, which belongs to the copper-containing proteins in the cupredoxin superfamily. In other bacteria, azurin is an electron donor to nitrite reductase, an important enzyme in the denitrifying process. It is not known whether Laz could function as an electron transfer protein in this important pathogen. Laz protein was heterologously expressed in Escherichia coli and purified. Electrospray mass spectrometry indicated that the Laz protein contains one copper ion. Laz was shown to be redox-active in the presence of its redox center copper ion. When oxidized, Laz exhibits an intense blue colour and absorbs visible light around 626 nm. The absorption is lost when exposed to diethyldithiocarbamate, a copper chelating agent. Polyclonal antibodies were raised against purified Laz for detecting expression of Laz under different growth conditions and to determine the orientation of Laz on the outer membrane. The expression of Laz under microaerobic and microaerobic denitrifying conditions was slightly higher than that under aerobic conditions. However, the expression of Laz was similar between the wild type strain and an fnr mutant, suggesting that Fumarate/Nitrate reduction regulator (FNR) does not regulate the expression of Laz despite the presence of a partial FNR box upstream of the laz gene. We propose that some Laz protein is exposed on the outer membrane surface of N. meningitidis as the αLaz antibodies can increase killing by complement in a capsule deficient N. meningitidis strain, in a dose-dependent fashion.