Phylodynamic signatures in the emergence of community-associated MRSA.

Community-associated, methicillin-resistant Staphylococcus aureus (MRSA) lineages have emerged in many geographically distinct regions around the world during the past 30 y. Here, we apply consistent phylodynamic methods across multiple community-associated MRSA lineages to describe and contrast their patterns of emergence and dissemination. We generated whole-genome sequencing data for the Australian sequence type (ST) ST93-MRSA-IV from remote communities in Far North Queensland and Papua New Guinea, and the Bengal Bay ST772-MRSA-V clone from metropolitan communities in Pakistan. Increases in the effective reproduction number (Re) and sustained transmission (Re > 1) coincided with spread of progenitor methicillin-susceptible S. aureus (MSSA) in remote northern Australian populations, dissemination of the ST93-MRSA-IV genotype into population centers on the Australian East Coast, and subsequent importation into the highlands of Papua New Guinea and Far North Queensland. Applying the same phylodynamic methods to existing lineage datasets, we identified common signatures of epidemic growth in the emergence and epidemiological trajectory of community-associated S. aureus lineages from America, Asia, Australasia, and Europe. Surges in Re were observed at the divergence of antibiotic-resistant strains, coinciding with their establishment in regional population centers. Epidemic growth was also observed among drug-resistant MSSA clades in Africa and northern Australia. Our data suggest that the emergence of community-associated MRSA in the late 20th century was driven by a combination of antibiotic-resistant genotypes and host epidemiology, leading to abrupt changes in lineage-wide transmission dynamics and sustained transmission in regional population centers.

Oral subchronic toxicity study and genetic toxicity evaluation of mitoquinone mesylate.

Mitochondrial dysfunction and excessive reactive oxygen species production contributes to the pathophysiology of aging. Coenzyme Q10 is thought to protect mitochondria from oxidative damage; thus, mitoquinone was developed as mitochondria-targeted analogue with similar antioxidant activity. Mitoquinone is the oxidized form of mitoquinol. Mitoquinone/mitoquinol mesylate has been proposed as a food ingredient. As part of the safety analysis, we performed genotoxicity assays and a 39-week toxicity study to determine overall toxicity potential. Mitoquinone mesylate showed no evidence of genotoxic potential in two in vitro assays, bacterial reverse mutation and human lymphocyte chromosome aberration, nor in the in vivo micronucleus test in rats. In the 39-week study in dogs, there were no findings observed, which were considered to represent adverse systemic toxicity; therefore, the high dose level (40 mg/kg/day) was considered the NOAEL. The principal findings in this study were fecal disturbances and vomiting. These findings were considered to be due to a local, possibly irritant effect of the test substance on the gastrointestinal tract and were not considered adverse as there were no impacts on clinical or histopathology. This highest dose exceeds the expected daily human intake more than 100-fold. Data from well-designed clinical trials actively collecting safety endpoints corroborate that 20 mg/day can be safely consumed and is not likely to result in significant gastrointestinal complaints. These results support the conclusion that the use of mitoquinone/mitoquinol mesylate as a food ingredient is safe.

Photographs as an essential biodiversity resource: drivers of gaps in the vascular plant photographic record.

The photographic record is increasingly becoming an important biodiversity resource for primary research and conservation monitoring. However, globally, there are important gaps in this record even in relatively well-researched floras. To quantify the gaps in the Australian native vascular plant photographic record, we systematically surveyed 33 sources of well-curated species photographs, assembling a list of species with accessible and verifiable photographs, as well as the species for which this search failed. Of 21 077 Australian native species, 3715 lack a verifiable photograph across our 33 surveyed resources. There are three major geographic hotspots of unphotographed species in Australia, all far from current population centres. Many unphotographed species are small in stature or uncharismatic, and many are also recently described. The large number of recently described species without accessible photographs was surprising. There are longstanding efforts in Australia to organise the plant photographic record, but in the absence of a global consensus to treat photographs as an essential biodiversity resource, this has not become common practice. Many recently described species are small-range endemics and some have special conservation status. Completing the botanical photographic record across the globe will facilitate a virtuous feedback loop of more efficient identification, monitoring and conservation.

Leaf relative water content at 50% stomatal conductance measured by noninvasive NMR is linked to climate of origin in nine species of eucalypt.

Stomata are the gatekeepers of plant water use and must quickly respond to changes in plant water status to ensure plant survival under fluctuating environmental conditions. The mechanism for their closure is highly sensitive to disturbances in leaf water status, which makes isolating their response to declining water content difficult to characterise and to compare responses among species. Using a small-scale non-destructive nuclear magnetic resonance spectrometer as a leaf water content sensor, we measure the stomatal response to rapid induction of water deficit in the leaves of nine species of eucalypt from contrasting climates. We found a strong linear correlation between relative water content at 50% stomatal conductance (RWCgs50 ) and mean annual temperature at the climate of origin of each species. We also show evidence for stomata to maintain control over water loss well below turgor loss point in species adapted to warmer climates and secondary increases in stomatal conductance despite declining water content. We propose that RWCgs50 is a promising trait to guide future investigations comparing stomatal responses to water deficit. It may provide a useful phenotyping trait to delineate tolerance and adaption to hot temperatures and high leaf-to-air vapour pressure deficits.

Genomic analysis of 600 vancomycin-resistant Enterococcus faecium reveals a high prevalence of ST80 and spread of similar vanA regions via IS1216E and plasmid transfer in diverse genetic lineages in Ireland.

BACKGROUND: Vancomycin-resistant Enterococcus faecium (VREfm) cause a wide range of hospital infections. Ireland has had one of the highest invasive VREfm infection rates in Europe over the last decade, yet little is known about Irish VREfm. OBJECTIVES: To investigate the population structure of Irish VREfm, explore diversity by analysing the vanA transposon region and compare Irish, Danish and global isolates. METHODS: E. faecium (n = 648) from five Irish hospitals were investigated, including VREfm [547 rectal screening and 53 bloodstream infection (BSI)] isolates and 48 vancomycin-susceptible (VSEfm) BSI isolates recovered between June 2017 and December 2019. WGS and core-genome MLST (cgMLST) were used to assess population structure. Genetic environments surrounding vanA were resolved by hybrid assembly of short-read (Illumina) and long-read (Oxford Nanopore Technologies) sequences. RESULTS: All isolates belonged to hospital-adapted clade A1 and the majority (435/648) belonged to MLST ST80. The population structure was highly polyclonal; cgMLST segregated 603/648 isolates into 51 clusters containing mixtures of screening and BSI isolates, isolates from different hospitals, and VREfm and VSEfm. Isolates within clusters were closely related (mean average ≤16 allelic differences). The majority (96.5%) of VREfm harboured highly similar vanA regions located on circular or linear plasmids with multiple IS1216E insertions, variable organization of vanA operon genes and 78.6% harboured a truncated tnpA transposase. Comparison of 648 Irish isolates with 846 global E. faecium from 30 countries using cgMLST revealed little overlap. CONCLUSIONS: Irish VREfm are polyclonal, yet harbour a characteristic plasmid-located vanA region with multiple IS1216E insertions that may facilitate spread.

Linezolid resistance in Enterococcus faecium and Enterococcus faecalis from hospitalized patients in Ireland: high prevalence of the MDR genes optrA and poxtA in isolates with diverse genetic backgrounds.

OBJECTIVES: To investigate the prevalence of the optrA, poxtA and cfr linezolid resistance genes in linezolid-resistant enterococci from Irish hospitals and to characterize associated plasmids. METHODS: One hundred and fifty-four linezolid-resistant isolates recovered in 14 hospitals between June 2016 and August 2019 were screened for resistance genes by PCR. All isolates harbouring resistance genes, and 20 without, underwent Illumina MiSeq WGS. Isolate relatedness was assessed using enterococcal whole-genome MLST. MinION sequencing (Oxford Nanopore) and hybrid assembly were used to resolve genetic environments/plasmids surrounding resistance genes. RESULTS: optrA and/or poxtA were identified in 35/154 (22.7%) isolates, the highest prevalence reported to date. Fifteen isolates with diverse STs harboured optrA only; one Enterococcus faecium isolate harboured optrA (chromosome) and poxtA (plasmid). Seven Enterococcus faecalis and one E. faecium harboured optrA on a 36 331 bp plasmid with 100% identity to the previously described optrA-encoding conjugative plasmid pE349. Variations around optrA were also observed, with optrA located on plasmids in five isolates and within the chromosome in three isolates. Nine E. faecium and 10 E. faecalis harboured poxtA, flanked by IS1216E, within an identical 4001 bp region on plasmids exhibiting 72.9%-100% sequence coverage to a 21 849 bp conjugative plasmid. E. faecalis isolates belonged to ST480, whereas E. faecium isolates belonged to diverse STs. Of the remaining 119 linezolid-resistant isolates without linezolid resistance genes, 20 investigated representatives all harboured the G2576T 23S RNA gene mutation associated with linezolid resistance. CONCLUSIONS: This high prevalence of optrA and poxtA in diverse enterococcal lineages in Irish hospitals indicates significant selective pressure(s) for maintenance.

The challenge of de-labeling penicillin allergy.

BACKGROUND: Even though 8%-25% of most populations studied globally are labeled as penicillin allergic, most diagnoses of penicillin allergy are made in childhood and relate to events that are either not allergic in nature, are low risk for immediate hypersensitivity, or are a potential true allergy that has waned over time. Penicillin allergy labels directly impact antimicrobial stewardship by leading to use of less effective and broader spectrum antimicrobials and are associated with antimicrobial resistance. They may also delay appropriate antimicrobial therapy and lead to increased risk of specific adverse healthcare outcomes. Operationalizing penicillin allergy de-labeling into a new arm of antimicrobial stewardship programs (ASPs) has become an increasing global focus. METHODS: We performed an evidence-based narrative review of the literature of penicillin allergy label carriage, the adverse effects of penicillin allergy labels, and current approaches and barriers to penicillin allergy de-labeling. Over the period 1928-2018 in Pubmed and Medline, search terms used included "penicillin allergy" or "penicillin hypersensitivity" alone or in combination with "adverse events," "testing," "evaluation," "effects," "label," "de-labeling," "prick or epicutaneous," and "intradermal" skin testing, "oral challenge or provocation," "cross-reactivity," and "antimicrobial stewardship". RESULTS: Penicillin allergy labels are highly prevalent, largely inaccurate and their carriage may lead to unnecessary treatment and inferior outcomes with alternative agents as well as adverse public health outcomes such as antibiotic resistance. CONCLUSIONS: Operationalizing penicillin allergy de-labeling as an aspect of ASP has become an increasing global focus. There is a need for validated approaches that optimally combine the use of history and ingestion challenge with or without proceeding formal skin testing to tackle penicillin allergy efficiently within complex healthcare systems. At the same time, there is great promise for penicillin allergy evaluation and de-labeling as an individual and public health strategy to reduce adverse healthcare outcomes, improve antimicrobial stewardship, and decrease healthcare costs.

Frustration between two- and three-dimensional smectic ordering leads to a biaxial nematic phase.

The bola-amphiphilic, T-shaped mesogen CT2 has an aromatic, biphenyl core terminated on both ends by hydrophilic groups and a semi-perfluorinated, aliphatic side chain. Upon cooling from the isotropic phase, the fluorinated tails and the polar, rod-like cores nanophase-segregate to form a fluid lamellar phase. At high temperatures, the biphenyl cores are orientationally disordered in two dimensions (2D) in the lamellar planes but on further cooling the cores order orientationally, giving a biaxial lamellar phase with 2D nematic in-plane ordering. At lower temperature, the aromatic and hydrophilic parts of the cores nanosegregate within the lamellae and 2D smectic correlations of the head groups develop. X-ray diffraction shows that this 2D smectic ordering is incompatible with the initial lamellar structure, with both structures becoming short-ranged, resulting in a 3D biaxial nematic phase with macroscopic orthorhombic symmetry featuring strong smectic correlations in two orthogonal spatial dimensions. Freeze-fracture transmission electron microscopy enables direct visualization of the resulting short-ranged periodic structures.

An epidemic CC1-MRSA-IV clone yields false-negative test results in molecular MRSA identification assays: a note of caution, Austria, Germany, Ireland, 2020.

We investigated why a clinical meticillin-resistant Staphylococcus aureus (MRSA) isolate yielded false-negative results with some commercial PCR tests for MRSA detection. We found that an epidemic European CC1-MRSA-IV clone generally exhibits this behaviour. The failure of the assays was attributable to a large insertion in the orfX/SCCmec integration site. To ensure the reliability of molecular MRSA tests, it is vital to monitor emergence of new SCCmec types and junction sites.

A molecular epidemiological investigation of methicillin-susceptible Staphylococcus aureus causing bloodstream infections in Ireland, 2006-2017.

The prevalence of methicillin-susceptible Staphylococcus aureus (MSSA) bloodstream infections (BSIs) has increased in many countries, including Ireland. This study aimed to investigate the molecular epidemiology of MSSA causing BSIs in Irish hospitals between 2006 and 2017, when MSSA BSIs increased, to identify any potential patient or pathogen contributing factors. A total of 252 MSSA isolates from patients in Irish hospitals in 2006/2007, 2011 and 2017 underwent spa typing and DNA microarray profiling. Each patient's gender, age, 14-day mortality and epidemiological context of infection were recorded. Significant increases in community-onset (CO) MSSA BSIs and the average patient's age and decreases in hospital-onset (HO) MSSA were identified. Although, extensive genetic diversity was detected amongst the isolates, i.e. 24 multilocus sequence type clonal complexes (CCs)/sequence types and 124 spa types, three CCs (CC30, CC45, CC5) dominated, albeit in different proportions, during the study periods. CC30 declined significantly, in particular spa type t021, and was more common amongst HO-MSSA and CC45 and CC8 increased, particularly spa types t015 and t008, respectively, and were more common amongst CO-MSSA. Five of the seven most frequent spa types were more common amongst CO-MSSA. Although overall multidrug resistance decreased, the prevalence of erm(C) increased significantly and virulence genes decreased, mostly notably egc, tst, scn, sep and fnbB. This study highlights the threat posed by the increasing prevalence of CO-MSSA BSIs and suggests an association with the increasing prevalence of CC45 CO-MSSA, decreasing prevalence of CC30 HO-MSSA, an ageing population and an overall decrease in virulence and resistance genes.

Macrophage-Associated Lipin-1 Enzymatic Activity Contributes to Modified Low-Density Lipoprotein-Induced Proinflammatory Signaling and Atherosclerosis.

OBJECTIVE: Macrophage proinflammatory responses induced by modified low-density lipoproteins (modLDL) contribute to atherosclerotic progression. How modLDL causes macrophages to become proinflammatory is still enigmatic. Macrophage foam cell formation induced by modLDL requires glycerolipid synthesis. Lipin-1, a key enzyme in the glycerolipid synthesis pathway, contributes to modLDL-elicited macrophage proinflammatory responses in vitro. The objective of this study was to determine whether macrophage-associated lipin-1 contributes to atherogenesis and to assess its role in modLDL-mediated signaling in macrophages. APPROACH AND RESULTS: We developed mice lacking lipin-1 in myeloid-derived cells and used adeno-associated viral vector 8 expressing the gain-of-function mutation of mouse proprotein convertase subtilisin/kexin type 9 (adeno-associated viral vector 8-proprotein convertase subtilisin/kexin type 9) to induce hypercholesterolemia and plaque formation. Mice lacking myeloid-associated lipin-1 had reduced atherosclerotic burden compared with control mice despite similar plasma lipid levels. Stimulation of bone marrow-derived macrophages with modLDL activated a persistent protein kinase Cα/ßII-extracellular receptor kinase1/2-jun proto-oncogene signaling cascade that contributed to macrophage proinflammatory responses that was dependent on lipin-1 enzymatic activity. CONCLUSIONS: Our data demonstrate that macrophage-associated lipin-1 is atherogenic, likely through persistent activation of a protein kinase Cα/ßII-extracellular receptor kinase1/2-jun proto-oncogene signaling cascade that contributes to foam cell proinflammatory responses. Taken together, these results suggest that modLDL-induced foam cell formation and modLDL-induced macrophage proinflammatory responses are not independent consequences of modLDL stimulation but rather are both directly influenced by enhanced lipid synthesis.

Molecular Characterization of Nasal Methicillin-Resistant Staphylococcus aureus Isolates Showing Increasing Prevalence of Mupirocin Resistance and Associated Multidrug Resistance following Attempted Decolonization.

Sequential methicillin-resistant Staphylococcus aureus (MRSA) isolates from patients following attempted mupirocin nasal decolonization showed an increase in mupirocin resistance (MR) from 6.6% to 20%. MR isolates from patients who failed decolonization yielded indistinguishable spa types and carried multiple antimicrobial and antiseptic resistance genes, which may guide infection control and prevention.

First Detailed Genetic Characterization of the Structural Organization of Type III Arginine Catabolic Mobile Elements Harbored by Staphylococcus epidermidis by Using Whole-Genome Sequencing.

The type III arginine catabolic mobile element (ACME) was detected in three Staphylococcus epidermidis oral isolates recovered from separate patients (one healthy, one healthy with dental implants, and one with periodontal disease) based on ACME-arc-operon- and ACME-opp3-operon-directed PCR. These isolates were subjected to whole-genome sequencing to characterize the precise structural organization of ACME III for the first time, which also revealed that all three isolates were the same sequence type, ST329.

High-dose rifapentine with moxifloxacin for pulmonary tuberculosis.

BACKGROUND: Tuberculosis regimens that are shorter and simpler than the current 6-month daily regimen are needed. METHODS: We randomly assigned patients with newly diagnosed, smear-positive, drug-sensitive tuberculosis to one of three regimens: a control regimen that included 2 months of ethambutol, isoniazid, rifampicin, and pyrazinamide administered daily followed by 4 months of daily isoniazid and rifampicin; a 4-month regimen in which the isoniazid in the control regimen was replaced by moxifloxacin administered daily for 2 months followed by moxifloxacin and 900 mg of rifapentine administered twice weekly for 2 months; or a 6-month regimen in which isoniazid was replaced by daily moxifloxacin for 2 months followed by one weekly dose of both moxifloxacin and 1200 mg of rifapentine for 4 months. Sputum specimens were examined on microscopy and after culture at regular intervals. The primary end point was a composite treatment failure and relapse, with noninferiority based on a margin of 6 percentage points and 90% confidence intervals. RESULTS: We enrolled a total of 827 patients from South Africa, Zimbabwe, Botswana, and Zambia; 28% of patients were coinfected with the human immunodefiency virus. In the per-protocol analysis, the proportion of patients with an unfavorable response was 4.9% in the control group, 3.2% in the 6-month group (adjusted difference from control, -1.8 percentage points; 90% confidence interval [CI], -6.1 to 2.4), and 18.2% in the 4-month group (adjusted difference from control, 13.6 percentage points; 90% CI, 8.1 to 19.1). In the modified intention-to-treat analysis these proportions were 14.4% in the control group, 13.7% in the 6-month group (adjusted difference from control, 0.4 percentage points; 90% CI, -4.7 to 5.6), and 26.9% in the 4-month group (adjusted difference from control, 13.1 percentage points; 90% CI, 6.8 to 19.4). CONCLUSIONS: The 6-month regimen that included weekly administration of high-dose rifapentine and moxifloxacin was as effective as the control regimen. The 4-month regimen was not noninferior to the control regimen. (Funded by the European and Developing Countries Clinical Trials Partnership and the Wellcome Trust; RIFAQUIN Current Controlled Trials number, ISRCTN44153044.).

Use of whole-genome sequencing to distinguish relapse from reinfection in a completed tuberculosis clinical trial.

BACKGROUND: RIFAQUIN was a tuberculosis chemotherapy trial in southern Africa including regimens with high-dose rifapentine with moxifloxacin. Here, the application of whole-genome sequencing (WGS) is evaluated within RIFAQUIN for identifying new infections in treated patients as either relapses or reinfections. WGS is further compared with mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR) typing. This is the first report of WGS being used to evaluate new infections in a completed clinical trial for which all treatment and epidemiological data are available for analysis. METHODS: DNA from 36 paired samples of Mycobacterium tuberculosis cultured from patients before and after treatment was typed using 24-loci MIRU-VNTR, in silico spoligotyping and WGS. Following WGS, the sequences were mapped against the reference strain H37Rv, the single-nucleotide polymorphism (SNP) differences between pairs were identified, and a phylogenetic reconstruction was performed. RESULTS: WGS indicated that 32 of the paired samples had a very low number of SNP differences (0-5; likely relapses). One pair had an intermediate number of SNP differences, and was likely the result of a mixed infection with a pre-treatment minor genotype that was highly related to the post-treatment genotype; this was reclassified as a relapse, in contrast to the MIRU-VNTR result. The remaining three pairs had very high SNP differences (>750; likely reinfections). CONCLUSIONS: WGS and MIRU-VNTR both similarly differentiated relapses and reinfections, but WGS provided significant extra information. The low proportion of reinfections seen suggests that in standard chemotherapy trials with up to 24 months of follow-up, typing the strains brings little benefit to an analysis of the trial outcome in terms of differentiating relapse and reinfection. However, there is a benefit to using WGS as compared to MIRU-VNTR in terms of the additional genotype information obtained, in particular for defining the presence of mixed infections and the potential to identify known and novel drug-resistance markers.

Novel multiresistance cfr plasmids in linezolid-resistant methicillin-resistant Staphylococcus epidermidis and vancomycin-resistant Enterococcus faecium (VRE) from a hospital outbreak: co-location of cfr and optrA in VRE.

BACKGROUND: Linezolid is often the drug of last resort to treat infections caused by Gram-positive cocci. Linezolid resistance can be mutational (23S rRNA or L-protein) or, less commonly, acquired [predominantly cfr, conferring resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins and streptogramin A compounds (PhLOPSA) or optrA, encoding oxazolidinone and phenicol resistance]. OBJECTIVES: To investigate the clonality and genetic basis of linezolid resistance in 13 linezolid-resistant (LZDR) methicillin-resistant Staphylococcus epidermidis (MRSE) isolates recovered during a 2013/14 outbreak in an ICU in an Irish hospital and an LZDR vancomycin-resistant Enterococcus faecium (VRE) isolate from an LZDR-MRSE-positive patient. METHODS: All isolates underwent PhLOPSA susceptibility testing, 23S rRNA sequencing, DNA microarray profiling and WGS. RESULTS: All isolates exhibited the PhLOPSA phenotype. The VRE harboured cfr and optrA on a novel 73 kb plasmid (pEF12-0805) also encoding erm(A), erm(B), lnu(B), lnu(E), aphA3 and aadE. One MRSE (M13/0451, from the same patient as the VRE) harboured cfr on a novel 8.5 kb plasmid (pSEM13-0451). The remaining 12 MRSE lacked cfr but exhibited linezolid resistance-associated mutations and were closely related to (1-52 SNPs) but distinct from M13/0451 (202-223 SNPs). CONCLUSIONS: Using WGS, novel and distinct cfr and cfr/optrA plasmids were identified in an MRSE and VRE isolate, respectively, as well as a cfr-negative LZDR-MRSE ICU outbreak and a distinct cfr-positive LZDR-MRSE from the same ICU. To our knowledge, this is the first report of cfr and optrA on a single VRE plasmid. Ongoing surveillance of linezolid resistance is essential to maintain its therapeutic efficacy.

Thrombotic Microangiopathy: A Multidisciplinary Team Approach.

Thrombotic microangiopathy (TMA) is characterized by the presence of microangiopathic hemolytic anemia and thrombocytopenia along with organ dysfunction, and pathologically, by the presence of microthrombi in multiple microvascular beds. Delays in diagnosis and initiation of therapy are common due to the low incidence, variable presentation, and poor awareness of these diseases, underscoring the need for interdisciplinary approaches to clinical care for TMA. We describe a new approach to improve clinical management via a TMA team that originally stemmed from an Affinity Research Collaborative team focused on thrombosis and hemostasis. The TMA team consists of clinical faculty from different disciplines who together are charged with the responsibility to quickly analyze clinical presentations, guide laboratory testing, and streamline prompt institution of treatment. The TMA team also includes faculty members from a broad range of disciplines collaborating to elucidate the pathogenesis of TMA. To this end, a clinical database and biorepository have been constructed. TMA leaders educate front-line providers from other departments through presentations in various forums across multiple specialties. Facilitated by an Affinity Research Collaborative mechanism, we describe an interdisciplinary team dedicated to improving both clinical care and translational research in TMA.

Extensive coronary and systemic arterial aneurysm development in severe refractory Kawasaki disease.

We describe the case of an 8-week-old infant with late presentation of severe refractory atypical Kawasaki disease. In addition to developing giant coronary arterial aneurysms and coronary thrombosis, she formed extensive bilateral arterial aneurysms throughout her systemic circulation.