The mechanisms responsible for 2-dimensional pattern formation in bacterial macrofiber populations grown on solid surfaces: fiber joining and the creation of exclusion zones

BACKGROUND: When Bacillus subtilis is cultured in a complex fluid medium under conditions where cell separation is suppressed, populations of multicellular macrofibers arise that mature into ball-like structures. The final sedentary forms are found distributed in patterns on the floor of the growth chamber although individual cells have no flagellar-driven motility. The nature of the patterns and their mode of formation are described in this communication. RESULTS: Time-lapse video films reveal that fiber-fiber contact in high density populations of macrofibers resulted in their joining either by entwining or supercoiling. Joining led to the production of aggregate structures that eventually contained all of the fibers located in an initial area. Fibers were brought into contact by convection currents and motions associated with macrofiber self-assembly such as walking, pivoting and supercoiling. Large sedentary aggregate structures cleared surrounding areas of other structures by dragging them into the aggregate using supercoiling of extended fibers to power dragging. The spatial distribution of aggregate structures in 6 mature patterns containing a total of 637 structures was compared to that expected in random theoretical populations of the same size distributed in the same surface area. Observed and expected patterns differ significantly. The distances separating all nearest neighbors from one another in observed populations were also measured. The average distance obtained from 1451 measurements involving 519 structures was 0.73 cm. These spacings were achieved without the use of flagella or other conventional bacterial motility mechanisms. A simple mathematical model based upon joining of all structures within an area defined by the minimum observed distance between structures in populations explains the observed distributions very well. CONCLUSIONS: Bacterial macrofibers are capable of colonizing a solid surface by forming large multicellular aggregate structures that are distributed in unique two-dimensional patterns. Cell growth geometry governs in an hierarchical way the formation of these patterns using forces associated with twisting and supercoiling to drive motions and the joining of structures together. Joining by entwining, supercoiling or dragging all require cell growth in a multicellular form, and all result in tightly fused aggregate structures

The dynamic behavior of bacterial macrofibers growing with one end prevented from rotating: variation in shaft rotation along the fiber's length, and supercoil movement on a solid surface toward the constrained end

BACKGROUND: Bacterial macrofibers twist as they grow, writhe, supercoil and wind up into plectonemic structures (helical forms the individual filaments of which cannot be taken apart without unwinding) that eventually carry loops at both of their ends. Terminal loops rotate about the axis of a fiber's shaft in contrary directions at increasing rate as the shaft elongates. Theory suggests that rotation rates should vary linearly along the length of a fiber ranging from maxima at the loop ends to zero at an intermediate point. Blocking rotation at one end of a fiber should lead to a single gradient: zero at the blocked end to maximum at the free end. We tested this conclusion by measuring directly the rotation at various distances along fiber length from the blocked end. The movement of supercoils over a solid surface was also measured in tethered macrofibers. RESULTS: Macrofibers that hung down from a floating wire inserted through a terminal loop grew vertically and produced small plectonemic structures by supercoiling along their length. Using these as markers for shaft rotation we observed a uniform gradient of initial rotation rates with slopes of 25.6°/min. mm. and 36.2°/min. mm. in two different fibers. Measurements of the distal tip rotation in a third fiber as a function of length showed increases proportional to increases in length with constant of proportionality 79.2 rad/mm. Another fiber tethered to the floor grew horizontally with a length-doubling time of 74 min, made contact periodically with the floor and supercoiled repeatedly. The supercoils moved over the floor toward the tether at approximately 0.06 mm/min, 4 times faster than the fiber growth rate. Over a period of 800 minutes the fiber grew to 23 mm in length and was entirely retracted back to the tether by a process involving 29 supercoils. CONCLUSIONS: The rate at which growing bacterial macrofibers rotated about the axis of the fiber shaft measured at various locations along fibers in structures prevented from rotating at one end reveal that the rate varied linearly from zero at the blocked end to maximum at the distal end. The increasing number of twisting cells in growing fibers caused the distal end to continuously rotate faster. When the free end was intermittently prevented from rotating a torque developed which was relieved by supercoiling. On a solid surface the supercoils moved toward the end permanently blocked from rotating as a result of supercoil rolling over the surface and the formation of new supercoils that reduced fiber length between the initial supercoil and the wire tether. All of the motions are ramifications of cell growth with twist and the highly ordered multicellular state of macrofibers

Whole-genome sequencing shows that patient-to-patient transmission rarely accounts for acquisition of Staphylococcus aureus in an intensive care unit

BACKGROUND  Strategies to prevent Staphylococcus aureus infection in hospitals focus on patient-to-patient transmission. We used whole-genome sequencing to investigate the role of colonized patients as the source of new S. aureus acquisitions, and the reliability of identifying patient-to-patient transmission using the conventional approach of spa typing and overlapping patient stay. METHODS Over 14 months, all unselected patients admitted to an adult intensive care unit (ICU) were serially screened for S. aureus. All available isolates (n = 275) were spa typed and underwent whole-genome sequencing to investigate their relatedness at high resolution. RESULTS Staphylococcus aureus was carried by 185 of 1109 patients sampled within 24 hours of ICU admission (16.7%); 59 (5.3%) patients carried methicillin-resistant S. aureus (MRSA). Forty-four S. aureus (22 MRSA) acquisitions while on ICU were detected. Isolates were available for genetic analysis from 37 acquisitions. Whole-genome sequencing indicated that 7 of these 37 (18.9%) were transmissions from other colonized patients. Conventional methods (spa typing combined with overlapping patient stay) falsely identified 3 patient-to-patient transmissions (all MRSA) and failed to detect 2 acquisitions and 4 transmissions (2 MRSA). CONCLUSIONS Only a minority of S. aureus acquisitions can be explained by patient-to-patient transmission. Whole-genome sequencing provides the resolution to disprove transmission events indicated by conventional methods and also to reveal otherwise unsuspected transmission events. Whole-genome sequencing should replace conventional methods for detection of nosocomial S. aureus transmission

The management of Staphylococcus aureus bacteremia in the United Kingdom and Vietnam: a multi-centre evaluation.

Background: Staphylococcus aureus bacteremia is a common and serious infection worldwide and although treatment guidelines exist, there is little consensus on optimal management. In this study we assessed the variation in management and adherence to treatment guidelines of S. aureus bacteremia. Methodology/Principal Findings: We prospectively recorded baseline clinical characteristics, management, and in-hospital outcome of all adults with S. aureus bacteremia treated consecutively over one year in eight centres in the United Kingdom, three in Vietnam and one in Nepal. 630 adults were treated for S. aureus bacteremia: 549 in the UK (21% methicillin-resistant), 80 in Vietnam (19% methicillin-resistant) and 1 in Nepal. In the UK, 41% had a removable infection focus (50% intravenous catheter-related), compared to 12% in Vietnam. Significantly (p50% of treatment with oral antibiotics alone (25% versus 4%). UK centres varied significantly (p50% of treatment (range 12-40%), in those treated for longer than 28 days (range 13-54%), and in those given combination therapy (range 14-94%). 24% died during admission: older age, time in hospital before bacteremia, and an unidentified infection focus were independent predictors of in-hospital death (p<0.001). Conclusions/Significance: The management of S. aureus bacteremia varies widely between the UK and Vietnam and between centres in the UK with little adherence to published guidelines. Controlled trials defining optimal therapy are urgently required

Global hepatitis C elimination: an investment framework

WHO has set global targets for the elimination of hepatitis B and hepatitis C as a public health threat by 2030. However, investment in elimination programmes remains low. To help drive political commitment and catalyse domestic and international financing, we have developed a global investment framework for the elimination of hepatitis B and hepatitis C. The global investment framework presented in this Health Policy paper outlines national and international activities that will enable reductions in hepatitis C incidence and mortality, and identifies potential sources of funding and tools to help countries build the economic case for investing in national elimination activities. The goal of this framework is to provide a way for countries, particularly those with minimal resources, to gain the substantial economic benefit and cost savings that come from investing in hepatitis C elimination

THE CONCISE GUIDE TO PHARMACOLOGY 2021/22:Transporters

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15543. Transporters are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors and enzymes. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate

New Variant of Multidrug-Resistant Salmonella enterica Serovar Typhimurium Associated with Invasive Disease in Immunocompromised Patients in Vietnam.

Nontyphoidal Salmonella (NTS), particularly Salmonella enterica serovar Typhimurium, is among the leading etiologic agents of bacterial enterocolitis globally and a well-characterized cause of invasive disease (iNTS) in sub-Saharan Africa. In contrast, S Typhimurium is poorly defined in Southeast Asia, a known hot spot for zoonotic disease with a recently described burden of iNTS disease. Here, we aimed to add insight into the epidemiology and potential impact of zoonotic transfer and antimicrobial resistance (AMR) in S Typhimurium associated with iNTS and enterocolitis in Vietnam. We performed whole-genome sequencing and phylogenetic reconstruction on 85 human (enterocolitis, carriage, and iNTS) and 113 animal S Typhimurium isolates isolated in Vietnam. We found limited evidence for the zoonotic transmission of S Typhimurium. However, we describe a chain of events where a pandemic monophasic variant of S Typhimurium (serovar I:4,[5],12:i:- sequence type 34 [ST34]) has been introduced into Vietnam, reacquired a phase 2 flagellum, and acquired an IncHI2 multidrug-resistant plasmid. Notably, these novel biphasic ST34 S Typhimurium variants were significantly associated with iNTS in Vietnamese HIV-infected patients. Our study represents the first characterization of novel iNTS organisms isolated outside sub-Saharan Africa and outlines a new pathway for the emergence of alternative Salmonella variants into susceptible human populations.IMPORTANCESalmonella Typhimurium is a major diarrheal pathogen and associated with invasive nontyphoid Salmonella (iNTS) disease in vulnerable populations. We present the first characterization of iNTS organisms in Southeast Asia and describe a different evolutionary trajectory from that of organisms causing iNTS in sub-Saharan Africa. In Vietnam, the globally distributed monophasic variant of Salmonella Typhimurium, the serovar I:4,[5],12:i:- ST34 clone, has reacquired a phase 2 flagellum and gained a multidrug-resistant plasmid to become associated with iNTS disease in HIV-infected patients. We document distinct communities of S Typhimurium and I:4,[5],12:i:- in animals and humans in Vietnam, despite the greater mixing of these host populations here. These data highlight the importance of whole-genome sequencing surveillance in a One Health context in understanding the evolution and spread of resistant bacterial infections

Adjunctive rifampicin to reduce early mortality from Staphylococcus aureus bacteraemia: the ARREST RCT.

BACKGROUND: Staphylococcus aureus bacteraemia is a common and frequently fatal infection. Adjunctive rifampicin may enhance early S. aureus killing, sterilise infected foci and blood faster, and thereby reduce the risk of dissemination, metastatic infection and death. OBJECTIVES: To determine whether or not adjunctive rifampicin reduces bacteriological (microbiologically confirmed) failure/recurrence or death through 12 weeks from randomisation. Secondary objectives included evaluating the impact of rifampicin on all-cause mortality, clinically defined failure/recurrence or death, toxicity, resistance emergence, and duration of bacteraemia; and assessing the cost-effectiveness of rifampicin. DESIGN: Parallel-group, randomised (1 : 1), blinded, placebo-controlled multicentre trial. SETTING: UK NHS trust hospitals. PARTICIPANTS: Adult inpatients (≥ 18 years) with meticillin-resistant or susceptible S. aureus grown from one or more blood cultures, who had received < 96 hours of antibiotic therapy for the current infection, and without contraindications to rifampicin. INTERVENTIONS: Adjunctive rifampicin (600-900 mg/day, oral or intravenous) or placebo for 14 days in addition to standard antibiotic therapy. Investigators and patients were blinded to trial treatment. Follow-up was for 12 weeks (assessments at 3, 7, 10 and 14 days, weekly until discharge and final assessment at 12 weeks post randomisation). MAIN OUTCOME MEASURES: The primary outcome was all-cause bacteriological (microbiologically confirmed) failure/recurrence or death through 12 weeks from randomisation. RESULTS: Between December 2012 and October 2016, 758 eligible participants from 29 UK hospitals were randomised: 370 to rifampicin and 388 to placebo. The median age was 65 years [interquartile range (IQR) 50-76 years]. A total of 485 (64.0%) infections were community acquired and 132 (17.4%) were nosocomial; 47 (6.2%) were caused by meticillin-resistant S. aureus. A total of 301 (39.7%) participants had an initial deep infection focus. Standard antibiotics were given for a median of 29 days (IQR 18-45 days) and 619 (81.7%) participants received flucloxacillin. By 12 weeks, 62 out of 370 (16.8%) patients taking rifampicin versus 71 out of 388 (18.3%) participants taking the placebo experienced bacteriological (microbiologically confirmed) failure/recurrence or died [absolute risk difference -1.4%, 95% confidence interval (CI) -7.0% to 4.3%; hazard ratio 0.96, 95% CI 0.68 to 1.35; p = 0.81]. There were 4 (1.1%) and 5 (1.3%) bacteriological failures (p = 0.82) in the rifampicin and placebo groups, respectively. There were 3 (0.8%) versus 16 (4.1%) bacteriological recurrences (p = 0.01), and 55 (14.9%) versus 50 (12.9%) deaths without bacteriological failure/recurrence (p = 0.30) in the rifampicin and placebo groups, respectively. Over 12 weeks, there was no evidence of differences in clinically defined failure/recurrence/death (p = 0.84), all-cause mortality (p = 0.60), serious (p = 0.17) or grade 3/4 (p = 0.36) adverse events (AEs). However, 63 (17.0%) participants in the rifampicin group versus 39 (10.1%) participants in the placebo group experienced antibiotic or trial drug-modifying AEs (p = 0.004), and 24 (6.5%) participants in the rifampicin group versus 6 (1.5%) participants in the placebo group experienced drug-interactions (p = 0.0005). Evaluation of the costs and health-related quality-of-life impacts revealed that an episode of S. aureus bacteraemia costs an average of £12,197 over 12 weeks. Rifampicin was estimated to save 10% of episode costs (p = 0.14). After adjustment, the effect of rifampicin on total quality-adjusted life-years (QALYs) was positive (0.004 QALYs), but not statistically significant (standard error 0.004 QALYs). CONCLUSIONS: Adjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S. aureus bacteraemia. FUTURE WORK: Given the substantial mortality, other antibiotic combinations or improved source management should be investigated. TRIAL REGISTRATIONS: Current Controlled Trials ISRCTN37666216, EudraCT 2012-000344-10 and Clinical Trials Authorisation 00316/0243/001. FUNDING: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 22, No. 59. See the NIHR Journals Library website for further project information.NIHR HTA Programm

Inactivation of Staphylococcal Phenol Soluble Modulins by Serum Lipoprotein Particles

Staphylococcus aureus virulence has been associated with the production of phenol soluble modulins (PSM). PSM are known to activate, attract and lyse neutrophils. However, the functional characterizations were generally performed in the absence of human serum. Here, we demonstrate that human serum can inhibit all the previously-described activities of PSM. We observed that serum can fully block both the cell lysis and FPR2 activation of neutrophils. We show a direct interaction between PSM and serum lipoproteins in human serum and whole blood. Subsequent analysis using purified high, low, and very low density lipoproteins (HDL, LDL, and VLDL) revealed that they indeed neutralize PSM. The lipoprotein HDL showed highest binding and antagonizing capacity for PSM. Furthermore, we show potential intracellular production of PSM by S. aureus upon phagocytosis by neutrophils, which opens a new area for exploration of the intracellular lytic capacity of PSM. Collectively, our data show that in a serum environment the function of PSM as important extracellular toxins should be reconsidered

Empagliflozin in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial

BackgroundEmpagliflozin has been proposed as a treatment for COVID-19 on the basis of its anti-inflammatory, metabolic, and haemodynamic effects. The RECOVERY trial aimed to assess its safety and efficacy in patients admitted to hospital with COVID-19.MethodsIn the randomised, controlled, open-label RECOVERY trial, several possible treatments are compared with usual care in patients hospitalised with COVID-19. In this analysis, we assess eligible and consenting adults who were randomly allocated in a 1:1 ratio to either usual standard of care alone or usual standard of care plus oral empagliflozin 10 mg once daily for 28 days or until discharge (whichever came first) using web-based simple (unstratified) randomisation with allocation concealment. The primary outcome was 28-day mortality; secondary outcomes were duration of hospitalisation and (among participants not on invasive mechanical ventilation at baseline) the composite of invasive mechanical ventilation or death. On March 3, 2023 the independent data monitoring committee recommended that the investigators review the data and recruitment was consequently stopped on March 7, 2023. The ongoing RECOVERY trial is registered with ISRCTN (50189673) and ClinicalTrials.gov(NCT04381936)FindingsBetween July 28, 2021 and March 6, 2023, 4271 patients were randomly allocated to receive either empagliflozin (2113 patients) or usual care alone (2158 patients). Primary and secondary outcome data were known for greater than 99% of randomly assigned patients. Overall, 289 (14%) of 2113 patients allocated to empagliflozin and 307 (14%) of 2158 patients allocated to usual care died within 28 days (rate ratio 0·96 [95% CI 0·82–1·13]; p=0·64). There was no evidence of significant differences in duration of hospitalisation (median 8 days for both groups) or the proportion of patients discharged from hospital alive within 28 days (1678 [79%] in the empagliflozin group vs 1677 [78%] in the usual care group; rate ratio 1·03 [95% CI 0·96–1·10]; p=0·44). Among those not on invasive mechanical ventilation at baseline, there was no evidence of a significant difference in the proportion meeting the composite endpoint of invasive mechanical ventilation or death (338 [16%] of 2084 vs 371 [17%] of 2143; risk ratio 0·95 [95% CI 0·84–1·08]; p=0·44). Two serious adverse events believed to be related to empagliflozin were reported: both were ketosis without acidosis.InterpretationIn adults hospitalised with COVID-19, empagliflozin was not associated with reductions in 28-day mortality, duration of hospital stay, or risk of progressing to invasive mechanical ventilation or death so is not indicated for the treatment of such patients unless there is an established indication due to a different condition such as diabetes