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ABSTRACT With the development of sequencing technology and analytic tools, studying within-species variations enhances the understanding of microbial biological processes. Nevertheless, most existing methods designed for strain-level analysis lack the capability to concurrently assess both strain proportions and genome-wide single nucleotide variants (SNVs) across longitudinal metagenomic samples. In this study, we introduce LongStrain, an integrated pipeline for the analysis of large-scale metagenomic data from individuals with longitudinal or repeated samples. In LongStrain, we first utilize two efficient tools, Kraken2 and Bowtie2, for the taxonomic classification and alignment of sequencing reads, respectively. Subsequently, we propose to jointly model strain proportions and shared haplotypes across samples within individuals. This approach specifically targets tracking a primary strain and a secondary strain for each subject, providing their respective proportions and SNVs as output. With extensive simulation studies of a microbial community and single species, our results demonstrate that LongStrain is superior to two genotyping methods and two deconvolution methods across a majority of scenarios. Furthermore, we illustrate the potential applications of LongStrain in the real data analysis of The Environmental Determinants of Diabetes in the Young study and a gastric intestinal metaplasia microbiome study. In summary, the proposed analytic pipeline demonstrates marked statistical efficiency over the same type of methods and has great potential in understanding the genomic variants and dynamic changes at strain level. LongStrain and its tutorial are freely available online at https://github.com/BoyanZhou/LongStrain.IMPORTANCEThe advancement in DNA-sequencing technology has enabled the high-resolution identification of microorganisms in microbial communities. Since different microbial strains within species may contain extreme phenotypic variability (e.g., nutrition metabolism, antibiotic resistance, and pathogen virulence), investigating within-species variations holds great scientific promise in understanding the underlying mechanism of microbial biological processes. To fully utilize the shared genomic variants across longitudinal metagenomics samples collected in microbiome studies, we develop an integrated analytic pipeline (LongStrain) for longitudinal metagenomics data. It concurrently leverages the information on proportions of mapped reads for individual strains and genome-wide SNVs to enhance the efficiency and accuracy of strain identification. Our method helps to understand strains’ dynamic changes and their association with genome-wide variants. Given the fast-growing longitudinal studies of microbial communities, LongStrain which streamlines analyses of large-scale raw sequencing data should be of great value in microbiome research communities.

ABSTRACT Severe COVID-19 has been associated with coinfections with bacterial and fungal pathogens. Notably, patients with COVID-19 who develop Staphylococcus aureus bacteremia exhibit higher rates of mortality than those infected with either pathogen alone. To understand this clinical scenario, we collected and examined S. aureus blood and respiratory isolates from a hospital in New York City during the early phase of the pandemic from both SARS-CoV-2+ and SARS-CoV-2− patients. Whole genome sequencing of these S. aureus isolates revealed broad phylogenetic diversity in both patient groups, suggesting that SARS-CoV-2 coinfection was not associated with a particular S. aureus lineage. Phenotypic characterization of the contemporary collection of S. aureus isolates from SARS-CoV-2+ and SARS-CoV-2− patients revealed no notable differences in several virulence traits examined. However, we noted a trend toward overrepresentation of S. aureus bloodstream strains with low cytotoxicity in the SARS-CoV-2+ group. We observed that patients coinfected with SARS-CoV-2 and S. aureus were more likely to die during the acute phase of infection when the coinfecting S. aureus strain exhibited high or low cytotoxicity. To further investigate the relationship between SARS-CoV-2 and S. aureus infections, we developed a murine coinfection model. These studies revealed that infection with SARS-CoV-2 renders mice susceptible to subsequent superinfection with low cytotoxicity S. aureus. Thus, SARS-CoV-2 infection sensitizes the host to coinfections, including S. aureus isolates with low intrinsic virulence.IMPORTANCEThe COVID-19 pandemic has had an enormous impact on healthcare across the globe. Patients who were severely infected with SARS-CoV-2, the virus causing COVID-19, sometimes became infected with other pathogens, which is termed coinfection. If the coinfecting pathogen is the bacterium Staphylococcus aureus, there is an increased risk of patient death. We collected S. aureus strains that coinfected patients with SARS-CoV-2 to study the disease outcome caused by the interaction of these two important pathogens. We found that both in patients and in mice, coinfection with an S. aureus strain lacking toxicity resulted in more severe disease during the early phase of infection, compared with infection with either pathogen alone. Thus, SARS-CoV-2 infection can directly increase the severity of S. aureus infection.

Background: The frequency of Staphylococcus aureus transmission in hospitals is unknown: symptomatic infection may occur months after transmission and colonization, and infection prevention efforts rely on indirect measurements, rather than direct detection of transmission events. We implemented a hospital-based S. aureus screening program, combined with whole genome sequencing of S. aureus surveillance and clinical cultures and data extracted from the electronic health record, to identify S. aureus clonal complex-, patient- and location-specific factors associated with S. aureus transmission in our health system. Methods: Screening S. aureus cultures were obtained at admission by nasal swab for adults admitted to Medicine, Transplant, Oncology and intensive care, and weekly by swab of nares, axilla and groin for children admitted to intensive care and Oncology at NYU Langone Health in New York City. All methicillin-resistant S. aureus (MRSA) from screening and clinical (blood, wound, sputum) cultures and all methicillin-susceptible S. aureus (MSSA) from screening and blood cultures underwent whole genome sequencing. Isolates from distinct patients with < 2 0 single nucleotide pair differences were considered genetically related. Electronic health data was extracted for descriptive statistics and for spatiotemporal plots to assess plausible transmissions. We used REDCap electronic data capture tools hosted at NYU Grossman School of Medicine and SAS software for data analysis to evaluate S. aureus transmissions between November 2022 and November 2023. Results: We analyzed 8,567 S. aureus isolates: including 6,552 screening cultures, 1,008 blood cultures, and 1,007 clinical cultures. We found 424 plausible S. aureus hospital transmissions using sequencing and electronic health data. Screening cultures identified 75% of transmissions that would have otherwise been missed with blood and clinical cultures alone. The majority of positive screening cultures isolated MSSA, but the proportion of transmissions due to MSSA differed by age. In children, MSSA colonization accounted for 62% of transmissions. In adults, only 15% of transmissions were due to MSSA colonization, whereas MRSA colonization accounted for 56% of transmissions. Analysis of adult MRSA isolates by clonal complex found that 45% of transmissions were due to CC8, higher than the 17% among isolates agnostic of transmissions. Emergency departments and the neonatal intensive care unit had the highest number of transmissions. Patients involved in transmissions had longer lengths of stay and frequent hospitalizations. Conclusions: A S. aureus screening program, coupled with genome sequencing and electronic health data, can identify patient group, hospital locations and clonal complexes that are at high risk for S. aureus transmissions.

Abstract Aim Acyl ghrelin increases cardiac output (CO) in heart failure with reduced ejection fraction (HFrEF). This could impair the right ventricular‐pulmonary arterial coupling (RVPAC), both through an increased venous return and right ventricular afterload. We aim to investigate if acyl ghrelin increases CO with or without worsening the right‐sided haemodynamics in HFrEF assessed by RVPAC. Methods and results The Karolinska Acyl ghrelin Trial was a randomized double‐blind placebo‐controlled trial of acyl ghrelin versus placebo (120‐min intravenous infusion) in HFrEF. RVPAC was assessed echocardiographically at baseline and 120 min. ANOVA was used for difference in change between acyl ghrelin versus placebo, adjusted for baseline values. Of the 30 randomized patients, 22 had available RVPAC (acyl ghrelin n = 12, placebo n = 10). Despite a 15% increase in CO in the acyl ghrelin group (from 4.0 (3.5–4.6) to 4.6 (3.9–6.1) L/min, P = 0.003), RVPAC remained unchanged; 5.9 (5.3–7.6) to 6.3 (4.8–7.5) mm·(m/s)−1, P = 0.372, while RVPAC was reduced in the placebo group, 5.2 (4.3–6.4) to 4.8 (4.2–5.8) mm·(m/s)−1, P = 0.035. Comparing change between groups, CO increased in the acyl ghrelin group versus placebo (P = 0.036) while RVPAC and the right ventricular pressure gradient remained unchanged. Conclusion Treatment with acyl ghrelin increases CO while preserving or even improving RVPAC in HFrEF, possibly due to increased contractility, reduced PVR and/or reduced left sided filling pressures. These potential effects strengthen the role of acyl ghrelin therapy in HFrEF with right ventricular failure.

The agr quorum-sensing system links Staphylococcus aureus metabolism to virulence, in part by increasing bacterial survival during exposure to lethal concentrations of H2O2, a crucial host defense against S. aureus. We now report that protection by agr surprisingly extends beyond post-exponential growth to the exit from stationary phase when the agr system is no longer turned on. Thus, agr can be considered a constitutive protective factor. Deletion of agr resulted in decreased ATP levels and growth, despite increased rates of respiration or fermentation at appropriate oxygen tensions, suggesting that Δagr cells undergo a shift towards a hyperactive metabolic state in response to diminished metabolic efficiency. As expected from increased respiratory gene expression, reactive oxygen species (ROS) accumulated more in the agr mutant than in wild-type cells, thereby explaining elevated susceptibility of Δagr strains to lethal H2O2 doses. Increased survival of wild-type agr cells during H2O2 exposure required sodA, which detoxifies superoxide. Additionally, pretreatment of S. aureus with respiration-reducing menadione protected Δagr cells from killing by H2O2. Thus, genetic deletion and pharmacologic experiments indicate that agr helps control endogenous ROS, thereby providing resilience against exogenous ROS. The long-lived ‘memory’ of agr-mediated protection, which is uncoupled from agr activation kinetics, increased hematogenous dissemination to certain tissues during sepsis in ROS-producing, wild-type mice but not ROS-deficient (Cybb−/−) mice. These results demonstrate the importance of protection that anticipates impending ROS-mediated immune attack. The ubiquity of quorum sensing suggests that it protects many bacterial species from oxidative damage.

Nine isolates of Trichoderma were obtained from the diverse phases of compost preparation and the production of different commercial varieties of oyster mushrooms Pleurotus spp. with the apparent presence of green mould disease. The isolates were morphologically and genetically characterized. Molecular fingerprinting indicated that the isolates belonged to the species T. pleuroticola and T. harzianum. In order to identify control measures, changes in temperature, pH, and application of fungicides were tested on the present isolates, using known Trichoderma biocontrol strains as a reference. Fungicide effects on the growth of Pleurotus isolates were also assessed. The optimal growth temperatures were 25 °C for Trichoderma isolates and 28 °C for Pleurotus isolates, and Trichoderma always grew faster than Pleurotus. In particular, a reduction of about 30% was recorded for some of the Trichoderma isolates when comparing the colony growth at 25 and 28 °C. Trichoderma isolates developed well within a wide pH range, with the best growth occurring between pH 5 and 7, whereas Pleurotus preferred more alkaline conditions (pH 8 to 9). Prochloraz and metrafenone were found to inhibit Trichoderma growth with different dose responses that did not affect the growth of Pleurotus spp. In particular, metrafenone was the most effective active ingredient that inhibited the majority of the Trichoderma isolates (1–25% growth reduction).

Abstract Background Total hip arthroplasty (THA) is the most common treatment for primary and secondary end-stage hip osteoarthritis (OA). Almost 20% of all patients undergoing primary THA suffer from bilateral hip OA and, consequently, will need a contralateral procedure to be performed in the following years. The aim of this study is to evaluate the cost-effectiveness and the reliability of one-stage bilateral THA (1-BTHA) compared to two-stage bilateral THA (2-BTHA), in low-risk patients, performed with anterior minimally invasive surgery (AMIS). Methods Single patient’s costs were obtained by dividing the annual costs report by the number of hospitalizations, considering the diagnosis related group (DRG) of the two procedures. Then, 16 patients undergoing 1-BTHA and 8 undergoing 2-BTHA were examined. Hemoglobin (Hb) values before surgery and before discharge, transfusion rate and the occurrence of post-operative complications were observed. Results Procedural costs were divided in different subgroups: pre-hospitalization, operating room, hospital stay, post-operative follow-up and other costs. 1-BTHA total costs amount to 5.754,82€, while performing 2-BTHA costs 7.624,32€. However, considering DRG reimbursement, the hospital’s profit margin following 1-BTHA is lower than that following 2-BTHA (6.346,18€ versus 9.261,68€). Surgical time was found not to be significantly different between 1-BTHA and 2-BTHA (141,13 ± 26,1 min vs 164,8 ± 44,3 min; p = 0,111). The two groups showed a statistically significant difference in Hb decrease (4,8 ± 1,3 g/dl vs 3,3 ± 0,9; p = 0,001), despite no variances in transfusion rate. No further complications were observed in either group. Conclusions This study demonstrates how, in carefully selected patients, 1-BTHA performed with AMIS is a cost-effective and safe technique compared to 2-BTHA, resulting in a shorter OR time, LOS and lower overall costs. Level of evidence III