Clinical and in vitro models identify distinct adaptations enhancing Staphylococcus aureus pathogenesis in human macrophages.

Staphylococcus aureus is a facultative intracellular pathogen of human macrophages, which facilitates chronic infection. The genotypes, pathways, and mutations influencing that phenotype remain incompletely explored. Here, we used two distinct strategies to ascertain S. aureus gene mutations affecting pathogenesis in macrophages. First, we analyzed isolates collected serially from chronic cystic fibrosis (CF) respiratory infections. We found that S. aureus strains evolved greater macrophage invasion capacity during chronic human infection. Bacterial genome-wide association studies (GWAS) identified 127 candidate genes for which mutation was significantly associated with macrophage pathogenesis in vivo. In parallel, we passaged laboratory S. aureus strains in vitro to select for increased infection of human THP-1 derived macrophages, which identified 15 candidate genes by whole-genome sequencing. Functional validation of candidate genes using isogenic transposon mutant knockouts and CRISPR interference (CRISPRi) knockdowns confirmed virulence contributions from 37 of 39 tested genes (95%) implicated by in vivo studies and 7 of 10 genes (70%) ascertained from in vitro selection, with one gene in common to the two strategies. Validated genes included 17 known virulence factors (39%) and 27 newly identified by our study (61%), some encoding functions not previously associated with macrophage pathogenesis. Most genes (80%) positively impacted macrophage invasion when disrupted, consistent with the phenotype readily arising from loss-of-function mutations in vivo. This work reveals genes and mechanisms that contribute to S. aureus infection of macrophages, highlights differences in mutations underlying convergent phenotypes arising from in vivo and in vitro systems, and supports the relevance of S. aureus macrophage pathogenesis during chronic respiratory infection in CF. Additional studies will be needed to illuminate the exact mechanisms by which implicated mutations affect their phenotypes.

2023 Neer Award for Basic Science: Genetics of Cutibacterium acnes in revision shoulder arthroplasty: a large-scale bacterial whole-genome sequencing study.

BACKGROUND: Cutibacterium acnes is the bacterium most commonly responsible for shoulder periprosthetic joint infection (PJI) and is often cultured from samples obtained at the time of revision for failed shoulder arthroplasty. We sought to determine whether these bacteria originate from the patient or from exogenous sources. We also sought to identify which C. acnes genetic traits were associated with the development of shoulder PJI. METHODS: We performed bacterial whole-genome sequencing of C. acnes from a single-institution repository of cultures obtained before or during primary and revision shoulder arthroplasty and correlated the molecular epidemiology and genetic content of strains with clinical features of infection. RESULTS: A total of 341 isolates collected over a 4-year period from 88 patients were sequenced. C. acnes cultured from surgical specimens demonstrated significant similarity to the strains colonizing the skin of the same patient (P < .001). Infrequently, there was evidence of strains shared across unrelated patients, suggesting that exogenous sources of C. acnes culture-positivity were uncommon. Phylotypes IB and II were modestly associated with clinical features of PJI, but all phylotypes appeared inherently capable of causing disease. Chronic shoulder PJI was associated with the absence of common C. acnes genes involved in bacterial quorum-sensing (luxS, tqsA). CONCLUSION: C. acnes strains cultured from deep intraoperative sources during revision shoulder arthroplasty demonstrate strong genetic similarity to the strains colonizing a patient's skin. Some phylotypes of C. acnes commonly colonizing human skin are modestly more virulent than others, but all phylotypes have a capacity for PJI. C. acnes cultured from cases of PJI commonly demonstrated genetic hallmarks associated with adaptation from acute to chronic phases of infection. This is the strongest evidence to date supporting the role of the patient's own, cutaneous C. acnes strains in the pathogenesis of shoulder arthroplasty infection. Our findings support the importance of further research focused on perioperative decolonization and management of endogenous bacteria that are likely to be introduced into the arthroplasty wound at the time of skin incision.

Transposon sequencing identifies genes impacting Staphylococcus aureus invasion in a human macrophage model.

Staphylococcus aureus is a facultative intracellular pathogen in many host cell types, facilitating its persistence in chronic infections. The genes contributing to intracellular pathogenesis have not yet been fully enumerated. Here, we cataloged genes influencing S. aureus invasion and survival within human THP-1 derived macrophages using two laboratory strains (ATCC2913 and JE2). We developed an in vitro transposition method to produce highly saturated transposon mutant libraries in S. aureus and performed transposon insertion sequencing (Tn-Seq) to identify candidate genes with significantly altered abundance following macrophage invasion. While some significant genes were strain-specific, 108 were identified as common across both S. aureus strains, with most (n = 106) being required for optimal macrophage infection. We used CRISPR interference (CRISPRi) to functionally validate phenotypic contributions for a subset of genes. Of the 20 genes passing validation, seven had previously identified roles in S. aureus virulence, and 13 were newly implicated. Validated genes frequently evidenced strain-specific effects, yielding opposing phenotypes when knocked down in the alternative strain. Genomic analysis of de novo mutations occurring in groups (n = 237) of clonally related S. aureus isolates from the airways of chronically infected individuals with cystic fibrosis (CF) revealed significantly greater in vivo purifying selection in conditionally essential candidate genes than those not associated with macrophage invasion. This study implicates a core set of genes necessary to support macrophage invasion by S. aureus, highlights strain-specific differences in phenotypic effects of effector genes, and provides evidence for selection of candidate genes identified by Tn-Seq analyses during chronic airway infection in CF patients in vivo.

In Vitro Selection Identifies Staphylococcus aureus Genes Influencing Biofilm Formation.

Staphylococcus aureus generates biofilms during many chronic human infections, which contributes to its growth and persistence in the host. Multiple genes and pathways necessary for S. aureus biofilm production have been identified, but knowledge is incomplete, and little is known about spontaneous mutations that increase biofilm formation as infection progresses. Here, we performed in vitro selection of four S. aureus laboratory strains (ATCC 29213, JE2, N315, and Newman) to identify mutations associated with enhanced biofilm production. Biofilm formation increased in passaged isolates from all strains, exhibiting from 1.2- to 5-fold the capacity of parental lines. Whole-genome sequencing identified nonsynonymous mutations affecting 23 candidate genes and a genomic duplication encompassing sigB. Six candidate genes significantly impacted biofilm formation as isogenic transposon knockouts: three were previously reported to impact S. aureus biofilm formation (icaR, spdC, and codY), while the remaining three (manA, narH, and fruB) were newly implicated by this study. Plasmid-mediated genetic complementation of manA, narH, and fruB transposon mutants corrected biofilm deficiencies, with high-level expression of manA and fruB further enhancing biofilm formation over basal levels. This work recognizes genes not previously identified as contributing to biofilm formation in S. aureus and reveals genetic changes able to augment biofilm production by that organism.

Dynamics of Cutibacterium repopulation onto the skin surface of the shoulder after chlorhexidine application.

PURPOSE: The objective of this study was to characterize the temporal dynamics of Cutibacterium repopulation of the skin surface after application of chlorhexidine to the shoulder. METHODS: Ten shoulders in five male subjects were used. A skin swab was taken prior to (0 minutes) and then at three, 30, 60, 120, and 240 minutes after skin preparation with 2% chlorhexidine gluconate and 70% isopropyl alcohol. Semi-quantitative bacterial load was measured for each timepoint. RESULTS: From zero minutes (pre-treatment) to three minutes, chlorhexidine-isopropyl alcohol reduced the skin bacterial load in eight out of ten shoulders. Of these eight shoulders, four (50%) had growth by 30 minutes, seven (88%) had growth by 60 minutes, and all eight (100%) had growth by 240 minutes. Compared to the three minutes after chlorhexidine application, bacterial load had significantly increased by 60 minutes but were still significantly lower than the pre-prep bacterial load (0 minutes). CONCLUSION: Following standard surgical skin preparation with chlorhexidine-isopropyl alcohol, the surface of the shoulder is repopulated with Cutibacterium within one hour, presumably from reservoirs in sebaceous glands not penetrated by topical antiseptic agents. Since these dermal glands are transected by skin incision for shoulder arthroplasty, this study suggests that they may be sources of wound contamination during surgery in spite of skin preparation with chlorhexidine.

Intraoperative prediction of postanaesthesia care unit hypotension.

BACKGROUND: Postoperative hypotension is associated with adverse outcomes, but intraoperative prediction of postanaesthesia care unit (PACU) hypotension is not routine in anaesthesiology workflow. Although machine learning models may support clinician prediction of PACU hypotension, clinician acceptance of prediction models is poorly understood. METHODS: We developed a clinically informed gradient boosting machine learning model using preoperative and intraoperative data from 88 446 surgical patients from 2015 to 2019. Nine anaesthesiologists each made 192 predictions of PACU hypotension using a web-based visualisation tool with and without input from the machine learning model. Questionnaires and interviews were analysed using thematic content analysis for model acceptance by anaesthesiologists. RESULTS: The model predicted PACU hypotension in 17 029 patients (area under the receiver operating characteristic [AUROC] 0.82 [95% confidence interval {CI}: 0.81-0.83] and average precision 0.40 [95% CI: 0.38-0.42]). On a random representative subset of 192 cases, anaesthesiologist performance improved from AUROC 0.67 (95% CI: 0.60-0.73) to AUROC 0.74 (95% CI: 0.68-0.79) with model predictions and information on risk factors. Anaesthesiologists perceived more value and expressed trust in the prediction model for prospective planning, informing PACU handoffs, and drawing attention to unexpected cases of PACU hypotension, but they doubted the model when predictions and associated features were not aligned with clinical judgement. Anaesthesiologists expressed interest in patient-specific thresholds for defining and treating postoperative hypotension. CONCLUSIONS: The ability of anaesthesiologists to predict PACU hypotension was improved by exposure to machine learning model predictions. Clinicians acknowledged value and trust in machine learning technology. Increasing familiarity with clinical use of model predictions is needed for effective integration into perioperative workflows.

Polyclonality, Shared Strains, and Convergent Evolution in Chronic Cystic Fibrosis Staphylococcus aureus Airway Infection.

Rationale:Staphylococcus aureus is the most common respiratory pathogen isolated from patients with cystic fibrosis (CF) in the United States. Although modes of acquisition and genetic adaptation have been described for Pseudomonas aeruginosa, resulting in improved diagnosis and treatment, these features remain more poorly defined for S. aureus.Objectives: To characterize the molecular epidemiology and genetic adaptation of S. aureus during chronic CF airway infection and in response to antibiotic therapy.Methods: We performed whole-genome sequencing of 1,382 S. aureus isolates collected longitudinally over a mean 2.2 years from 246 children with CF at five U.S. centers between 2008 and 2017. Results were integrated with clinical and demographic data to characterize bacterial population dynamics and identify common genetic targets of in vivo adaptation.Measurements and Main Results: Results showed that 45.5% of patients carried multiple, coexisting S. aureus lineages, often having different antibiotic susceptibility profiles. Adaptation during the course of infection commonly occurred in a set of genes related to persistence and antimicrobial resistance. Individual sequence types demonstrated wide geographic distribution, and we identified limited strain-sharing among children linked by common household or clinical exposures. Unlike P. aeruginosa, S. aureus genetic diversity was unconstrained, with an ongoing flow of new genetic elements into the population of isolates from children with CF.Conclusions: CF airways are frequently coinfected by multiple, genetically distinct S. aureus lineages, indicating that current clinical procedures for sampling isolates and selecting antibiotics are likely inadequate. Strains can be shared by patients in close domestic or clinical contact and can undergo convergent evolution in key persistence and antimicrobial-resistance genes, suggesting novel diagnostic and therapeutic approaches for future study.

Occurrence and Timing of Subsequent Severe Acute Respiratory Syndrome Coronavirus 2 Reverse-transcription Polymerase Chain Reaction Positivity Among Initially Negative Patients.

Using data for 20 912 patients from 2 large academic health systems, we analyzed the frequency of severe acute respiratory syndrome coronavirus 2 reverse-transcription polymerase chain reaction test discordance among individuals initially testing negative by nasopharyngeal swab who were retested on clinical grounds within 7 days. The frequency of subsequent positivity within this window was 3.5% and was similar across institutions.

Identifying Optimal Loci for the Molecular Diagnosis of Microsatellite Instability.

BACKGROUND: Microsatellite instability (MSI) predicts oncological response to checkpoint blockade immunotherapies. Although microsatellite mutation is pathognomonic for the condition, loci have unequal diagnostic value for predicting MSI within and across cancer types. METHODS: To better inform molecular diagnosis of MSI, we examined 9438 tumor-normal exome pairs and 901 whole genome sequence pairs from 32 different cancer types and cataloged genome-wide microsatellite instability events. Using a statistical framework, we identified microsatellite mutations that were predictive of MSI within and across cancer types. The diagnostic accuracy of different subsets of maximally informative markers was estimated computationally using a dedicated validation set. RESULTS: Twenty-five cancer types exhibited hypermutated states consistent with MSI. Recurrently mutated microsatellites associated with MSI were identifiable in 15 cancer types, but were largely specific to individual cancer types. Cancer-specific microsatellite panels of 1 to 7 loci were needed to attain ≥95% diagnostic sensitivity and specificity for 11 cancer types, and in 8 of the cancer types, 100% sensitivity and specificity were achieved. Breast cancer required 800 loci to achieve comparable performance. We were unable to identify recurrent microsatellite mutations supporting reliable MSI diagnosis in ovarian tumors. Features associated with informative microsatellites were cataloged. CONCLUSIONS: Most microsatellites informative for MSI are specific to particular cancer types, requiring the use of tissue-specific loci for optimal diagnosis. Limited numbers of markers are needed to provide accurate MSI diagnosis in most tumor types, but it is challenging to diagnose breast and ovarian cancers using predefined microsatellite locus panels.

Impact of COVID-19 response on global surgical volumes: an ongoing observational study.

OBJECTIVE: To determine whether location-linked anaesthesiology calculator mobile application (app) data can serve as a qualitative proxy for global surgical case volumes and therefore monitor the impact of the coronavirus disease 2019 (COVID-19) pandemic. METHODS: We collected data provided by users of the mobile app "Anesthesiologist" during 1 October 2018-30 June 2020. We analysed these using RStudio and generated 7-day moving-average app use plots. We calculated country-level reductions in app use as a percentage of baseline. We obtained data on COVID-19 case counts from the European Centre for Disease Prevention and Control. We plotted changing app use and COVID-19 case counts for several countries and regions. FINDINGS: A total of 100 099 app users within 214 countries and territories provided data. We observed that app use was reduced during holidays, weekends and at night, correlating with expected fluctuations in surgical volume. We observed that the onset of the pandemic prompted substantial reductions in app use. We noted strong cross-correlation between COVID-19 case count and reductions in app use in low- and middle-income countries, but not in high-income countries. Of the 112 countries and territories with non-zero app use during baseline and during the pandemic, we calculated a median reduction in app use to 73.6% of baseline. CONCLUSION: App data provide a proxy for surgical case volumes, and can therefore be used as a real-time monitor of the impact of COVID-19 on surgical capacity. We have created a dashboard for ongoing visualization of these data, allowing policy-makers to direct resources to areas of greatest need.

Initial Clinical Impressions of the Critical Care of COVID-19 Patients in Seattle, New York City, and Chicago.

Since the first recognition of a cluster of novel respiratory viral infections in China in late December 2019, intensivists in the United States have watched with growing concern as infections with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus-now named coronavirus disease of 2019 (COVID-19)-have spread to hospitals in the United States. Because COVID-19 is extremely transmissible and can progress to a severe form of respiratory failure, the potential to overwhelm available critical care resources is high and critical care management of COVID-19 patients has been thrust into the spotlight. COVID-19 arrived in the United States in January and, as anticipated, has dramatically increased the usage of critical care resources. Three of the hardest-hit cities have been Seattle, New York City, and Chicago with a combined total of over 14,000 cases as of March 23, 2020.In this special article, we describe initial clinical impressions of critical care of COVID-19 in these areas, with attention to clinical presentation, laboratory values, organ system effects, treatment strategies, and resource management. We highlight clinical observations that align with or differ from already published reports. These impressions represent only the early empiric experience of the authors and are not intended to serve as recommendations or guidelines for practice, but rather as a starting point for intensivists preparing to address COVID-19 when it arrives in their community.

The Impact of Postoperative Intensive Care Unit Admission on Postoperative Hospital Length of Stay and Costs: A Prespecified Propensity-Matched Cohort Study.

BACKGROUND: In this prespecified cohort study, we investigated the influence of postoperative admission to the intensive care unit versus surgical ward on health care utilization among patients undergoing intermediate-risk surgery. METHODS: Of adult surgical patients who underwent general anesthesia without an absolute indication for postoperative intensive care unit admission, 3530 patients admitted postoperatively to an intensive care unit were matched to 3530 patients admitted postoperatively to a surgical ward using a propensity score based on 23 important preoperative and intraoperative predictor variables. Postoperative hospital length of stay and hospital costs were defined as primary and secondary end points, respectively. RESULTS: Among patients with low propensity for postoperative intensive care unit admission, initial triage to an intensive care unit was associated with increased postoperative length of stay (incidence rate ratio, 1.69 [95% CI, 1.59-1.79]; P < .001) and hospital costs (incidence rate ratio, 1.92 [95% CI, 1.81-2.03]; P < .001). By contrast, postoperative intensive care unit admission of patients with high propensity was associated with decreased postoperative length of stay (incidence rate ratio, 0.90 [95% CI, 0.85-0.95]; P < .001) and costs (incidence rate ratio, 0.92 [95% CI, 0.88-0.97]; P = .001). Decisions regarding postoperative intensive care unit resource utilization were influenced by individual preferences of anesthesiologists and surgeons. CONCLUSIONS: In patients with an unclear indication for postoperative critical care, intensive care unit admission may negatively impact postoperative hospital length of stay and costs. Postoperative discharge disposition varies substantially based on anesthesia and surgical provider preferences but should optimally be driven by an objective assessment of a patient's status at the end of surgery.

Effects of Intraoperative Fluid Management on Postoperative Outcomes: A Hospital Registry Study.

OBJECTIVE: Evaluate the dose-response relationship between intraoperative fluid administration and postoperative outcomes in a large cohort of surgical patients. BACKGROUND: Healthy humans may live in a state of fluid responsiveness without the need for fluid supplementation. Goal-directed protocols driven by such measures are limited in their ability to define the optimal fluid state during surgery. METHODS: This analysis of data on file included 92,094 adult patients undergoing noncardiac surgery with endotracheal intubation between 2007 and 2014 at an academic tertiary care hospital and two affiliated community hospitals. The primary exposure variable was total intraoperative volume of crystalloid and colloid administered. The primary outcome was 30-day survival. Secondary outcomes were respiratory complications within three postoperative days (pulmonary edema, reintubation, pneumonia, or respiratory failure) and acute kidney injury. Exploratory outcomes were postoperative length of stay and total cost of care. Our models were adjusted for patient-, procedure-, and anesthesia-related factors. RESULTS: A U-shaped association was observed between the volume of fluid administered intraoperatively and 30-day mortality, costs, and postoperative length of stay. Liberal fluid volumes (highest quintile of fluid administration practice) were significantly associated with respiratory complications whereas both liberal and restrictive (lowest quintile) volumes were significantly associated with acute kidney injury. Moderately restrictive volumes (second quintile) were consistently associated with optimal postoperative outcomes and were characterized by volumes approximately 40% less than traditional textbook estimates: infusion rates of approximately 6-7 mL/kg/hr or 1 L of fluid for a 3-hour case. CONCLUSIONS: Intraoperative fluid dosing at the liberal and restrictive margins of observed practice is associated with increased morbidity, mortality, cost, and length of stay.

Preventing Surgical Site Infections in the Era of Escalating Antibiotic Resistance and Antibiotic Stewardship.

Importance: According to the Centers for Disease Control and Prevention and governing bodies within the American College of Surgeons, the administration of antibiotics as prophylaxis against infection prior to a planned elective procedure is, with rare exception, routinely recommended. The goal of "getting to zero" infections remains a high priority for policymakers, practitioners, and certainly for patients. Observations: Despite the many advances in surgical technique, skin decontamination, sterile procedure, and enhanced recovery programs, surgical site infections continue to adversely affect procedures as diverse as dental implant surgery, joint arthroplasty, and major abdominal surgery. Although surgical site infection rates are at historically low levels, progress has stalled in recent reporting periods and such infections remain disabling, costly, and occasionally lethal. Stakeholders in the field, including surgeons, infectious diseases specialists, and industry, advocate for strategies emphasizing greater levels of intraoperative sterility or broader-spectrum antibiotic coverage as the most appropriate path forward. Conclusions and Relevance: The current emphasis on ever-increasing levels of intraoperative sterility and extended-spectrum antibiotic use are not sustainable long-term solutions. Continuing to escalate these approaches may contribute to unintended consequences including antimicrobial resistance. Principles of antimicrobial stewardship and microbiome sciences can be applied to inform a more effective and sustainable approach to infection prevention in the field of surgery.