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A retrospective observational study was performed to assess the relationship between diagnostic method (traditional work-up [TW], multiplex PCR panel with < 12 target pathogens [PCR

Background Little is known about the epidemiology and burden of acute infectious gastroenteritis (AIG) among adult outpatients treated in United States (US) hospitals. We assessed the demographic and clinical characteristics, common pathogens detected, healthcare resource use (HRU), and cost among adult outpatients in US hospitals. Methods Using large geographically diverse PINC AI Healthcare Database data during January 1, 2016–June 30, 2021, we analyzed adult patients (≥ 18-year-old) with a principal diagnosis of AIG during an outpatient visit. Patients with a history of AIG within 30 days prior to the index visit and visits for diagnostic testing or surgery were excluded. Pathogen analysis was performed among those with microbiology data available. Results Among 248,896 eligible patients, the mean age was 44.3 years (range 18-89+), 62.9% were female, and 68.5% were White (Table 1). Most patients visited small (48.7%, 1-299 beds), non-teaching (60.4%), and urban (77.9%) hospitals. Common comorbid conditions included hypertension (27.5%), chronic pulmonary disease (19.4%), diabetes (16.1%), and obesity (7.6%). However, 62.0% of the patients did not have any pre-existing comorbidity. Among all patients with an AIG diagnosis, only 15.3% underwent stool work-up at the hospital. The majority of patients (84.7%) were seen in the emergency room, and most (96.4%) were discharged home (Table 2). Within 30 days of discharge, < 1.0% underwent ancillary abdominal/gastrointestinal diagnostic testing, 1.0% were hospitalized and 2.8% had another outpatient visit due to AIG at the same hospital. Mean cost of the index visit plus 30-day follow-up was $1,338 per patient, amounting to $333,060,182 for the total study population. Among the subgroup of patients with microbiology data available (n=12,469), common pathogens detected were Clostridium difficile (32.2%), Norovirus (6.3%), and Campylobacter spp. (4.0%). No pathogen was detected in 47.5%. Table 1.Patient, clinical, and hospital characteristics of hospital-based infectious gastroenteritis outpatients during January 1, 2016 - June 30, 2021Table 2.Outcomes of hospital-based infectious gastroenteritis outpatients during January 1, 2016 - June 30, 2021 Conclusion Acute infectious gastroenteritis is a common and costly disease affecting adults of all ages and more females than males, including individuals either with or without baseline conditions in a hospital-based outpatient setting. Clostridium difficile was the most common pathogen detected. Disclosures Rena Moon, MD, MPH, Premier Inc: Employee Tammy C. Bleak, PharmD, MSc, bioMerieux: Employee of bioMerieux Ning Rosenthal, MD, PhD, MPH, Premier Inc: Employee|Premier Inc: Stocks/Bonds Brianne Couturier, PhD, bioMerieux: Employee of bioMerieux Rachael Hemmert, MSPH, bioMerieux: Employee of bioMerieux Tristan T. Timbrook, PharmD, bioMerieux: Employee of bioMerieux Harold Brown, MBA, MHA, Premier Inc: Employee Ferric C. Fang, MD, bioMerieux: Advisor/Consultant.

Background Multiplex polymerase chain reaction (PCR) gastroenteritis testing panels simultaneously detect multiple pathogens with rapid turnaround, allowing more accurate and timely diagnosis and treatment. This retrospective observational study assessed the association between stool test type (i.e., multiplex PCR panel with < 12 target pathogens [PCR< 12], multiplex PCR panel with ≥ 12 target pathogens [PCR12], traditional work-up [TW]) and healthcare resource use (HRU), cost, and diagnostic yield in adult outpatients treated in U.S. hospitals for acute infectious gastroenteritis (AIG). Methods Using large geographically diverse PINC AI Healthcare Database data from January 1, 2016–June 30, 2021, we analyzed adults (≥ 18-years) with an AIG diagnosis and stool test performed during an outpatient visit. Pathogen analysis was performed in those with microbiology data available. Results Among 36,787 patients, TW was most often performed (57.0%), followed by PCR12 (30.2%) and PCR< 12 (12.8%). Patients with PCR12 were more likely younger, Hispanic/Latino, and uninsured, and less likely socially vulnerable compared to TW (all p< 0.01, Table 1). PCR12 was more common in patients from large (500+ beds), teaching, urban hospitals in the Midwest, compared to patients with TW (all p< 0.01, Table 2). Patients with PCR12 were more likely to have a history of transplantation or HIV/AIDS (both p< 0.01). PCR12 was associated with a higher mean index visit cost (by $101) but lower mean 30-day follow-up cost (by $130) than TW (Table 3). Patients with PCR12 had lower 30-day AIG-related admission risk than TW (1.7% vs. 2.7% p< 0.01). Among 8,451 patients with microbiology data, PCR12 was associated with fewer stool tests per patient (mean:1.61 vs. 1.26), faster turnaround time (mean: 6.3 vs. 25.7 hours) and less likelihood of receiving in-hospital antibiotics (39.4% vs. 47.1%, all p< 0.01) than TW. A higher percentage of patients with PCR12 had a target pathogen detected (73.1%) compared to PCR< 12 (63.6%) or TW (45.4%, p< 0.01). Table 1.Patient characteristics of hospital-based infectious gastroenteritis outpatients during January 1, 2016 - June 30, 2021, stratified by type of stool testTable 2.Clinical and hospital characteristics of hospital-based infectious gastroenteritis outpatients during January 1, 2016 - June 30, 2021, stratified by type of stool testTable 3.Outcomes of hospital-based infectious gastroenteritis outpatients during January 1, 2016 - June 30, 2021, stratified by the type of stool test Conclusion PCR12 multiplex panels were associated with lower 30-day follow-up cost and risk of IG-related hospitalization. PCR12 was also associated with less secondary testing, reduced in-hospital antibiotic use, faster turnaround, and increased diagnostic yield compared to TW. Disclosures Rena Moon, MD, MPH, Premier Inc: Employee Tammy C. Bleak, PharmD, MSc, bioMerieux: Employee of bioMerieux Ning Rosenthal, MD, PhD, MPH, Premier Inc: Employee|Premier Inc: Stocks/Bonds Brianne Couturier, PhD, bioMerieux: Employee of bioMerieux Rachael Hemmert, MSPH, bioMerieux: Employee of bioMerieux Tristan T. Timbrook, PharmD, bioMerieux: Employee of bioMerieux Harold Brown, MBA, MHA, Premier Inc: Employee Ferric C. Fang, MD, bioMerieux: Advisor/Consultant.

The sequestration of iron in case of infection, termed nutritional immunity, is an established strategy of host defense. However, the interaction between pathogens and the mammalian iron storage protein ferritin is hitherto not completely understood. To better characterize the function of ferritin in Gram-negative infections, we incubated iron-starved cultures of Salmonella Typhimurium and knockout mutant strains defective for major iron uptake pathways or Escherichia coli with horse spleen ferritin or ionic iron as the sole iron source. Additionally, we added bovine superoxide dismutase and protease inhibitors to the growth medium to assess the effect of superoxide and bacterial proteases, respectively, on Salmonella proliferation and reductive iron release. Compared to free ionic iron, ferritin-bound iron was less available to Salmonella, but was still sufficient to significantly enhance the growth of the bacteria. In the absence of various iron acquisition genes, the availability of ferritin iron further decreased. Supplementation with superoxide dismutase significantly reduced the growth of the ΔentC knockout strain with holoferritin as the sole iron source in comparison with ionic ferrous iron. In contrast, this difference was not observed in the wildtype strain, suggesting that superoxide dismutase undermines bacterial iron uptake from ferritin by siderophore-independent mechanisms. Ferritin seems to diminish iron availability for bacteria in comparison to ionic iron, and its iron sequestering effect could possibly be enhanced by host superoxide dismutase activity.

In this article, the editors of Clinical Infectious Diseases review some of the most important lessons they have learned about the epidemiology, clinical features, diagnosis, treatment and prevention of SARS-CoV-2 infection and identify essential questions about COVID-19 that remain to be answered., This article reviews important lessons learned to this day, October 15, 2020, about the epidemiology, clinical virology, presentation, complications, diagnosis, treatment and prevention of SARS-CoV-2 infection and identifies essential questions about the COVID-19 pandemic that remain to be answered.

Nitric oxide (NO˙) is a radical molecule produced by mammalian phagocytic cells as part of the innate immune response to bacterial pathogens. It exerts its antimicrobial activity in part by impairing the function of metalloproteins, particularly those containing iron and zinc cofactors. The pathogenic Gram-negative bacterium Salmonella enterica serovar typhimurium undergoes dynamic changes in its cellular content of the four most common metal cofactors following exposure to NO˙ stress. Zinc, iron and magnesium all decrease in response to NO˙ while cellular manganese increases significantly. Manganese acquisition is driven primarily by increased expression of the mntH and sitABCD transporters following derepression of MntR and Fur. ZupT also contributes to manganese acquisition in response to nitrosative stress. S. Typhimurium mutants lacking manganese importers are more sensitive to NO˙, indicating that manganese is important for resistance to nitrosative stress.

Diagnostic testing has played and will continue to play a major role in the coronavirus disease 2019 (COVID-19) pandemic. The ability to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in respiratory secretions is essential to determine when an individual is infected and potentially infectious to others. Viral detection is used for the identification, management, and isolation of individual patients. Viral detection is also used to determine when the virus has entered a community and how rapidly it is spreading. As communities attempt to reopen following periods of shutdown, the detection of both SARS-CoV-2 and specific antibodies that recognize the virus will become increasingly important as a means to assess infection and immunity in individuals and communities. Here, we discuss questions commonly asked by clinicians about COVID-19 diagnostic testing.

Gene duplication facilitates the evolution of biological complexity, as one copy of a gene retains its original function while a duplicate copy can acquire mutations that would otherwise diminish fitness. Duplication has played a particularly important role in the evolution of regulatory networks by permitting novel regulatory interactions and responses to stimuli. The diverse MarR family of transcription factors (MFTFs) illustrates this concept, ranging from highly specific repressors of single operons to pleiotropic global regulators controlling hundreds of genes. MFTFs are often genetically and functionally linked to antimicrobial efflux systems. However, the SlyA MFTF lineage in the Enterobacteriaceae plays little or no role in regulating efflux but rather functions as transcriptional counter-silencers, which alleviate xenogeneic silencing of horizontally-acquired genes and facilitate bacterial evolution by horizontal gene transfer. This review will explore recent advances in our understanding of MFTF traits that have contributed to their functional evolution.

A variety of eubacteria, plants and protozoa can modify membrane lipids by cyclopropanation, which is reported to modulate membrane permeability and fluidity. The ability to cyclopropanate membrane lipids has been associated with resistance to oxidative stress in Mycobacterium tuberculosis, organic solvent stress in Escherichia coli, and acid stress in E. coli and Salmonella. In bacteria, the cfa gene encoding cyclopropane fatty acid (CFA) synthase is induced during the stationary phase of growth. In the present study we constructed a cfa mutant of Salmonella enterica serovar Typhimurium 14028s (S. Typhimurium) and determined the contribution of CFA-modified lipids to stress resistance and virulence in mice. Cyclopropane fatty acid content was quantified in wild-type and cfa mutant S. Typhimurium. CFA levels in a cfa mutant were greatly reduced compared to wild-type, indicating that CFA synthase is the major enzyme responsible for cyclopropane modification of lipids in Salmonella. S. Typhimurium cfa mutants were more sensitive to extreme acid pH, the protonophore CCCP, and hydrogen peroxide, compared to wild-type. In addition, cfa mutants exhibited reduced viability in murine macrophages and could be rescued by addition of the NADPH phagocyte oxidase inhibitor diphenyleneiodonium (DPI) chloride. S. Typhimurium lacking cfa was also attenuated for virulence in mice. These observations indicate that CFA modification of lipids makes an important contribution to Salmonella virulence.

A variety of eubacteria, plants, and protozoa can modify membrane lipids by cyclopropanation, which is reported to modulate membrane permeability and fluidity. The ability to cyclopropanate membrane lipids has been associated with resistance to oxidative stress in Mycobacterium tuberculosis, organic solvent stress in Escherichia coli, and acid stress in E. coli and Salmonella. In bacteria, the cfa gene encoding cyclopropane fatty acid (CFA) synthase is induced during the stationary phase of growth. In the present study, we constructed a cfa mutant of Salmonella enterica serovar Typhimurium 14028s (S. Typhimurium) and determined the contribution of CFA-modified lipids to stress resistance and virulence in mice. Cyclopropane fatty acid content was quantified in wild-type and cfa mutant S. Typhimurium. CFA levels in the cfa mutant were greatly reduced compared to CFA levels in the wild type, indicating that CFA synthase is the major enzyme responsible for cyclopropane modification of lipids in Salmonella. S. Typhimurium cfa mutants were more sensitive to extreme acid pH, the protonophore CCCP, and hydrogen peroxide compared to the wild type. In addition, cfa mutants exhibited reduced viability in murine macrophages and could be rescued by the addition of the NADPH phagocyte oxidase inhibitor diphenyleneiodonium (DPI) chloride. S. Typhimurium lacking cfa was also attenuated for virulence in mice. These observations indicate that CFA modification of lipids makes an important contribution to Salmonella virulence.

The intracellular pathogen concept classifies pathogenic microbes on the basis of their site of replication and dependence on host cells. This concept played a fundamental role in establishing the field of cellular microbiology, founded in part by Dr. Pascale Cossart, whose seminal contributions are honored in this issue of Molecular Microbiology. The recognition that microbes can access and replicate in privileged compartments within host cells has led to many new and fruitful lines of investigation into the biology of the cell and mechanisms of cell-mediated immunity. However, like any scientific concept, the intracellular pathogen concept can become a dogma that constrains thinking and oversimplifies complex and dynamic host-pathogen interactions. Growing evidence has blurred the distinction between "intracellular" and "extracellular" pathogens and demonstrated that many pathogens can exist both within and outside of cells. Although the intracellular pathogen concept remains useful, it should not be viewed as a rigid classification of pathogenic microbes, which exhibit remarkable variation and complexity in their behavior in the host.

Background Campylobacter species are among the most common causes of enteric bacterial infections worldwide. Men who have sex with men (MSM) are at increased risk for sexually transmitted enteric infections, including globally distributed strains of multidrug-resistant Shigella species. Methods This was a retrospective study of MSM-associated Campylobacter in Seattle, Washington and Montréal, Québec with phenotypic antimicrobial resistance profiles and whole genome sequencing (WGS). Results We report the isolation of 2 clonal lineages of multidrug-resistant Campylobacter coli from MSM in Seattle and Montréal. WGS revealed nearly identical strains obtained from the 2 regions over a 4-year period. Comparison with the National Center for Biotechnology Information’s Pathogen Detection database revealed extensive Campylobacter species clusters carrying multiple drug resistance genes that segregated with these isolates. Examination of the genetic basis of antimicrobial resistance revealed multiple macrolide resistance determinants including a novel ribosomal RNA methyltransferase situated in a CRISPR (clustered regularly interspaced short palindromic repeats) array locus in a C. coli isolate. Conclusions As previously reported for Shigella, specific multidrug-resistant strains of Campylobacter are circulating by sexual transmission in MSM populations across diverse geographic locations, suggesting a need to incorporate sexual behavior in the investigation of clusters of foodborne pathogens revealed by WGS data., We show defined clonal lineages of likely sexually transmitted Campylobacter species associated with spread in men who have sex with men between Seattle and Montréal. We also report the first CRISPR-acquired antibiotic resistance gene in a clinical isolate.

Two near-identical clinical Streptococcus pyogenes isolates of emm subtype emm43.4 with a pbp2x missense mutation (T553K) were detected. Minimum inhibitory concentrations (MICs) for ampicillin and amoxicillin were 8-fold higher, and the MIC for cefotaxime was 3-fold higher than for near-isogenic control isolates, consistent with a first step in developing β-lactam resistance.

BackgroundMen who have sex with men (MSM) are at risk for sexual transmission of enteric pathogens. The microbiology of gastroenteritis in MSM has not been examined since the advent of antiretroviral therapy and molecular diagnostics. Our objective was to assess the causes of gastroenteritis among MSM living with and without human immunodeficiency virus (HIV) coinfection in Seattle, Washington.MethodsWe conducted a retrospective cohort study of 235 MSM who underwent multiplex stool polymerase chain reaction (PCR) testing between 1 January 2017 and 1 June 2018. We abstracted clinical and laboratory data from electronic medical records. Parallel or reflexive culture and susceptibility testing were performed when PCR detected cultivable pathogens.ResultsAmong 235 MSM tested (268 episodes), 131 had 151 episodes with positive test results. 148 (63.0%) individuals were living with HIV. Among positive tests, 88.7% detected a bacterial pathogen, 26% a virus, and 40% a parasite. Diarrheagenic Escherichia coli (enteroaggretative, enteropathogenic), Shigella, and Campylobacter were the most commonly detected bacteria (33.1%, 30.5%, and 17.2% of positive samples, respectively). Forty-three percent of positive specimens had ≥2 pathogens. Etiologies and clinical presentations were similar between men living with and without HIV. Cultured Shigella and Campylobacter isolates were frequently resistant to multiple antibiotics.ConclusionsMSM present with gastroenteritis from varied pathogens, including some not detected by conventional stool culture. High levels of antibiotic resistance are consistent with frequent antibiotic exposure in this population and the transmission of multiresistant strains. New approaches are needed to detect, treat, and prevent enteric infections in MSM.

Medicopsis species are rare fungal pathogens that frequently resist common antifungal therapies and are difficult to identify morphologically as conidia are produced in pycnidia, a key feature of coelomycetes. Immunocompromised patients are at risk of these infections, even after remote exposure, and typically present with phaeohyphomycoses without dissemination. We present the case of a renal transplant recipient 6.5 years post-transplant who developed a slowly progressive soft tissue infection mimicking a synovial cyst. A cultured isolate was identified as Medicopsis romeroi by sequencing of multiple ribosomal loci. The patient responded well to debridement and posaconazole therapy. Solid-organ transplant patients are at risk of opportunistic fungal infection long after transplant, and molecular methods are often required for definitive identification.

Salmonella enterica serovar Typhimurium is an intracellular pathogen that elicits nitric oxide (NO·) production by host macrophages. NO· is a potent antimicrobial mediator with diverse targets, including protein thiols and metal centers. The mobilization of zinc from metalloproteins by NO· increases the availability of free intracellular zinc, which is detrimental to bacterial cells, but the precise mechanism of zinc cytotoxicity is uncertain. Here, we show that excess zinc results in the mismetallation of the essential iron-containing enzyme peptide deformylase (PDF), thereby diminishing its activity. PDF mismetallation is observed in zinc-treated bacteria lacking the zinc exporters ZntA and ZitB and is also observed during nitrosative stress, suggesting that NO·-mediated zinc mobilization results in PDF mismetallation. However, NO· also inhibits PDF directly by S-nitrosylating the metal-binding Cys90 residue. These observations identify PDF as an essential bacterial protein that is subject to both direct and indirect inactivation by NO·, providing a novel mechanism of zinc toxicity and NO·-mediated antibacterial activity. IMPORTANCE We have previously shown that the host-derived antimicrobial mediator nitric oxide (NO·) mobilizes zinc from bacterial metalloproteins. The present study demonstrates that NO· inactivates the essential iron-containing enzyme peptide deformylase, both by promoting its mismetallation by zinc and by directly modifying its metal-binding site. We explain how free intracellular zinc is detrimental for cells and reveal a new mechanism of NO·-mediated bacterial growth inhibition that is distinct from previously known targets.

Background An elevated white blood cell count (WBC; >15 000/μL) is an established prognostic marker in patients with Clostridium difficile infection (CDI). Small observational studies have suggested that a markedly elevated WBC should prompt consideration of CDI. However, there is limited evidence correlating WBC elevation with the results of C. difficile nucleic acid amplification testing (NAAT). Methods Retrospective review of laboratory testing, outcomes, and treatment of 16 568 consecutive patients presenting to 4 hospitals over 4 years with NAAT and WBC testing on the same day. Results No significant relationship between C. difficile NAAT results and concurrent WBC in the inpatient setting was observed. Although an elevated WBC did predict NAAT results in the outpatient and emergency department populations (P 15 000/μL) in CDI was associated with a longer median hospital length of stay (15.5 vs 11.0 days; P Conclusions Although WBC is an important prognostic indicator in patients with CDI, an isolated WBC elevation has low sensitivity and specificity as a predictor of fecal C. difficile NAAT positivity in the inpatient setting. A high or rising WBC in isolation is not a sufficient indication for CDI testing.

Answer: Chronic pulmonary coccidioidomycosis. An IgG antibody to Aspergillus fumigatus was not present in the patient’s serum, and Aspergillus fumigatus was not detected in either BAL sample by PCR. However, a serum Coccidioides IgG complement fixation titer was positive at 1:32. Coccidioides

A 51-year-old Spanish-speaking female with a history of diabetes mellitus and hypertension presented to the emergency department with dyspnea, pleuritic chest pain, night sweats, and fever. She noted 10 years of intermittent cough, productive of clear phlegm, that had worsened over the past year.

We are learning that the host response to severe acute respiratory syndrome coronavirus 2 ( SARS-CoV-2) infection is complex and highly dynamic. Effective initial host defense in the lung is associated with mild symptoms and disease resolution. Viral evasion of the immune response can lead to refractory alveolar damage, ineffective lung repair mechanisms, and systemic inflammation with associated organ dysfunction. The immune response in these patients is highly variable and can include moderate to severe systemic inflammation and/or marked systemic immune suppression. There is unlikely to be a "one size fits all" approach to immunomodulation in patients with coronavirus disease 2019 (COVID-19). We believe that a personalized, immunophenotype-driven approach to immunomodulation that may include anticytokine therapy in carefully selected patients and immunostimulatory therapies in others is the shortest path to success in the study and treatment of patients with critical illness due to COVID-19.

With news on many fronts seeming to get inexorably worse, it is worth pausing to raise our glasses to celebrate some recent evidence of progress. In this issue of the Journal, Jernigan and colleagu...

Recent technological advances are revolutionizing clinical microbiology. In place of the traditional diagnostic approach in which a clinician suspecting an infectious disease is required to test for the most likely etiology, perform follow-up testing for rarer causes, and then perhaps be left with a diagnosis of exclusion, many laboratories are now offering multiplex syndromic panels to provide a comprehensive diagnostic approach to bloodstream, gastrointestinal, respiratory, and central nervous system infections. In addition to simplifying testing and laboratory workflow, multiplex panels can improve patient care, promote antimicrobial stewardship, and assist with infection control and prevention. This review discusses the growing body of evidence demonstrating the clinical impact and cost-effectiveness of multiplex panels.

We aim to describe the characteristics, risk factors, and clinical outcomes associated with NAP1 strain Clostridioides difficile infection (CDI) in this single-center, retrospective, case–control (1:1) study. We found that the NAP1 strain accounted for 19.7% of CDI, and risk factors for acquisition included residence in skilled nursing facilities, previous CDI, and proton pump inhibitor use.

Background Men-who-have-sex-with-men (MSM) are at increased risk for infection with enteric pathogens such as Shigella through sexual transmission. The etiology of acute gastroenteritis in this population has not been examined since the advent of molecular diagnostics. This study describes the causes of acute gastroenteritis in MSM in Seattle, Washington. Methods Clinical and laboratory data were obtained for 226 MSM individuals seen in University of Washington-affiliated hospitals and clinics who underwent multiplex stool PCR testing (FilmArray GI Panel, BioFire Diagnostics, Salt Lake City, UT) from January 1, 2017 to June 1, 2018. Student’s t-test and chi-square were used to determine significant differences between HIV-positive and HIV-negative men. Results Of 226 individuals tested, 130 (57.5%) had at least one positive stool test. Of individuals with a positive test result, 70% were HIV-positive. Sixty-one percent of HIV-negative patients were using PrEP. Of the positive samples, 88.7% detected a bacterial etiology, 26% detected a virus, and 40% a protozoal pathogen. Shigella, Campylobacter and diarrheagenic E. coli were the most commonly-detected bacteria (30.5%, 17.2% and 33.1% of positive samples, respectively), while norovirus was the most commonly-detected virus (15.2%), and Giardia was the most common parasite (20.5%). The etiologies of gastroenteritis were similar between HIV-positive and HIV-negative men. Cultured Shigella and Campylobacter isolates were frequently resistant to multiple antibiotics. Conclusion MSM present with acute gastroenteritis caused by a range of pathogens, including some not detected by conventional stool culture, as well as sexually-transmitted pathogens such as Shigella, Campylobacter and Giardia. PrEP may be a risk factor for sexually-transmitted enteric infections as well as other STIs due to risk compensation. High levels of resistance to antibiotics used to treat gastroenteritis is consistent with a high rate of antibiotic exposure in this population and/or transmission of multi-resistant strains. New approaches may be needed to detect, treat and prevent enteric infections in MSM. Disclosure No significant relationships.

Bacteria primarily evolve via horizontal gene transfer, acquiring new traits such as virulence and antibiotic resistance in single transfer events. However, newly acquired genes must be integrated into existing regulatory networks to allow appropriate expression in new hosts. This is accommodated in part by the opposing mechanisms of xenogeneic silencing and countersilencing. An understanding of these mechanisms is necessary to understand the relationship between gene regulation and bacterial evolution. Here we examine the functional evolution of an important lineage of countersilencers belonging to the ancient MarR family of classical transcriptional repressors. We show that although members of the SlyA lineage retain some ancestral features associated with the MarR family, their cis-regulatory sequences have evolved significantly to support their new function. Understanding the mechanistic requirements for countersilencing is critical to understanding the pathoadaptation of emerging pathogens and also has practical applications in synthetic biology., Gene duplication and subsequent evolutionary divergence have allowed conserved proteins to develop unique roles. The MarR family of transcription factors (TFs) has undergone extensive duplication and diversification in bacteria, where they act as environmentally responsive repressors of genes encoding efflux pumps that confer resistance to xenobiotics, including many antimicrobial agents. We have performed structural, functional, and genetic analyses of representative members of the SlyA/RovA lineage of MarR TFs, which retain some ancestral functions, including repression of their own expression and that of divergently transcribed multidrug efflux pumps, as well as allosteric inhibition by aromatic carboxylate compounds. However, SlyA and RovA have acquired the ability to countersilence horizontally acquired genes, which has greatly facilitated the evolution of Enterobacteriaceae by horizontal gene transfer. SlyA/RovA TFs in different species have independently evolved novel regulatory circuits to provide the enhanced levels of expression required for their new role. Moreover, in contrast to MarR, SlyA is not responsive to copper. These observations demonstrate the ability of TFs to acquire new functions as a result of evolutionary divergence of both cis-regulatory sequences and in trans interactions with modulatory ligands.

Here we show that dopamine increases bacterial iron incorporation and promotes Salmonella Typhimurium growth both in vitro and in vivo. These observations suggest the potential hazards of pharmacological catecholamine administration in patients with bacterial sepsis but also suggest that the inhibition of bacterial iron acquisition might provide a useful approach to antimicrobial therapy., We have recently shown that the catecholamine dopamine regulates cellular iron homeostasis in macrophages. As iron is an essential nutrient for microbes, and intracellular iron availability affects the growth of intracellular bacteria, we studied whether dopamine administration impacts the course of Salmonella infections. Dopamine was found to promote the growth of Salmonella both in culture and within bone marrow-derived macrophages, which was dependent on increased bacterial iron acquisition. Dopamine administration to mice infected with Salmonella enterica serovar Typhimurium resulted in significantly increased bacterial burdens in liver and spleen, as well as reduced survival. The promotion of bacterial growth by dopamine was independent of the siderophore-binding host peptide lipocalin-2. Rather, dopamine enhancement of iron uptake requires both the histidine sensor kinase QseC and bacterial iron transporters, in particular SitABCD, and may also involve the increased expression of bacterial iron uptake genes. Deletion or pharmacological blockade of QseC reduced but did not abolish the growth-promoting effects of dopamine. Dopamine also modulated systemic iron homeostasis by increasing hepcidin expression and depleting macrophages of the iron exporter ferroportin, which enhanced intracellular bacterial growth. Salmonella lacking all central iron uptake pathways failed to benefit from dopamine treatment. These observations are potentially relevant to critically ill patients, in whom the pharmacological administration of catecholamines to improve circulatory performance may exacerbate the course of infection with siderophilic bacteria.

PCR ribotyping and antimicrobial susceptibility testing were used to characterize 940 Clostridioides (Clostridium) difficile isolates collected from 26 U S. hospitals over three time periods from 2011 to 2017. The proportion of ribotype (RT) 027 isolated during the three surveys decreased significantly over time from 31% in 2011-2012, to 22% in 2013-2014, and to 14% in 2015-2017 (p 0.001 and p = 0.010, respectively), while we observed an increase in prevalence of RT106, that rose from 7% in our first survey to 19% of isolates in our last survey (p 0.001). In addition, both RT056 and RT002 rose from 3% to 10% (p 0.001). The proportions of all other ribotypes remained steady over time, and RT014/020 was the third most common strain type in our convenience sample in the final survey. Overall, resistance to moxifloxacin, rifampin, and vancomycin decreased during our studies, mainly due to the decline in RT027 isolates. A decrease in moxifloxacin resistance and an increase in tetracycline resistance were found among RT027 strains isolated in the last survey. Although the proportion of RT027 isolates declined, multidrug resistance among this ribotype continues to be common.