Most microorganisms from all taxonomic levels are uncultured. Single-cell genomes and metagenomes continue to increase the known diversity of Bacteria and Archaea; however, while 'omics can be used ...to infer physiological or ecological roles for species in a community, most of these hypothetical roles remain unvalidated. Here, we report an approach to capture specific microorganisms from complex communities into pure cultures using genome-informed antibody engineering. We apply our reverse genomics approach to isolate and sequence single cells and to cultivate three different species-level lineages of human oral Saccharibacteria (TM7). Using our pure cultures, we show that all three Saccharibacteria species are epibionts of diverse Actinobacteria. We also isolate and cultivate human oral SR1 bacteria, which are members of a lineage of previously uncultured bacteria. Reverse-genomics-enabled cultivation of microorganisms can be applied to any species from any environment and has the potential to unlock the isolation, cultivation and characterization of species from as-yet-uncultured branches of the microbial tree of life.
Polymorphisms in ATP2B4 coding for PMCA4b, the primary regulator of erythrocyte calcium concentration, have been shown by GWAS and cross-sectional studies to protect against severe malaria but the ...mechanism remains unknown.
Using a recall-by-genotype design, we investigated the impact of a common haplotype variant in ATP2B4 using in vitro assays that model erythrocyte stage malaria pathogenesis. Ninety-six donors representing homozygote (carriers of the minor allele, C/C), heterozygote (T/C) and wildtype (T/T) carriers of the tagging SNP rs1541252 were selected from a cohort of over 12,000 participants in the Keneba Biobank.
Red blood cells (RBCs) from homozygotes showed reduced PMCA4b protein expression (mean fluorescence intensities (MFI = 2428 ± 124, 3544 ± 159 and 4261 ± 283, for homozygotes, heterozygotes and wildtypes respectively, p < 0.0001) and slower rates of calcium expulsion (calcium t
± SD = 4.7 ± 0.5, 1.8 ± 0.3 and 1.9 ± 0.4 min, p < 0.0001). Growth of a Plasmodium falciparum laboratory strain (FCR3) and two Gambian field isolates was decreased in RBCs from homozygotes compared to heterozygotes and wildtypes (p < 0.01). Genotype group did not affect parasite adhesion in vitro or var-gene expression in malaria-infected RBCs. Parasite growth was inhibited by a known inhibitor of PMCA4b, aurintricarboxylic acid (IC
= 122uM CI: 110-134) confirming its sensitivity to calcium channel blockade.
The data support the hypothesis that this ATP2B4 genotype, common in The Gambia and other malaria-endemic areas, protects against severe malaria through the suppression of parasitaemia during an infection. Reduction in parasite density plays a pivotal role in disease outcome by minimizing all aspects of malaria pathogenesis. Follow up studies are needed to further elucidate the mechanism of protection and to determine if this ATP2B4 genotype carries a fitness cost or increases susceptibility to other human disease.
Iron deficiency is the most common nutrient deficiency worldwide and routine supplementation is standard policy for pregnant mothers and children in most low-income countries. However, iron lies at ...the center of host-pathogen competition for nutritional resources and recent trials of iron administration in African and Asian children have resulted in significant excesses of serious adverse events including hospitalizations and deaths. Increased rates of malaria, respiratory infections, severe diarrhea and febrile illnesses of unknown origin have all been reported, but the mechanisms are unclear. We here investigated the ex vivo growth characteristics of exemplar sentinel bacteria in adult sera collected before and 4 h after oral supplementation with 2 mg/kg iron as ferrous sulfate. Escherichia coli, Yersinia enterocolitica and Salmonella enterica serovar Typhimurium (all gram-negative bacteria) and Staphylococcus epidermidis (gram-positive) showed markedly elevated growth in serum collected after iron supplementation. Growth rates were very strongly correlated with transferrin saturation (p < 0.0001 in all cases). Growth of Staphylococcus aureus, which preferentially scavenges heme iron, was unaffected. These data suggest that even modest oral supplements with highly soluble (non-physiological) iron, as typically used in low-income settings, could promote bacteremia by accelerating early phase bacterial growth prior to the induction of immune defenses.
Human neonates elicit a profound hypoferremia which may protect against bacterial sepsis. We examined the transience of this hypoferremia by measuring iron and its chaperone proteins, inflammatory ...and haematological parameters over the first post-partum week. We prospectively studied term, normal weight Gambian newborns. Umbilical cord vein and artery, and serial venous blood samples up to day 7 were collected. Hepcidin, serum iron, transferrin, transferrin saturation, haptoglobin, c-reactive protein, α1-acid-glycoprotein, soluble transferrin receptor, ferritin, unbound iron-binding capacity and full blood count were assayed. In 278 neonates we confirmed the profound early postnatal decrease in serum iron (22.7 ± 7.0 µmol/L at birth to 7.3 ± 4.6 µmol/L during the first 6-24 h after birth) and transferrin saturation (50.2 ± 16.7% to 14.4 ± 6.1%). Both variables increased steadily to reach 16.5 ± 3.9 µmol/L and 36.6 ± 9.2% at day 7. Hepcidin increased rapidly during the first 24 h of life (19.4 ± 14.4 ng/ml to 38.9 ± 23.9 ng/ml) and then dipped (32.7 ± 18.4 ng/ml) before rising again at one week after birth (45.2 ± 19.1 ng/ml). Inflammatory markers increased during the first week of life. The acute postnatal hypoferremia in human neonates on the first day of life is highly reproducible but transient. The rise in serum iron during the first week of life occurs despite very high hepcidin levels indicating partial hepcidin resistance.Trial Registration: clinicaltrials.gov (NCT03353051). Registration date: November 27, 2017.
The COVID-19 pandemic in 2020 has resulted in widespread training disruption in many sports. Some athletes have access to facilities and equipment, while others have limited or no access, severely ...limiting their training practices. A primary concern is that the maintenance of key physical qualities (e. g. strength, power, high-speed running ability, acceleration, deceleration and change of direction), game-specific contact skills (e. g. tackling) and decision-making ability, are challenged, impacting performance and injury risk on resumption of training and competition. In extended periods of reduced training, without targeted intervention, changes in body composition and function can be profound. However, there are strategies that can dramatically mitigate potential losses, including resistance training to failure with lighter loads, plyometric training, exposure to high-speed running to ensure appropriate hamstring conditioning, and nutritional intervention. Athletes may require psychological support given the challenges associated with isolation and a change in regular training routine. While training restrictions may result in a decrease in some physical and psychological qualities, athletes can return in a positive state following an enforced period of rest and recovery. On return to training, the focus should be on progression of all aspects of training, taking into account the status of individual athletes.
Inadequate supply of one or more mineral elements can slow the growth of animal consumers and alter their physiology, life history and behaviour. A key concept for understanding nutrient deficiency ...in animals is the threshold elemental ratio (TER), at which growth limitation switches from one element to another. We used a stoichiometric model that coupled animal bioenergetics and body elemental composition to estimate TER of carbon and phosphorus (TERC:P) for 41 aquatic consumer taxa. We found a wide range in TERC:P (77-3086, ratio by atoms), which was generated by interspecific differences in body C : P ratios and gross growth efficiencies of C. TERC:P also varied among aquatic invertebrates having different feeding strategies, such that detritivores had significantly higher threshold ratios than grazers and predators. The higher TERC:P in detritivores resulted not only from lower gross growth efficiencies of carbon but also reflected lower body P content in these consumers. Supporting previous stoichiometric theory, we found TERC:P to be negatively correlated with the maximum growth rate of invertebrate consumers. By coupling bioenergetics and stoichiometry, this analysis revealed strong linkages among the physiology, ecology and evolution of nutritional demands for animal growth.
Background Thirty million small and sick newborns worldwide require inpatient care each year. Many receive antibiotics for clinically diagnosed infections without blood cultures, the current 'gold ...standard' for neonatal infection detection. Low neonatal blood culture use hampers appropriate antibiotic use, fuelling antimicrobial resistance (AMR) which threatens newborn survival. This study analysed the gap between blood culture use and antibiotic prescribing in hospitals implementing with Newborn Essential Solutions and Technologies (NEST360) in Kenya, Malawi, Nigeria, and Tanzania. Methods Inpatient data from every newborn admission record (July 2019-August 2022) were included to describe hospital-level blood culture use and antibiotic prescription. Health Facility Assessment data informed performance categorisation of hospitals into four tiers: (Tier 1) no laboratory, (Tier 2) laboratory but no microbiology, (Tier 3) neonatal blood culture use < 50% of newborns receiving antibiotics, and (Tier 4) neonatal blood culture use > 50%. Results A total of 144,146 newborn records from 61 hospitals were analysed. Mean hospital antibiotic prescription was 70% (range = 25-100%), with 6% mean blood culture use (range = 0-56%). Of the 10,575 blood cultures performed, only 24% (95%CI 23-25) had results, with 10% (10-11) positivity. Overall, 40% (24/61) of hospitals performed no blood cultures for newborns. No hospitals were categorised as Tier 1 because all had laboratories. Of Tier 2 hospitals, 87% (20/23) were District hospitals. Most hospitals could do blood cultures (38/61), yet the majority were categorised as Tier 3 (36/61). Only two hospitals performed > 50% blood cultures for newborns on antibiotics (Tier 4). Conclusions The two Tier 4 hospitals, with higher use of blood cultures for newborns, underline potential for higher blood culture coverage in other similar hospitals. Understanding why these hospitals are positive outliers requires more research into local barriers and enablers to performing blood cultures. Tier 3 facilities are missing opportunities for infection detection, and quality improvement strategies in neonatal units could increase coverage rapidly. Tier 2 facilities could close coverage gaps, but further laboratory strengthening is required. Closing this culture gap is doable and a priority for advancing locally-driven antibiotic stewardship programmes, preventing AMR, and reducing infection-related newborn deaths. Keywords: Newborn, Neonatal, Blood culture, Antibiotics, Low- and middle-income countries, Inpatient care, Antimicrobial resistance, Small and sick newborn care, Quality of care, Infection, Sepsis
Every Newborn Action Plan (ENAP) coverage target 4 necessitates national scale-up of Level-2 Small and Sick Newborn Care (SSNC) (with Continuous Positive Airway Pressure (CPAP)) in 80% of districts ...by 2025. Routine neonatal inpatient data is important for improving quality of care, targeting equity gaps, and enabling data-driven decision-making at individual, district, and national-levels. Existing neonatal inpatient datasets vary in purpose, size, definitions, and collection processes. We describe the co-design and operationalisation of a core inpatient dataset for use to track outcomes and improve quality of care for small and sick newborns in high-mortality settings.
A three-step systematic framework was used to review, co-design, and operationalise this novel neonatal inpatient dataset in four countries (Malawi, Kenya, Tanzania, and Nigeria) implementing with the Newborn Essential Solutions and Technologies (NEST360) Alliance. Existing global and national datasets were identified, and variables were mapped according to categories. A priori considerations for variable inclusion were determined by clinicians and policymakers from the four African governments by facilitated group discussions. These included prioritising clinical care and newborn outcomes data, a parsimonious variable list, and electronic data entry. The tool was designed and refined by > 40 implementers and policymakers during a multi-stakeholder workshop and online interactions.
Identified national and international datasets (n = 6) contained a median of 89 (IQR:61-154) variables, with many relating to research-specific initiatives. Maternal antenatal/intrapartum history was the largest variable category (21, 23.3%). The Neonatal Inpatient Dataset (NID) includes 60 core variables organised in six categories: (1) birth details/maternal history; (2) admission details/identifiers; (3) clinical complications/observations; (4) interventions/investigations; (5) discharge outcomes; and (6) diagnosis/cause-of-death. Categories were informed through the mapping process. The NID has been implemented at 69 neonatal units in four African countries and links to a facility-level quality improvement (QI) dashboard used in real-time by facility staff.
The NEST360 NID is a novel, parsimonious tool for use in routine information systems to inform inpatient SSNC quality. Available on the NEST360/United Nations Children's Fund (UNICEF) Implementation Toolkit for SSNC, this adaptable tool enables facility and country-level comparisons to accelerate progress toward ENAP targets. Additional linked modules could include neonatal at-risk follow-up, retinopathy of prematurity, and Level-3 intensive care.
PDF
