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Abstract Introduction International guidelines recommend definitive combination antibiotic therapy for the management of serious infections involving carbapenem-resistant Acinetobacter (CRAB) species. The commonly available combination options include high-dose sulbactam, polymyxins, tetracyclines, and cefiderocol. Scanty prospective data exist to support this approach. Methods Patients with CRAB bacteraemia, ventilator-associated pneumonia (VAP), or both were categorized based on whether they received combination therapy or monotherapy. The 30-day mortality was compared between the two groups. Inverse probability treatment weighting (IPTW) was done using propensity score (PS) for a balanced comparison between groups. Results Between January 2021 and May 2023, of the 161 patients with CRAB bacteraemia (n = 55, 34.2%), VAP (n = 46, 28.6%), or both (n = 60, 37.3%) who received appropriate intravenous antibiotic therapy, 70% (112/161) received monotherapy, and the rest received combination therapy. The overall 30-day mortality was 62% (99/161) and not different (p = 0.76) between the combination therapy (31/49, 63.3%) and monotherapy (68/112, 60.7%) groups. The propensity score matching using IPTW did not show a statistical difference (p = 0.47) in 30-day mortality for receiving combination therapy with an adjusted odds ratio (OR) P of 1.29 (0.64, 2.58). Conclusion Combination therapy for CRAB infections needs further study in a randomised controlled trial, as this observational study showed no difference in 30-day mortality between monotherapy and combination therapy.

Abstract Introduction Blood cultures have low sensitivity for candidemia. Sensitivity can be improved by the culture-independent system T2 Magnetic Resonance (T2). SeptiCyte RAPID is a host response assay quantifying the risk of infection-related inflammation through a scoring system (SeptiScore). We investigate the performance of SeptiScore in detecting persistent candidemia as defined by conventional cultures and T2. Methods This is a prospective multicentre observational study on patients with candidemia. Blood cultures and blood samples for assessment by T2 and SeptiCyte were collected for 4 consecutive days after the index culture. The performance of SeptiScore was explored to predict persistent candidemia as defined by (1) positive follow-up blood culture (2) either positive follow-up blood culture or T2 sample. Results 10 patients were enrolled including 34 blood collections assessed with the 3 methods. Overall, 4/34 (12%) follow-up blood cultures and 6/34 (18%) T2 samples were positive. A mixed model showed significantly higher SeptiScores associated with persistent candidemia when this was defined as either a positive follow-up blood culture or T2 sample (0.82, 95%CI 0.06 to 1.58) but not when this was defined as a positive follow-up blood culture only (-0.57, 95%CI -1.28 to 0.14). ROC curve for detection of persistent candidemia by SeptiScore at day 1 follow-up showed an AUC of 0.85 (95%CI 0.52-1.00) when candidemia was defined by positive follow-up blood culture, and an AUC of 1.00 (95%CI 1.00–1.00) when candidemia was defined according to both methods. Conclusion Integrating transcriptome profiling with culture-independent systems and conventional cultures may increase our ability to diagnose persistent candidemia.

Objectives: To report trends in carbapenem resistance and difficult-to-treat resistance (DTR) among clinical isolates of Gram-negative priority pathogens collected by the ATLAS global surveillance program from 2018 to 2022. Methods: Reference broth microdilution testing was performed in a central laboratory for 79,214 Enterobacterales, 30,504 Pseudomonas aeruginosa, and 13,500 Acinetobacter baumannii-calcoaceticus complex isolates collected by a constant set of 157 medical centres in 49 countries in Asia Pacific (APAC), Europe (EUR), Latin America (LATAM), Middle East-Africa (MEA), and North America (NA) regions. MICs were interpreted by 2023 CLSI M100 breakpoints. β-lactamase genes were identified for meropenem-nonsusceptible (MIC ≥2 mg/L) Enterobacterales isolates. Results: Carbapenem-resistant Enterobacterales (CRE) detection increased (P < 0.05) in APAC, EUR, LATAM, and MEA regions and decreased in NA, while annual DTR percentages increased in all five regions. Carbapenem-resistant P. aeruginosa (CRPA; decreased in MEA region) and carbapenem-resistant A. baumannii-calcoaceticus complex (CRAB; decreased in MEA region and increased in EUR) remained relatively stable over time in all regions, although notably, annual percentages of CRAB and DTR A. baumannii-calcoaceticus complex isolates were consistently >25 percentage points lower in NA than in other regions. For all regions except NA, the majority of changes in CRE percentages could be attributed to hospital-acquired infections. Among meropenem-nonsusceptible Enterobacterales, KPC was the most frequent carbapenemase in NA and EUR each year. NDM was the most prevalent carbapenemase detected in 2022 in other global regions. Conclusion: CRE, CRPA, CRAB, and DTR rates vary among global regions over time highlighting the need for continuing surveillance to inform treatment strategies and antimicrobial stewardship.

Metal‐based nanoparticles (NPs) have entered clinical use for enhancing radiotherapy, but the underlying mechanisms remain ambiguous. Herein, single‐cell analysis of two cell lines in response to megavolt irradiation and a radiomimetic drug, neocarzinostatin (NCS) after coculture with gold NPs with different surface coatings, polyethylene glycol (AuPEG), PEG, and transferrin (AuT) or silica (AuSiO2), is reported. Different surface chemistry presents a major challenge for objective comparison between the biological impacts where major differences in cell‐uptake exist. AuSiO2 NPs are the most efficient for promoting radiosensitization despite being associated with cells 10 times less than the actively targeted AuT NPs. Conversely, for cells exposed to NCS, AuSiO2 NPs impede the radiomimetic action and promote cell survival. AuT NPs enhance death of cells in combination with NCS showing that NPs can sensitize against cytotoxic agents in addition to radiation. While NPs contribute to radiosensitization (or enhancing/impeding chemotherapeutic drug activity), due to cell and cell line heterogeneity, the ultimate radiosensitivity of a cell appears to be dominated by its inherent radiosensitivity and how this cell‐regulated response is manipulated by NPs. This is evidenced through comparison of radiobiological response of cells with equivalent NP association rather than equivalent coculture conditions.

Objectives: This population-based study aimed to investigate the risk factors and effect of extended-spectrum beta-lactamase (ESBL) production on clinical outcomes in Escherichia coli bloodstream infection (BSI) patients. Methods: The study population was defined as patients aged ≥15 years with E. coli BSI in Queensland, Australia, from 2000 to 2019. Outcomes were defined as 30-day case fatality, hospital length of stay (LOS), and recurrent E. coli BSI. Results: A total of 27,796 E. coli BSIs were identified, of which 1112 (4.0%) were ESBL-producers. Patients with ESBL-Ec BSI were more frequently older, male, with comorbidity, recurrent E. coli BSI, and less likely with community-associated community-onset infections as compared to non-ESBL-Ec BSI patients. The standardized mortality rate of ESBL-Ec BSI increased 8-fold from 2000 to 2019 (1 to 8 per million residents) and case fatality was 12.8% (n = 142) at 30 days from positive blood culture. Patients with ESBL-Ec BSI were not at higher risk of 30-day case fatality (adjusted hazard ratio [HR] = 0.98, 95% CI: 0.83-1.17), but had higher risk of recurring episodes (adjusted subdistribution HR = 1.58, 95% CI: 1.29-1.92) and observed 14% longer LOS (adjusted incidence rate ratio = 1.14, 95% CI: 1.10-1.18) than non-ESBL-Ec BSI patients. Conclusion: In this large patient cohort, ESBL-Ec BSI did not increase case fatality risk but observed higher hospital LOS and recurrent E. coli BSI than non-ESBL-Ec BSI. Clinical resources are warranted to account for the higher morbidity risk associated with ESBL production and incidence.

Placental health and foetal development are dependent upon element homeostasis. Analytical techniques such as mass spectroscopy can provide quantitative data on element concentrations in placental tissue but do not show spatial distribution or co-localisation of elements that may affect placental function. The present study used synchrotron-based X-ray fluorescence microscopy to elucidate element content and distribution in healthy and pathological placental tissue. The X-ray fluorescence microscopy (XFM) beamline at the Australian Synchrotron was used to image trace metal content of 19 placental sections from healthy term (n = 5, 37–39 weeks), foetal growth-restricted (n = 3, n = 7, >41 completed weeks), and stillbirth-complicated pregnancies (n = 4, 37–40 weeks). Samples were cryo-sectioned and freeze-dried. The concentration and distribution of fourteen elements were detected in all samples: arsenic, bromine, calcium, chlorine, copper, iron, molybdenum, phosphorous, potassium, rubidium, selenium, strontium, sulphur, and zinc. The elements zinc, calcium, phosphorous, and strontium were significantly increased in stillbirth placental tissue in comparison to healthy-term controls. Strontium, zinc, and calcium were found to co-localise in stillbirth tissue samples, and calcium and strontium concentrations were correlated in all placental groups. Molybdenum was significantly decreased in stillbirth, foetal growth-restricted, and postdate placental tissue in comparison to healthy-term samples (p < 0.0001). Synchrotron-based XFM reveals elemental distribution within biological samples such as the placenta, allowing for the co-localisation of metal deposits that may have a pathological role. Our pilot study further indicates low concentrations of placental molybdenum in pregnancies complicated by foetal growth restriction, postdate delivery, and stillbirth.

Al–Zn–Mg–Si alloy coatings have been developed to inhibit the corrosion of cold-rolled steel sheets by offering galvanic and barrier protection to the substrate steel. It is known that Fe deposited from the steel strip modifies the microstructure of the alloy. We cast samples of Al–Zn–Mg–Si coating alloys containing 0.4 wt% Fe and directionally solidified them using a Bridgman furnace to quantify the effect of this Fe addition between 600 °C and 240 °C. By applying a temperature gradient, growth is encouraged, and by then quenching the sample in coolant, the microstructure may be frozen. These samples were analysed using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) to determine the morphological effects of the Fe distribution across the experimental temperature range. However, due to the sub 1 wt% concentration of Fe, synchrotron X-ray fluorescence microscopy (XFM) was applied to quantitatively confirm the Fe distribution. Directionally solidified samples were scanned at 7.05 keV and 18.5 keV using X-ray fluorescence at the Australian Synchrotron using the Maia array detector. It was found that a mass nucleation event of the Fe-based τ6 phase occurred at 495 °C following the nucleation of the primary α-Al phase as a result of a peritectic reaction with remaining liquid.

Abstract While trimethoprim-sulfamethoxazole (TMP-SMX) is the first-line therapy of Stenotrophomonas maltophilia infections, colistin is one of the therapeutic options in cases of allergy or resistance to TMP-SMX. However, understanding the global status of resistance to colistin amongst S. maltophilia isolates could be helpful for appropriate antibiotic prescription. This study aimed to conduct a systematic review and meta-analysis to examine the prevalence of colistin resistance in clinical S. maltophilia isolates worldwide. According to eligibility criteria, a total of 61 studies were included in the analysis. The pooled prevalence for colistin resistance was 42% (95% CI: 35-49%), ranging from 0.1 to 97%. Subgroups analysis indicated that, the pooled prevalence of colistin resistance was 44% (95% CI: 29-60%) in 15 studies during 2000–2010, and it was estimated to be 41% (95% CI: 33-50%) in 46 articles from 2011 to 2021. It was 46% (95% CI: 35-58%) in the studies that used broth microdilution method, and 39% (95% CI: 30-49%) in the studies with other used methods. The resistance rate in Asian countries was 45% (95% CI: 31-60%), in European countries was 45% (95% CI: 34-56%) and in the countries of North and South America was 33% (95% CI: 20-46%). Our review showed notable resistance to colistin in clinical S. maltophilia isolates. Given the estimated resistance rates, alternative antibiotics could be preferred to treat serious infections due to S. maltophilia.

Abstract Microbiological weathering of coarse residue deposit (CRD) kimberlite produced by the Venetia Diamond Mine, Limpopo, South Africa enhanced mineral carbonation relative to untreated material. Cultures of photosynthetically enriched biofilm produced maximal carbonation conditions when mixed with kimberlite and incubated under near surface conditions. Interestingly, mineral carbonation also occurred in the dark, under water-saturated conditions. The examination of mineralized biofilms in ca. 150 µm-thick-sections using light microscopy, X-ray fluorescence microscopy (XFM) and backscatter electron—scanning electron microscopy-energy dispersive x-ray spectrometry demonstrated that microbiological weathering aided in producing secondary calcium/magnesium carbonates on silicate grain boundaries. Calcium/magnesium sulphate(s) precipitated under vadose conditions demonstrating that evaporites formed upon drying. In this system, mineral carbonation was only observed in regions possessing bacteria, preserved within carbonate as cemented microcolonies. 16S rDNA molecular diversity of bacteria in kimberlite and in natural biofilms growing on kimberlite were dominated by Proteobacteria that are active in nitrogen, phosphorus and sulphur cycling. Cyanobacteria based enrichment cultures provided with nitrogen & phosphorus (nutrients) to enhance growth, possessed increased diversity of bacteria, with Proteobacteria re-establishing themselves as the dominant bacterial lineage when incubated under dark, vadose conditions consistent with natural kimberlite. Overall, 16S rDNA analyses revealed that weathered kimberlite hosts a diverse microbiome consistent with soils, metal cycling and hydrocarbon degradation. Enhanced weathering and carbonate-cemented microcolonies demonstrate that microorganisms are key to mineral carbonation of kimberlite.

ABSTRACTWe aimed to evaluate the performance of Oxford Nanopore Technologies (ONT) sequencing from positive blood culture (BC) broths for bacterial identification and antimicrobial susceptibility prediction. Patients with suspected sepsis in four intensive care units were prospectively enrolled. Human-depleted DNA was extracted from positive BC broths and sequenced using ONT (MinION). Species abundance was estimated using Kraken2, and a cloud-based system (AREScloud) provided in silico predictive antimicrobial susceptibility testing (AST) from assembled contigs. Results were compared to conventional identification and phenotypic AST. Species-level agreement between conventional methods and AST predicted from sequencing was 94.2% (49/52), increasing to 100% in monomicrobial infections. In 262 high-quality AREScloud AST predictions across 24 samples, categorical agreement (CA) was 89.3%, with major error (ME) and very major error (VME) rates of 10.5% and 12.1%, respectively. Over 90% CA was achieved for some taxa (e.g., Staphylococcus aureus) but was suboptimal for Pseudomonas aeruginosa. In 470 AST predictions across 42 samples, with both high quality and exploratory-only predictions, overall CA, ME, and VME rates were 87.7%, 8.3%, and 28.4%. VME rates were inflated by false susceptibility calls in a small number of species/antibiotic combinations with few representative resistant isolates. Time to reporting from sequencing could be achieved within 8–16 h from BC positivity. Direct sequencing from positive BC broths is feasible and can provide accurate predictive AST for some species. ONT-based approaches may be faster but significant improvements in accuracy are required before it can be considered for clinical use.IMPORTANCESepsis and bloodstream infections carry a high risk of morbidity and mortality. Rapid identification and susceptibility prediction of causative pathogens, using Nanopore sequencing direct from blood cultures, may offer clinical benefit. We assessed this approach in comparison to conventional phenotypic methods and determined the accuracy of species identification and susceptibility prediction from genomic data. While this workflow holds promise, and performed well for some common bacterial species, improvements in sequencing accuracy and more robust predictive algorithms across a diverse range of organisms are required before this can be considered for clinical use. However, results could be achieved in timeframes that are faster than conventional phenotypic methods.

Severe sepsis has a high mortality rate. Here, the authors provide genomic, transcriptomic, proteomic and metabolomic data across four sepsis-causing pathogens and identify a signature of global increase in fatty acid and lipid biosynthesis as well as cholesterol acquisition.

The World Health Organization (WHO)’s list of neglected tropical diseases (NTDs) highlights conditions that are responsible for devastating health, social and economic consequences, and yet, they are overlooked and poorly resourced. The NTD list does not include conditions caused by Gram-negative bacilli (GNB). Infections due to GNB cause significant morbidity and mortality and are prevalent worldwide. Southeast Asia is a WHO region of low- and middle-income countries carrying the largest burden of NTDs. Two significant health threats in Southeast Asia are Burkholderia pseudomallei (causing melioidosis) and hypervirulent Klebsiella pneumoniae (HvKp). Both diseases have high mortality and increasing prevalence, yet both suffer from a lack of awareness, significant under-resourcing, incomplete epidemiological data, limited diagnostics, and a lack of evidence-based treatment. Emerging evidence shows that both melioidosis and HvKp are spreading globally, including in high-income countries, highlighting the potential future global threat they pose. In this article, we review both conditions, identifying current trends and challenges in Southeast Asia and areas for future research. We also argue that melioidosis and HvKp merit inclusion as NTDs, and that mandatory global surveillance and reporting systems should be established, and we make an urgent call for research to better understand, detect, and treat these neglected diseases.

IntroductionCarbapenem-resistant Enterobacterales (CRE) and multidrug-resistant Pseudomonas aeruginosa (MDR-PA) infections are associated with a high risk of morbidity, mortality, and treatment costs. We aimed to evaluate in vitro, in vivo and clinical studies comparing the efficacy of ceftazidime-avibactam (CZA) combination regimens with CZA alone against CRE and/or MDR-PA isolates or infections.MethodsWe systematically reviewed the relevant literature in CINAHL/MEDLINE, Pubmed, Cochrane, Web of Science, Embase, and Scopus until December 1, 2022. Review articles, grey literature, abstracts, comments, editorials, non-peer reviewed articles, non-English articles, and in vitro synergy studies conducted on single isolates were excluded.Results22 in vitro, 7 in vivo and 20 clinical studies were evaluated. In vitro studies showed reliable synergy between CZA and aztreonam against metallo-β-lactamase (MBL)-producing isolates. Some studies indicated good in vitro synergy between CZA and amikacin, meropenem, fosfomycin and polymyxins against CRE isolates. For MDR-PA isolates, there are comparatively fewer in vitro or in vivo studies. In observational clinical studies, mortality, clinical cure, adverse events, and development of CZA resistance after exposure were generally similar in monotherapy and combination therapy groups. However, antibiotic-related nephrotoxicity and infection relapses were higher in patients receiving CZA combination therapies.DiscussionThe benefit, if any, of CZA combination regimens in MDR-PA infections is elusive, as very few clinical studies have included these infections. There is no currently documented clinical benefit for the use of CZA combination regimens rather than CZA monotherapy. CZA combined with aztreonam for serious infections due to MBL producers should be evaluated by randomized controlled trials.Systematic review registrationhttps://www.crd.york.ac.uk/prospero/display_record.php?RecordID=278552, CRD42021278552.

Abstract Background Early identification of bloodstream pathogens and their associated antimicrobial resistance may shorten time to optimal therapy in patients with sepsis. The BioFire Blood Culture Identification 2 Panel (BCID2) is a novel multiplex PCR detecting 43 targets directly from positive blood cultures, reducing turnaround times. Methods We have performed a systematic review and meta-analysis of diagnostic test accuracy studies to assess the BCID2 performance for pathogen identification and resistance markers detection compared to gold standard culture-based methods (including phenotypic and/or genotypic characterization). Results Nine studies were identified reporting data to build 2 × 2 tables for each BCID2 target, including 2005 blood cultures. The pooled specificity of the assay was excellent (> 97%) across most subgroups of targets investigated, with a slightly broader confidence interval for S. epidermidis (98.1%, 95% CI 93.1 to 99.5). Pooled sensitivity was also high for the major determinants of bloodstream infection, including Enterobacterales (98.2%, 95% CI 96.3 to 99.1), S. aureus (96.0%, 95% CI 90.4 to 98.4), Streptococcus spp. (96.7%, 95% CI 92.8 to 98.5), P. aeruginosa (92.7%, 95% CI 83.1 to 97.0), E. faecalis (92.3%, 95% CI 83.5 to 96.6), as well as bla CTX-M (94.9, 95% CI 85.7 to 98.3), carbapenemases (94.9%, 95% CI 83.4 to 98.6) and mecA/C & MREJ (93.9%, 95% CI 83.0 to 98.0). Sensitivity for less common targets was slightly lower, possibly due to their under-representation in the included studies. Conclusions BCID2 showed good performance for detecting major determinants of bloodstream infection and could support early antimicrobial treatment, especially for ESBL or carbapenemase-producing Gram-negative bacilli and methicillin-resistant S. aureus.

Background: Antimicrobial resistance surveillance is essential for empiric antibiotic prescribing, infection prevention and control policies and to drive novel antibiotic discovery. However, most existing surveillance systems are isolate-based without supporting patient-based clinical data, and not widely implemented especially in low- and middle-income countries (LMICs). Methods: A Clinically-Oriented Antimicrobial Resistance Surveillance Network (ACORN) II is a large-scale multicentre protocol which builds on the WHO Global Antimicrobial Resistance and Use Surveillance System to estimate syndromic and pathogen outcomes along with associated health economic costs. ACORN-healthcare associated infection (ACORN-HAI) is an extension study which focuses on healthcare-associated bloodstream infections and ventilator-associated pneumonia. Our main aim is to implement an efficient clinically-oriented antimicrobial resistance surveillance system, which can be incorporated as part of routine workflow in hospitals in LMICs. These surveillance systems include hospitalised patients of any age with clinically compatible acute community-acquired or healthcare-associated bacterial infection syndromes, and who were prescribed parenteral antibiotics. Diagnostic stewardship activities will be implemented to optimise microbiology culture specimen collection practices. Basic patient characteristics, clinician diagnosis, empiric treatment, infection severity and risk factors for HAI are recorded on enrolment and during 28-day follow-up. An R Shiny application can be used offline and online for merging clinical and microbiology data, and generating collated reports to inform local antibiotic stewardship and infection control policies. Discussion: ACORN II is a comprehensive antimicrobial resistance surveillance activity which advocates pragmatic implementation and prioritises improving local diagnostic and antibiotic prescribing practices through patient-centred data collection. These data can be rapidly communicated to local physicians and infection prevention and control teams. Relative ease of data collection promotes sustainability and maximises participation and scalability. With ACORN-HAI as an example, ACORN II has the capacity to accommodate extensions to investigate further specific questions of interest.

There is an urgent need to address coastal dynamics as a fundamental interaction between physical and biological processes, particularly when trying to predict future biological–physical linkages under anticipated changes in environmental forcing. More integrated modelling, support for observational networks and the use of management interventions as controlled experimental exercises should now be vigorously pursued.

Plasmids are major drivers of increasing antibiotic resistance, necessitating an urgent need to understand their biology. Here we describe a detailed dissection of the molecular components controlling the genetics of I-complex plasmids, a group of antibiotic resistance plasmids found frequently in pathogenic Escherichia coli and other Enterobacteriaceae that cause significant human disease. We show these plasmids cluster into four distinct subgroups, with the prototype IncI1 plasmid R64 subgroup displaying low nucleotide sequence conservation to other I-complex plasmids. Using pMS7163B, an I-complex plasmid distantly related to R64, we performed a high-resolution transposon-based genetic screen and defined genes involved in replication, stability, and conjugative transfer. We identified the replicon and a partitioning system as essential for replication/stability. Genes required for conjugation included the type IV secretion system, relaxosome, and several uncharacterised genes located in the pMS7163B leading transfer region that exhibited an upstream strand-specific transposon insertion bias. The overexpression of these genes severely impacted host cell growth or reduced fitness during mixed competitive growth, demonstrating that their expression must be controlled to avoid deleterious impacts. These genes were present in >80% of all I-complex plasmids and broadly conserved across multiple plasmid incompatibility groups, implicating an important role in plasmid dissemination. Author summary Antimicrobial resistance is one of the greatest threats to human health. Left unchecked, we risk a rapid escalation of untreatable infections. Plasmids are one of the most important vehicles for resistance gene carriage and transmission between bacteria, and thus an understanding of plasmid biology is crucial to controlling the spread antimicrobial resistance. Here, we combine advanced bioinformatics and a state-of-the-art genetic screen to understand the molecular mechanisms involved in the maintenance and spread of a group of plasmids strongly associated with antibiotic resistance among bacteria that cause human infection. We characterised genes involved in the replication and maintenance of these plasmids, and experimentally demonstrated that plasmid spread is dependent on a well-conserved secretion system. Our genetic screen also discovered a set of broadly conserved, uncharacterised genes that adversely impact host fitness and plasmid spread under dysregulation. Taken together, these findings describe a molecular blueprint for the biology of a group of clinically relevant antibiotic resistance plasmids found frequently in Gram-negative bacterial pathogens.

ABSTRACT To investigate an outbreak of vancomycin-resistant Enterococcus faecium (VREfm) sequence type 78 (ST78) in a large tertiary Australian hospital. A collection of 63 VREfm ST78 isolates, identified during a routine genomic surveillance program, were subjected to genomic epidemiological analysis based on whole-genome sequencing (WGS) data. The population structure was reconstructed using phylogenetic analysis, and a collection of publicly available VREfm ST78 genomes were used to provide global context. Core genome single nucleotide polymorphism (SNP) distances and available clinical metadata were used to characterize outbreak clusters and reconstruct transmission events. In silico genotyping confirmed that all study isolates were vanB-type VREfm carrying virulence characteristics of the hospital-associated E. faecium. Phylogenetic analysis identified two distinct phylogenetic clades, only one of which was responsible for a hospital outbreak. Four outbreak subtypes could be defined with examples of recent transmissions. Inference on transmission trees suggested complex transmission routes with unknown environmental reservoirs mediating the outbreak. WGS-based cluster analysis with publicly available genomes identified closely related Australian ST78 and ST203 isolates, highlighting the capacity for WGS to resolve complex clonal relationships between the VREfm lineages. Whole genome-based analysis has provided a high-resolution description of an outbreak of vanB-type VREfm ST78 in a Queensland hospital. Combined routine genomic surveillance and epidemiological analysis have facilitated better understanding of the local epidemiology of this endemic strain, providing valuable insight for better targeted control of VREfm. IMPORTANCE Vancomycin-resistant Enterococcus faecium (VREfm) is a leading cause of health care-associated infections (HAIs) globally. In Australia, the spread of hospital-adapted VREfm is largely driven by a single clonal group (clonal complex [CC]), CC17, to which the lineage ST78 belongs. While implementing a genomic surveillance program in Queensland, we observed increased incidence of ST78 colonizations and infections among patients. Here, we demonstrate the use of real-time genomic surveillance as a tool to support and enhance infection control (IC) practices. Our results show that real-time whole-genome sequencing (WGS) can efficiently disrupt outbreaks by identifying transmission routes that in turn can be targeted using resource-limited interventions. Additionally, we demonstrate that by placing local outbreaks in a global context, high-risk clones can be identified and targeted prior to them becoming established within clinical environments. Finally, the persistence of these organism within the hospital highlights the need for routine genomic surveillance as a management tool to control VRE transmission.

Abstract Background The incidence of Gram-negative bacteraemia is rising globally and remains a major cause of morbidity and mortality. The majority of patients with Gram-negative bacteraemia initially receive intravenous (IV) antibiotic therapy. However, it remains unclear whether patients can step down to oral antibiotics after appropriate clinical response has been observed without compromising outcomes. Compared with IV therapy, oral therapy eliminates the risk of catheter-associated adverse events, enhances patient quality of life and reduces healthcare costs. As current management of Gram-negative bacteraemia entails a duration of IV therapy with limited evidence to guide oral conversion, we aim to evaluate the clinical efficacy and economic impact of early stepdown to oral antibiotics. Methods This is an international, multicentre, randomised controlled, open-label, phase III, non-inferiority trial. To be eligible, adult participants must be clinically stable / non-critically ill inpatients with uncomplicated Gram-negative bacteraemia. Randomisation to the intervention or standard arms will be performed with 1:1 allocation ratio. Participants randomised to the intervention arm (within 72 h from index blood culture collection) will be immediately switched to an oral fluoroquinolone or trimethoprim-sulfamethoxazole. Participants randomised to the standard arm will continue to receive IV therapy for at least 24 h post-randomisation before clinical re-assessment and decision-making by the treating doctor. The recommended treatment duration is 7 days of active antibiotics (including empiric therapy), although treatment regimen may be longer than 7 days if clinically indicated. Primary outcome is 30-day all-cause mortality, and the key secondary outcome is health economic evaluation, including estimation of total healthcare cost as well as assessment of patient quality of life and number of quality-adjusted life years saved. Assuming a 30-day mortality of 8% in the standard and intervention arms, with 6% non-inferiority margin, the target sample size is 720 participants which provides 80% power with a one-sided 0.025 α-level after adjustment for 5% drop-out. Discussion A finding of non-inferiority in efficacy of oral fluoroquinolones or trimethoprim-sulfamethoxazole versus IV standard of care antibiotics may hypothetically translate to wider adoption of a more cost-effective treatment strategy with better quality of life outcomes. Trial registration ClinicalTrials.gov NCT05199324 . Registered 20 January 2022.

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Background: Small case series and reports suggest that Sphingomonas paucimobilis is predominantly a cause of nosocomial bloodstream infections (BSI) with very low associated mortality. Our objective was to describe the epidemiology and outcome of Sphingomonas species BSI in a large Australian population. Methods: We included all residents of Queensland, Australia, with BSI because of Sphingomonas species identified within the publicly funded system from 2000 to 2019. Results: A total of 282 incident episodes of Sphingomonas species BSI were identified for an age- and sex-adjusted incidence of 3.2 per million population annually. Incidence rates were highest in the tropical regions of the state. Most (94%) of the isolates were confirmed as Sphingomonas paucimobilis. In addition, 77% of the infections were community-onset, of which 48% were community-associated, and 30% were healthcare-associated. The very young, the old, and male patients were at the highest risk. Patients with community-associated disease were, on average, younger, had fewer co-morbidities, and were less likely to have polymicrobial infections. At least 1 co-morbidity was identified in 62% of patients, with malignancy, diabetes, and lung disease most prevalent. The overall all-cause 30-day case-fatality rate was 6%. Conclusion: Sphingomonas paucimobilis BSI is a predominantly community-onset disease associated with a significant risk of death.

Abstract Background Inappropriate antibiotic use can cause harm and promote antimicrobial resistance, which has been declared a major health challenge by the World Health Organization. In Australian residential aged care facilities (RACFs), the most common indications for antibiotic prescribing are for infections of the urinary tract, respiratory tract and skin and soft tissue. Studies indicate that a high proportion of these prescriptions are non-compliant with best prescribing guidelines. To date, a variety of interventions have been reported to address inappropriate prescribing and overuse of antibiotics but with mixed outcomes. This study aims to identify the impact of a set of sustainable, multimodal interventions in residential aged care targeting three common infection types. Methods This protocol details a 20-month stepped-wedge cluster-randomised trial conducted across 18 RACFs (as 18 clusters). A multimodal multi-disciplinary set of interventions, the ‘AMS ENGAGEMENT bundle’, will be tailored to meet the identified needs of participating RACFs. The key elements of the intervention bundle include education for nurses and general practitioners, telehealth support and formation of an antimicrobial stewardship team in each facility. Prior to the randomised sequential introduction of the intervention, each site will act as its own control in relation to usual care processes for antibiotic use and stewardship. The primary outcome for this study will be antibiotic consumption measured using defined daily doses (DDDs). Cluster-level rates will be calculated using total occupied bed numbers within each RACF during the observation period as the denominator. Results will be expressed as rates per 1000 occupied bed days. An economic analysis will be conducted to compare the costs associated with the intervention to that of usual care. A comprehensive process evaluation will be conducted using the REAIM Framework, to enable learnings from the trial to inform sustainable improvements in this field. Discussion A structured AMS model of care, incorporating targeted interventions to optimise antimicrobial use in the RACF setting, is urgently needed and will be delivered by our trial. The trial will aim to empower clinicians, residents and families by providing a robust AMS programme to improve antibiotic-related health outcomes. Trial registration US National Library of Medicine Clinical Trials.gov ( NCT04705259 ). Prospectively registered in 12th of January 2021.

Abstract Background The purpose of this study was to investigate the use of routinely available rectal swabs as a surrogate sample type for testing the gut microbiome and monitoring antibiotic effects on key gut microorganisms, of patients hospitalised in an intensive care unit. A metagenomic whole genome sequencing approach was undertaken to determine the diversity of organisms as well as resistance genes and to compare findings between the two sampling techniques. Results No significant difference was observed in overall diversity between the faeces and rectal swabs and sampling technique was not demonstrated to predict microbial community variation. More human DNA was present in the swabs and some differences were observed only for a select few anaerobes and bacteria also associated with skin and/or the female genitourinary system, possibly reflecting sampling site or technique. Antibiotics and collections at different times of admission were both considered significant influences on microbial community composition alteration. Detection of antibiotic resistance genes between rectal swabs and faeces were overall not significantly different, although some variations were detected with a potential association with the number of human sequence reads in a sample. Conclusion Testing the gut microbiome using standard rectal swab collection techniques currently used for multi-resistant organism screening has been demonstrated to have utility in gut microbiome monitoring in intensive care. The use of information from this article, in terms of methodology as well as near equivalence demonstrated between rectal swabs and faeces will be able to support and potentially facilitate the introduction into clinical practice.

The epidemiology of bloodstream infections caused by Shewanella spp. is not well defined. Our objective was to define the incidence and determinants of Shewanella spp. bloodstream infections by using population-based surveillance in Queensland, Australia during 2000‒2019. The incidence was 1.0 cases/1 million persons annually and was highest during summer and in the tropical Torres and Cape region. Older persons and male patients were at highest risk. At least 1 concurrent condition was documented in 75% of case-patients, and 30-day all cause case-fatality rate was 15%. Aging populations in warm climates might expect an increasing burden of these infections.

Abstract Cultural ecosystem services (CES), a key aspect of nature's contributions to people, remain a challenge to incorporate into decision making. One contributing factor is the difficulty of defining and describing these, due partly to: ongoing poor understanding of what drives people to interact with nature, a lack of appropriate data to quantify these interactions, and basic difficulties in measuring and modelling the complex array of social, psychological and behavioural attributes which help explain people's actions. In this study we present a framework which develops the concepts of cultural capital, social capital and human capital as specific forms of human‐centred capital, in the context of their contribution to understanding CES. Each form of capital encompasses separate attributes of beneficiaries. Testing the framework with data from a separate trans‐disciplinary study illustrated that the framework was readily applicable to specific situations. A measure of cultural capital, EcoCentrism, explained more variation than a suite of seven demographic variables. Applying the framework also showed that despite using a wide range of explanatory variables, a large proportion of observed variation remained unaccounted for. This suggests that more work is needed to understand and to develop metrics which can measure additional factors which underlie peoples’ motivations to engage with nature. The framework is applicable to other types of ecosystem service, and may also be useful for exploring relational values. A free Plain Language Summary can be found within the Supporting Information of this article.

Objectives: Understand the long-term mortality, risk of readmission for sepsis and cause of death following a gram-negative bloodstream infection (GN-BSI). Methods: This was a propensity-matched study using data linkage of Queensland hospital data, Australia. GN-BSIs were collected from 2005 to 2010 and matched 1:1 to hospital admissions without BSI for age, gender, year of culture collection, frequency of admissions in the prior year and Charlson-Deyo Comorbidity score and each comorbidity within the Charlson-Deyo score. Readmissions for sepsis, mortality and causes of death were evaluated. Results: Cases of GN-BSI were propensity-matched 1:1 to culture-negative hospital admissions (n = 14016). Readmissions for sepsis were higher in the GN-BSI cohort from 91 to 365 days (P

Objectives: The dissemination of Escherichia coli–producing extended-spectrum β-lactamase (ESBL-Ec) is evident in the community. In this population-based spatial analysis, we sought to describe the distribution of ESBL-Ec and to identify predictors of incidence in the community. Methods: The study population was defined as individuals with the ESBL-Ec isolate in Queensland, Australia, from 2010 to 2019. Annual choropleth maps and a global Moran index were constructed to describe ESBL-Ec distribution. Getis-Ord Gi* was performed to identify “hot spots” of statistical significance. Using demographic factors and incidence per postal area from 2016, multivariable analyses with or without spatially structured random effects were performed. Results: In total, 12,786 individuals with ESBL-Ec isolate were identified. The incidence rate increased annually from 9.1 per 100,000 residents in 2010 to 49.8 per 100,000 residents in 2019. The geographical distribution changed from random to clustered in 2014. Hot spots were more frequently identified in the Outback and Far North Queensland, where remote communities and hotter weather are prevalent. Multivariable spatial analysis suggests that communities with higher socioeconomic status (RR, 0.66; 95% CI, 0.55–0.79 per 100 units) and employment in the agricultural industry (RR, 0.79; 95% CI, 0.67–0.95 per 10%) were protective of lower ESBL-Ec incidence. After accounting for multiple demographic factors, the residual, structured, random-effects model indicated that hot spots were still detected in more remote communities but also in several city regions. Conclusions: The change in distribution of ESBL-Ec across Queensland suggests the presence of area-level specific risk factors that enhance spread in the community. Risk factors for spread appear different between remote and city settings, and future research should be tailored to understand the respective area-level risk factors. Factors such as local temperature, antibiotic consumption, and access to services should be validated. Future public health measures to reduce transmission should be focused on the identified hot spots.

Abstract Background Increasing rates of antibiotic resistance in Gram-negative organisms due to the presence of extended-spectrum beta-lactamases (ESBL), hyperproduction of AmpC enzymes, carbapenemases and other mechanisms of resistance are identified in common hospital- and healthcare-associated pathogens including Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii. Cefiderocol is a novel siderophore cephalosporin antibiotic with a catechol moiety on the 3-position side chain. Cefiderocol has been shown to be potent in vitro against a broad range of Gram-negative organisms, including carbapenem-resistant Enterobacteriaceae (CRE) and multi-drug-resistant (MDR) P. aeruginosa and A. baumannii. Recent clinical data has shown cefiderocol to be effective in the setting of complicated urinary tract infections and nosocomial pneumonia, but it has not yet been studied as treatment of bloodstream infection. Methods This study will use a multicentre, open-label non-inferiority trial design comparing cefiderocol and standard of care antibiotics. Eligible participants will be adult inpatients who are diagnosed with a bloodstream infection with a Gram-negative organism on the basis of a positive blood culture result where the acquisition meets the definition for healthcare-associated or hospital-acquired. It will compare cefiderocol with the current standard of care (SOC) antibiotic regimen according to the patient’s treating clinician. Eligible participants will be randomised 1:1 to cefiderocol or SOC and receive 5–14 days of antibiotic therapy. Trial recruitment will occur in at least 20 sites in ten countries (Australia, Malaysia, Singapore, Thailand, Turkey and Greece). The sample size has been derived from an estimated 14 day, all-cause mortality rate of 10% in the control group, and a non-inferiority margin of 10% difference in the two groups. A minimum of 284 patients are required in total to achieve 80% power with a two-sided alpha level of 0.05. Data describing demographic information, risk factors, concomitant antibiotics, illness scores, microbiology, multidrug-resistant organism screening, discharge and mortality will be collected. Discussion With increasing antimicrobial resistance, there is a need for the development of new antibiotics with broad activity against Gram-negative pathogens such as cefiderocol. By selecting a population at risk for multi-drug-resistant pathogens and commencing study treatment early in the clinical illness (within 48 h of index blood culture) the trial hopes to provide guidance to clinicians of the efficacy of this novel agent. Trial registration The GAME CHANGER trial is registered under the US National Institute of Health ClinicalTrials.gov register, reference number NCT03869437 . Registered on March 11, 2019.

Abstract Background Convalescent plasma has been widely used to treat COVID-19 and is under investigation in numerous randomized clinical trials, but results are publicly available only for a small number of trials. The objective of this study was to assess the benefits of convalescent plasma treatment compared to placebo or no treatment and all-cause mortality in patients with COVID-19, using data from all available randomized clinical trials, including unpublished and ongoing trials (Open Science Framework, https://doi.org/10.17605/OSF.IO/GEHFX ). Methods In this collaborative systematic review and meta-analysis, clinical trial registries (ClinicalTrials.gov, WHO International Clinical Trials Registry Platform), the Cochrane COVID-19 register, the LOVE database, and PubMed were searched until April 8, 2021. Investigators of trials registered by March 1, 2021, without published results were contacted via email. Eligible were ongoing, discontinued and completed randomized clinical trials that compared convalescent plasma with placebo or no treatment in COVID-19 patients, regardless of setting or treatment schedule. Aggregated mortality data were extracted from publications or provided by investigators of unpublished trials and combined using the Hartung–Knapp–Sidik–Jonkman random effects model. We investigated the contribution of unpublished trials to the overall evidence. Results A total of 16,477 patients were included in 33 trials (20 unpublished with 3190 patients, 13 published with 13,287 patients). 32 trials enrolled only hospitalized patients (including 3 with only intensive care unit patients). Risk of bias was low for 29/33 trials. Of 8495 patients who received convalescent plasma, 1997 died (23%), and of 7982 control patients, 1952 died (24%). The combined risk ratio for all-cause mortality was 0.97 (95% confidence interval: 0.92; 1.02) with between-study heterogeneity not beyond chance (I2 = 0%). The RECOVERY trial had 69.8% and the unpublished evidence 25.3% of the weight in the meta-analysis. Conclusions Convalescent plasma treatment of patients with COVID-19 did not reduce all-cause mortality. These results provide strong evidence that convalescent plasma treatment for patients with COVID-19 should not be used outside of randomized trials. Evidence synthesis from collaborations among trial investigators can inform both evidence generation and evidence application in patient care.

Paleocene deep-sea limestones from New Zealand contain densely packed micro-scale dissolution seams which potentially released carbon into the ocean that was previously thought to be stored, according to high-resolution multimodal X-ray microscopy.

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is routinely used for bacterial identification. It would be highly beneficial to also be able to use the technology as a fast way to detect clinically relevant clones of bacterial species. However, studies to this aim have often had limited success. The methods used for data acquisition, processing and data interpretation are highly diverse amongst studies on MALDI-TOF MS sub-species typing. In addition to this, feasibility may depend on the bacterial species and strains investigated, making it difficult to determine what methods may or may not work. In our paper, we have reviewed recent research on MALDI-TOF MS typing of bacterial strains. Although we found a lot of variation amongst the methods used, there were approaches shared by multiple research groups. Multiple spectra of the same isolate were often combined before further analysis for strain distinction. Many groups used a protein extraction step to increase resolution in their MALDI-TOF MS results. Peaks at a high mass range were often excluded for data interpretation. Three groups have found ways to determine feasibility of MALDI-TOF MS typing for their set of strains at an early stage of their project.

Abstract Background Oxford Nanopore Technology (ONT) long-read sequencing has become a popular platform for microbial researchers due to the accessibility and affordability of its devices. However, easy and automated construction of high-quality bacterial genomes using nanopore reads remains challenging. Here we aimed to create a reproducible end-to-end bacterial genome assembly pipeline using ONT in combination with Illumina sequencing. Results We evaluated the performance of several popular tools used during genome reconstruction, including base-calling, filtering, assembly, and polishing. We also assessed overall genome accuracy using ONT both natively and with Illumina. All steps were validated using the high-quality complete reference genome for the Escherichia coli sequence type (ST)131 strain EC958. Software chosen at each stage were incorporated into our final pipeline, MicroPIPE. Further validation of MicroPIPE was carried out using 11 additional ST131 E. coli isolates, which demonstrated that complete circularised chromosomes and plasmids could be achieved without manual intervention. Twelve publicly available Gram-negative and Gram-positive bacterial genomes (with available raw ONT data and matched complete genomes) were also assembled using MicroPIPE. We found that revised basecalling and updated assembly of the majority of these genomes resulted in improved accuracy compared to the current publicly available complete genomes. Conclusions MicroPIPE is built in modules using Singularity container images and the bioinformatics workflow manager Nextflow, allowing changes and adjustments to be made in response to future tool development. Overall, MicroPIPE provides an easy-access, end-to-end solution for attaining high-quality bacterial genomes. MicroPIPE is available at https://github.com/BeatsonLab-MicrobialGenomics/micropipe .

Sympathetic neurons are powerful drivers of cardiac excitability. In the early stages of hypertension, sympathetic hyperactivity is underpinned by down regulation of M current and increased activity of Cav2.2 that is associated with greater intracellular calcium transients and enhanced neurotransmission. Emerging evidence suggests that retrograde signaling from the myocyte itself can modulate synaptic plasticity. Here we tested the hypothesis that cross culturing healthy myocytes onto diseased stellate neurons could influence sympathetic excitability. We employed neuronal mono-cultures, co-cultures of neonatal ventricular myocytes and sympathetic stellate neurons, and mono-cultures of sympathetic neurons with media conditioned by myocytes from normal (Wistar) and pre-hypertensive (SHR) rats, which have heightened sympathetic responsiveness. Neuronal firing properties were measured by current-clamp as a proxy for neuronal excitability. SHR neurons had a maximum higher firing rate, and reduced rheobase compared to Wistar neurons. There was no difference in firing rate or other biophysical properties in Wistar neurons when they were co-cultured with healthy myocytes. However, the firing rate decreased, phenocopying the Wistar response when either healthy myocytes or media in which healthy myocytes were grown was cross-cultured with SHR neurons. This supports the idea of a paracrine signaling pathway from the healthy myocyte to the diseased neuron, which can act as a modulator of sympathetic excitability.

Background: Conventional blood cultures methods are associated with long turnaround times, preventing early treatment optimization in bloodstream infections. The BioFire Blood Culture Identification 2 (BCID2) Panel is a new multiplex PCR applied on positive blood cultures, reducing time to pathogen identification and resistant markers detection. Methods: We conducted a prospective observational study including positive blood cultures from Intensive Care Units and Emergency Departments and performed BCID2 in addition to conventional testing. Concordance between the two methods was assessed and BCID2 performance characteristics were evaluated. Resistance markers detected by BCID2 were confirmed by in-house PCR. Whole genome sequencing was performed in discordant cases. Results: Among 60 monomicrobial blood cultures, BCID2 correctly identified 55/56 (91.7%) on-panel pathogens, showing an overall concordance of 98%. In 4/60 cases BCID2 did not detect any target and these all grew BCID2 off-panel bacteria. Only one discordant case was found. Sensitivity and specificity for Gram-positive bacteria on monomicrobial samples were 100% (95% CI 85.8–100%) and 100% (95% CI 90.3–100%) respectively, while for Gram-negatives 100% (95% CI 87.7–100) and 96.9% (95% CI 83.8–99.9%), respectively. Among two polymicrobial blood cultures, full concordance was observed in one case only. BCID2 identified antimicrobial resistance genes in 6/62 samples, all confirmed by in-house PCR (3 mecA/C S. epidermidis, 3 blaCTX-M E. coli). Estimated time to results gained using BCID2 as compared to conventional testing was 9.69 h (95% CI: 7.85–11.53). Conclusions: BCID2 showed good agreement with conventional methods. Studies to assess its clinical impact are warranted.

Mineral carbonation of alkaline mine residues is a carbon dioxide removal (CDR) strategy that can be employed by the mining industry. Here, we describe the mineralogy and reactivity of processed kimberlites and kimberlite ore from Venetia (South Africa) and Gahcho Kué (Canada) diamond mines, which are smectite-rich (2.3–44.1 wt.%). Whereas, serpentines, olivines, hydrotalcites and brucite have been traditionally used for mineral carbonation, little is known about the reactivity of smectites to CO2. The smectite from both mines is distributed as a fine-matrix and is saponite, Mx/mm+Mg3(AlxSi4−x)O10(OH)2·nH2O, where the layer charge deficiency is balanced by labile, hydrated interlayer cations (Mm+). A positive correlation between cation exchange capacity and saponite content indicates that smectite is the most reactive phase within these ultramafic rocks and that it can be used as a source of labile Mg2+ and Ca2+ for carbonation reactions. Our work shows that smectites provide the fast reactivity of kimberlite to CO2 in the absence of the highly reactive mineral brucite [Mg(OH)2]. It opens up the possibility of using other, previously inaccessible rock types for mineral carbonation including tailings from smectite-rich sediment-hosted metal deposits and oil sands tailings. We present a decision tree for accelerated mineral carbonation at mines based on this revised understanding of mineralogical controls on carbonation potential.

BackgroundBurkholderia pseudomallei is an environmental gram-negative bacterium that causes the disease melioidosis and is endemic in many countries of the Asia-Pacific region. In Australia, the mortality rate remains high at approximately 10%, despite curative antibiotic treatment being available. The bacterium is almost exclusively found in the endemic region, which spans the tropical Northern Territory and North Queensland, with clusters occasionally present in more temperate climates. Despite being endemic to North Queensland, these infections remain understudied compared to those of the Northern Territory.Methodology/principal findingsThis study aimed to assess the prevalence of central nervous system (CNS) disease associated variant bimABm, identify circulating antimicrobial resistance mutations and genetically distinct strains from Queensland, via comparative genomics. From 76 clinical isolates, we identified the bimABm variant in 20 (26.3%) isolates and in 9 (45%) of the isolates with documented CNS infection (n = 18). Explorative analysis suggests a significant association between isolates carrying the bimABm variant and CNS disease (OR 2.8, 95% CI 1.3-6.0, P = 0.009) compared with isolates carrying the wildtype bimABp. Furthermore, 50% of isolates were identified as novel multi-locus sequence types, while the bimABm variant was more commonly identified in isolates with novel sequence types, compared to those with previously described. Additionally, mutations associated with acquired antimicrobial resistance were only identified in 14.5% of all genomes.Conclusions/significanceThe findings of this research have provided clinically relevant genomic data of B. pseudomallei in Queensland and suggest that the bimABm variant may enable risk stratification for the development CNS complications and be a potential therapeutic target.

The relationship between dietary NaCl intake and high blood pressure is well-established, and occurs primarily through activation of the sympathetic nervous system. Nax, a Na+-sensitive Na+ channel, plays a pivotal role in driving sympathetic excitability, which is thought to originate from central regions controlling neural outflow. We investigated whether post-ganglionic sympathetic neurons from different ganglia innervating cardiac and vasculature tissue can also directly sense extracellular Na+. Using whole-cell patch clamp recordings we demonstrate that sympathetic neurons from three sympathetic ganglia (superior cervical, stellate and superior mesenteric/coeliac) respond to elevated extracellular NaCl concentration. In sympathetic stellate ganglia neurons, we established that the effect of NaCl was dose-dependent and independent of osmolarity, Cl− and membrane Ca2+ flux, and critically dependent on extracellular Na+ concentration. We show that Nax is expressed in sympathetic stellate ganglia neurons at a transcript and protein level using single-cell RNA-sequencing and immunohistochemistry respectively. Additionally, the response to NaCl was prevented by siRNA-mediated knockdown of Nax, but not by inhibition of other membrane Na+ pathways. Together, these results demonstrate that post-ganglionic sympathetic neurons are direct sensors of extracellular Na+via Nax, which could contribute to sympathetic driven hypertension.

ABSTRACT Debate continues as to the role of combination antibiotic therapy for the management of Pseudomonas aeruginosa infections. We studied the extent of bacterial killing by and the emergence of resistance to meropenem and amikacin as monotherapies and as a combination therapy against susceptible and resistant P. aeruginosa isolates from bacteremic patients using the dynamic in vitro hollow-fiber infection model. Three P. aeruginosa isolates (meropenem MICs of 0.125, 0.25, and 64 mg/L) were used, simulating bacteremia with an initial inoculum of ~1 × 105 CFU/mL and the expected pharmacokinetics of meropenem and amikacin in critically ill patients. For isolates susceptible to amikacin and meropenem (isolates 1 and 2), the extent of bacterial killing was increased with the combination regimen compared with the killing by monotherapy of either antibiotic. Both the combination and meropenem monotherapy were able to sustain bacterial killing throughout the 7-day treatment course, whereas regrowth of bacteria occurred with amikacin monotherapy after 12 h. For the meropenem-resistant P. aeruginosa isolate (isolate 3), only the combination regimen demonstrated bacterial killing. Given that tailored antibiotic regimens can maximize potential synergy against some isolates, future studies should explore the benefit of combination therapy against resistant P. aeruginosa. IMPORTANCE Current guidelines recommend that aminoglycosides should be used in combination with β-lactam antibiotics as initial empirical therapy for serious infections, and otherwise, patients should receive β-lactam antibiotic monotherapy. Given the challenges associated with studying the clinical effect of different antibiotic strategies on patient outcomes, useful data for subsequent informed clinical testing can be obtained from in vitro models like the hollow-fiber infection model (HFIM). Based on the findings of our HFIM, we propose that the initial use of combination therapy with meropenem and amikacin provides some bacterial killing against carbapenem-resistant P. aeruginosa isolates. For susceptible isolates, combination therapy may only be of benefit in specific patient populations, such as critically ill or immunocompromised patients. Therefore, clinicians may want to consider using the combination therapy for the initial management and ceasing the aminoglycosides once antibiotic susceptibility results have been obtained.

ABSTRACT Although recent reports of extensively antibiotic-resistant strains have highlighted the importance of Morganella morganii as an emerging pathogen, the epidemiology of serious infections due to this organism is not well defined. The objective of this study was to determine the incidence, determinants, and outcomes of Morganella morganii bloodstream infections (BSIs). Retrospective, population-based surveillance for Morganella morganii BSIs was conducted in Queensland, Australia, in 2000 to 2019; 709 cases were identified, for an annual incidence of 9.2 cases per million population. Most cases were of community onset, with 280 (39.5%) community-associated cases and 226 (31.9%) health care-associated cases. Morganella morganii BSIs were rare in children and young adults, and the incidence increased markedly with advancing age. The most common foci of infection were skin and soft tissue (131 cases [18.5%]), genitourinary (97 cases [13.7%]), and intraabdominal (90 cases [12.7%]). Most patients (580 cases [81.8%]) had at least one comorbid medical illness, with diabetes mellitus (250 cases [35.3%]), renal disease (208 cases [29.3%]), and congestive heart failure (167 cases [23.6%]) being most prevalent. Resistance to one or more of quinolones, co-trimoxazole, aminoglycosides, or carbapenems was observed in 67 cases (9.5%), and this did not change significantly over the study. The 30-day all-cause case fatality rate was 21.2%, and increasing age, nonfocal infection, heart failure, dementia, and cancer were independently associated with increased risk of death. Morganella morganii BSIs are increasing in our population, and elderly male subjects and individuals with comorbidities are at highest risk. Although antibiotic resistance is not a major contributor to the current burden in Queensland, ongoing surveillance is warranted. IMPORTANCE Recent reports of extensively antibiotic-resistant strains have highlighted the importance of Morganella morganii as an emerging pathogen. Despite its present and evolving importance as an agent of human disease, there is a limited body of literature detailing the epidemiology of serious infections due to Morganella morganii. Therefore, the objectives of this study were to examine the incidence and determinants of Morganella morganii BSIs and to examine risk factors for death in a large Australian population in 2000 to 2019.

Abstract Background Extended-spectrum beta-lactamase (ESBL) and AmpC-producing Enterobacterales are common causes of bloodstream infection. ESBL-producing bacteria are typically resistant to third-generation cephalosporins and result in a sizeable economic and public health burden. AmpC-producing Enterobacterales may develop third-generation cephalosporin resistance through enzyme hyper-expression. In no observational study has the outcome of treatment of these infections been surpassed by carbapenems. Widespread use of carbapenems may drive the development of carbapenem-resistant Gram-negative bacilli. Methods This study will use a multicentre, parallel group open-label non-inferiority trial design comparing ceftolozane-tazobactam and meropenem in adult patients with bloodstream infection caused by ESBL or AmpC-producing Enterobacterales. Trial recruitment will occur in up to 40 sites in six countries (Australia, Singapore, Italy, Spain, Saudi Arabia and Lebanon). The sample size is determined by a predefined quantity of ceftolozane-tazobactam to be supplied by Merck, Sharpe and Dohme (MSD). We anticipate that a trial with 600 patients contributing to the primary outcome analysis would have 80% power to declare non-inferiority with a 5% non-inferiority margin, assuming a 30-day mortality of 5% in both randomised groups. Once randomised, definitive treatment will be for a minimum of 5 days and a maximum of 14 days with the total duration determined by treating clinicians. Data describing demographic information, risk factors, concomitant antibiotics, illness scores, microbiology, multidrug-resistant organism screening, discharge and mortality will be collected. Discussion Participants will have bloodstream infection due to third-generation cephalosporin non-susceptible E. coli and Klebsiella spp. or Enterobacter spp., Citrobacter freundii, Morganella morganii, Providencia spp. or Serratia marcescens. They will be randomised 1:1 to ceftolozane-tazobactam 3 g versus meropenem 1 g, both every 8 h. Secondary outcomes will be a comparison of 14-day all-cause mortality, clinical and microbiological success at day 5, functional bacteraemia score, microbiological relapse, new bloodstream infection, length of hospital stay, serious adverse events, C. difficile infection, multidrug-resistant organism colonisation. The estimated trial completion date is December 2024. Trial registration The MERINO-3 trial is registered under the US National Institute of Health ClinicalTrials.gov register, reference number: NCT04238390 . Registered on 23 January 2020.

Foliar zinc (Zn) fertilization is an important approach for overcoming crop Zn deficiency, yet little is known regarding the subsequent translocation of this foliar-applied Zn. Using synchrotron-based X-ray fluorescence microscopy (XFM) and transcriptome analysis, the present study examined the translocation of foliar absorbed Zn in sunflower (Helianthus annuus) leaves. Although bulk analyses showed that there had been minimal translocation of the absorbed Zn out of the leaf within 7 days, in situ analyses showed that the distribution of Zn in the leaf had changed with time. Specifically, when Zn was applied to the leaf for 0.5 h and then removed, Zn primarily accumulated within the upper and lower epidermal layers (when examined after 3 h), but when examined after 24 h, the Zn had moved to the vascular tissues. Transcriptome analyses identified a range of genes involved in stress response, cell wall reinforcement, and binding that were initially upregulated following foliar Zn application, whereas they were downregulated after 24 h. These observations suggest that foliar Zn application caused rapid stress to the leaf, with the initial Zn accumulation in the epidermis as a detoxification strategy, but once this stress decreased, Zn was then moved to the vascular tissues. Overall, this study has shown that despite foliar Zn application causing rapid stress to the leaf and that most of the Zn stayed within the leaf over 7 days, the distribution of Zn in the leaf had changed, with Zn mostly located in the vascular tissues 24 h after the Zn had been applied. Not only do the data presented herein provide new insight for improving the efficiency of foliar Zn fertilizers, but our approach of combining XFM with a transcriptome methodological system provides a novel approach for the study of element translocation in plants.

Antibiotic-resistant bacteria are an urgent threat to human health. Here, Roberts et al. characterise and monitor an ongoing hospital outbreak of carbapenemase-producing Enterobacter hormaechei by integrating several technologies for whole-genome sequencing and shotgun metagenomics.

ABSTRACT Acinetobacter baumannii causes high mortality in ventilator-associated pneumonia patients, and antibiotic treatment is compromised by multidrug-resistant strains resistant to β-lactams, carbapenems, cephalosporins, polymyxins, and tetracyclines. Among COVID-19 patients receiving ventilator support, a multidrug-resistant A. baumannii secondary infection is associated with a 2-fold increase in mortality. Here, we investigated the use of the 8-hydroxyquinoline ionophore PBT2 to break the resistance of A. baumannii to tetracycline class antibiotics. In vitro, the combination of PBT2 and zinc with either tetracycline, doxycycline, or tigecycline was shown to be bactericidal against multidrug-resistant A. baumannii, and any resistance that did arise imposed a fitness cost. PBT2 and zinc disrupted metal ion homeostasis in A. baumannii, increasing cellular zinc and copper while decreasing magnesium accumulation. Using a murine model of pulmonary infection, treatment with PBT2 in combination with tetracycline or tigecycline proved efficacious against multidrug-resistant A. baumannii. These findings suggest that PBT2 may find utility as a resistance breaker to rescue the efficacy of tetracycline-class antibiotics commonly employed to treat multidrug-resistant A. baumannii infections. IMPORTANCE Within intensive care unit settings, multidrug-resistant (MDR) Acinetobacter baumannii is a major cause of ventilator-associated pneumonia, and hospital-associated outbreaks are becoming increasingly widespread. Antibiotic treatment of A. baumannii infection is often compromised by MDR strains resistant to last-resort β-lactam (e.g., carbapenems), polymyxin, and tetracycline class antibiotics. During the on-going COVID-19 pandemic, secondary bacterial infection by A. baumannii has been associated with a 2-fold increase in COVID-19-related mortality. With a rise in antibiotic resistance and a reduction in new antibiotic discovery, it is imperative to investigate alternative therapeutic regimens that complement the use of current antibiotic treatment strategies. Rescuing the efficacy of existing therapies for the treatment of MDR A. baumannii infection represents a financially viable pathway, reducing time, cost, and risk associated with drug innovation.

Hypertension is a major health burden worldwide with many cases resistant to current treatments. Hyperactivity of the sympathetic nervous contributes to the etiology and progression of the disease, where emerging evidence suggests that inflammation may underpin the development of sympathetic dysautonomia. This study examined whether macrophages could drive the sympathetic phenotype in Spontaneously Hypertensive Rats (SHR) before animals develop high pressure. Stellate neurons from wild-type control Wistar rats and SHRs were co-cultured with blood leukocytes from their own strain, and also crossed cultured between strains. The calcium transient response to nicotinic stimulation was recorded using Fura-2 calcium imaging, where SHR neurons had a greater calcium transient compared with Wistar neurons. However, when co-cultured with leukocytes, Wistar neurons began to phenocopy the SHR sympathetic hyperactivity, while the SHR neurons themselves were unaltered. Resident leukocyte populations of the SHR and Wistar stellate ganglia were then compared using flow cytometry, where there was a shift in monocyte-macrophage subset proportions. While classical monocyte-macrophages were predominant in the Wistar, there were relatively more of the non-classical subset in the SHR, which have been implicated in pro-inflammatory roles in a number of diseases. When bone marrow-derived macrophages (BMDMs) were co-cultured with stellate neurons, they made Wistar neurons recapitulate the SHR nicotinic stimulated calcium transient. Wistar BMDMs however, had no effect on SHR neurons, even though SHR BMDMs increased SHR neuron responsiveness further above their hyper-responsive state. Taken together, these findings show that macrophages can be potent enhancers of sympathetic neuronal calcium responsiveness, and thus could conceivably play a role in peripheral sympathetic hyperactivity observed in the early stages of hypertension.

BackgroundNeonatal sepsis is a significant global health issue associated with marked regional disparities in mortality. Antimicrobial resistance (AMR) is a growing concern in Gram-negative organisms, which increasingly predominate in neonatal sepsis, and existing WHO empirical antibiotic recommendations may no longer be appropriate. Previous systematic reviews have been limited to specific low- and middle-income countries. We therefore completed a systematic review and meta-analysis of available data from all low- and lower-middle-income countries (LLMICs) since 2010, with a focus on regional differences in Gram-negative infections and AMR.Methods and findingsAll studies published from 1 January 2010 to 21 April 2021 about microbiologically confirmed bloodstream infections or meningitis in neonates and AMR in LLMICs were assessed for eligibility. Small case series, studies with a small number of Gram-negative isolates (ConclusionsGram-negative bacteria are an important cause of neonatal sepsis in LLMICs and are associated with significant rates of resistance to WHO-recommended first- and second-line empirical antibiotics. AMR surveillance should underpin region-specific empirical treatment recommendations. Meanwhile, a significant global commitment to accessible and effective antimicrobials for neonates is required.

ABSTRACT Extended-spectrum-beta-lactamase (ESBL)-producing Enterobacterales continue to pose a major threat to human health worldwide. Given the limited therapeutic options available to treat infections caused by these pathogens, identifying additional effective antimicrobials or revisiting existing drugs is important. Ceftriaxone-resistant Escherichia coli and Klebsiella pneumoniae containing CTX-M-type ESBLs or AmpC, in addition to narrow-spectrum OXA and SHV enzymes, were selected from blood culture isolates obtained from the MERINO trial. Isolates had previously undergone whole-genome sequencing (WGS) to identify antimicrobial resistance genes. Cefotetan MICs were determined by broth microdilution (BMD) testing with a concentration range of 0.125 to 64 mg/liter; CLSI breakpoints were used for susceptibility interpretation. BMD was performed using an automated digital antibiotic dispensing platform (Tecan D300e). One hundred ten E. coli and 40 K. pneumoniae isolates were used. CTX-M-15 and CTX-M-27 were the most common beta-lactamases present; only 7 isolates had coexistent ampC genes. Overall, 98.7% of isolates were susceptible, with MIC50s and MIC90s of 0.25 mg/liter and 2 mg/liter (range, ≤0.125 to 64 mg/liter), respectively. MICs appeared higher among isolates with ampC genes present, with an MIC50 of 16 mg/liter, than among those containing CTX-M-15, which had an MIC50 of only 0.5 mg/liter. Isolates with an ampC gene exhibited an overall susceptibility of 85%. Presence of a narrow-spectrum OXA beta-lactamase did not appear to alter the cefotetan MIC distribution. Cefotetan demonstrated favorable in vitro efficacy against ESBL-producing E. coli and K. pneumoniae bloodstream isolates. IMPORTANCE Carbapenem antibiotics remain the treatment of choice for severe infection due to ESBL- and AmpC-producing Enterobacterales. The use of carbapenems is a major driver of the emergence of carbapenem-resistant Gram-negative bacilli, which are often resistant to most available antimicrobials. Cefotetan is a cephamycin antibiotic developed in the 1980s that demonstrates enhanced resistance to beta-lactamases and has a broad spectrum of activity against Gram-negative bacteria. Cefotetan holds potential to be a carbapenem-sparing treatment option. Data on the in vitro activity of cefotetan against ESBL-producing Enterobacterales remain scarce. Our study assessed the in vitro activity of cefotetan against ceftriaxone-nonsusceptible blood culture isolates obtained from patients enrolled in the MERINO trial.

Although new-generation antimicrobials, in particular β-lactam/β-lactamase inhibitors, have largely replaced polymyxins in carbapenem-resistant Gram-negative bacterial infections, polymyxins are still needed for carbapanem-resistant Acinetobacter baumannii infections and in settings where novel agents are not readily available. Despite their potent in vitro activity, the clinical utility of polymyxins is significantly limited by their pharmacokinetic properties and nephrotoxicity risk. There is significant interest, therefore, in developing next-generation polymyxins with activity against colistin-resistant strains and lower toxicity than existing polymyxins. In this review, we aim to present the antibacterial activity mechanisms, in vitro and in vivo efficacy data, and toxicity profiles of new-generation polymyxins, including SPR206, MRX-8, and QPX9003, as well as the general characteristics of old polymyxins. Considering the emergence of colistin-resistant strains particularly in endemic regions, the restoration of the antimicrobial activity of polymyxins via PBT2 is also described in this review.

Negative associations of prenatal tobacco and alcohol exposure (PTE and PAE) on birth outcomes and childhood development have been well documented, but less is known about underlying mechanisms. A possible pathway for the adverse fetal outcomes associated with PTE and PAE is the alteration of fetal autonomic nervous system development. This study assessed PTE and PAE effects on measures of fetal autonomic regulation, as quantified by heart rate (HR), heart rate variability (SD-HR), movement, and HR-movement coupling in a population of fetuses at ≥ 34 weeks gestational age. Participants are a subset of the Safe Passage Study, a prospective cohort study that enrolled pregnant women from clinical sites in Cape Town, South Africa, and the Northern Plains region, United States. PAE was defined by six levels: no alcohol, low quit early, high quit early, low continuous, moderate continuous, and high continuous; while PTE by 4 levels: no smoking, quit early, low continuous, and moderate/high continuous. Linear regression analyses of autonomic measures were employed controlling for fetal sex, gestational age at assessment, site, maternal education, household crowding, and depression. Analyses were also stratified by sleep state (1F and 2F) and site (South Africa, N = 4025, Northern Plains, N = 2466). The final sample included 6491 maternal-fetal-dyad assessed in the third trimester [35.21 ± 1.26 (mean ± SD) weeks gestation]. PTE was associated with a decrease in mean HR in state 2F, in a dose dependent fashion, only for fetuses of mothers who continued smoking after the first trimester. In state 1F, there was a significant increase in mean HR in fetuses whose mother quit during the first trimester. This effect was driven by the Norther Plains cohort. PTE was also associated with a significant reduction in fetal movement in the most highly exposed group. In South Africa a significant increase in mean HR both for the high quit early and the high continuous group was observed. In conclusion, this investigation addresses a critical knowledge gap regarding the relationship between PTE and PAE and fetal autonomic regulation. We believe these results can contribute to elucidating mechanisms underlying risk for adverse outcomes.

Abstract Background Carbapenemase-producing organisms (CPOs) have emerged as antibiotic-resistant bacteria of global concern. Here we assessed the performance of the Carba (beta) assay, a multiplex real-time PCR assay developed by SpeeDx for the detection of key carbapenemase-encoding genes: KPC, NDM, OXA-48-like, IMP-4-like, and VIM. Methods DNA extracts of 180 isolates were tested with the Carba (beta) assay, using previously validated in-house TaqMan probe assays for the relevant carbapenemase genes as the reference standard. The Carba (beta) assay was then directly used to screen 460 DNA extracts of faecal specimens, with positive results subjected to the aforementioned in-house assays plus Sanger sequencing. Results The Carba (beta) assay correctly identified the presence of the respective carbapenemase genes in 154 of 156 isolates and provided negative results for all 24 non-CPO isolates. Two isolates provided positive results for OXA-48-like carbapenemase by the Carba (beta) assay only. The Carba (beta) assay had sensitivities of 100% for all targets, and specificities of 100% for KPC, NDM, IMP-4-like, and VIM targets, and 98.5% for OXA-48-like targets. When applied directly to faecal specimens, eight samples were positive by the Carba (beta) assay, two of which were confirmed by in-house TaqMan probe PCR or DNA sequencing. Conclusions The Carba (beta) assay is highly sensitive and specific for detecting key carbapenemase genes in isolates. Further testing is required to assess this assay’s suitability for direct screening of clinical specimens.