Small protein modules dictate prophage fates during polylysogeny.

Most bacteria in the biosphere are predicted to be polylysogens harbouring multiple prophages1-5. In studied systems, prophage induction from lysogeny to lysis is near-universally driven by DNA-damaging agents6. Thus, how co-residing prophages compete for cell resources if they respond to an identical trigger is unknown. Here we discover regulatory modules that control prophage induction independently of the DNA-damage cue. The modules bear little resemblance at the sequence level but share a regulatory logic by having a transcription factor that activates the expression of a neighbouring gene that encodes a small protein. The small protein inactivates the master repressor of lysis, which leads to induction. Polylysogens that harbour two prophages exposed to DNA damage release mixed populations of phages. Single-cell analyses reveal that this blend is a consequence of discrete subsets of cells producing one, the other or both phages. By contrast, induction through the DNA-damage-independent module results in cells producing only the phage sensitive to that specific cue. Thus, in the polylysogens tested, the stimulus used to induce lysis determines phage productivity. Considering the lack of potent DNA-damaging agents in natural habitats, additional phage-encoded sensory pathways to lysis likely have fundamental roles in phage-host biology and inter-prophage competition.

A major lineage of non-tailed dsDNA viruses as unrecognized killers of marine bacteria.

The most abundant viruses on Earth are thought to be double-stranded DNA (dsDNA) viruses that infect bacteria. However, tailed bacterial dsDNA viruses (Caudovirales), which dominate sequence and culture collections, are not representative of the environmental diversity of viruses. In fact, non-tailed viruses often dominate ocean samples numerically, raising the fundamental question of the nature of these viruses. Here we characterize a group of marine dsDNA non-tailed viruses with short 10-kb genomes isolated during a study that quantified the diversity of viruses infecting Vibrionaceae bacteria. These viruses, which we propose to name the Autolykiviridae, represent a novel family within the ancient lineage of double jelly roll (DJR) capsid viruses. Ecologically, members of the Autolykiviridae have a broad host range, killing on average 34 hosts in four Vibrio species, in contrast to tailed viruses which kill on average only two hosts in one species. Biochemical and physical characterization of autolykiviruses reveals multiple virion features that cause systematic loss of DJR viruses in sequencing and culture-based studies, and we describe simple procedural adjustments to recover them. We identify DJR viruses in the genomes of diverse major bacterial and archaeal phyla, and in marine water column and sediment metagenomes, and find that their diversity greatly exceeds the diversity that is currently captured by the three recognized families of such viruses. Overall, these data suggest that viruses of the non-tailed dsDNA DJR lineage are important but often overlooked predators of bacteria and archaea that impose fundamentally different predation and gene transfer regimes on microbial systems than on tailed viruses, which form the basis of all environmental models of bacteria-virus interactions.

Natural resource landscapes of a marine bacterium reveal distinct fitness-determining genes across the genome.

Heterotrophic bacteria exploit diverse microhabitats in the ocean, from particles to transient gradients. Yet the degree to which genes and pathways can contribute to an organism's fitness on such complex and variable natural resource landscapes remains poorly understood. Here, we determine the gene-by-gene fitness of a generalist saprophytic marine bacterium (Vibrio sp. F13 9CS106) on complex resources derived from its natural habitats - copepods (Apocyclops royi) and brown algae (Fucus vesiculosus) - and as reference substrates, glucose and the polysaccharide alginate, derived from brown algal cell walls. We find that resource complexity strongly buffers fitness costs of mutations, and that anabolic rather than catabolic pathways are more stringently required, likely due to functional redundancy in the latter. Moreover, while carbohydrate-rich algae requires several synthesis pathways, protein-rich Apocyclops does not, suggesting this ancestral habitat for Vibrios is a replete medium with metabolically redundant substrates. We also identify a candidate fitness trade-off for algal colonization: deletion of mshA increases mutant fitness. Our results demonstrate that gene fitness depends on habitat composition, and suggest that this generalist uses distinct resources in different natural habitats. The results further indicate that substrate replete conditions may lead to relatively relaxed selection on catabolic genes.

Patients with atrial fibrillation and diabetes mellitus affected by nonalcoholic fatty liver disease have a greater risk of mortality and worse clinical outcomes.

Background: Nonalcoholic fatty liver disease (NAFLD) is associated with several adverse clinical outcomes. In this study, we assessed the association between NAFLD and several clinical outcome measures in patients with diabetes mellitus (DM) and atrial fibrillation (AF). Methods: We queried the National Inpatient Sample (NIS) between 2016 and 2019 for adult patients who were hospitalized with DM and AF. NAFLD was the independent variable. The primary outcome was inpatient mortality. The secondary outcomes were cardiogenic shock, cardiac arrest, gastrointestinal bleeding (GIB), invasive mechanical ventilation, length of stay, and total hospital charges. A multivariable logistic regression model was used to estimate odds ratios with a 95% confidence interval (CI) and a P value of less than 0.05 was considered significant. Results: There were 6 723 293 hospitalizations with AF and DM and 253 639 (3.7%) had NAFLD. NAFLD and non-NAFLD cohorts had a mean age of 70.4 vs. 73.8 years, respectively. Overall, 55.6% were male and 73.8% were White. NAFLD was found to be significantly associated with in-hospital mortality [adjusted odds ratio (AOR), 4.2; 95% CI, 4.08-4.32], cardiogenic shock (AOR, 4.78; 95% CI, 4.59-4.98), cardiac arrest (AOR, 3.43; 95% CI, 3.27-3.59), GIB (AOR, 1.92; 95% CI, 1.86-1.98), length of stay, and total hospital charges. Conclusion: In patients with AF and DM patients, the presence of NAFLD was associated with significantly worse clinical outcomes and higher resource utilization. Adverse cardiovascular events were common as well as GIB. Screening and prevention strategies modifying the risk and disease severity of NAFLD are needed.

COVID-19 stressors and mental health problems amongst women who arrived as refugees and those born in Australia.

Women from refugee background residing in high income countries are at greater mental health risk during the COVID-19 pandemic given their higher baseline prevalence of mental disorders, trauma exposures and social adversities. During the COVID-19 pandemic we drew on data from wave-4 of the WATCH cohort study, collected between October 2019 and June 2021. We conducted a cross-sectional analysis to compare the prevalence of common mental disorders (CMDs) from the sample of 650 consecutively recruited women, 339 (52.2%) from the refugee-background who were resettled in Australia and 311 (48.8%) randomly and contemporaneously selected Australian born women. We assessed COVID-19 psychosocial stressors: 1. COVID-related material hardship and 2. COVID-related fear and stress. We examined for associations between scores on these two items and CMDs in each group respectively. Compared to Australian-born woman, women from refugee background recorded a significantly higher prevalence of Major Depressive Disorder (MDD) (19.8% vs 13.5%), PTSD (9.7% vs 5.1%), Separation Anxiety Disorder (SEPAD) (19.8% vs 13.5%) and Persistent Complicated Bereavement Disorder (PCBD) (6.5% vs 2.9%). In refugee women, associations were found between COVID-related material hardship and CMDs [MDD, Relative Risk (RR) = 1.39, 95%CI: 1.02-1.89, p = 0.02] as well as between COVID-related fear and stress and CMDs (MDD, RR = 1.74, 95%CI: 1.04-2.90, p = 0.02 p = 0.02). For Australian-born women, associations were more commonly found between CMDs and material hardship. Our study demonstrates that both women from refugee background and those born in Australia are experiencing significant rates of CMD during the pandemic and that material hardship is an associated factor. We found that women from refugee background are at greater risk for mental health problems and are more likely to report an association of those problems with fear and stress related to COVID_19. All women, and particularly those from refugee background, require urgent and specialised attention to their mental health and psychosocial problems during this pandemic.

Correction: COVID-19 stressors and mental health problems amongst women who arrived as refugees and those born in Australia.

[This corrects the article DOI: 10.1371/journal.pgph.0002073.].

Correction: COVID-19 stressors and mental health problems amongst women who arrived as refugees and those born in Australia.

[This corrects the article DOI: 10.1371/journal.pgph.0002073.].

Vaginal Lactobacillus fatty acid response mechanisms reveal a novel strategy for bacterial vaginosis treatment.

Bacterial vaginosis (BV), a common syndrome characterized by Lactobacillus-deficient vaginal microbiota, is associated with adverse health outcomes. BV often recurs after standard antibiotic therapy in part because antibiotics promote microbiota dominance by Lactobacillus iners instead of Lactobacillus crispatus, which has more beneficial health associations. Strategies to promote L. crispatus and inhibit L. iners are thus needed. We show that oleic acid (OA) and similar long-chain fatty acids simultaneously inhibit L. iners and enhance L. crispatus growth. These phenotypes require OA-inducible genes conserved in L. crispatus and related species, including an oleate hydratase (ohyA) and putative fatty acid efflux pump (farE). FarE mediates OA resistance, while OhyA is robustly active in the human vaginal microbiota and sequesters OA in a derivative form that only ohyA-harboring organisms can exploit. Finally, OA promotes L. crispatus dominance more effectively than antibiotics in an in vitro model of BV, suggesting a novel approach for treatment.

Adsorption of perfluorooctanoic acid from water by pH-modulated Brönsted acid and base sites in mesoporous hafnium oxide ceramics.

Per- and polyfluoroalkyl substances (PFAS) are increasingly appearing in drinking water sources globally. Our work focuses specifically on the adsorption of the legacy perfluorooctanoic acid (PFOA) using mesoporous hafnium oxide (MHO) ceramic synthesized via a sol-gel process. Experiments were performed at varying pH to determine the effect of surface charge on adsorption capacity of PFOA by MHO, and to postulate adsorption behavior. At pH 2.3, the adsorption capacity of PFOA on MHO was 20.9 mg/g, whereas at a higher pH of 6.3, it was much lower at 9.2 mg/g. This was due to increased coulombic attractions at lower pH between the positively charged conjugate acid active sites on MHO surface and negatively charged deprotonated PFOA anion in solution. After adsorption, the solid MHO was regenerated via calcination, reducing the amount of toxic solid waste to be disposed since the adsorbent is regenerated, and the PFOA is completely removed.

Resolving the structure of phage-bacteria interactions in the context of natural diversity.

Microbial communities are shaped by viral predators. Yet, resolving which viruses (phages) and bacteria are interacting is a major challenge in the context of natural levels of microbial diversity. Thus, fundamental features of how phage-bacteria interactions are structured and evolve in the wild remain poorly resolved. Here we use large-scale isolation of environmental marine Vibrio bacteria and their phages to obtain estimates of strain-level phage predator loads, and use all-by-all host range assays to discover how phage and host genomic diversity shape interactions. We show that lytic interactions in environmental interaction networks (as observed in agar overlay) are sparse-with phage predator loads being low for most bacterial strains, and phages being host-strain-specific. Paradoxically, we also find that although overlap in killing is generally rare between tailed phages, recombination is common. Together, these results suggest that recombination during cryptic co-infections is an important mode of phage evolution in microbial communities. In the development of phages for bioengineering and therapeutics it is important to consider that nucleic acids of introduced phages may spread into local phage populations through recombination, and that the likelihood of transfer is not predictable based on lytic host range.

Appropriateness of antifungal prescribing in Oman.

Background: The inappropriate use of antimicrobials has substantially contributed to the development of antimicrobial drug resistance. Appropriate antibacterial prescribing has been emphasised, with minimal focus on appropriate prescribing of antifungals. Evaluation of antifungal use in the clinical setting is essential to prevent unnecessary drug exposure, development of resistance, adverse effects, and high hospitalisation costs. Objective: The purpose of this study was to assess the appropriateness of antifungal prescribing among adult patients at the Sultan Qaboos University Hospital (SQUH) in Oman. Methods: In this retrospective, observational study, the study population comprised adult patients treated with oral or intravenous antifungals between July 2018 and December 2019. The appropriateness of treatment was assessed using guidelines from the Infectious Diseases Society of America (IDSA) and the National Comprehensive Cancer Network (NCCN), as well as a set of literature-based criteria that were modified by SQUH infectious diseases team to suit local practices. These criteria included indication, dosage, and potential drug interactions. The primary outcome was the frequency of adherence to the treatment guidelines for fungal infections. Descriptive statistics were used for data analysis. Results: A total of 400 prescriptions were collected, of which 158 (39.5%) were for empirical therapy, 135 (33.8%) for targeted therapy, 69 (17.3%) for prophylactic therapy, and 38 (9.5%) for pre-emptive therapy. The overall appropriateness was 74.8%. The indication, dosage, and potential for antifungal-drug interactions were considered appropriate in 391 (97.8%), 314 (78.5%), and 381 (95.3%) prescriptions, respectively. Anidulafungin was the most prescribed antifungal agent, with 210 prescriptions (52.5%), followed by fluconazole with 102 prescriptions (25.5%), and voriconazole with 48 prescriptions (12%). Conclusion: In comparison with publised literature, our study revealed appropriate antifungal drug prescribing practices. However, studies with larger sample size in various hospital settings are necessary to confirm our findings on a national scale, and to obtain better statistical inferences and generalisability.

Screening and characterization of vaginal fluid donations for vaginal microbiota transplantation.

Bacterial vaginosis (BV), the overgrowth of diverse anaerobic bacteria in the vagina, is the most common cause of vaginal symptoms worldwide. BV frequently recurs after antibiotic therapy, and the best probiotic treatments only result in transient changes from BV-associated states to "optimal" communities dominated by a single species of Lactobacillus. Therefore, additional treatment strategies are needed to durably alter vaginal microbiota composition for patients with BV. Vaginal microbiota transplantation (VMT), the transfer of vaginal fluid from a healthy person with an optimal vaginal microbiota to a recipient with BV, has been proposed as one such alternative. However, VMT carries potential risks, necessitating strict safety precautions. Here, we present an FDA-approved donor screening protocol and detailed methodology for donation collection, storage, screening, and analysis of VMT material. We find that Lactobacillus viability is maintained for over six months in donated material stored at - 80 °C without glycerol or other cryoprotectants. We further show that species-specific quantitative PCR for L. crispatus and L. iners can be used as a rapid initial screening strategy to identify potential donors with optimal vaginal microbiomes. Together, this work lays the foundation for designing safe, reproducible trials of VMT as a treatment for BV.

Phage-host coevolution in natural populations.

Coevolution between bacteriophages (phages) and their bacterial hosts occurs through changes in resistance and counter-resistance mechanisms. To assess phage-host evolution in wild populations, we isolated 195 Vibrio crassostreae strains and 243 vibriophages during a 5-month time series from an oyster farm and combined these isolates with existing V. crassostreae and phage isolates. Cross-infection studies of 81,926 host-phage pairs delineated a modular network where phages are best at infecting co-occurring hosts, indicating local adaptation. Successful propagation of phage is restricted by the ability to adsorb to closely related bacteria and further constrained by strain-specific defence systems. These defences are highly diverse and predominantly located on mobile genetic elements, and multiple defences are active within a single genome. We further show that epigenetic and genomic modifications enable phage to adapt to bacterial defences and alter host range. Our findings reveal that the evolution of bacterial defences and phage counter-defences is underpinned by frequent genetic exchanges with, and between, mobile genetic elements.

Cysteine dependence of Lactobacillus iners is a potential therapeutic target for vaginal microbiota modulation.

Vaginal microbiota composition affects many facets of reproductive health. Lactobacillus iners-dominated microbial communities are associated with poorer outcomes, including higher risk of bacterial vaginosis (BV), compared with vaginal microbiota rich in L. crispatus. Unfortunately, standard-of-care metronidazole therapy for BV typically results in dominance of L. iners, probably contributing to post-treatment relapse. Here we generate an L. iners isolate collection comprising 34 previously unreported isolates from 14 South African women with and without BV and 4 previously unreported isolates from 3 US women. We also report an associated genome catalogue comprising 1,218 vaginal Lactobacillus isolate genomes and metagenome-assembled genomes from >300 women across 4 continents. We show that, unlike L. crispatus, L. iners growth is dependent on L-cysteine in vitro and we trace this phenotype to the absence of canonical cysteine biosynthesis pathways and a restricted repertoire of cysteine-related transport mechanisms. We further show that cysteine concentrations in cervicovaginal lavage samples correlate with Lactobacillus abundance in vivo and that cystine uptake inhibitors selectively inhibit L. iners growth in vitro. Combining an inhibitor with metronidazole promotes L. crispatus dominance of defined BV-like communities in vitro by suppressing L. iners growth. Our findings enable a better understanding of L. iners biology and suggest candidate treatments to modulate the vaginal microbiota to improve reproductive health for women globally.

Rapid evolutionary turnover of mobile genetic elements drives bacterial resistance to phages.

Although it is generally accepted that phages drive bacterial evolution, how these dynamics play out in the wild remains poorly understood. We found that susceptibility to viral killing in marine Vibrio is mediated by large and highly diverse mobile genetic elements. These phage defense elements display exceedingly fast evolutionary turnover, resulting in differential phage susceptibility among clonal bacterial strains while phage receptors remain invariant. Protection is cumulative, and a single bacterial genome can harbor 6 to 12 defense elements, accounting for more than 90% of the flexible genome among close relatives. The rapid turnover of these elements decouples phage resistance from other genomic features. Thus, resistance to phages in the wild follows evolutionary trajectories alternative to those predicted from laboratory-based evolutionary experiments.

Discovering the Molecular Determinants of Phaeobacter inhibens Susceptibility to Phaeobacter Phage MD18.

Bacteriophages have immense potential as antibiotic therapies and in genetic engineering. Understanding the mechanisms that bacteriophages implement to infect their hosts will allow researchers to manipulate these systems and adapt them to specific bacterial targets. In this study, we isolated a bacteriophage capable of infecting the marine alphaproteobacterium Phaeobacter inhibens and determined its mechanism of infection. Phaeobacter virus MD18, a novel species of bacteriophage isolated in Woods Hole, MA, exhibits potent lytic ability against P. inhibens and appears to be of the Siphoviridae morphotype. The genomic sequence of MD18 displayed significant similarity to another siphophage, the recently discovered Roseobacter phage DSS3P8, but genomic and phylogenetic analyses, assessing host range and a search of available metagenomes are all consistent with the conclusion that Phaeobacter phage MD18 is a novel lytic phage. We incubated MD18 with a library of barcoded P. inhibens transposon insertion mutants and identified 22 genes that appear to be required for phage predation of this host. Network analysis of these genes using genomic position, Gene Ontology (GO) term enrichment, and protein associations revealed that these genes are enriched for roles in assembly of a type IV pilus (T4P) and regulators of cellular morphology. Our results suggest that T4P serve as receptors for a novel marine virus that targets P. inhibens.IMPORTANCE Bacteriophages are useful nonantibiotic therapeutics for bacterial infections as well as threats to industries utilizing bacterial agents. This study identified Phaeobacter virus MD18, a phage antagonist of Phaeobacter inhibens, a bacterium with promising use as a probiotic for aquatic farming industries. Genomic analysis suggested that Phaeobacter phage MD18 has evolved to enhance its replication in P. inhibens by adopting favorable tRNA genes as well as through genomic sequence adaptation to resemble host codon usage. Lastly, a high-throughput analysis of P. inhibens transposon insertion mutants identified genes that modulate host susceptibility to phage MD18 and implicated the type IV pilus as the likely receptor recognized for adsorption. This study marks the first characterization of the relationship between P. inhibens and an environmentally sampled phage, which informs our understanding of natural threats to the bacterium and may promote the development of novel phage technologies for genetic manipulation of this host.

Development and characterization of cross-linked enzyme aggregates of thermotolerant alkaline protease from Bacillus licheniformis.

An alkaline protease was produced by B. licheniformis with 132.43±3.4U/mL activity in LSF which was further enhanced by optimizing culture conditions. The optimum enzyme activity (148.9±4.1U/mL) was harvested at pH7.5; temperature, 40°C and inoculum, 1.5mL after 48h incubation. Alkaline protease was immobilization by forming cross linked enzyme aggregates (CLEAs) and the processes of CLEAs formation was also optimized. The protease CLEAs developed using 80% ammonium sulfate, 65mM glutaraldehyde and 0.11mM BSA showed best activity recovery (39.76%). Free protease and CLEAs were characterized and compared. It was observed that CLEAs of protease exhibited broad pH range with best activity at pH10. The immobilized protease was also thermo-tolerant with optimum activity at 65°C temperature. The Vmax and Km of protease-CLEAs were 125.5U/mL and 18.97µM, respectively as compared to 104.9U/mL and 29µM, respectively for free protease. It was concluded that immobilized enzyme in the form of CLEAs is a valuable catalyst for potential industrial applications.

Feasibility of a Post-Auricle Wireless Power System for Pediatric Mechanical Circulatory Support Pumps.

Heart failure (HF) affects approximately 12,000-35,000 children each year in the United States. The development of blood pumps has provided circulatory support for many adults suffering with HF until they receive a heart transplant. However, while the development of blood pumps for adults has led to fullyimplantable continuous flow devices, blood pump technology for children has lagged significantly behind. One area for improving blood pump implantability in children is the use of wireless powering transfer systems (WPTS). These systems eliminate the power cord connecting the implanted blood pump to the external power supply. In adults, WPTS have decreased the number of power cord-related infections and have improved patient outcomes after pump implantation. Unfortunately, the components of these wireless systems are too large for children. In this paper we describe the preliminary work to develop a fully implantable WPTS specifically designed to power the Jarvik 2000 Child. Specifically, we design planar coils 36 um in thickness to be implanted in behind-the-ear fashion. An amplifier and rectifier circuit were also built to provide 15.7V and 0.5A of voltage and current to the pump.

Widespread distribution of prophage-encoded virulence factors in marine Vibrio communities.

Prophages are known to encode important virulence factors in the human pathogen Vibrio cholerae. However, little is known about the occurrence and composition of prophage-encoded traits in environmental vibrios. A database of 5,674 prophage-like elements constructed from 1,874 Vibrio genome sequences, covering sixty-four species, revealed that prophage-like elements encoding possible properties such as virulence and antibiotic resistance are widely distributed among environmental vibrios, including strains classified as non-pathogenic. Moreover, we found that 45% of Vibrio species harbored a complete prophage-like element belonging to the Inoviridae family, which encode the zonula occludens toxin (Zot) previously described in the V. cholerae. Interestingly, these zot-encoding prophages were found in a variety of Vibrio strains covering both clinical and marine isolates, including strains from deep sea hydrothermal vents and deep subseafloor sediments. In addition, the observation that a spacer from the CRISPR locus in the marine fish pathogen V. anguillarum strain PF7 had 95% sequence identity with a zot gene from the Inoviridae prophage found in V. anguillarum strain PF4, suggests acquired resistance to inoviruses in this species. Altogether, our results contribute to the understanding of the role of prophages as drivers of evolution and virulence in the marine Vibrio bacteria.

Immobilized lignin peroxidase from Ganoderma lucidum IBL-05 with improved dye decolorization and cytotoxicity reduction properties.

Use of free microbial enzymes for bioremediation and other industrial applications has several disadvantages like low stability and non-reusability in repeated batch operations. Immobilized enzymes are stable, recoverable and reusable in industrial processes. In this scenario G. lucidum IBL-05 LiP was entrapped in Ca-alginate beads using optimum concentrations of Na-alginate (4%), calcium chloride (0.2M) and glutraldehyde (0.02%). Optimum pH (pH 5) and temperature (55°C) for entrapped LiP were improved as compared to free LiP. Catalytic behavior of LiP also significantly enhanced, as Km value (0.25mM) decreased and Vmax value (868.6µmol/min) increased after ca-alginate entrapment of LiP. Decolorization efficiencies of Sandal reactive dyes after treating with immobilized LiP were in the range of 80-93%. A significant reduction was observed in water quality parameters including, BOD (66.44-98.22%), COD (81.34-98.82%) and TOC (80.21-97.77%) values. The cytotoxicity values for heamolytic and brine shrimp lethality of dye solutions treated with Ca-alginate immobilized LiP reduced up to 2.10-5.06% and 5.43-9.23%, respectively. On the basis of reduced toxicity and cytotoxicity values, it was concluded that Ca-alginate beads entrapped LiP may be an effective biocatalyst for bioremediation of dye based textile industry effluents.