How SWAT Changed the Conversation: Implementation of a Discharge SWAT Team Improves Morale and Facilitates Communication During the COVID-19 Pandemic.

PURPOSE OF STUDY: Hospital overcrowding and delays in discharge are serious issues in the modern health care landscape and can lead to poor patient outcomes and health care personnel (HCP) burnout. The goal of this project was to develop a collaborative forum where HCP representing the entire spectrum of the inpatient care team, including case management team members, could connect to discuss challenges and barriers to patient discharge. The following describes the development, implementation, and outcomes of the discharge SWAT (Solutions, Wins, Actions, and Tactics) team, which is a 30-min virtual daily meeting where discussion is primarily centered around challenges in discharging individual patients and addressing case manager needs. The primary aim of SWAT meetings is fostering a positive atmosphere to address barriers to discharge while prioritizing patient care and outcomes. PRIMARY PRACTICE SETTING: This study was conducted in a 40-hospital academic health system in the United States. METHODOLOGY AND SAMPLE: SWAT meetings were first implemented at a representative flagship facility in a health system. HCP at this first facility were surveyed to assess satisfaction with SWAT meetings. SWAT meetings then were implemented at the majority of facilities in a 40-hospital academic health system. During SWAT implementation, average inpatient length of stay (LOS) and patient care transitions were monitored for participating and nonparticipating service lines. RESULTS: Among surveyed HCP, the majority view SWAT meetings favorably and reported that it was a valuable use of their time and positively impacted their work in the patient discharge space. Nonprovider and case management staff in particular valued the SWAT meetings and found them beneficial. LOS remained stable for patients under the care of participating providers, despite the upheaval of the ongoing COVID-19 pandemic, and the research team also observed a positive impact of SWAT meetings on appropriate inpatient care transitions.

Prospective assessment of humoral and cellular immune responses to a 3rd COVID-19 mRNA vaccine dose among immunocompromised individuals.

BACKGROUND: Improved COVID-19 prevention is needed for immunocompromised individuals. METHODS: Prospective study of healthcare workers (HCW) and immunocompromised participants with baseline serology following 2 mRNA vaccines and who were retested after dose 3 (D3); multivariable regression was used to identify predictors of serological responses. IFNγ/TNFα T-cell responses were assessed in a subset. RESULTS: 536 participants were included: 492 immunocompromised [(206 solid organ transplant (SOT), 128 autoimmune, 80 hematologic malignancy (HM), 48 solid tumor, 25 HIV], 44 HCW. D3 significantly increased Spike IgG levels among all, but SOT and HM participants had the lowest median antibody levels post-D3 (increase from 0.09 to 0.83 and 0.27 to 1.92, respectively), versus HCW and persons with HIV, autoimmune conditions, and solid tumors (increases from 4.44 to 19.79, 2.9 to 15.75, 3.82 to 16.32, and 4.1 to 25.54, respectively). Seropositivity post-D3 was lowest for SOT (49.0%) and HM (57.8%), versus others (>90% seropositive). Neutralization post-D3 was lowest among SOT and HM. Predictors of lower antibody levels included low baseline levels and shorter intervals between vaccines. T-cell responses against Spike increased significantly among HCW and non-significantly among immunocompromised individuals. CONCLUSIONS: D3 significantly improves serological but not T-cell responses among immunocompromised individuals. SOT and HM patients have suboptimal responses to D3.

Implementing a Discharge Follow-up Phone Call Program Reduces Readmission Rates in an Integrated Health System.

ABSTRACT: In this study, we sought to determine the effect of implementing a large-scale discharge follow-up phone call program on hospital readmission rates. Previous work has shown that patients with unaddressed concerns during discharge have significantly higher rates of care complications and hospital readmissions. This study is an observational quality improvement project completed from April 17, 2020 to January 31, 2022 at 22 hospitals in a large, integrated academic health system. A nurse-led scripted discharge follow-up phone call program was implemented to contact all patients discharged from inpatient care within 72 hours of discharge. Readmission rates were tracked before and after project implementation. Over a 21-month span, 137,515 phone calls were placed, and 57.92% of patients were successfully contacted within 7 days of discharge. The 7-day readmission rate for contacted patients was 2.91% compared with 4.73% for noncontacted patients. The 30-day readmission rate for contacted patients was 11.00% compared with 12.17% for noncontacted patients. We have found that discharge follow-up phone calls targeting patients decreases risk of readmission, which improves overall patient outcomes.

Implementing a Serious Illness Risk Prediction Model: Impact on Goals of Care Documentation.

CONTEXT: Goals of care conversations can promote high value care for patients with serious illness, yet documented discussions infrequently occur in hospital settings. OBJECTIVES: We sought to develop a quality improvement initiative to improve goals of care documentation for hospitalized patients. METHODS: Implementation occurred at an academic medical center in Pittsburgh, Pennsylvania. Intervention included integration of a 90-day mortality prediction model grouping patients into low, intermediate, and high risk; a centralized goals of care note; and automated notifications and targeted palliative consults. We compared documented goals of care discussions by risk score before and after implementation. RESULTS: Of the 12,571 patients hospitalized preimplementation and 10,761 postimplementation, 1% were designated high risk and 11% intermediate risk of mortality. Postimplementation, goals of care documentation increased for high (17.6%-70.8%, P< 0.0001) and intermediate risk patients (9.6%-28.0%, P < 0.0001). For intermediate risk patients, the percentage of goals of care documentation performed by palliative medicine specialists increased from pre- to postimplementation (52.3%-71.2%, P = 0.0002). For high-risk patients, the percentage of goals of care documentation completed by the primary service increased from pre-to postimplementation (36.8%-47.1%, P = 0.5898, with documentation performed by palliative medicine specialists slightly decreasing from pre- to postimplementation (63.2%-52.9%, P = 0.5898). CONCLUSIONS: Implementation of a goals of care initiative using a mortality prediction model significantly increased goals of care documentation especially among high-risk patients. Further study to assess strategies to increase goals of care documentation for intermediate risk patients is needed especially by nonspecialty palliative care.

A Current State Assessment on Nursing Work Activities: An Observational Study.

BACKGROUND: Nationwide nursing shortages have led to higher patient-to-nurse ratios, nursing burnout, and decreased quality of care. LOCAL PROBLEM: Staffing challenges and nursing burnout were becoming growing concerns and success was contingent upon efficient use of existing resources. METHODS: Direct observation current state assessment was completed on medical-surgical specialty units to better understand work activities of registered nurses (RNs) and unlicensed assistive personnel (UAPs). RESULTS: RNs spent more time performing indirect care (eg, documentation) than direct patient care. Interruptions and problems consumed 17.4% and 5.6% of their time, respectively. UAPs performed more direct patient care but had a higher proportion of downtime. RNs underdelegated nonclinical tasks. CONCLUSIONS: Direct observation current state assessment offers a better understanding of workflow and workload inefficiencies. This information is critical to provide informed, evidence-based recommendations to develop future patient care models with more capacity to deliver high-quality care with greater efficiency and lessen nursing burden and burnout during the nursing shortage crisis.

Healthcare-associated infections during the coronavirus disease 2019 (COVID-19) pandemic and the modulating effect of centralized surveillance.

We analyzed efficacy of a centralized surveillance infection prevention (CSIP) program in a healthcare system on healthcare-associated infection (HAI) rates amid the coronavirus disease 2019 (COVID-19) pandemic. HAI rates were variable in CSIP and non-CSIP facilities. Central-line-associated bloodstream infection (CLABSI), C. difficile infection (CSI), and surgical-site infection (SSI) rates were negatively correlated with COVID-19 intensity in CSIP facilities.

Development and implementation of a centralized surveillance infection prevention program in a multi-facility health system: A quality improvement project.

Objective: To develop, implement, and evaluate the effectiveness of a unique centralized surveillance infection prevention (CSIP) program. Design: Observational quality improvement project. Setting: An integrated academic healthcare system. Intervention: The CSIP program comprises senior infection preventionists who are responsible for healthcare-associated infection (HAI) surveillance and reporting, allowing local infection preventionists (LIPs) a greater portion of their time to non-surveillance patient safety activities. Four CSIP team members accrued HAI responsibilities at 8 facilities. Methods: We evaluated the effectiveness of the CSIP program using 4 measures: recovery of LIP time, efficiency of surveillance activities by LIPs and CSIP staff, surveys characterizing LIP perception of their effectiveness in HAI reduction, and nursing leaders' perception of LIP effectiveness. Results: The amount of time spent by LIP teams on HAI surveillance was highly variable, while CSIP time commitment and efficiency was steady. Post-CSIP implementation, 76.9% of LIPs agreed that they spend adequate time on inpatient units, compared to 15.4% pre-CSIP; LIPs also reported more time to allot to non-surveillance activities. Nursing leaders reported greater satisfaction with LIP involvement with HAI reduction practices. Conclusion: CSIP programs are a little-reported strategy to ease burden on LIPs with reallocation of HAI surveillance. The analyses presented here will aid health systems in anticipating the benefit of CSIP programs.

COVID-19 contagious health care personnel 5-day early return-to-work program.

BACKGROUND: COVID-19 contagious health care personnel (HCP) who are self-isolating for a 10-day period increases burden to workforce shortages. Implementation of a 5-day early return-to-work (RTW) program may reduce self-isolation periods, without increasing transmission risk, during the COVID-19 pandemic. DESIGN AND METHODS: This observational cohort quality improvement study included newly diagnosed COVID-19 HCP at a multifacility health care system. The program allowed HCP to return to work 6 days after date of a positive test result if they were not immunocompromised, had mild and improving symptoms, and self-reported a SARS-CoV-2 antigen negative test on day 5. RESULTS: Between January 4 and April 3, 2022, 1,023 HCP self-enrolled and 344 (33.6%) self-reported negative test results. Among these, 161 (46.8%) self-reported negative test results on day 5 and were eligible for early RTW on day 6. A total of 714 days were saved from missed work in self-isolation. The number of tests purchased, dispensed, and reported per day of HCP time saved was 4.4. No transmission events were observed originating from HCP who participated in early RTW. CONCLUSION: Implementing a 5-day early RTW program that includes HCP self-reporting SARS-CoV-2 antigen test results can increase staffing availability, while maintaining a low risk of SARS-CoV-2 transmission.

Assessment of the effectiveness of ultraviolet-C disinfection on transmission of hospital-acquired pathogens from prior room occupants.

Objective: To evaluate the effectiveness of ultraviolet-C (UV-C) disinfection as an adjunct to standard chlorine-based disinfectant terminal room cleaning in reducing transmission of hospital-acquired multidrug-resistant organisms (MDROs) from a prior room occupant. Design: A retrospective cohort study was conducted to compare rates of MDRO transmission by UV-C status from January 1, 2016, through December 31, 2018. Setting: Acute-care, single-patient hospital rooms at 6 hospitals within an academic healthcare system in Pennsylvania. Methods: Transmission of hospital-acquired MDRO infection was assessed in patients subsequently assigned to a single-patient room of a source occupant with carriage of 1 or more MDROs on or during admission. Acquisition of 5 pathogens was compared between exposed patients in rooms with standard-of-care chlorine-based disinfectant terminal cleaning with or without adjunct UV-C disinfection. Logistic regression analysis was used to estimate the adjusted risk of pathogen transfer with adjunctive use of UV-C disinfection. Results: In total, 33,771 exposed patient admissions were evaluated; the source occupants carried 46,688 unique pathogens. Prior to the 33,771 patient admissions, 5,802 rooms (17.2%) were treated with adjunct UV-C disinfection. After adjustment for covariates, exposed patients in rooms treated with adjunct UV-C were at comparable risk of transfer of any pathogen (odds ratio, 1.06; 95% CI, 0.84-1.32; P = .64). Conclusion: Our analysis does not support the use of UV-C in addition to post-discharge cleaning with chlorine-based disinfectant to lower the risk of prior room occupant pathogen transfer.

The Small RNA Teg41 Is a Pleiotropic Regulator of Virulence in Staphylococcus aureus.

Previously, our group demonstrated a role for the small RNA (sRNA) Teg41 in regulating production of the alpha phenol-soluble modulin toxins (αPSMs) in Staphylococcus aureus. Overexpressing Teg41 increased αPSM production while deleting the 3' end of Teg41 (Teg41Δ3' strain) resulted in a decrease in αPSM production, reduced hemolytic activity of S. aureus culture supernatants, and attenuated virulence in a murine abscess model of infection. In this study, we further explore the attenuation of virulence in the Teg41Δ3' strain. Using both localized and systemic models of infection, we demonstrate that the Teg41Δ3' strain is more severely attenuated than an ΔαPSM mutant, strongly suggesting that Teg41 influences more than the αPSMs. Proteomic and transcriptomic analysis of the wild-type and Teg41Δ3' strains reveals widespread alterations in transcript abundance and protein production in the absence of Teg41, confirming that Teg41 has pleiotropic effects in the cell. We go on to investigate the molecular mechanism underlying Teg41-mediated gene regulation. Surprisingly, results demonstrate that certain Teg41 target genes, including the αPSMs and ßPSMs, are transcriptionally altered in the Teg41Δ3' strain, while other targets, specifically spa (encoding surface protein A), are regulated at the level of transcript stability. Collectively, these data demonstrate that Teg41 is a pleiotropic RNA regulator in S. aureus that influences expression of a variety of genes using multiple different mechanisms.

Effectiveness of Casirivimab-Imdevimab and Sotrovimab During a SARS-CoV-2 Delta Variant Surge: A Cohort Study and Randomized Comparative Effectiveness Trial.

Importance: The effectiveness of monoclonal antibodies (mAbs), casirivimab-imdevimab and sotrovimab, is unknown in patients with mild to moderate COVID-19 caused by the SARS-CoV-2 Delta variant. Objective: To evaluate the effectiveness of mAb against the Delta variant compared with no mAb treatment and to ascertain the comparative effectiveness of casirivimab-imdevimab and sotrovimab. Design, Setting, and Participants: This study comprised 2 parallel studies: (1) a propensity score-matched cohort study of mAb treatment vs no mAb treatment and (2) a randomized comparative effectiveness trial of casirivimab-imdevimab and sotrovimab. The cohort consisted of patients who received mAb treatment at the University of Pittsburgh Medical Center outpatient infusion centers and emergency departments from July 14 to September 29, 2021. Participants were patients with a positive SARS-CoV-2 test result who were eligible to receive mAbs according to emergency use authorization criteria. Exposure: For the trial, patients were randomized to either intravenous casirivimab-imdevimab or sotrovimab according to a system therapeutic interchange policy. Main Outcomes and Measures: For the cohort study, risk ratio (RR) estimates for the primary outcome of hospitalization or death by 28 days were compared between mAb treatment and no mAb treatment using propensity score-matched models. For the comparative effectiveness trial, the primary outcome was hospital-free days (days alive and free of hospitalization) within 28 days after mAb treatment, where patients who died were assigned -1 day in a bayesian cumulative logistic model adjusted for treatment location, age, sex, and time. Inferiority was defined as a 99% posterior probability of an odds ratio (OR) less than 1. Equivalence was defined as a 95% posterior probability that the OR was within a given bound. Results: A total of 3069 patients (1023 received mAb treatment: mean [SD] age, 53.2 [16.4] years; 569 women [56%]; 2046 had no mAb treatment: mean [SD] age, 52.8 [19.5] years; 1157 women [57%]) were included in the prospective cohort study, and 3558 patients (mean [SD] age, 54 [18] years; 1919 women [54%]) were included in the randomized comparative effectiveness trial. In propensity score-matched models, mAb treatment was associated with reduced risk of hospitalization or death (RR, 0.40; 95% CI, 0.28-0.57) compared with no treatment. Both casirivimab-imdevimab (RR, 0.31; 95% CI, 0.20-0.50) and sotrovimab (RR, 0.60; 95% CI, 0.37-1.00) were associated with reduced hospitalization or death compared with no mAb treatment. In the clinical trial, 2454 patients were randomized to receive casirivimab-imdevimab and 1104 patients were randomized to receive sotrovimab. The median (IQR) hospital-free days were 28 (28-28) for both mAb treatments, the 28-day mortality rate was less than 1% (n = 12) for casirivimab-imdevimab and less than 1% (n = 7) for sotrovimab, and the hospitalization rate by day 28 was 12% (n = 291) for casirivimab-imdevimab and 13% (n = 140) for sotrovimab. Compared with patients who received casirivimab-imdevimab, those who received sotrovimab had a median adjusted OR for hospital-free days of 0.88 (95% credible interval, 0.70-1.11). This OR yielded 86% probability of inferiority for sotrovimab vs casirivimab-imdevimab and 79% probability of equivalence. Conclusions and Relevance: In this propensity score-matched cohort study and randomized comparative effectiveness trial, the effectiveness of casirivimab-imdevimab and sotrovimab against the Delta variant was similar, although the prespecified criteria for statistical inferiority or equivalence were not met. Both mAb treatments were associated with a reduced risk of hospitalization or death in nonhospitalized patients with mild to moderate COVID-19 caused by the Delta variant. Trial Registration: ClinicalTrials.gov Identifier: NCT04790786.

The comparative effectiveness of COVID-19 monoclonal antibodies: A learning health system randomized clinical trial.

BACKGROUND: Monoclonal antibodies (mAb) that neutralize SARS-CoV-2 decrease hospitalization and death compared to placebo in patients with mild to moderate COVID-19; however, comparative effectiveness is unknown. We report the comparative effectiveness of bamlanivimab, bamlanivimab-etesevimab, and casirivimab-imdevimab. METHODS: A learning health system platform trial in a U.S. health system enrolled patients meeting mAb Emergency Use Authorization criteria. An electronic health record-embedded application linked local mAb inventory to patient encounters and provided random mAb allocation. Primary outcome was hospital-free days to day 28. Primary analysis was a Bayesian model adjusting for treatment location, age, sex, and time. Inferiority was defined as 99% posterior probability of an odds ratio < 1. Equivalence was defined as 95% posterior probability the odds ratio is within a given bound. FINDINGS: Between March 10 and June 25, 2021, 1935 patients received treatment. Median hospital-free days were 28 (IQR 28, 28) for each mAb. Mortality was 0.8% (1/128), 0.8% (7/885), and 0.7% (6/922) for bamlanivimab, bamlanivimab-etesevimab, and casirivimab-imdevimab, respectively. Relative to casirivimab-imdevimab (n = 922), median adjusted odds ratios were 0.58 (95% credible interval [CI] 0.30-1.16) and 0.94 (95% CI 0.72-1.24) for bamlanivimab (n = 128) and bamlanivimab-etesevimab (n = 885), respectively. These odds ratios yielded 91% and 94% probabilities of inferiority of bamlanivimab versus bamlanivimab-etesevimab and casirivimab-imdevimab, and an 86% probability of equivalence between bamlanivimab-etesevimab and casirivimab-imdevimab. INTERPRETATION: Among patients with mild to moderate COVID-19, bamlanivimab-etesevimab or casirivimab-imdevimab treatment resulted in 86% probability of equivalence. No treatment met prespecified criteria for statistical equivalence. Median hospital-free days to day 28 were 28 (IQR 28, 28) for each mAb. FUNDING AND REGISTRATION: This work received no external funding. The U.S. government provided the reported mAb. This trial is registered at ClinicalTrials.gov, NCT04790786.

RNase III CLASH in MRSA uncovers sRNA regulatory networks coupling metabolism to toxin expression.

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen responsible for significant human morbidity and mortality. Post-transcriptional regulation by small RNAs (sRNAs) has emerged as an important mechanism for controlling virulence. However, the functionality of the majority of sRNAs during infection is unknown. To address this, we performed UV cross-linking, ligation, and sequencing of hybrids (CLASH) in MRSA to identify sRNA-RNA interactions under conditions that mimic the host environment. Using a double-stranded endoribonuclease III as bait, we uncovered hundreds of novel sRNA-RNA pairs. Strikingly, our results suggest that the production of small membrane-permeabilizing toxins is under extensive sRNA-mediated regulation and that their expression is intimately connected to metabolism. Additionally, we also uncover an sRNA sponging interaction between RsaE and RsaI. Taken together, we present a comprehensive analysis of sRNA-target interactions in MRSA and provide details on how these contribute to the control of virulence in response to changes in metabolism.

Healthcare personnel early return-to-work program after higher-risk SARS-CoV-2 exposure: A learning health system quality improvement project.

BACKGROUND: Incidence of health care personnel (HCP) with a higher-risk SARS-CoV-2 exposure and subsequent 14-day quarantine period adds substantial burden on the workforce. Implementation of an early return-to-work (RTW) program may reduce quarantine periods for asymptomatic HCP and reduce workforce shortages during the COVID-19 pandemic. METHODS: This observational quality improvement study included asymptomatic HCP of a multi-facility health care system with higher-risk workplace or non-household community SARS-CoV-2 exposure ≤4 days. The program allowed HCP to return to work 8 days after exposure if they remained asymptomatic through day 7 with day 5-7 SARS-CoV-2 nucleic acid amplification test result negative. RESULTS: Between January 4 and June 25, 2021, 384 HCP were enrolled, 333 (86.7%) remained asymptomatic and of these, 323 (97%) tested negative and were early RTW eligible. Mean days in quarantine was 8.16 (SD 2.40). Median day of early RTW was 8 (range 6-9, IQR 8-8). Mean days saved from missed work was 1.84 (SD 0.52). A total of 297 (92%) HCP did RTW ≤10 days from exposure and days saved from missed work was 546.48. CONCLUSIONS: Implementing an HCP early RTW program is a clinical approach for COVID-19 workplace safety that can increase staffing availability, while maintaining a low risk of SARS-CoV-2 transmission.

Global Annotation, Expression Analysis, and Stability of Candidate sRNAs in Group B Streptococcus.

Small, noncoding RNAs (sRNAs) are being increasingly identified as important regulatory molecules in prokaryotes. Due to the prevalence of next-generation sequencing-based techniques, such as RNA sequencing (RNA-seq), there is potential for increased discovery of sRNAs within bacterial genomes; however, these elements are rarely included in annotation files. Consequently, expression values for sRNAs are omitted from most transcriptomic analyses, and mechanistic studies have lagged behind those of protein regulators in numerous bacteria. Two previous studies have identified sRNAs in the human pathogen group B Streptococcus (GBS). Here, we utilize the data from these studies to create updated genome annotation files for the model GBS strains NEM316 and COH1. Using the updated COH1 annotation file, we reanalyze publicly available GBS RNA-seq whole-transcriptome data from GenBank to monitor GBS sRNA expression under a variety of conditions and genetic backgrounds. This analysis generated expression values for 232 putative sRNAs that were overlooked in previous transcriptomic analyses in 21 unique comparisons. To demonstrate the utility of these data, we identify an sRNA that is upregulated during vaginal colonization and demonstrate that overexpression of this sRNA leads to increased bacterial invasion into host epithelial cells. Finally, to monitor RNA degradation, we perform a transcript stability assay to identify highly stable sRNAs and compare stability profiles of sRNA- and protein-coding genes. Collectively, these data provide a wealth of transcriptomic data for putative sRNAs in GBS and a platform for future mechanistic studies. IMPORTANCE In recent years, sRNAs have emerged as potent regulatory molecules in bacteria, including numerous streptococcal species, and contribute to diverse processes, including stress response, metabolism, housekeeping, and virulence regulation. Improvements in sequencing technologies and in silico analyses have facilitated identification of these regulatory molecules as well as improved attempts to determine the location of sRNA genes on the genome. However, despite these advancements, sRNAs are rarely included in genome annotation files. Consequently, these molecules are often omitted from transcriptomic data analyses and are commonly repeat identified across multiple studies. Updating current genomes to include sRNA genes is therefore critical for better understanding bacterial regulation.

Staphylococcus aureus Responds to Physiologically Relevant Temperature Changes by Altering Its Global Transcript and Protein Profile.

Staphylococcus aureus is an opportunistic pathogen that colonizes the anterior nares of 30 to 50% of the population. Colonization is most often asymptomatic; however, self-inoculation can give rise to potentially fatal infections of the deeper tissues and blood. Like all bacteria, S. aureus can sense and respond to environmental cues and modify gene expression to adapt to specific environmental conditions. The transition of S. aureus from the nares to the deeper tissues and blood is accompanied by changes in environmental conditions, such as nutrient availability, pH, and temperature. In this study, we perform transcriptomics and proteomics on S. aureus cultures growing at three physiologically relevant temperatures, 34°C (nares), 37°C (body), and 40°C (pyrexia), to determine if small scale, biologically meaningful alterations in temperature impact S. aureus gene expression. Results show that small but definite temperature changes elicit a large-scale restructuring of the S. aureus transcriptome and proteome in a manner that, most often, inversely correlates with increasing temperature. We also provide evidence that a large majority of these changes are modulated at the posttranscriptional level, possibly by sRNA regulatory elements. Phenotypic analyses were also performed to demonstrate that these changes have physiological relevance. Finally, we investigate the impact of temperature-dependent alterations in gene expression on S. aureus pathogenesis and demonstrate decreased intracellular invasion of S. aureus grown at 34°C. Collectively, our results demonstrate that small but biologically meaningful alterations in temperature influence S. aureus gene expression, a process that is likely a major contributor to the transition from a commensal to pathogen.IMPORTANCE Enteric bacterial pathogens, like Escherichia coli, are known to experience large temperature differences as they are transmitted through the fecal oral route. This change in temperature has been demonstrated to influence bacterial gene expression and facilitate infection. Staphylococcus aureus is a human-associated pathogen that can live as a commensal on the skin and nares or cause invasive infections of the deeper tissues and blood. Factors influencing S. aureus nasal colonization are not fully understood; however, individuals colonized with S. aureus are at increased risk of invasive infections through self-inoculation. The transition of S. aureus from the nose (colonization) to the body (infection) is accompanied by a modest but definite temperature increase, from 34°C to 37°C. In this study, we investigate whether these host-associated small temperature changes can influence S. aureus gene expression. Results show widespread changes in the bacterial transcriptome and proteome at three physiologically relevant temperatures (34°C, 37°C, and 40°C).

Staphylococcus aureus Trigger Factor Is Involved in Biofilm Formation and Cooperates with the Chaperone PpiB.

Peptidyl-prolyl cis/trans isomerases (PPIases) are enzymes that assist in protein folding around proline-peptide bonds, and they often possess chaperone activity. Staphylococcus aureus encodes three PPIases, i.e., PrsA, PpiB, and trigger factor (TF). Previous work by our group demonstrated a role for both PrsA and PpiB in S. aureus; however, TF remains largely unstudied. Here, we identify a role for TF in S. aureus biofilm formation and demonstrate cooperation between TF and the cytoplasmic PPIase PpiB. Mutation of the tig gene (encoding TF) led to reduced biofilm development in vitro but no significant attenuation of virulence in a mouse model of infection. To investigate whether TF possesses chaperone activity, we analyzed the ability of a tig mutant to survive acid and base stress. While there was no significant decrease for a tig mutant, a ppiBtig double mutant exhibited significant decreases in cell viability after acid and base challenges. We then demonstrated that a ppiB tig double mutant had exacerbated phenotypes in vitro and in vivo, compared to either single mutant. Finally, in vivo immunoprecipitation of epitope-tagged PpiB revealed that PpiB interacted with 4 times the number of proteins when TF was absent from the cell, suggesting that it may be compensating for the loss of TF. Interestingly, the only proteins found to interact with TF were TF itself, fibronectin-binding protein B (FnBPB), and the chaperone protein ClpB. Collectively, these results support the first phenotype for S. aureus TF and demonstrate a greater network of cooperation between chaperone proteins in Staphylococcus aureusIMPORTANCES. aureus encodes a large number of virulence factors that aid the bacterium in survival and pathogenesis. These virulence factors have a wide variety of functions; however, they must all be properly secreted in order to be functional. Bacterial chaperone proteins often assist in secretion by trafficking proteins to secretion machinery or assisting in proper protein folding. Here, we report that the S. aureus chaperone TF contributes to biofilm formation and cooperates with the chaperone PpiB to regulate S. aureus virulence processes. These data highlight the first known role for TF in S. aureus and suggest that S. aureus chaperone proteins may be involved in a greater regulatory network in the cell.

Novel Regulation of Alpha-Toxin and the Phenol-Soluble Modulins by Peptidyl-Prolyl cis/trans Isomerase Enzymes in Staphylococcus aureus.

Peptidyl-prolyl cis/trans isomerases (PPIases) are enzymes that catalyze the cis-to-trans isomerization around proline bonds, allowing proteins to fold into their correct confirmation. Previously, we identified two PPIase enzymes in Staphylococcus aureus (PpiB and PrsA) that are involved in the regulation of virulence determinants and have shown that PpiB contributes to S. aureus virulence in a murine abscess model of infection. Here, we further examine the role of these PPIases in S. aureus virulence and, in particular, their regulation of hemolytic toxins. Using murine abscess and systemic models of infection, we show that a ppiB mutant in a USA300 background is attenuated for virulence but that a prsA mutant is not. Deletion of the ppiB gene leads to decreased bacterial survival in macrophages and nasal epithelial cells, while there is no significant difference when prsA is deleted. Analysis of culture supernatants reveals that a ppiB mutant strain has reduced levels of the phenol-soluble modulins and that both ppiB and prsA mutants have reduced alpha-toxin activity. Finally, we perform immunoprecipitation to identify cellular targets of PpiB and PrsA. Results suggest a novel role for PpiB in S. aureus protein secretion. Collectively, our results demonstrate that PpiB and PrsA influence S. aureus toxins via distinct mechanisms, and that PpiB but not PrsA contributes to disease.

MroQ Is a Novel Abi-Domain Protein That Influences Virulence Gene Expression in Staphylococcus aureus via Modulation of Agr Activity.

Numerous factors have, to date, been identified as playing a role in the regulation of Agr activity in Staphylococcus aureus, including transcription factors, antisense RNAs, and host elements. Herein we investigated the product of SAUSA300_1984 (termed MroQ), a transmembrane Abi-domain/M79 protease-family protein, as a novel effector of this system. Using a USA300 mroQ mutant, we observed a drastic reduction in proteolysis, hemolysis, and pigmentation that was fully complementable. This appears to result from diminished agr activity, as transcriptional analysis revealed significant decreases in expression of both RNAII and RNAIII in the mroQ mutant. Such effects appear to be direct, rather than indirect, as known agr effectors demonstrated limited alterations in their activity upon mroQ disruption. A comparison of RNA sequencing data sets for both mroQ and agr mutants revealed a profound overlap in their regulomes, with the majority of factors affected being known virulence determinants. Importantly, the preponderance of alterations in expression were more striking in the agr mutant, indicating that MroQ is necessary, but not sufficient, for Agr function. Mechanism profiling revealed that putative residues for metalloprotease activity within MroQ are required for its Agr-controlling effect; however, this was not wielded at the level of AgrD processing. Virulence assessment demonstrated that both mroQ and agr mutants exhibited increased formation of renal abscesses but decreased skin abscess formation alongside diminished dermonecrosis. Collectively, we present the characterization of a novel agr effector in S. aureus which would appear to be a direct regulator, potentially functioning via interaction with the AgrC histidine kinase.

The Small RNA Teg41 Regulates Expression of the Alpha Phenol-Soluble Modulins and Is Required for Virulence in Staphylococcus aureus.

Small RNAs (sRNAs) remain an understudied class of regulatory molecules in bacteria in general and in Gram-positive bacteria in particular. In the major human pathogen Staphylococcus aureus, hundreds of sRNAs have been identified; however, only a few have been characterized in detail. In this study, we investigate the role of the sRNA Teg41 in S. aureus virulence. We demonstrate that Teg41, an sRNA divergently transcribed from the locus that encodes the cytolytic alpha phenol-soluble modulin (αPSM) peptides, plays a critical role in αPSM production. Overproduction of Teg41 leads to an increase in αPSM levels and a corresponding increase in hemolytic activity from S. aureus cells and cell-free culture supernatants. To identify regions of Teg41 important for its function, we performed an in silico RNA-RNA interaction analysis which predicted an interaction between the 3' end of Teg41 and the αPSM transcript. Deleting a 24-nucleotide region from the S. aureus genome, corresponding to the 3' end of Teg41, led to a 10-fold reduction in αPSM-dependent hemolytic activity and attenuation of virulence in a murine abscess model of infection. Restoration of hemolytic activity in the Teg41Δ3' strain was possible by expressing full-length Teg41 in trans Restoration of hemolytic activity was also possible by expressing the 3' end of Teg41, suggesting that this region of Teg41 is necessary and sufficient for αPSM-dependent hemolysis. Our results show that Teg41 is positively influencing αPSM production, demonstrating for the first time regulation of the αPSM peptides by an sRNA in S. aureusIMPORTANCE The alpha phenol-soluble modulins (αPSMs) are among the most potent toxins produced by Staphylococcus aureus Their biological role during infection has been studied in detail; however, the way they are produced by the bacterial cell is not well understood. In this work, we identify a small RNA molecule called Teg41 that plays an important role in αPSM production by S. aureus Teg41 positively influences αPSM production. The importance of Teg41 is highlighted by the fact that a strain containing a deletion in the 3' end of Teg41 produces significantly less αPSMs and is attenuated for virulence in a mouse abscess model of infection. As the search for new therapeutic strategies to combat S. aureus infection proceeds, Teg41 may represent a novel target.