Is the Success of Cefazolin plus Ertapenem in Methicillin-Susceptible Staphylococcus aureus Bacteremia Based on Release of Interleukin-1 Beta?

Cefazolin and ertapenem have been shown to be an effective salvage regimen for refractory methicillin-susceptible Staphylococcus aureus bacteremia. Our findings suggest cefazolin plus ertapenem in vitro stimulates interleukin-1ß release from peripheral blood monocytes both with and without S. aureus presence. This IL-1ß augmentation was primarily driven by ertapenem. These findings support further exploration of cefazolin plus ertapenem in MSSA bacteremia and may partially explain its marked potency in vivo despite modest synergy in vitro.

The HCM-linked W792R mutation in cardiac myosin-binding protein C reduces C6 FnIII domain stability.

Cardiac myosin-binding protein C (cMyBP-C) is a functional sarcomeric protein that regulates contractility in response to contractile demand, and many mutations in cMyBP-C lead to hypertrophic cardiomyopathy (HCM). To gain insight into the effects of disease-causing cMyBP-C missense mutations on contractile function, we expressed the pathogenic W792R mutation (substitution of a highly conserved tryptophan residue by an arginine residue at position 792) in mouse cardiomyocytes lacking endogenous cMyBP-C and studied the functional effects using three-dimensional engineered cardiac tissue constructs (mECTs). Based on complete conservation of tryptophan at this location in fibronectin type II (FnIII) domains, we hypothesized that the W792R mutation affects folding of the C6 FnIII domain, destabilizing the mutant protein. Adenoviral transduction of wild-type (WT) and W792R cDNA achieved equivalent mRNA transcript abundance, but not equivalent protein levels, with W792R compared with WT controls. mECTs expressing W792R demonstrated abnormal contractile kinetics compared with WT mECTs that were nearly identical to cMyBP-C-deficient mECTs. We studied whether common pathways of protein degradation were responsible for the rapid degradation of W792R cMyBP-C. Inhibition of both ubiquitin-proteasome and lysosomal degradation pathways failed to increase full-length mutant protein abundance to WT equivalence, suggesting rapid cytosolic degradation. Bacterial expression of WT and W792R protein fragments demonstrated decreased mutant stability with altered thermal denaturation and increased susceptibility to trypsin digestion. These data suggest that the W792R mutation destabilizes the C6 FnIII domain of cMyBP-C, resulting in decreased full-length protein expression. This study highlights the vulnerability of FnIII-like domains to mutations that alter domain stability and further indicates that missense mutations in cMyBP-C can cause disease through a mechanism of haploinsufficiency. NEW & NOTEWORTHY This study is one of the first to describe a disease mechanism for a missense mutation in cardiac myosin-binding protein C linked to hypertrophic cardiomyopathy. The mutation decreases stability of the fibronectin type III domain and results in substantially reduced mutant protein expression dissonant to transcript abundance.

Diverse Role of blaCTX-M and Porins in Mediating Ertapenem Resistance among Carbapenem-Resistant Enterobacterales.

Among carbapenem-resistant Enterobacterales (CRE) are diverse mechanisms, including those that are resistant to meropenem but susceptible to ertapenem, adding further complexity to the clinical landscape. This study investigates the emergence of ertapenem-resistant, meropenem-susceptible (ErMs) Escherichia coli and Klebsiella pneumoniae CRE across five hospitals in San Antonio, Texas, USA, from 2012 to 2018. The majority of the CRE isolates were non-carbapenemase producers (NCP; 54%; 41/76); 56% of all NCP isolates had an ErMs phenotype. Among ErMs strains, E. coli comprised the majority (72%). ErMs strains carrying blaCTX-M had, on average, 9-fold higher copies of blaCTX-M than CP-ErMs strains as well as approximately 4-fold more copies than blaCTX-M-positive but ertapenem- and meropenem-susceptible (EsMs) strains (3.7 vs. 0.9, p < 0.001). Notably, carbapenem hydrolysis was observed to be mediated by strains harboring blaCTX-M with and without a carbapenemase(s). ErMs also carried more mobile genetic elements, particularly IS26 composite transposons, than EsMs (37 vs. 0.2, p < 0.0001). MGE- ISVsa5 was uniquely more abundant in ErMs than either EsMs or ErMr strains, with over 30 more average ISVsa5 counts than both phenotype groups (p < 0.0001). Immunoblot analysis demonstrated the absence of OmpC expression in NCP-ErMs E. coli, with 92% of strains lacking full contig coverage of ompC. Overall, our findings characterize both collaborative and independent efforts between blaCTX-M and OmpC in ErMs strains, indicating the need to reappraise the term "non-carbapenemase (NCP)", particularly for strains highly expressing blaCTX-M. To improve outcomes for CRE-infected patients, future efforts should focus on mechanisms underlying the emerging ErMs subphenotype of CRE strains to develop technologies for its rapid detection and provide targeted therapeutic strategies.

Oxygen dose responsiveness of human fetal airway smooth muscle cells.

Maintenance of blood oxygen saturation dictates supplemental oxygen administration to premature infants, but hyperoxia predisposes survivors to respiratory diseases such as asthma. Although much research has focused on oxygen effects on alveoli in the setting of bronchopulmonary dysplasia, the mechanisms by which oxygen affects airway structure or function relevant to asthma are still under investigation. We used isolated human fetal airway smooth muscle (fASM) cells from 18-20 postconceptual age lungs (canalicular stage) to examine oxygen effects on intracellular Ca(2+) ([Ca(2+)](i)) and cellular proliferation. fASM cells expressed substantial smooth muscle actin and myosin and several Ca(2+) regulatory proteins but not fibroblast or epithelial markers, profiles qualitatively comparable to adult human ASM. Fluorescence Ca(2+) imaging showed robust [Ca(2+)](i) responses to 1 µM acetylcholine (ACh) and 10 µM histamine (albeit smaller and slower than adult ASM), partly sensitive to zero extracellular Ca(2+). Compared with adult, fASM showed greater baseline proliferation. Based on this validation, we assessed fASM responses to 10% hypoxia through 90% hyperoxia and found enhanced proliferation at <60% oxygen but increased apoptosis at >60%, effects accompanied by appropriate changes in proliferative vs. apoptotic markers and enhanced mitochondrial fission at >60% oxygen. [Ca(2+)](i) responses to ACh were enhanced for <60% but blunted at >60% oxygen. These results suggest that hyperoxia has dose-dependent effects on structure and function of developing ASM, which could have consequences for airway diseases of childhood. Thus detrimental effects on ASM should be an additional consideration in assessing risks of supplemental oxygen in prematurity.

Thymic stromal lymphopoietin in cigarette smoke-exposed human airway smooth muscle.

Thymic stromal lymphopoietin (TSLP) is a newly identified IL-7-like cytokine known to be expressed in airway biopsies of patients with asthma and chronic obstructive pulmonary disease. As both diseases may be induced or exacerbated by cigarette smoking, it is possible that TSLP represents an important link between cigarette smoke exposure and inflammatory signaling in the airways. In this regard, TSLP appears to also be expressed in airway smooth muscle (ASM); however, its role is unknown. In the current study, we examined TSLP and the TSLP receptor (TSLP-R) expression and function in human ASM cells under normal conditions and following exposure to cigarette smoke extract (CSE). Western blot analysis of human ASM cells showed significant expression of TSLP and TSLP-R, with increased expression of both by overnight exposure to 1 or 2% CSE. Furthermore, CSE increased TSLP release by ASM. In parallel experiments using enzymatically dissociated human ASM cells loaded with the Ca(2+) indicator fura 2-AM and imaged using fluorescence microscopy, we evaluated the effects of CSE exposure on intracellular Ca(2+) ([Ca(2+)](i)) responses to agonist stimulation. [Ca(2+)](i) responses to histamine were increased with overnight CSE exposure. Exposure to TSLP also resulted in elevated responses, which were blunted by TSLP and TSLP-R Abs. Importantly, the enhancing effects of CSE on [Ca(2+)](i) responses were also blunted by these Abs. These effects were associated with CSE- and TSLP-induced changes in STAT5 phosphorylation. Overall, these novel data suggest that cigarette smoke, TSLP, and ASM are functionally linked and that cigarette smoke-induced increase in airway contractility may be mediated via ASM-derived increases in TSLP signaling.

Potent Activity of Ertapenem Plus Cefazolin Within Staphylococcal Biofilms: A Contributing Factor in the Treatment of Methicillin-Susceptible Staphylococcus aureus Endocarditis.

Background: Besides antistaphylococcal beta-lactams and source control, there are limited validated antimicrobial salvage options in patients with prolonged methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia, including infective endocarditis (IE). Methods: MSSA IE cases treated with ertapenem (ETP) plus cefazolin (CZ) were compared with matched IE cases treated with standard beta-lactam monotherapy. The bactericidal activity of ETP plus CZ was also compared with nafcillin (NAF), CZ, and ETP alone using an in vitro MSSA biofilm model. Results: The median duration of bacteremia experienced by patients (n = 12) while on CZ or NAF was 4 days (range 1-16 days) compared with 1 day (range 1-3 days) for patients (n = 5) treated with ETP + CZ (P = .01, Mann-Whitney U test). Cefazolin and NAF alone or in combination did not achieve biofilm eradication at clinically relevant concentrations. However, the addition of ETP to CZ led to bactericidal eradication within biofilms at standard dosing. Conclusions: Ertapenem reduces CZ concentrations required to eradicate MSSA biofilms to those achievable in vivo by standard dosing, translating into shorter bacteremia duration in patients with MSSA endocarditis. Larger studies are needed to investigate ETP plus CZ therapy in the treatment of biofilm-related MSSA infections such as endocarditis.

Long-Acting Lipoglycopeptides Can Interfere With Vancomycin Therapeutic Drug Monitoring.

Oritavancin and dalbavancin are long-acting lipoglycopeptides with activity against susceptible gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. Though similar in structure to traditional glycopeptide antibiotics like vancomycin, these antibiotics have terminal half-lives >10 days, and, as a result, there is potential for administration of vancomycin to a patient while oritavancin or dalbavancin are still appreciably present in serum. Given the structural similarities, this creates an opportunity for lab assay interference when performing therapeutic drug monitoring for vancomycin. Following higher-than-expected serum vancomycin concentrations in a patient who received both oritavancin and vancomycin within a short time frame, we evaluated the potential for lipoglycopeptide interference with clinical vancomycin assays. Five platforms covering 3 immunoassay technologies were used to quantify vancomycin concentrations in serum spiked with oritavancin or dalbavancin. Oritavancin generated spurious vancomycin concentrations (20%-84% increase) in both enzyme-multiplied immunoassay technique and a particle-enhanced turbidimetric inhibition immunoassay. However, the improper detection of oritavancin was not consistent across all particle-enhanced turbidimetric inhibition immunoassay platforms. Dalbavancin interference was not detected on any of the platforms tested. The interference from oritavancin may result in falsely elevated vancomycin concentrations and, subsequently, inappropriately adjusted vancomycin doses.

Neurokinin-neurotrophin interactions in airway smooth muscle.

Neurally derived tachykinins such as substance P (SP) play a key role in modulating airway contractility (especially with inflammation). Separately, the neurotrophin brain-derived neurotrophic factor (BDNF; potentially derived from nerves as well as airway smooth muscle; ASM) and its tropomyosin-related kinase receptor, TrkB, are involved in enhanced airway contractility. In this study, we hypothesized that neurokinins and neurotrophins are linked in enhancing intracellular Ca(2+) concentration ([Ca(2+)](i)) regulation in ASM. In rat ASM cells, 24 h exposure to 10 nM SP significantly increased BDNF and TrkB expression (P < 0.05). Furthermore, [Ca(2+)](i) responses to 1 µM ACh as well as BDNF (30 min) effects on [Ca(2+)](i) regulation were enhanced by prior SP exposure, largely via increased Ca(2+) influx (P < 0.05). The enhancing effect of SP on BDNF signaling was blunted by the neurokinin-2 receptor antagonist MEN-10376 (1 µM, P < 0.05) to a greater extent than the neurokinin-1 receptor antagonist RP-67580 (5 nM). Chelation of extracellular BDNF (chimeric TrkB-F(c); 1 µg/ml), as well as tyrosine kinase inhibition (100 nM K252a), substantially blunted SP effects (P < 0.05). Overnight (24 h) exposure of ASM cells to 50% oxygen increased BDNF and TrkB expression and potentiated both SP- and BDNF-induced enhancement of [Ca(2+)](i) (P < 0.05). These results suggest a novel interaction between SP and BDNF in regulating agonist-induced [Ca(2+)](i) regulation in ASM. The autocrine mechanism we present here represents a new area in the development of bronchoconstrictive reflex response and airway hyperreactive disorders.

Role of Hypoxia-Induced Brain Derived Neurotrophic Factor in Human Pulmonary Artery Smooth Muscle.

BACKGROUND: Hypoxia effects on pulmonary artery structure and function are key to diseases such as pulmonary hypertension. Recent studies suggest that growth factors called neurotrophins, particularly brain-derived neurotrophic factor (BDNF), can influence lung structure and function, and their role in the pulmonary artery warrants further investigation. In this study, we examined the effect of hypoxia on BDNF in humans, and the influence of hypoxia-enhanced BDNF expression and signaling in human pulmonary artery smooth muscle cells (PASMCs). METHODS AND RESULTS: 48h of 1% hypoxia enhanced BDNF and TrkB expression, as well as release of BDNF. In arteries of patients with pulmonary hypertension, BDNF expression and release was higher at baseline. In isolated PASMCs, hypoxia-induced BDNF increased intracellular Ca2+ responses to serotonin: an effect altered by HIF1α inhibition or by neutralization of extracellular BDNF via chimeric TrkB-Fc. Enhanced BDNF/TrkB signaling increased PASMC survival and proliferation, and decreased apoptosis following hypoxia. CONCLUSIONS: Enhanced expression and signaling of the BDNF-TrkB system in PASMCs is a potential mechanism by which hypoxia can promote changes in pulmonary artery structure and function. Accordingly, the BDNF-TrkB system could be a key player in the pathogenesis of hypoxia-induced pulmonary vascular diseases, and thus a potential target for therapy.

Brain-derived neurotrophic factor enhances calcium regulatory mechanisms in human airway smooth muscle.

Neurotrophins (NTs), which play an integral role in neuronal development and function, have been found in non-neuronal tissue (including lung), but their role is still under investigation. Recent reports show that NTs such as brain-derived neurotrophic factor (BDNF) as well as NT receptors are expressed in human airway smooth muscle (ASM). However, their function is still under investigation. We hypothesized that NTs regulate ASM intracellular Ca(2+) ([Ca(2+)](i)) by altered expression of Ca(2+) regulatory proteins. Human ASM cells isolated from lung samples incidental to patient surgery were incubated for 24 h (overnight) in medium (control) or 1 nM BDNF in the presence vs. absence of inhibitors of signaling cascades (MAP kinases; PI3/Akt; NFκB). Measurement of [Ca(2+)](i) responses to acetylcholine (ACh) and histamine using the Ca(2+) indicator fluo-4 showed significantly greater responses following BDNF exposure: effects that were blunted by pathway inhibitors. Western analysis of whole cell lysates showed significantly higher expression of CD38, Orai1, STIM1, IP(3) and RyR receptors, and SERCA following BDNF exposure, effects inhibited by inhibitors of the above cascades. The functional significance of BDNF effects were verified by siRNA or pharmacological inhibition of proteins that were altered by this NT. Overall, these data demonstrate that NTs activate signaling pathways in human ASM that lead to enhanced [Ca(2+)](i) responses via increased regulatory protein expression, thus enhancing airway contractility.