Systematic Analysis of Mobile Genetic Elements Mediating ß-Lactamase Gene Amplification in Noncarbapenemase-Producing Carbapenem-Resistant Enterobacterales Bloodstream Infections.

Noncarbapenemase-producing carbapenem-resistant Enterobacterales (non-CP-CRE) are increasingly recognized as important contributors to prevalent carbapenem-resistant Enterobacterales (CRE) infections. However, there is limited understanding of mechanisms underlying non-CP-CRE causing invasive disease. Long- and short-read whole-genome sequencing was used to elucidate carbapenem nonsusceptibility determinants in Enterobacterales bloodstream isolates at MD Anderson Cancer Center in Houston, Texas. We investigated carbapenem nonsusceptible Enterobacterales (CNSE) mechanisms (i.e., isolates with carbapenem intermediate resistance phenotypes or greater) through a combination of phylogenetic analysis, antimicrobial resistance gene detection/copy number quantification, porin assessment, and mobile genetic element (MGE) characterization. Most CNSE isolates sequenced were non-CP-CRE (41/79; 51.9%), whereas 25.3% (20/79) were Enterobacterales with intermediate susceptibility to carbapenems (CIE), and 22.8% (18/79) were carbapenemase-producing Enterobacterales (CPE). Statistically significant copy number variants (CNVs) of extended-spectrum ß-lactamase (ESBL) genes (Wilcoxon Test; P-value < 0.001) were present in both non-CP-CR E. coli (median CNV = 2.6×; n = 17) and K. pneumoniae (median CNV = 3.2×, n = 17). All non-CP-CR E. coli and K. pneumoniae had predicted reduced expression of at least one outer membrane porin gene (i.e., ompC/ompF or ompK36/ompK35). Completely resolved CNSE genomes revealed that IS26 and ISEcp1 structures harboring blaCTX-M variants along with other antimicrobial resistance elements were associated with gene amplification, occurring in mostly IncFIB/IncFII plasmid contexts. MGE-mediated ß-lactamase gene amplifications resulted in either tandem arrays, primarily mediated by IS26 translocatable units, or segmental duplication, typically due to ISEcp1 transposition units. Non-CP-CRE strains were the most common cause of CRE bacteremia with carbapenem nonsusceptibility driven by concurrent porin loss and MGE-mediated amplification of blaCTX-M genes. IMPORTANCE Carbapenem-resistant Enterobacterales (CRE) are considered urgent antimicrobial resistance (AMR) threats. The vast majority of CRE research has focused on carbapenemase-producing Enterobacterales (CPE) even though noncarbapenemase-producing CRE (non-CP-CRE) comprise 50% or more of isolates in some surveillance studies. Thus, carbapenem resistance mechanisms in non-CP-CRE remain poorly characterized. To address this problem, we applied a combination of short- and long-read sequencing technologies to a cohort of CRE bacteremia isolates and used these data to unravel complex mobile genetic element structures mediating ß-lactamase gene amplification. By generating complete genomes of 65 carbapenem nonsusceptible Enterobacterales (CNSE) covering a genetically diverse array of isolates, our findings both generate novel insights into how non-CP-CRE overcome carbapenem treatments and provide researchers scaffolds for characterization of their own non-CP-CRE isolates. Improved recognition of mechanisms driving development of non-CP-CRE could assist with design and implementation of future strategies to mitigate the impact of these increasingly recognized AMR pathogens.

The role of endometrial staining for CD138 as a marker of chronic endometritis in predicting live birth.

PURPOSE: Chronic endometritis (CE) is diagnosed via endometrial biopsy and staining for plasma cells. A threshold plasma cell count that identifies CE and predicts pregnancy outcomes has not been established, and the prevalence of plasma cells in the general infertile population is unknown. The purpose of this study was to determine the prevalence of plasma cells in the general infertile population and whether a threshold exists which predicts live birth. METHODS: Endometrial samples were obtained prospectively from 80 women undergoing IVF, embedded in paraffin, and stained for plasma cells using mouse mono-clonal antibody for CD138. Slides were reviewed at 20× magnification and 10 random images captured. Three reviewers graded each image for plasma cells. Participants underwent single, euploid, and frozen blastocyst transfer. RESULTS: Forty-nine percent of samples had ≥1 plasma cell across 10 HPFs, 11% had ≥5 cells across 10 HPFs, and 4% had ≥10 cells across 10 HPFs. There was no difference in prevalence between those who did and did not achieve live birth. Using thresholds of 1, 5, and 10 plasma cells per 10 HPFs, there were no differences in implantation, clinical pregnancy, clinical pregnancy loss, or live birth rates between patients with and without CE. CONCLUSION: Endometrial plasma cells are present in half the general infertile population and do not predict implantation, clinical pregnancy, clinical pregnancy loss, or live birth rates at low levels.

Emerging evidence for a mechanistic link between low-frequency oscillation of ventricular repolarization measured from the electrocardiogram T-wave vector and arrhythmia.

Strong recent clinical evidence links the presence of prominent oscillations of ventricular repolarization in the low-frequency range (0.04-0.15 Hz) to the incidence of ventricular arrhythmia and sudden death in post-MI patients and patients with ischaemic and non-ischaemic cardiomyopathy. It has been proposed that these oscillations reflect oscillations of ventricular action potential duration at the sympathetic nerve frequency. Here we review emerging evidence to support that contention and provide insight into possible underlying mechanisms for this association.

Persistent confirmed low-grade dysplasia in Barrett's esophagus is a risk factor for progression to high-grade dysplasia and adenocarcinoma in a US Veterans cohort.

Patients with Barrett's esophagus (BE) and low-grade dysplasia (LGD) are at increased risk of esophageal adenocarcinoma (EAC), although many regress to nondysplastic BE. This has significant clinical importance for patients being considered for endoscopic eradication therapy. Our aim is to determine the risk for progression in patients with confirmed persistent LGD. We performed a single-center retrospective cohort study of patients with BE and confirmed LGD between 2006 and 2016. Confirmed LGD was defined as LGD diagnosed by consensus conference with an expert GI pathologist or review by an expert GI pathologist and persistence as LGD present on subsequent endoscopic biopsy. The primary outcome was the incidence rate of HGD (high-grade dysplasia)/EAC. Secondary outcomes included risk factors for dysplastic progression. Risk factors for progression were assessed using univariate and multivariate analysis with logistic regression. Of 69 patients (mean age 65.2 years) with confirmed LGD were included. In total, 16 of 69 patients (23.2%) with LGD developed HGD/EAC during a median follow-up of 3.74 years (IQR, 1.24-5.45). For persistent confirmed LGD, the rate was 6.44 (95% confidence interval (CI), 2.61-13.40) compared to 2.61 cases per 100 patient-years (95% CI, 0.83-6.30) for nonpersistent LGD. Persistent LGD was found in only 29% of patients. Persistent LGD was an independent risk factor for the development of HGD/EAC (OR 4.18; [95% CI, 1.03-17.1]). Persistent confirmed LGD, present in only 1/3 of patients, was an independent risk factor for the development of HGD/EAC. Persistence LGD may be useful in decision making regarding the management of BE.

A study of dysphagia symptoms and esophageal body function in children undergoing anti-reflux surgery.

BACKGROUND: The role of high-resolution esophageal impedance manometry (HRIM) for establishing risk for dysphagia after anti-reflux surgery is unclear. We conducted a prospective study of children with primary gastroesophageal reflux (GER) disease, for whom symptoms of dysphagia were determined pre-operatively and then post-operatively and we examined for features that may predict post-operative dysphagia. METHODS: Thirteen children (aged 6.8-15.5 years) undergoing work-up prior to 360o Nissen fundoplication were included in the study. A dysphagia score assessed symptoms at pre-operative study and post-operatively (mean 1.4 years). A HRIM procedure recorded 5-ml liquid, 5-ml viscous and 2-cm solid boluses. We assessed esophageal motility, esophago-gastric junction (EGJ) morphology, EGJ contractility and pressure-flow variables indicative of bolus distension pressures and bolus clearance pressures. A composite pressure-flow index score was also derived. RESULTS: Pre-operative pressure-flow index was positively correlated with post-operative dysphagia score (viscous bolus r = 0.771, p < 0.005). Of three variables that comprise the pressure-flow index, the ramp pressure measured during bolus clearance was the main driver of the effect seen (viscous bolus r = 0.819, p < 0.005). CONCLUSIONS: In order to mitigate symptoms in relation to anti-reflux surgery, dysphagia symptoms and esophageal function need to be pre-operatively assessed. In patients with normal motility, an elevated pressure-flow index may predict post-operative dysphagia.

Staphylococcus aureus in the nose and throat of Iowan families.

The study objective was to determine the prevalence of Staphylococcus aureus colonisation in the nares and oropharynx of healthy persons and identify any risk factors associated with such S. aureus colonisation. In total 263 participants (177 adults and 86 minors) comprising 95 families were enrolled in a year-long prospective cohort study from one urban and one rural county in eastern Iowa, USA, through local newspaper advertisements and email lists and through the Keokuk Rural Health Study. Potential risk factors including demographic factors, medical history, farming and healthcare exposure were assessed. Among the participants, 25.4% of adults and 36.1% minors carried S. aureus in their nares and 37.9% of adults carried it in their oropharynx. The overall prevalence was 44.1% among adults and 36.1% for minors. Having at least one positive environmental site for S. aureus in the family home was associated with colonisation (prevalence ratio: 1.34, 95% CI: 1.07-1.66). The sensitivity of the oropharyngeal cultures was greater than that of the nares cultures (86.1% compared with 58.2%, respectively). In conclusion, the nares and oropharynx are both important colonisation sites for healthy community members and the presence of S. aureus in the home environment is associated with an increased probability of colonisation.

Examining the sustainability potential of a multisite pilot to integrate alcohol screening and brief intervention within three primary care systems.

The U.S. Preventive Services Task Force recommends that clinicians adopt universal alcohol screening and brief intervention as a routine preventive service for adults, and efforts are underway to support its widespread dissemination. The likelihood that healthcare systems will sustain this change, once implemented, is under-reported in the literature. This article identifies factors that were important to postimplementation sustainability of an evidence-based practice change to address alcohol misuse that was piloted within three diverse primary care organizations. The Centers for Disease Control and Prevention funded three academic teams to pilot and evaluate implementation of alcohol screening and brief intervention within multiclinic healthcare systems in their respective regions. Following the completion of the pilots, teams used the Program Sustainability Assessment Tool to retrospectively describe and compare differences across eight sustainability domains, identify strengths and potential threats to sustainability, and make recommendations for improvement. Health systems varied across all domains, with greatest differences noted for Program Evaluation, Strategic Planning, and Funding Stability. Lack of funding to sustain practice change, or data monitoring to promote fit and fidelity, was an indication of diminished Organizational Capacity in systems that discontinued the service after the pilot. Early assessment of sustainability factors may identify potential threats that could be addressed prior to, or during implementation to enhance Organizational Capacity. Although this study provides a retrospective assessment conducted by external academic teams, it identifies factors that may be relevant for translating evidence-based behavioral interventions in a way that assures that they are sustained within healthcare systems.

Effects of Extracellular Polymeric Substance Composition on Bacteria Disinfection by Monochloramine: Application of MALDI-TOF/TOF-MS and Multivariate Analysis.

In our previous study, we reported that the transport of monochloramine is affected by the extracellular polymeric substance (EPS) composition, which in turn affects the cell viability of both biofilm and detached clusters.11 However, although the transport and reaction of monochloramine in biofilm could be observed, the specific biomolecules reacting with the disinfectant and the mechanism of disinfection remains elusive. In this study, the impact of EPS composition on bacteria disinfection by monochloramine was qualitatively determined using both wild-type and isogenic mutant Pseudomonas strains with different EPS-secretion capacity and composition. To evaluate their EPS reactivity and contribution to susceptibility to monochloramine, we investigated the bacteria disinfection process using Fourier transform infrared spectroscopy (FTIR) and matrix-assisted laser desorption-ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Canonical correlation analysis and partial least-squares regression modeling were employed to explore the changes that EPS underwent during the monochloramine disinfection process. The analyses results suggested significant reactions of the monochloramine with peptide fragments of proteins that are associated with carbohydrate utilization. Selected enzymes also showed different levels of inhibition by monochloramine when tested.

Visualizing the Bohr effect in hemoglobin: neutron structure of equine cyanomethemoglobin in the R state and comparison with human deoxyhemoglobin in the T state.

Neutron crystallography provides direct visual evidence of the atomic positions of deuterium-exchanged H atoms, enabling the accurate determination of the protonation/deuteration state of hydrated biomolecules. Comparison of two neutron structures of hemoglobins, human deoxyhemoglobin (T state) and equine cyanomethemoglobin (R state), offers a direct observation of histidine residues that are likely to contribute to the Bohr effect. Previous studies have shown that the T-state N-terminal and C-terminal salt bridges appear to have a partial instead of a primary overall contribution. Four conserved histidine residues [αHis72(EF1), αHis103(G10), αHis89(FG1), αHis112(G19) and ßHis97(FG4)] can become protonated/deuterated from the R to the T state, while two histidine residues [αHis20(B1) and ßHis117(G19)] can lose a proton/deuteron. αHis103(G10), located in the α1:ß1 dimer interface, appears to be a Bohr group that undergoes structural changes: in the R state it is singly protonated/deuterated and hydrogen-bonded through a water network to ßAsn108(G10) and in the T state it is doubly protonated/deuterated with the network uncoupled. The very long-term H/D exchange of the amide protons identifies regions that are accessible to exchange as well as regions that are impermeable to exchange. The liganded relaxed state (R state) has comparable levels of exchange (17.1% non-exchanged) compared with the deoxy tense state (T state; 11.8% non-exchanged). Interestingly, the regions of non-exchanged protons shift from the tetramer interfaces in the T-state interface (α1:ß2 and α2:ß1) to the cores of the individual monomers and to the dimer interfaces (α1:ß1 and α2:ß2) in the R state. The comparison of regions of stability in the two states allows a visualization of the conservation of fold energy necessary for ligand binding and release.

Effective emotion regulation strategies improve fMRI and ECG markers of psychopathology in panic disorder: implications for psychological treatment action.

Impairments in emotion regulation are thought to have a key role in the pathogenesis of anxiety disorders, but the neurobiological underpinnings contributing to vulnerability remain poorly understood. It has been a long-held view that exaggerated fear is linked to hyperresponsivity of limbic brain areas and impaired recruitment of prefrontal control. However, increasing evidence suggests that prefrontal-cortical networks are hyperactive during threat processing in anxiety disorders. This study directly explored limbic-prefrontal neural response, connectivity and heart-rate variability (HRV) in patients with a severe anxiety disorder during incidental versus intentional emotion regulation. During 3 Tesla functional magnetic resonance imaging, 18 participants with panic disorder and 18 healthy controls performed an emotion regulation task. They either viewed negative images naturally (Maintain), or they were instructed to intentionally downregulate negative affect using previously taught strategies of cognitive reappraisal (Reappraisal). Electrocardiograms were recorded throughout to provide a functional measure of regulation and emotional processing. Compared with controls, patients showed increased neural activation in limbic-prefrontal areas and reduced HRV during incidental emotion regulation (Maintain). During intentional regulation (Reappraisal), group differences were significantly attenuated. These findings emphasize patients' ability to regulate negative affect if provided with adaptive strategies. They also bring prefrontal hyperactivation forward as a potential mechanism of psychopathology in anxiety disorders. Although these results challenge models proposing impaired allocation of prefrontal resources as a key characteristic of anxiety disorders, they are in line with more recent neurobiological frameworks suggesting that prefrontal hyperactivation might reflect increased utilisation of maladaptive regulation strategies quintessential for anxiety disorders.

Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography.

Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pKa values of key side chains during catalysis. However, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD=pH+0.4) values. The general acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. We performed continuum electrostatics calculations to estimate the pKa values of the catalytic Glu residues in both the apo- and substrate-bound states of the enzyme. The calculated pKa of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. These findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen.

Molecular typing of antibiotic-resistant Staphylococcus aureus in Nigeria.

BACKGROUND: Antibiotic-resistant Staphylococcus aureus including methicillin-resistant strains (MRSA) are a major concern in densely populated urban areas. Initial studies of S. aureus in Nigeria indicated existence of antibiotic-resistant S. aureus strains in clinical and community settings. METHODS: 73 biological samples (40 throat, 23 nasal, 10 wound) were collected from patients and healthcare workers in three populations in Nigeria: Lagos University Teaching Hospital, Nigerian Institute of Medical Research, and Owerri General Hospital. RESULTS: S. aureus was isolated from 38 of 73 samples (52%). Of the 38 S. aureus samples, 9 (24%) carried the Panton-Valentine leukocidin gene (PVL) while 16 (42%) possessed methicillin resistance genes (mecA). Antibiotic susceptibility profiles indicated resistance to several broad-spectrum antibiotics. CONCLUSION: Antibiotic-resistant S. aureus isolates were recovered from clinical and community settings in Nigeria. Insight about S. aureus in Nigeria may be used to improve antibiotic prescription methods and minimize the spread of antibiotic-resistant organisms in highly populated urban communities similar to Lagos, Nigeria.

L-Arabinose binding, isomerization, and epimerization by D-xylose isomerase: X-ray/neutron crystallographic and molecular simulation study.

D-xylose isomerase (XI) is capable of sugar isomerization and slow conversion of some monosaccharides into their C2-epimers. We present X-ray and neutron crystallographic studies to locate H and D atoms during the respective isomerization and epimerization of L-arabinose to L-ribulose and L-ribose, respectively. Neutron structures in complex with cyclic and linear L-arabinose have demonstrated that the mechanism of ring-opening is the same as for the reaction with D-xylose. Structural evidence and QM/MM calculations show that in the reactive Michaelis complex L-arabinose is distorted to the high-energy (5)S1 conformation; this may explain the apparent high KM for this sugar. MD-FEP simulations indicate that amino acid substitutions in a hydrophobic pocket near C5 of L-arabinose can enhance sugar binding. L-ribulose and L-ribose were found in furanose forms when bound to XI. We propose that these complexes containing Ni(2+) cofactors are Michaelis-like and the isomerization between these two sugars proceeds via a cis-ene-diol mechanism.

Slow adaptation of ventricular repolarization as a cause of arrhythmia?

INTRODUCTION: This article is part of the Focus Theme of Methods of Information in Medicine on "Biosignal Interpretation: Advanced Methods for Studying Cardiovascular and Respiratory Systems". BACKGROUND: Adaptation of the QT-interval to changes in heart rate reflects on the body-surface electrocardiogram the adaptation of action potential duration (APD) at the cellular level. The initial fast phase of APD adaptation has been shown to modulate the arrhythmia substrate. Whether the slow phase is potentially proarrhythmic remains unclear. OBJECTIVES: To analyze in-vivo human data and use computer simulations to examine effects of the slow APD adaptation phase on dispersion of repolarization and reentry in the human ventricle. METHODS: Electrograms were acquired from 10 left and 10 right ventricle (LV/RV) endocardial sites in 15 patients with normal ventricles during RV pacing. Activation-recovery intervals, as a surrogate for APD, were measured during a sustained increase in heart rate. Observed dynamics were studied using computer simulations of human tissue electrophysiology. RESULTS: Spatial heterogeneity of rate adaptation was observed in all patients. Inhomogeneity in slow APD adaptation time constants (Δτ(s)) was greater in LV than RV (Δτ(s)(LV) = 31.8 ± 13.2, Δτ(s)(RV) = 19.0 ± 12.8 s , P< 0.01). Simulations showed that altering local slow time constants of adaptation was sufficient to convert partial wavefront block to block with successful reentry. CONCLUSIONS: Using electrophysiological data acquired in-vivo in human and computer simulations, we identify heterogeneity in the slow phase of APD adaptation as an important component of arrhythmogenesis.

Label-free electronic detection of bio-toxins using aligned carbon nanotubes.

A facile route for sensitive label-free detection of bio-toxins using aligned single walled carbon nanotubes is described. This approach involves patterning of a catalyst on the surface of a quartz substrate using a sub-100 µm stripe-patterned polydimethylsiloxane stamp for aligned carbon nanotube generation followed by fabrication of field effect transistor (FET). Atomic force microscopy, field emission scanning electron microscopy and Raman spectroscopy are employed to characterize the synthesized nanotubes. Unlike previous reports, the adopted approach enables direct electronic detection of bio-toxins with sensitivities comparable to ELISA. As a proof of concept, the fabricated FET responds to nM concentration levels (with a LOD of ∼2 nM) of epsilon toxin produced by Clostridium perfringens and a prominent food toxin. This facile approach could be customized to detect other classes of toxins and biomarkers upon appropriate functionalization of the aligned carbon nanotubes. Finally, we demonstrate the use of the FET-platform for detection of toxin in more complex matrices such as orange juice.

Prevalence and characteristics of Staphylococcus aureus in Connecticut swine and swine farmers.

This study explores the characteristics of Staphylococcus aureus (S. aureus) in swine and their human handlers in a convenience sample of 35 farms in Connecticut. Husbandry practices are clearly different from better-known concentrated animal feeding operations (CAFOs) with less intensive rearing conditions. Nasal samples were collected from 263 pigs and nine humans on 35 farms during the 2010 rearing season. Samples were analysed using established microbiology methods, and resulting methicillin-sensitive (MSSA) and resistant (MRSA) isolates were typed by pulsed-field gel electrophoresis (PFGE) and spa typing. PCR was used to detect the presence of the Panton-Valentine Leukocidin (PVL) gene, a cytotoxin usually associated with CA-MRSA infection. A farm assessment form and questionnaire were used to obtain the information about husbandry practices and human exposure risk, respectively. Staphylococcus aureus colonized swine and humans were found in 51% (18/35) of the farms sampled at a rate of 30% (85/259) and 22% (2/9), respectively. Eight pigs and two humans were MRSA positive on five farms. MRSA in swine was related to healthcare-associated (HA), community-associated (CA) or livestock-associated (LA) MRSA strains, whereas humans were colonized with HA-MRSA. On the basis of spa typing, there was evidence of human-animal transmission thereby signifying humanosis/reverse zoonoses. The PVL gene was found in 88% (7/8) of MRSA swine isolates, the first time this gene has been seen in colonized pigs sampled on US farm. MSSA isolates belonged to six spa types: t337 (41%), t034 (12%), t334 (12%), t4529 (12%), t8760 (18%) and t1166 (6%) including LA strains. This is the first time spa type t8760 has been reported and the only MSSA with the PVL gene. In summary, MRSA including LA strains (LA-MRSA) can be found on small farms with different husbandry practices from CAFOs, suggesting that preventive measures for zoonotic MRSA infection should address a range of animal production.

Inhibition of D-xylose isomerase by polyols: atomic details by joint X-ray/neutron crystallography.

D-Xylose isomerase (XI) converts the aldo-sugars xylose and glucose to their keto analogs xylulose and fructose, but is strongly inhibited by the polyols xylitol and sorbitol, especially at acidic pH. In order to understand the atomic details of polyol binding to the XI active site, a 2.0 Å resolution room-temperature joint X-ray/neutron structure of XI in complex with Ni(2+) cofactors and sorbitol inhibitor at pH 5.9 and a room-temperature X-ray structure of XI containing Mg(2+) ions and xylitol at the physiological pH of 7.7 were obtained. The protonation of oxygen O5 of the inhibitor, which was found to be deprotonated and negatively charged in previous structures of XI complexed with linear glucose and xylulose, was directly observed. The Ni(2+) ions occupying the catalytic metal site (M2) were found at two locations, while Mg(2+) in M2 is very mobile and has a high B factor. Under acidic conditions sorbitol gains a water-mediated interaction that connects its O1 hydroxyl to Asp257. This contact is not found in structures at basic pH. The new interaction that is formed may improve the binding of the inhibitor, providing an explanation for the increased affinity of the polyols for XI at low pH.