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Central metabolic pathways controls virulence and antibiotic resistance, and constitute potential targets for antibacterial drugs. InStaphylococcus aureusthe role of the pentose phosphate pathway (PPP) remains largely unexplored. Mutation of the 6-phosphogluconolactonase genepgl,which encodes the only non-essential enzyme in the oxidative phase of the PPP, significantly increased MRSA resistance to β-lactam antibiotics, particularly in chemically defined media with glucose, and reduced oxacillin (OX)-induced lysis. Expression of the methicillin-resistance penicillin binding protein 2a and peptidoglycan architecture were unaffected. Carbon tracing and metabolomics revealed extensive metabolic reprogramming in thepglmutant including increased flux to glycolysis, the TCA cycle, and several cell envelope precursors, which was consistent with increased β-lactam resistance. Morphologically,pglmutant cells were smaller than wild-type with a thicker cell wall and ruffled surface when grown in OX. Further evidence of the pleiotropic effect of thepglmutation was reduced resistance to Congo Red, sulfamethoxazole and oxidative stress, and increased resistance to targocil, fosfomycin and vancomycin. Reduced binding of wheat germ agglutinin (WGA) topglwas indicative of lower wall teichoic acid/lipoteichoic acid levels or altered teichoic acid structures. Mutations in thevraFGorgraRSloci reversed the increased OX resistance phenotype and restored WGA binding to wild-type levels. VraFG/GraRS was previously implicated in susceptibility to cationic antimicrobial peptides and vancomycin, and these data reveal a broader role for this multienzyme membrane complex in the export of cell envelope precursors or modifying subunits required for resistance to diverse antimicrobial agents. Altogether our study highlights important roles for the PPP and VraFG/GraRS in β-lactam resistance, which will support efforts to identify new drug targets and reintroduce β-lactams in combination with adjuvants or other antibiotics for infections caused by MRSA and other β-lactam resistant pathogens.Author summaryHigh-level resistance to penicillin-type (β-lactam) antibiotics significantly limits the therapeutic options for patients with MRSA infections necessitating the use of newer agents, for which reduced susceptibility has already been described. Here we report for the first time that the central metabolism pentose phosphate pathway controls MRSA resistance to penicillin-type antibiotics. We comprehensively demonstrated that mutation of the PPP genepglperturbed metabolism in MRSA leading to increased flux to cell envelope precursors to drive increased antibiotic resistance. Moreover, increased resistance was dependent on the VraRG/GraRS multienzyme membrane complex previously implicated in resistance to antimicrobial peptides and vancomycin. Our data thus provide new insights on MRSA mechanisms of β-lactam resistance, which will support efforts to expand the treatment options for infections caused by this and other antimicrobial resistant pathogens.

Staphylococci, whether beneficial commensals or pathogens, often colonize human skin, potentially leading to competition for the same niche. In this multidisciplinary study we investigate the structure, binding specificity, and mechanism of adhesion of the Aap lectin domain required forStaphylococcus epidermidisskin colonization and compare its characteristics to the lectin domain from the orthologousStaphylococcus aureusadhesin SasG. The Aap structure reveals a legume lectin-like fold with atypical architecture, showing specificity for N-acetyllactosamine and sialyllactosamine. Bacterial adhesion assays using human corneocytes confirmed the biological relevance of these Aap-glycan interactions. Single-cell force spectroscopy experiments measured individual binding events between Aap and corneocytes, revealing an extraordinarily tight adhesion force of nearly 900 nN and a high density of receptors at the corneocyte surface. The SasG lectin domain shares similar structural features, glycan specificity, and corneocyte adhesion behavior. We observe cross-inhibition of Aap- and SasG-mediated staphylococcal adhesion to corneocytes. Together, these data provide insights into staphylococcal interspecies competition for skin colonization and suggest potential avenues for inhibition ofS. aureuscolonization.

Background The currently recommended HIV testing algorithm begins with a 4th generation antigen/antibody (Ag/Ab) combination immunoassay, which allows for early detection of HIV. However, this assay’s high sensitivity can result in false positives that must be distinguished from acute infections via an HIV nucleic acid test (NAT). At our medical center, HIV NATs are recommended in the lab report for anyone with a positive p24Ag component on the screening assay, but they are not done reflexively by the lab as it requires a different blood tube. Thus, difficulties arise in obtaining the NAT, such as providers failing to order the test or patients failing to return for another blood draw, jeopardizing the diagnosis of early HIV. Methods We conducted a retrospective chart review of adult patients at a Midwest academic medical center with a positive p24Ag on the HIV-1/2 Ag/Ab combination assay between Jan. 1, 2017 and Feb. 21, 2021. We assessed if appropriate confirmatory testing was performed, identified barriers to missed opportunities for confirmatory testing, and used t-tests and Fisher’s exact tests to evaluate differences between those who did and did not receive confirmatory testing. Results Of 37 people with a positive p24Ag on the HIV Ag/Ab immunoassay, 6 (17%) did not have an HIV NAT performed within 30 days of their positive screening test. Of these 6 cases, the ordering provider did not acknowledge the need for a confirmatory test in 3 cases, the patient did not return for the test in 1 case, and other factors prevented follow-up in 2 cases. Of those who had an HIV NAT ordered, 7 (23%) had a positive HIV NAT. Younger age was significantly associated with completion of appropriate confirmatory testing (p=0.05), but other factors including sex, race, insurance status, having a primary care provider, specialty of ordering provider, encounter location, and test indication were not. Conclusion Our review identified multiple missed opportunities for confirmatory HIV testing following a positive p24Ag component on the HIV screening test. Provider lack of knowledge/awareness of the need for appropriate testing was the most common barrier. A quality improvement initiative will be undertaken to minimize future risk of missed opportunities for confirmatory testing. Disclosures Paul D. Fey, PhD, BioFire: Advisor/Consultant|BioFire: Grant/Research Support|Merck: Grant/Research Support Sara H. Bares, MD, Gilead Sciences: Expert Testimony|GSK ViiV Healthcare: Grant/Research Support|Janssen: Grant/Research Support.

Background Hospitalized COVID-19 patients with severe pneumonia are commonly treated for secondary bacterial pneumonia. The Biofire pneumonia panel, a rapid molecular diagnostic tool with 18 bacterial, 8 viral and 7 resistance gene targets, was made available to critical care and infectious disease clinicians in May 2020 at our institution. We sought to describe its utilization and influence on antibiotic use in patients hospitalized with COVID-19 lower respiratory tract infection (LRTI). Methods Eligible patients with COVID-19 LRTI (positive PCR test and abnormal chest imaging) had sputum or bronchoalveolar lavage pneumonia panel (PNP) paired with a respiratory tract culture between May 4 and Dec. 8, 2020, were included. Demographics, comorbidities, clinical data including microbiologic testing, PNP indication(s), and antibiotic use after testing were abstracted through chart review. Descriptive statistics were utilized. Results Characteristics of 133 patients are provided in Table 1. Median age was 61 years, 93 (70%) were male, 93 (70%) were mechanically ventilated, and 68 (51%) died within 30 days on PNP testing. PNP results, including culture results are listed in Table 2. No target was identified in 63 (47%) patients. Staphylococcus aureus was the most common bacterial isolate identified (MSSA in 32 [24%], MRSA in 8 [6%]) with culture growth in 21 specimens. More than 1 target was identified in 29 patients (22%). Empiric antibiotics and subsequent modifications within 24h hours of pneumonia panel are provided in Table 3. Vancomycin and cefepime were most frequently prescribed. Antibiotic modifications were made in 71/133 patients. Cessation of the anti-MRSA agent occurred in 39/72 (54%) of eligible patients and the anti-Pseudomonal agent in 21/78 (27%). Conclusion The PNP is a useful tool to evaluate secondary bacterial pneumonia in critically ill COVID-19 patients and may assist clinicians and antimicrobial stewardship programs in expedited antibiotic discontinuation or de-escalation particularly where rates of secondary bacterial infection are low, such as COVID-19 LRTI. Disclosures Erica J. Stohs, MD, MPH, bioMerieux: Grant/Research Support Paul D. Fey, PhD, BioFire: Advisor/Consultant|BioFire: Grant/Research Support|Merck: Grant/Research Support Trevor C. Van Schooneveld, MD, bioMerieux: Advisor/Consultant|bioMerieux: Grant/Research Support|Insmed: Grant/Research Support|Merck: Grant/Research Support|Thermo-Fischer: Advisor/Consultant.

West Nile virus (WNv) is a major cause of viral encephalitis in the United States. WNv infection is usually asymptomatic or a limited febrile illness in the immunocompetent hosts, although a small percentage can develop neuroinvasive disease. Neuroinvasive disease due to WNv in solid organ transplant recipients occurs at higher rates than observed in the general population and can have long term neurological sequalae.We retrospectively reviewed medical records of all solid organ transplant recipients at our institution who tested positive for WNv from 2010 to 2018. Two reviewers performed electronic searches of Medline, Embase, Cochrane Library of literature of WNv infections in SOT. Descriptive statistics were performed on key variables.Eight recipients (mean age 54, five males) were diagnosed with neuroinvasive WNv infection at our institution. Distribution of infection was as follows: five kidney transplants, one in each kidney-pancreas, liver, and lung. Diagnoses included meningitis (3), encephalitis (1), meningo-encephalitis (4). Median time from transplant to infection was 49.8 months (2.7-175.4). No infections were considered donor-derived. Five patients received treatment with IVIG. Six patients were alive at median follow-up of 49.5 months (21.7-116.8). We identified 29 studies published from 2002 to 2019. Median time from transplant to infection was 14.2 months, with similar allograft distribution; 53% were donor-derived infections.WNv infections in solid organ transplant recipients can be a consequence of organ donation or can be acquired via the community. Infections can be more severe in SOT recipients and lead to neuroinvasive disease.

Staphylococcus aureus colonizes or causes infection in a multitude of niches within a mammalian host. Many of these niches are acidic, yet specific pH resistance mechanisms that facilitate survival have not been thoroughly investigated. This review discusses recent studies documenting known acid resistance mechanisms in S. aureus and other staphylococcal species. However, studies that clearly define the regulation of the acid resistance regulon and potential interactions with weak organic acids in specific niches of the host including the skin and gut are yet to be defined.

Proline acquired via specific transporters can serve as a proteinogenic substrate, carbon and nitrogen source, or osmolyte. Previous reports have documented that Staphylococcus aureus, a major community and nosocomial pathogen, encodes at least four proline transporters, PutP, OpuC, OpuD, and ProP. A combination of genetic approaches and 3H-proline transport assays reveal that a previously unrecognized transporter, ProT, in addition to PutP, are the major proline transporters in S. aureus. Complementation experiments using constitutively expressed non-cognate promoters found that proline transport via OpuD, OpuC, and ProP is minimal. Both proline biosynthesis from arginine and proline transport via ProT are critical for growth when S. aureus is grown under conditions of high salinity. Further, proline transport mediated by ProT or PutP are required for growth in media with and without glucose, indicating both transporters function to acquire proline for proteinogenic purposes in addition to acquisition of proline as a carbon/nitrogen source. Lastly, inactivation of proT and putP resulted in a significant reduction (5 log10) of bacterial burden in murine skin-and-soft tissue infection and bacteremia models, suggesting that proline transport is required to establish a S. aureus infection.

Staphylococcus aureus is a major cause of skin and soft tissue infections as well as bloodstream infections worldwide. Here, we report the draft genome sequences of 18 deidentified S. aureus clinical strains collected from positive blood cultures.

Penicillin binding protein 2a (PBP2a)-dependent resistance to β-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA) is regulated by the activity of the tricarboxylic acid (TCA) cycle via a poorly understood mechanism. We report that mutations in sucC and sucD, but not other TCA cycle enzymes, negatively impact β-lactam resistance without changing PBP2a expression. Increased intracellular levels of succinyl coenzyme A (succinyl-CoA) in the sucC mutant significantly perturbed lysine succinylation in the MRSA proteome. Suppressor mutations in sucA or sucB, responsible for succinyl-CoA biosynthesis, reversed sucC mutant phenotypes. The major autolysin (Atl) was the most succinylated protein in the proteome, and increased Atl succinylation in the sucC mutant was associated with loss of autolytic activity. Although PBP2a and PBP2 were also among the most succinylated proteins in the MRSA proteome, peptidoglycan architecture and cross-linking were unchanged in the sucC mutant. These data reveal that perturbation of the MRSA succinylome impacts two interconnected cell wall phenotypes, leading to repression of autolytic activity and increased susceptibility to β-lactam antibiotics. IMPORTANCEmecA-dependent methicillin resistance in MRSA is subject to regulation by numerous accessory factors involved in cell wall biosynthesis, nucleotide signaling, and central metabolism. Here, we report that mutations in the TCA cycle gene, sucC, increased susceptibility to β-lactam antibiotics and was accompanied by significant accumulation of succinyl-CoA, which in turn perturbed lysine succinylation in the proteome. Although cell wall structure and cross-linking were unchanged, significantly increased succinylation of the major autolysin Atl, which was the most succinylated protein in the proteome, was accompanied by near complete repression of autolytic activity. These findings link central metabolism and levels of succinyl-CoA to the regulation of β-lactam antibiotic resistance in MRSA through succinylome-mediated control of two interlinked cell wall phenotypes. Drug-mediated interference of the SucCD-controlled succinylome may help overcome β-lactam resistance.

As the coronavirus disease 2019 (COVID-19) continues to circulate, testing strategies are of the utmost importance. Given national shortages of testing supplies, personal protective equipment, and other hospital resources, diagnostic stewardship is necessary to aid in resource management. We report the low utility of serial testing in a low-prevalence setting.

Reported cases of Lyme disease in Nebraska have been assumed to be imported from other endemic areas. Previous surveillance efforts provided no evidence of established populations as only individual specimens of Ixodes scapularis (Say) had been collected. In the winter of 2018, adult I. scapularis were found on a dog at Two Rivers State Recreation Area, Douglas County, prompting tick collection at the site and nearby natural areas. In May 2019, all life stages of host-seeking I. scapularis were collected using dragging and flagging techniques in sites located near the Platte River in Douglas, Sarpy, and Saunders counties. This is the first documentation of established populations of I. scapularis in Nebraska.

Zusammenfassung Hintergrund Acrylnagel beherbergen mehr Bakterien als Naturnagel, und die Nutzung wird fur Gesundheitspersonal (HCWs) nicht empfohlen. Uber die neuen und beliebten Gel-Nagelprodukte ist wenig bekannt. Diese Studie untersucht die bakterielle Belastung von Gel-Nageln, Standard-Nagellack und Naturnageln an den Handen von HCWs. Methode Die Studie wurde in 3 Gesundheitszentren durchgefuhrt. Nagel auf der dominierenden Hand von 88 HCWs waren lackiert mit Standard Nagellack oder Gel. Kulturen wurden an den Tagen 1, 7 und 14 vor und nach der Handehygiene mit Alkohol-Handgel abgenommen. Ergebnisse Insgesamt wurden 741 Kulturen gewonnen. Die bakterielle Belastung ist bei allen Nageltypen gestiegen (P ≤ .0001). Eine Reduktion der bakteriellen Belastung konnte nach der Handehygiene bei den Naturnageln und dem Standardnagellack, jedoch nicht bei Gel-Nageln nachgewiesen werden (P = .001, P = .0028 bzw. P = .98). Alle 3 Nageltypen waren im Laufe der Zeit mit mehr Bakterien kontaminiert. Standard-Nagellack und Naturnagel konnen fur die Handehygiene geeigneter sein als Gel-Nagel. Schlussfolgerungen Diese Studie zeigt keine erhohte Anzahl von Mikroorganismen auf Nageln mit Gel; jedoch konnen Gelnagel mit Alkohol-Handgel schwieriger zu desinfizieren sein.

Medical device-associated biofilm infections are a therapeutic challenge based on their antibiotic tolerance and ability to evade immune-mediated clearance. The virulence determinants responsible for bacterial biofilm to induce a maladaptive immune response remain largely unknown. This study identified a critical role for S. aureus ATP synthase in influencing the host immune response to biofilm infection. An S. aureus ATP synthase alpha subunit mutant (ΔatpA) elicited heightened proinflammatory cytokine production by leukocytes in vitro and in vivo, which coincided with improved biofilm clearance in a mouse model of prosthetic joint infection. The ability of S. aureus ΔatpA to augment host proinflammatory responses was cell lysis-dependent, as inhibition of bacterial lysis by polyanethole sodium sulfanate or a ΔatpAΔatl biofilm did not elicit heightened cytokine production. These studies reveal a critical role for AtpA in shaping the host immune response to S. aureus biofilm., Staphylococcus aureus is a major cause of prosthetic joint infection (PJI), which is characterized by biofilm formation. S. aureus biofilm skews the host immune response toward an anti-inflammatory profile by the increased recruitment of myeloid-derived suppressor cells (MDSCs) that attenuate macrophage proinflammatory activity, leading to chronic infection. A screen of the Nebraska Transposon Mutant Library identified several hits in the ATP synthase operon that elicited a heightened inflammatory response in macrophages and MDSCs, including atpA, which encodes the alpha subunit of ATP synthase. An atpA transposon mutant (ΔatpA) had altered growth kinetics under both planktonic and biofilm conditions, along with a diffuse biofilm architecture that was permissive for leukocyte infiltration, as observed by confocal laser scanning microscopy. Coculture of MDSCs and macrophages with ΔatpA biofilm elicited significant increases in the proinflammatory cytokines interleukin 12p70 (IL-12p70), tumor necrosis factor alpha (TNF-α), and IL-6. This was attributed to increased leukocyte survival resulting from less toxin and protease production by ΔatpA biofilm as determined by liquid chromatography with tandem mass spectrometry (LC-MS/MS). The enhanced inflammatory response elicited by ΔatpA biofilm was cell lysis-dependent since it was negated by polyanethole sodium sulfanate treatment or deletion of the major autolysin, Atl. In a mouse model of PJI, ΔatpA-infected mice had decreased MDSCs concomitant with increased monocyte/macrophage infiltrates and proinflammatory cytokine production, which resulted in biofilm clearance. These studies identify S. aureus ATP synthase as an important factor in influencing the immune response during biofilm-associated infection and bacterial persistence.

Staphylococcus aureus is a metabolically versatile pathogen that colonizes nearly all organs of the human body. A detailed and comprehensive knowledge of staphylococcal metabolism is essential to understand its pathogenesis. To this end, we have reconstructed and experimentally validated an updated and enhanced genome-scale metabolic model of S. aureus USA300_FPR3757. The model combined genome annotation data, reaction stoichiometry, and regulation information from biochemical databases and previous strain-specific models. Reactions in the model were checked and fixed to ensure chemical balance and thermodynamic consistency. To further refine the model, growth assessment of 1920 nonessential mutants from the Nebraska Transposon Mutant Library was performed, and metabolite excretion profiles of important mutants in carbon and nitrogen metabolism were determined. The growth and no-growth inconsistencies between the model predictions and in vivo essentiality data were resolved using extensive manual curation based on optimization-based reconciliation algorithms. Upon intensive curation and refinements, the model contains 863 metabolic genes, 1379 metabolites (including 1159 unique metabolites), and 1545 reactions including transport and exchange reactions. To improve the accuracy and predictability of the model to environmental changes, condition-specific regulation information curated from the existing knowledgebase was incorporated. These critical additions improved the model performance significantly in capturing gene essentiality, substrate utilization, and metabolite production capabilities and increased the ability to generate model-based discoveries of therapeutic significance. Use of this highly curated model will enhance the functional utility of omics data, and therefore, serve as a resource to support future investigations of S. aureus and to augment staphylococcal research worldwide., Systems Biology: Integrated study of Staphylococcus aureus metabolism Integration of in vivo experiment with a newly developed model of Staphylococcus aureus metabolism helps explore its metabolic versatility. A multidisciplinary team led by Rajib Saha at the University of Nebraska developed a new genome-scale metabolic model of the multi-drug resistant pathogen S. aureus by combining genome annotation data, reaction stoichiometry, and condition- and mutant-specific regulations from biochemical databases and previous strain-specific models. Extensive manual curation and incorporation of newly generated experimental data on growth and metabolite production improved the accuracy and predictability of the model and increased its ability to generate model-based discoveries of therapeutic significance. Use of this highly curated model will enhance the functional utility of omics data and, therefore, serve as a resource to support future investigations of S. aureus and to augment staphylococcal research worldwide.

A Staphylococcus aureus strain deleted for the c‐di‐AMP cyclase gene dacA is unable to survive in rich medium unless it acquires compensatory mutations. Previously identified mutations were in opuD, encoding the main glycine‐betaine transporter, and alsT, encoding a predicted amino acid transporter. Here, we show that inactivation of OpuD restores the cell size of a dacA mutant to near wild‐type (WT) size, while inactivation of AlsT does not. AlsT was identified as an efficient glutamine transporter, indicating that preventing glutamine uptake in rich medium rescues the growth of the S. aureus dacA mutant. In addition, GltS was identified as a glutamate transporter. By performing growth curves with WT, alsT and gltS mutant strains in defined medium supplemented with ammonium, glutamine or glutamate, we revealed that ammonium and glutamine, but not glutamate promote the growth of S. aureus. This suggests that besides ammonium also glutamine can serve as a nitrogen source under these conditions. Ammonium and uptake of glutamine via AlsT and hence likely a higher intracellular glutamine concentration inhibited c‐di‐AMP production, while glutamate uptake had no effect. These findings provide, besides the previously reported link between potassium and osmolyte uptake, a connection between nitrogen metabolism and c‐di‐AMP signalling in S. aureus., A large number of amino acid transporters and oligopeptide permeases are encoded in bacterial genomes. However, their actual substrate specificity and functions are hard to predict bioinformatically. In this study, we report that GltS and AlsT are main glutamate and glutamine transporters in Staphylococcus aureus, respectively, and show that glutamine and ammonium uptake inhibit the production of the nucleotide signalling molecule c‐di‐AMP.

Nontyphoidal Salmonella enterica is a common cause of illness in humans ranging from gastroenteritis to invasive disease. National surveillance programs continually monitor trends in antimicrobial resistance patterns and mechanisms of resistance to identify emerging public health threats. Our study shows the emergence of nonsusceptibility to both levofloxacin and ceftriaxone, a concerning phenotype that threatens first-line antibiotic therapy, in Salmonella isolates recovered between 2014 and 2015. From 2010 to 2013 the rate of resistance increased from 0.0% (0/1181) to 1.5% (9/593) in 2014 and 2015. The isolates with this phenotype were found to be from multiple serotypes, including Typhimurium, Newport, and Enteritidis. Resistance to ceftriaxone was attributed to the presence of either an AmpC or extended-spectrum β-lactamase, and resistance to fluoroquinolones was attributable to the presence of mutations in the quinolone resistance-determining region or the presence of plasmid-mediated quinolone resistance genes. As this resistance pattern was seen in a variety of Salmonella serotypes harboring varied resistance mechanisms, it indicates a worrying trend in the spread of isolates resistant to both first-line treatment options.

The cyclic lipodepsipeptides WAP-8294A are antibiotics with potent activity against methicillin-resistant Staphylococcus aureus (MRSA). One member of this family, WAP-8294A2 (Lotilibcin), was in clinical trials due to its high activity and distinct chemistry. However, WAP-8294A compounds are produced in a very low yield by Lysobacter and only under very stringent conditions. Improving WAP-8294A yield has become very critical for research and application of these anti-MRSA compounds. Here, we report a strategy to increase WAP-8294A production. We first used the CRISPR/dCas9 system to increase the expression of five cotranscribed genes (orf1–5) in the WAP gene cluster, by fusing the omega subunit of RNA polymerase with dCas9 that targets the operon’s promoter region. This led to the transcription of the genes increased by 5–48 folds in strain dCas9-ω3. We then refactored four putative self-protection genes (orf6, orf 7, orf 9 and orf10) by reorganizing them into an operon under the control of a strong Lysobacter promoter, PHSAF. The refactored operon was introduced into strain dCas9-ω3, and the transcription of the self-protection genes increased by 20–60 folds in the resultant engineered strains. The yield of the three main WAP-8294A compounds, WAP-8294A1, WAP-8294A2, and WAP-8294A4, increased by 6, 4, and 9 folds, respectively, in the engineered strains. The data also showed that the yield increase of WAP-8294A compounds was mainly due to the increase of the extracellular distribution. WAP-8294A2 exhibited potent (MIC 0.2–0.8 μg/mL) and specific activity against S. aureus among a battery of clinically relevant Gram-positive pathogens (54 isolates).

We aim to review the epidemiology of respiratory viral infections and the strengths and limitations of multiplex respiratory pathogen panels that are currently available along with their respective features and differences.We give particular emphasis to the pathogens included on each test and evaluate their performance in the hospital setting.We conclude with a discussion on the evidence for the clinical utility of respiratory pathogen multiplex panels in hospitalized patients, including the potential for coinfection with viral and bacterial pathogens.

The otherwise harmless skin inhabitant Staphylococcus epidermidis is a major cause of healthcare-associated medical device infections. The species' selective pathogenic potential depends on its production of surface adherent biofilms. The Cell wall-anchored protein Aap promotes biofilm formation in S. epidermidis, independently from the polysaccharide intercellular adhesin PIA. Aap requires proteolytic cleavage to act as an intercellular adhesin. Whether and which staphylococcal proteases account for Aap processing is yet unknown. Here, evidence is provided that in PIA-negative S. epidermidis 1457Δica, the metalloprotease SepA is required for Aap-dependent S. epidermidis biofilm formation in static and dynamic biofilm models. qRT-PCR and protease activity assays demonstrated that under standard growth conditions, sepA is repressed by the global regulator SarA. Inactivation of sarA increased SepA production, and in turn augmented biofilm formation. Genetic and biochemical analyses demonstrated that SepA-related induction of biofilm accumulation resulted from enhanced Aap processing. Studies using recombinant proteins demonstrated that SepA is able to cleave the A domain of Aap at residue 335 and between the A and B domains at residue 601. This study identifies the mechanism behind Aap-mediated biofilm maturation, and also demonstrates a novel role for a secreted staphylococcal protease as a requirement for the development of a biofilm.

Regulation of icaADBC -encoded polysaccharide intercellular adhesin (PIA)/poly- N -acetylglucosasmine (PNAG) production in staphylococci plays an important role in biofilm-associated medical-device-related infections. Here, we report that the AraC-type transcriptional regulator Rbf activates icaADBC operon transcription and PIA production in Staphylococcus epidermidis . Purified recombinant Rbf did not bind to the ica operon promoter region in electrophoretic mobility shift assays (EMSAs), indicating that Rbf regulates ica transcription indirectly. To identify the putative transcription factor(s) involved in Rbf-mediated icaADBC regulation, the ability of recombinant Rbf to interact with the promoter sequences of known icaADBC regulators was investigated. Recombinant Rbf bound to the sarR promoter and not the sarX , sarA , sarZ , spx , and srrA promoters. Reverse transcription (RT)-PCR demonstrated that Rbf acts as a repressor of sarR transcription. PIA expression and biofilm production were restored to wild-type levels in an rbf sarR double mutant grown in brain heart infusion (BHI) medium supplemented with NaCl, which is known to activate the ica locus, but not in BHI medium alone. RT-PCR further demonstrated that although Rbf does not bind the sarX promoter, it nevertheless exerted a negative effect on sarX expression. Apparently, direct downregulation of the SarR repressor by Rbf has a dominant effect over indirect repression of the SarX activator by Rbf in the control of S. epidermidis PIA production and biofilm formation. IMPORTANCE The importance of Staphylococcus epidermidis as an opportunistic pathogen in hospital patients with implanted medical devices derives largely from its capacity to form biofilm. Expression of the icaADBC -encoded extracellular polysaccharide is the predominant biofilm mechanism in S. epidermidis clinical isolates and is tightly regulated. Here, we report that the transcriptional regulator Rbf promotes icaADBC expression by negatively regulating expression of sarR , which encodes an ica operon repressor. Furthermore, Rbf indirectly represses the ica operon activator, SarX. The data reveal complicated interplay between Rbf and two Sar family proteins in fine-tuning regulation of the biofilm phenotype and indicate that in the hierarchy of biofilm regulators, IcaR is dominant over the Rbf-SarR-SarX axis.

Staphylococcus aureusis a metabolically versatile pathogen that colonizes nearly all organs of the human body. A detailed and comprehensive knowledge of staphylococcal metabolism is essential to understanding its pathogenesis. To this end, we have reconstructed and experimentally validated an updated and enhanced genome-scale metabolic model ofS. aureusUSA300_FPR3757. The model combined genome annotation data, reaction stoichiometry, and regulation information from biochemical databases and previous strain-specific models. Reactions in the model were checked and fixed to ensure chemical balance and thermodynamic consistency. To further refine the model, growth assessment of 1920 non-essential mutants from the Nebraska Transposon Mutant Library was performed and metabolite excretion profiles of important mutants in carbon and nitrogen metabolism were determined. The growth and no-growth inconsistencies between the model predictions andin vivoessentiality data were resolved using extensive manual curation based on optimization-based reconciliation algorithms. Upon intensive curation and refinements, the model contains 840 metabolic genes, 1442 metabolites, and 1566 reactions including transport and exchange reactions. To improve the accuracy and predictability of the model to environmental changes, condition-specific regulation information curated from the existing knowledgebase was incorporated. These critical additions improved the model performance significantly in capturing gene essentiality, substrate utilization, and metabolite production capabilities and increased the ability to generate model-based discoveries of therapeutic significance. Use of this highly curated model will enhance the functional utility of omics data and, therefore, serve as a resource to support future investigations ofS. aureusand to augment staphylococcal research worldwide.

Transposon mutagenesis is one of the most widely used strategies to generate a large number of random mutations within a bacterial genome and then to precisely identify the mutated sites. The generation of sequence-defined transposon mutant libraries that are composed of a collection of different mutants, each containing a single transposon insertion mutation within nearly all of the nonessential genes within the genome, is a rapid and reliable way to enhance the study of gene function. In this chapter, we describe the process to generate a sequence-defined transposon mutant library in Staphylococcus aureus utilizing the mariner-based bursa aurealis transposon.

Objective:To evaluate the impact of a hard stop in the electronic health record (EHR) on inappropriate gastrointestinal pathogen panel testing (GIPP).Design:We used a quasi-experimental study to evaluate testing before and after the implementation of an EHR alert to stop inappropriate GIPP ordering.Setting:Midwest academic medical center.Participants:Hospitalized patients with diarrhea for which GIPP testing was ordered, between January 2016 through March 2017 (period 1) and April 2017 through June 2018 (period 2).Intervention:A hard stop in the EHR prevented clinicians from ordering a GIPP more than once per admission or in patients hospitalized for >72 hours.Results:During period 1, 1,587 GIPP tests were ordered over 212,212 patient days, at a rate of 7.48 per 1,000 patient days. In period 2, 1,165 GIPP tests were ordered over 222,343 patient days, at a rate of 5.24 per 1,000 patient days. The Poisson model estimated a 30% reduction in total GIPP ordering rates between the 2 periods (relative risk, 0.70; 95% confidence interval [CI], 0.63–0.78;P< .001). The rate of inappropriate tests ordered decreased from 21.5% to 4.9% between the 2 periods (P< .001). The total savings calculated factoring only GIPP orders that triggered the hard stop was ∼$67,000, with potential savings of $168,000 when factoring silent best-practice alert data.Conclusions:A simple hard stop alert in the EHR resulted in significant reduction of inappropriate GIPP testing, which was associated with significant cost savings. Clinicians can practice diagnostic stewardship by avoiding ordering this test more than once per admission or in patients hospitalized >72 hours.

Prolonging the clinical effectiveness of {beta}-lactams, which remain first-line antibiotics for many infections, is an important part of efforts to address antimicrobial resistance. We report here that inactivation of the predicted D-cycloserine (DCS) transporter gene cycA gene re-sensitizes MRSA to {beta}-lactam antibiotics. The cycA mutation also resulted in hyper-susceptibility to DCS, an alanine analogue antibiotic that inhibits the alanine racemase and D-Alanine ligase enzymes required for D-Alanine incorporation into peptidoglycan. Amino acid transport studies showed that CycA functions as an alanine permease, and the cycA mutation was accompanied by increased accumulation of muropeptides with tripeptide stems lacking the terminal D-Ala-D-Ala, and reduced peptidoglycan cross linking. Exposure of MRSA to DCS was also associated with a dose-dependent accumulation of muropeptides with tripeptide stems and reduced PG crosslinking. Because impaired alanine transport or DCS-treatment have similar effects on PG structure, synergism between {beta}-lactams and DCS was investigated. Therapeutically achievable concentrations of DCS re-sensitised MRSA to {beta}-lactam antibiotics in vitro and significantly enhanced MRSA eradication in a mouse bacteraemia model. The susceptibility of several other ESKAPE group pathogens to {beta}-lactams was also increased by DCS. These data reveal the potential of DCS, or new drugs targeting CycA, to facilitate the reintroduction of {beta}-lactam antibiotics for the treatment of infections caused by MRSA.nnAuthor SummaryTreatment options for infections caused by ESKAPE group pathogens continue to dwindle with increasing antimicrobial resistance. Particularly important are the {beta}-lactams, which remain first line antibiotics for many infections, and finding new ways to maintain their clinical effectiveness is a priority. Here we report that mutation of cycA in MRSA, which was found to encode an alanine permease, was associated with hyper-susceptibility to {beta}-lactam antibiotics and D-cycloserine (DCS), used in the treatment of multi-drug resistant tuberculosis infections. DCS re-sensitised MRSA to oxacillin and other {beta}-lactams in vitro and significantly enhanced oxacillin activity in a mouse model of bacteraemia. DCS also sensitised other ESKAPE pathogens to {beta}-lactams revealing the therapeutic potential of DCS as an adjunct drug for these important antibiotics.

Staphylococcus aureus has the ability to cause infections in multiple organ systems, suggesting an ability to rapidly adapt to changing carbon and nitrogen sources. Although there is little information about the nutrients available at specific sites of infection, a mature skin abscess has been characterized as glucose depleted, indicating that peptides and free amino acids are an important source of nutrients for the bacteria. Our studies have found that mutations in enzymes necessary for growth on amino acids, including pyruvate carboxykinase (ΔpckA) and glutamate dehydrogenase (ΔgudB), reduced the ability of the bacteria to proliferate within a skin abscess, suggesting that peptides and free amino acids are important for S. aureus growth. Furthermore, we found that collagen, an abundant host protein that is present throughout a skin abscess, serves as a reservoir of peptides. To liberate peptides from the collagen, we identified that the host protease, MMP-9, as well as the staphylococcal proteases aureolysin and staphopain B function to cleave collagen into peptide fragments that can support S. aureus growth under nutrient-limited conditions. Moreover, the oligopeptide transporter Opp3 is the primary staphylococcal transporter responsible for peptide acquisition. Lastly, we observed that the presence of peptides (3-mer to 7-mer) induces the expression of aureolysin, suggesting that S. aureus has the ability to detect peptides in the environment. IMPORTANCEStaphylococcus aureus has the ability to cause infections in a variety of niches, suggesting a robust metabolic capacity facilitating proliferation under various nutrient conditions. The mature skin abscess is glucose depleted, indicating that peptides and free amino acids are important sources of nutrients for S. aureus. Our studies have found that mutations in both pyruvate carboxykinase and glutamate dehydrogenase, enzymes that function in essential gluconeogenesis reactions when amino acids serve as the major carbon source, reduce bacterial burden in a murine skin abscess model. Moreover, peptides liberated from collagen by host protease MMP-9 as well as the staphylococcal protease aureolysin support S. aureus growth in an Opp3-dependent manner under nutrient-limited conditions. Additionally, the presence of peptides induces aureolysin expression. Overall, these studies define one pathway by which S. aureus senses a nutrient-limiting environment and induces factors that function to acquire and utilize carbon from host-derived sources.