Programming of DNA methylation patterns.

DNA methylation represents a form of genome annotation that mediates gene repression by serving as a maintainable mark that can be used to reconstruct silent chromatin following each round of replication. During development, germline DNA methylation is erased in the blastocyst, and a bimodal pattern is established anew at the time of implantation when the entire genome gets methylated while CpG islands are protected. This brings about global repression and allows housekeeping genes to be expressed in all cells of the body. Postimplantation development is characterized by stage- and tissue-specific changes in methylation that ultimately mold the epigenetic patterns that define each individual cell type. This is directed by sequence information in DNA and represents a secondary event that provides long-term expression stability. Abnormal methylation changes play a role in diseases, such as cancer or fragile X syndrome, and may also occur as a function of aging or as a result of environmental influences.

Hepatocyte regeneration is driven by embryo-like DNA methylation reprogramming.

As a result of partial hepatectomy, the remaining liver tissue undergoes a process of renewed proliferation that leads to rapid regeneration of the liver. By following the early stages of this process, we observed dramatic programmed changes in the DNA methylation profile, characterized by both de novo and demethylation events, with a subsequent return to the original adult pattern as the liver matures. Strikingly, these transient alterations partially mimic the DNA methylation state of embryonic hepatoblasts (E16.5), indicating that hepatocytes actually undergo epigenetic dedifferentiation. Furthermore, Tet2/Tet3-deletion experiments demonstrated that these changes in methylation are necessary for carrying out basic embryonic functions, such as proliferation, a key step in liver regeneration. This implies that unlike tissue-specific regulatory regions that remain demethylated in the adult, early embryonic genes are programmed to first undergo demethylation, followed by remethylation as development proceeds. The identification of this built-in system may open targeting opportunities for regenerative medicine.

Muscle injury causes long-term changes in stem-cell DNA methylation.

Injury to muscle brings about the activation of stem cells, which then generate new myocytes to replace damaged tissue. We demonstrate that this activation is accompanied by a dramatic change in the stem-cell methylation pattern that prepares them epigenetically for terminal myocyte differentiation. These de- and de novo methylation events occur at regulatory elements associated with genes involved in myogenesis and are necessary for activation and regeneration. Local injury of one muscle elicits an almost identical epigenetic change in satellite cells from other muscles in the body, in a process mediated by circulating factors. Furthermore, this same methylation state is also generated in muscle stem cells (MuSCs) of female animals following pregnancy, even in the absence of any injury. Unlike the activation-induced expression changes, which are transient, the induced methylation profile is stably maintained in resident MuSCs and thus represents a molecular memory of previous physiological events that is probably programmed to provide a mechanism for long-term adaptation.

Is Carbapenem Therapy Necessary for the Treatment of Non-CTX-M ESBL-Producing Enterobacterales Bloodstream Infections?

BACKGROUND: Investigations into antibiotics for extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-E) bloodstream infections (BSIs) have focused on blaCTX-M genes. Outcomes of patients with non-CTX-M-producing ESBL-E BSIs and optimal treatment are unknown. METHODS: A multicenter observational study investigating 500 consecutive patients with ceftriaxone-resistant Enterobacterales BSIs during 2018-2022 was conducted. Broth microdilution and whole genome sequencing confirmed antibiotic susceptibilities and ESBL gene presence, respectively. Inverse probability weighting (IPW) using propensity scores was employed to ensure patients infected with non-CTX-M and CTX-M ESBL-E BSIs were similar prior to evaluation of outcomes. RESULTS: 396 patients (79.2%) were confirmed to have an ESBL-E BSI. ESBL gene family prevalence was as follows: blaCTX-M (n=370), blaSHV (n=16), blaOXY (n=12), and blaVEB (n=5). ESBL gene identification was not limited to Escherichia coli and Klebsiella species. In the IPW cohort, there was no difference in 30-day mortality or ESBL-E infection recurrence between the non-CTX-M and CTX-M groups (OR=.99, 95% CI 0.87-1.11; p=0.83) and (OR=1.10, 95% CI 0.85--1.42; p=0.47), respectively. In an exploratory analysis limited to the non-CTX-M group, 86% of the 21 patients receiving meropenem were alive on day 30; none of the 5 patients receiving piperacillin-tazobactam were alive on day 30. CONCLUSIONS: Our findings suggest that non-CTX-M and CTX-M ESBL-producing Enterobacterales BSIs are equally concerning and associated with similar clinical outcomes. Meropenem may be associated with improved survival in patients with non-CTX-M ESBL-E BSIs, underscoring the potential benefit of comprehensive molecular diagnostics to enable early antibiotic optimization for patients with ESBL-E BSI, beyond just blaCTX-M genes.

Validation and Application of Long-Read Whole-Genome Sequencing for Antimicrobial Resistance Gene Detection and Antimicrobial Susceptibility Testing.

Next-generation sequencing applications are increasingly used for detection and characterization of antimicrobial-resistant pathogens in clinical settings. Oxford Nanopore Technologies (ONT) sequencing offers advantages for clinical use compared with other sequencing methodologies because it enables real-time basecalling, produces long sequencing reads that increase the ability to correctly assemble DNA fragments, provides short turnaround times, and requires relatively uncomplicated sample preparation. A drawback of ONT sequencing, however, is its lower per-read accuracy than short-read sequencing. We sought to identify best practices in ONT sequencing protocols. As some variability in sequencing results may be introduced by the DNA extraction methodology, we tested three DNA extraction kits across three independent laboratories using a representative set of six bacterial isolates to investigate accuracy and reproducibility of ONT technology. All DNA extraction techniques showed comparable performance; however, the DNeasy PowerSoil Pro kit had the highest sequencing yield. This kit was subsequently applied to 42 sequentially collected bacterial isolates from blood cultures to assess Ares Genetics's pipelines for predictive whole-genome sequencing antimicrobial susceptibility testing (WGS-AST) performance compared to phenotypic triplicate broth microdilution results. WGS-AST results ranged across the organisms and resulted in an overall categorical agreement of 95% for penicillins, 82.4% for cephalosporins, 76.7% for carbapenems, 86.9% for fluoroquinolones, and 96.2% for aminoglycosides. Very major errors/major errors were 0%/16.7% (penicillins), 11.7%/3.6% (cephalosporins), 0%/24.4% (carbapenems), 2.5%/7.7% (fluoroquinolones), and 0%/4.1% (aminoglycosides), respectively. This work showed that, although additional refinements are necessary, ONT sequencing demonstrates potential as a method to perform WGS-AST on cultured isolates for patient care.

Multicenter Evaluation of an MIC-Based Aztreonam and Ceftazidime-Avibactam Broth Disk Elution Test.

Due to limited therapeutic options, there is a clinical need to assess the in vitro activity of the combination of aztreonam (ATM) and ceftazidime-avibactam (CZA) to guide the therapeutic management of multidrug-resistant (MDR) Gram-negative organism infections. We set out to develop a practical MIC-based broth disk elution (BDE) method to determine the in vitro activity of the combination ATM-CZA using readily available supplies and compare it to reference broth microdilution (BMD). For the BDE method, a 30-µg ATM disk, a 30/20-µg CZA disk, both disks in combination, and no disks were added to 4 separate 5-mL cation-adjusted Mueller-Hinton broth (CA-MHB) tubes, using various manufacturers. Three testing sites performed both BDE and reference BMD testing of bacterial isolates in parallel from a single 0.5 McFarland standard inoculum and after overnight incubation, assessed them for growth (not susceptible) or no growth (susceptible) at a final concentration of 6/6/4 µg/mL ATM-CZA. During the first phase, the precision and accuracy of the BDE were analyzed by testing 61 Enterobacterales isolates at all sites. This testing yielded 98.3% precision between sites, with 98.3% categorical agreement and 1.8% major errors (ME). During the second phase, at each site, we evaluated unique, clinical isolates of metallo-ß-lactamase (MBL)-producing Enterobacterales (n = 75), carbapenem-resistant Pseudomonas aeruginosa (n = 25), Stenotrophomonas maltophilia (n = 46), and Myroides sp. (n = 1). This testing resulted in 97.9% categorical agreement, with 2.4% ME. Different results were observed for different disk and CA-MHB manufacturers, requiring a supplemental ATM-CZA-not-susceptible quality control organism to ensure the accuracy of results. The BDE is a precise and effective methodology for determining susceptibility to the combination ATM-CZA.

A plasmid locus associated with Klebsiella clinical infections encodes a microbiome-dependent gut fitness factor.

Klebsiella pneumoniae (Kp) is an important cause of healthcare-associated infections, which increases patient morbidity, mortality, and hospitalization costs. Gut colonization by Kp is consistently associated with subsequent Kp disease, and patients are predominantly infected with their colonizing strain. Our previous comparative genomics study, between disease-causing and asymptomatically colonizing Kp isolates, identified a plasmid-encoded tellurite (TeO3-2)-resistance (ter) operon as strongly associated with infection. However, TeO3-2 is extremely rare and toxic to humans. Thus, we used a multidisciplinary approach to determine the biological link between ter and Kp infection. First, we used a genomic and bioinformatic approach to extensively characterize Kp plasmids encoding the ter locus. These plasmids displayed substantial variation in plasmid incompatibility type and gene content. Moreover, the ter operon was genetically independent of other plasmid-encoded virulence and antibiotic resistance loci, both in our original patient cohort and in a large set (n = 88) of publicly available ter operon-encoding Kp plasmids, indicating that the ter operon is likely playing a direct, but yet undescribed role in Kp disease. Next, we employed multiple mouse models of infection and colonization to show that 1) the ter operon is dispensable during bacteremia, 2) the ter operon enhances fitness in the gut, 3) this phenotype is dependent on the colony of origin of mice, and 4) antibiotic disruption of the gut microbiota eliminates the requirement for ter. Furthermore, using 16S rRNA gene sequencing, we show that the ter operon enhances Kp fitness in the gut in the presence of specific indigenous microbiota, including those predicted to produce short chain fatty acids. Finally, administration of exogenous short-chain fatty acids in our mouse model of colonization was sufficient to reduce fitness of a ter mutant. These findings indicate that the ter operon, strongly associated with human infection, encodes factors that resist stress induced by the indigenous gut microbiota during colonization. This work represents a substantial advancement in our molecular understanding of Kp pathogenesis and gut colonization, directly relevant to Kp disease in healthcare settings.

Progressive Development of Cefiderocol Resistance in Escherichia coli During Therapy is Associated With an Increase in blaNDM-5 Copy Number and Gene Expression.

BACKGROUND: As cefiderocol is increasingly being prescribed in clinical practice, it is critical that we understand key mechanisms contributing to acquired resistance to this agent. METHODS: We describe a patient with acute lymphoblastic leukemia and a New Delhi metallo-ß-lactamase (NDM)-5-producing Escherichia coli intra-abdominal infection in whom resistance to cefiderocol evolved approximately 2 weeks after the start of treatment. Through whole-genome sequencing (WGS), messenger RNA expression studies, and ethylenediaminetetraacetic acid inhibition analysis, we investigated the role of increased NDM-5 production and genetic mutations contributing to the development of cefiderocol resistance, using 5 sequential clinical E. coli isolates obtained from the patient. RESULTS: In all 5 isolates, blaNDM-5 genes were identified. The minimum inhibitory concentrations for cefiderocol were 2, 4, and >32 µg/mL for isolates 1-2, 3, and 4-5, respectively. WGS showed that isolates 1-3 contained a single copy of the blaNDM-5 gene, whereas isolates 4 and 5 had 5 and 10 copies of the blaNDM-5 gene, respectively, on an IncFIA/FIB/IncFII plasmid. These findings were correlated with those of blaNDM-5 messenger RNA expression analysis, in which isolates 4 and 5 expressed blaNDM-5 1.7- and 2.8-fold, respectively, compared to, isolate 1. Synergy testing with the combination of ceftazidime-avibactam and aztreonam demonstrated expansion of the zone of inhibition between the disks for all isolates. The patient was successfully treated with this combination and remained infection free 1 year later. CONCLUSIONS: The findings in our patient suggest that increased copy numbers of blaNDM genes through translocation events are used by Enterobacterales to evade cefiderocol-mediated cell death. The frequency of increased blaNDM-5 expression in contributing to cefiderocol resistance needs investigation.

Multicenter Evaluation of the Acuitas AMR Gene Panel for Detection of an Extended Panel of Antimicrobial Resistance Genes among Bacterial Isolates.

The Acuitas antimicrobial resistance (AMR) gene panel is a qualitative, multiplex, nucleic acid-based in vitro diagnostic test for the detection and differentiation of 28 antimicrobial resistance markers associated with not susceptible results (NS; i.e., intermediate or resistant) to one or more antimicrobial agents among cultured isolates of select Enterobacterales, Pseudomonas aeruginosa, and Enterococcus faecalis. This study was conducted at four sites and included testing of 1,224 deidentified stocks created from 584 retrospectively collected isolates and 83 prospectively collected clinical isolates. The Acuitas results were compared with a combined reference standard including whole-genome sequencing, organism identification, and phenotypic antimicrobial susceptibility testing. The positive percent agreement (PPA) for FDA-cleared AMR targets ranged from 94.4% for MCR-1 to 100% for armA, CTX-M-2, DHA, IMP, OXA-9, SHV, vanA, and VEB. The negative percent agreement (NPA) for the majority of targets was ≥99%, except for AAC, AAD, CMY-41, P. aeruginosa gyrA mutant, Sul1, Sul2, and TEM targets (range, 96.5% to 98.5%). Three AMR markers did not meet FDA inclusion criteria (GES, SPM, and MCR-2). For each organism, 1 to 22 AMR targets met the minimum reportable PPA/NPA and correlated with ≥80% positive predictive value with associated NS results for at least one agent (i.e., the probability of an organism carrying an AMR marker testing NS to the associated agent). We demonstrate that the Acuitas AMR gene panel is an accurate method to detect a broad array of AMR markers among cultured isolates. The AMR markers were further associated with expected NS results for specific agent-organism combinations.

Modifiable Risk Factors for the Emergence of Ceftolozane-tazobactam Resistance.

BACKGROUND: Ceftolozane-tazobactam (TOL-TAZ) affords broad coverage against Pseudomonas aeruginosa. Regrettably, TOL-TAZ resistance has been reported. We sought to identify modifiable risk factors that may reduce the emergence of TOL-TAZ resistance. METHODS: Twenty-eight consecutive patients infected with carbapenem-resistant P. aeruginosa isolates susceptible to TOL-TAZ, treated with ≥72 hours of TOL-TAZ , and with P. aeruginosa isolates available both before and after TOL-TAZ exposure between January 2018 and December 2019 in Baltimore, Maryland, were included. Cases were defined as patients with at least a 4-fold increase in P. aeruginosa TOL-TAZ MICs after exposure to TOL-TAZ. Independent risk factors for the emergence of TOL-TAZ resistance comparing cases and controls were investigated using logistic regression. Whole genome sequencing of paired isolates was used to identify mechanisms of resistance that emerged during TOL-TAZ therapy. RESULTS: Fourteen patients (50%) had P. aeruginosa isolates which developed at least a 4-fold increase in TOL-TAZ MICs(ie, cases). Cases were more likely to have inadequate source control (29% vs 0%, P = .04) and were less likely to receive TOL-TAZ as an extended 3-hour infusion (0% vs 29%; P = .04). Eighty-six percent of index isolates susceptible to ceftazidime-avibactam (CAZ-AVI) had subsequent P. aeruginosa isolates with high-level resistance to CAZ-AVI, after TOL-TAZ exposure and without any CAZ-AVI exposure. Common mutations identified in TOL-TAZ resistant isolates involved AmpC, a known binding site for both ceftolozane and ceftazidime, and DNA polymerase. CONCLUSIONS: Due to our small sample size, our results remain exploratory but forewarn of the potential emergence of TOL-TAZ resistance during therapy and suggest extending TOL-TAZ infusions may be protective. Larger studies are needed to investigate this association.

Combined selective culture and molecular methods for the detection of carbapenem-resistant organisms from fecal specimens.

Detection of patients with intestinal colonization of carbapenem-resistant organisms (CRO), or more specifically carbapenemase-producing (CP) CRO, can prevent their transmission in healthcare facilities and aid with outbreak investigations. The objective of this work was to further develop and compare methods that combine selective culture and/or PCR to rapidly detect and recover CRO from fecal specimens. Molecularly characterized Gram-negative bacilli (n = 62) were used to spike fecal samples to establish limit of detection (LOD; n = 12), sensitivity (n = 28), and specificity (n= 21) for 3 methods to detect CP-CRO: direct MacConkey (MAC) plate and Xpert Carba-R (Cepheid) on growth, MAC broth and Carba-R testing of the broth, and direct testing by Carba-R. This was followed by a clinical study comparing methods in parallel for 286 fecal specimens. The LOD ranged from 102-105 CFU/mL depending on the carbapenemase gene and method. Combined culture/PCR methods had a sensitivity of 100%, whereas direct Carba-R testing had a sensitivity of 96% for the detection of CP-CRO. All methods had specificities of 100%. The prevalence of CP-CRO (0.7%) and non-CP-CRO (5.2 %) were low in the clinical study, where all methods demonstrated 100% agreement. The three methods performed comparably in detecting CP-CRO. Direct Carba-R testing had a higher LOD than the combined selective culture methods, but this may be offset by its rapid turnaround time for detection of CP-CRO. The selective culture methods provide the benefit of simultaneously isolating CP-CRO in culture for follow-up testing and detecting non-CP-CRO.

Cefiderocol Antimicrobial Susceptibility Testing against Multidrug-Resistant Gram-Negative Bacilli: a Comparison of Disk Diffusion to Broth Microdilution.

Antimicrobial susceptibility testing (AST) of cefiderocol poses challenges because of its unique mechanism of action (i.e., requiring an iron-depleted state) and due to differences in interpretative criteria established by the Clinical and Laboratory Standards Institute (CLSI), U.S. Food and Drug Administration (FDA), and European Committee on Antimicrobial Susceptibility Testing (EUCAST). Our objective was to compare cefiderocol disk diffusion methods (DD) to broth microdilution (BMD) for AST of Gram-negative bacilli (GNB). Cefiderocol AST was performed on consecutive carbapenem-resistant Enterobacterales (CRE; 58 isolates) and non-glucose-fermenting GNB (50 isolates) by BMD (lyophilized panels; Sensititre; Thermo Fisher) and DD (30 µg; research-use-only [RUO] MASTDISCS and FDA-cleared HardyDisks). Results were interpreted using FDA (prior to 28 September 2020 update), EUCAST, and investigational CLSI breakpoints (BPs). Categorical agreement (CA), minor errors (mE), major errors (ME), and very major errors (VME) were calculated for DD methods. The susceptibilities of all isolates by BMD were 72% (FDA), 75% (EUCAST) and 90% (CLSI). For DD methods, EUCAST BPs demonstrated lower susceptibility at 65% and 66%, compared to 74% and 72% (FDA) and 87% and 89% (CLSI) by HardyDisks and MASTDISCS, respectively. CA ranged from 75% to 90%, with 8 to 25% mE, 0 to 19% ME, and 0 to 20% VME and varied based on disk, GNB, and BPs evaluated. Both DD methods performed poorly for Acinetobacter baumannii complex. There is considerable variability when cefiderocol ASTs are interpreted using CLSI, FDA, and EUCAST breakpoints. DD offers a convenient alternative approach to BMD methods for cefiderocol AST, with the exception of A. baumannii complex isolates.

Evaluation of the NG-Test MCR-1 Lateral Flow Assay and EDTA-Colistin Broth Disk Elution Methods To Detect Plasmid-Mediated Colistin Resistance among Gram-Negative Bacterial Isolates.

Plasmid-mediated colistin resistance (PMCR) is a global public health concern, given its ease of transmissibility. The purpose of this study was to evaluate two methods for the detection of PMCR from bacterial colonies: (i) the NG-Test MCR-1 lateral flow immunoassay (LFA; NG Biotech, Guipry, France) and (ii) the EDTA-colistin broth disk elution (EDTA-CBDE) screening test method. These methods were evaluated using a cohort of contemporary, clinical Gram-negative bacillus isolates from 3 U.S. academic medical centers (126 isolates of the Enterobacterales, 50 Pseudomonas aeruginosa isolates, and 50 Acinetobacter species isolates; 1 isolate was mcr positive) and 12 mcr-positive CDC-FDA Antibiotic Resistance (AR) Isolate Bank isolates for which reference broth microdilution colistin susceptibility results were available. Eleven (4.6%) isolates were strongly positive by the MCR-1 LFA, with an additional 8 (3.4%) isolates yielding faintly positive results. The positive percent agreement (PPA) and negative percent agreement (NPA) for MCR-1 detection were 100% and 96.1%, respectively. Upon repeat testing, only a single false-positive MCR-2 producer remained, as the isolates with initially faintly positive results were negative. The EDTA-CBDE screening method had an overall PPA and NPA of 100% and 94.3%, respectively. The NPA for the EDTA-CBDE method was slightly lower at 94.2% with Enterobacterales, whereas it was 96.0% with P. aeruginosa The MCR-1 LFA and EDTA-CBDE methods are both accurate and user-friendly methods for the detection of PMCR. Despite the rarity of PMCR among clinical isolates in the United States, these methods are valuable tools that may be implemented in public health and clinical microbiology laboratories to further discern the mechanism of resistance among colistin-resistant Gram-negative isolates and to detect PMCR for infection prevention and control purposes.

Evaluation of NG-Test Carba 5 for Rapid Phenotypic Detection and Differentiation of Five Common Carbapenemase Families: Results of a Multicenter Clinical Evaluation.

NG-Test Carba 5 is a rapid in vitro multiplex immunoassay for the phenotypic detection and differentiation of five common carbapenemase families (KPC, OXA-48-like, VIM, IMP, and NDM) directly from bacterial colonies. The assay is simple to perform and has recently received U.S. Food and Drug Administration clearance. A method comparison study was performed at geographically diverse medical centers (n = 3) in the United States, where 309 Enterobacterales and Pseudomonas aeruginosa isolates were evaluated by NG-Test Carba 5 (NG Biotech, Guipry, France), the Xpert Carba-R assay (Cepheid, Inc., Sunnyvale, CA), the modified carbapenem inactivation method (mCIM), the EDTA-modified carbapenem inactivation method, and disk diffusion with carbapenems. Colonies from tryptic soy agar with 5% sheep blood (blood agar) and MacConkey agar were tested, and the results were compared to those obtained by a composite reference method. Additionally, a fourth medical center performed a medium comparison study by evaluating the performance characteristics of NG-Test Carba 5 from blood, MacConkey, and Mueller-Hinton agars with 110 isolates of Enterobacterales and P. aeruginosa These results were compared to the expected genotypic and mCIM results. For the multicenter method comparison study, the overall positive percent agreement (PPA) and the overall negative percent agreement (NPA) of NG-Test Carba 5 with the composite reference method were 100% for both blood and MacConkey agars. The medium comparison study at the fourth site showed that the PPA ranged from 98.9% to 100% and that the NPA ranged from 95.2% to 100% for blood, MacConkey, and Mueller-Hinton agars. NG-Test Carba 5 accurately detected and differentiated five common carbapenemase families from Enterobacterales and P. aeruginosa colonies on commonly used agar media. The results of this test will support a streamlined laboratory work flow and will expedite therapeutic and infection control decisions.

The Likelihood of Developing a Carbapenem-Resistant Enterobacteriaceae Infection during a Hospital Stay.

Of 1,455 unique patients in U.S. intensive care units (ICUs), 4% were rectally colonized with CRE on admission. A total of 297 patients were initially negative for carbapenem-resistant Enterobacteriaceae (CRE) and remained in the ICU long enough to contribute additional swabs; 22% of these patients had a subsequent CRE-positive swab, with a median time to CRE colonization of 13 days (interquartile range, 7 to 21 days). Patients colonized with carbapenemase-producing CRE were more likely than those colonized with non-carbapenemase-producing CRE to develop CRE infections during their hospitalizations (36% versus 3%; P < 0.05).

Applying Rapid Whole-Genome Sequencing To Predict Phenotypic Antimicrobial Susceptibility Testing Results among Carbapenem-Resistant Klebsiella pneumoniae Clinical Isolates.

Standard antimicrobial susceptibility testing (AST) approaches lead to delays in the selection of optimal antimicrobial therapy. Here, we sought to determine the accuracy of antimicrobial resistance (AMR) determinants identified by Nanopore whole-genome sequencing in predicting AST results. Using a cohort of 40 clinical isolates (21 carbapenemase-producing carbapenem-resistant Klebsiella pneumoniae, 10 non-carbapenemase-producing carbapenem-resistant K. pneumoniae, and 9 carbapenem-susceptible K. pneumoniae isolates), three separate sequencing and analysis pipelines were performed, as follows: (i) a real-time Nanopore analysis approach identifying acquired AMR genes, (ii) an assembly-based Nanopore approach identifying acquired AMR genes and chromosomal mutations, and (iii) an approach using short-read correction of Nanopore assemblies. The short-read correction of Nanopore assemblies served as the reference standard to determine the accuracy of Nanopore sequencing results. With the real-time analysis approach, full annotation of acquired AMR genes occurred within 8 h from subcultured isolates. Assemblies sufficient for full resistance gene and single-nucleotide polymorphism annotation were available within 14 h from subcultured isolates. The overall agreement of genotypic results and anticipated AST results for the 40 K. pneumoniae isolates was 77% (range, 30% to 100%) and 92% (range, 80% to 100%) for the real-time approach and the assembly approach, respectively. Evaluating the patients contributing the 40 isolates, the real-time approach and assembly approach could shorten the median time to effective antibiotic therapy by 20 h and 26 h, respectively, compared to standard AST. Nanopore sequencing offers a rapid approach to both accurately identify resistance mechanisms and to predict AST results for K. pneumoniae isolates. Bioinformatics improvements enabling real-time alignment, coupled with rapid extraction and library preparation, will further enhance the accuracy and workflow of the Nanopore real-time approach.

Lysyl oxidase-like 2 depletion is protective in age-associated vascular stiffening.

Vascular stiffening and its sequelae are major causes of morbidity and mortality in the elderly. The increasingly accepted concept of "smooth muscle cell (SMC) stiffness syndrome" along with matrix deposition has emerged in vascular biology to account for the mechanical phenotype of arterial aging, but the molecular targets remain elusive. In this study, using an unbiased proteomic analysis, we identified lysyl oxidase-like 2 (LOXL2) as a critical SMC mediator for age-associated vascular stiffening. We tested the hypothesis that loss of LOXL2 function is protective in aging-associated vascular stiffening. We determined that exogenous and endogenous nitric oxide markedly decreased LOXL2 abundance and activity in the extracellular matrix of isolated SMCs and LOXL2 endothelial cells suppress LOXL2 abundance in the aorta. In a longitudinal study, LOXL2+/- mice were protected from age-associated increase in pulse-wave velocity, an index of vascular stiffening, as occurred in littermate wild-type mice. Using isolated aortic segments, we found that LOXL2 mediates vascular stiffening in aging by promoting SMC stiffness, augmented SMC contractility, and vascular matrix deposition. Together, these studies establish LOXL2 as a nodal point for a new therapeutic approach to treat age-associated vascular stiffening. NEW & NOTEWORTHY Increased central vascular stiffness augments risk of major adverse cardiovascular events. Despite significant advances in understanding the genetic and molecular underpinnings of vascular stiffening, targeted therapy has remained elusive. Here, we show that lysyl oxidase-like 2 (LOXL2) drives vascular stiffening during aging by promoting matrix remodeling and vascular smooth muscle cell stiffening. Reduced LOXL2 expression protects mice from age-associated vascular stiffening and delays the onset of isolated systolic hypertension, a major consequence of stiffening.

A Novel Phenotypic Method To Screen for Plasmid-Mediated Colistin Resistance among Enterobacteriales.

Plasmid-mediated colistin resistance (PMCR), a consequence of the mcr genes, is a significant public health concern given its potential to easily spread among clinical pathogens. Recently, it was discovered that MCR enzymes require zinc for activity. Thus, we modified the colistin broth-disk elution (CBDE) test to screen for plasmid-mediated colistin resistance (PMCR) genes based on any reduction of colistin MIC in the presence of EDTA. Eighty-five isolates of the order Enterobacteriales (12 mcr positive) were tested by CBDE ± EDTA. The sensitivity and specificity of the EDTA-CBDE method to detect PMCR compared to the molecular genotype results were 100% and 95.8%, respectively. Isolates positive by the EDTA-CBDE test should be further evaluated to confirm the presence of mcr genes.

Multicenter Evaluation of Colistin Broth Disk Elution and Colistin Agar Test: a Report from the Clinical and Laboratory Standards Institute.

Susceptibility testing of the polymyxins (colistin and polymyxin B) is challenging for clinical laboratories. The Clinical and Laboratory Standards Institute (CLSI) Antimicrobial Susceptibility Testing Subcommittee evaluated two methods to enable accurate testing of these agents. These methods were a colistin broth disk elution (CBDE) and a colistin agar test (CAT), the latter of which was evaluated using two inoculum volumes, 1 µl (CAT-1) and 10 µl (CAT-10). The methods were evaluated using a collection of 270 isolates of Enterobacterales, 122 Pseudomonas aeruginosa isolates, and 106 Acinetobacter spp. isolates. Overall, 94.4% of CBDE results were in essential agreement and 97.9% in categorical agreement (CA) with reference broth microdilution MICs. Nine very major errors (VME; 3.2%) and 3 major errors (ME; 0.9%) were observed. With the CBDE, 98.6% CA was observed for Enterobacterales (2.5% VME, 0% ME), 99.3% CA was observed for P. aeruginosa (0% VME, 0.7% ME), and 93.1% CA was observed for Acinetobacter spp. (5.6% VME, 3.3% ME). Overall, CA was 94.9% with 6.8% VME using CAT-1 and improved to 98.3% with 3.9% VME using CAT-10. No ME were observed using either CAT-1 or CAT-10. Using the CAT-1/CAT-10, the CA observed was 99.4%/99.7% for Enterobacterales (1%/0.5% VME), 98.7%/100% for P. aeruginosa (8.3%/0% VME), and 88.5%/92.3% for Acinetobacter spp. (21.4%/14.3% VME). Based on these data, the CLSI antimicrobial susceptibility testing (AST) subcommittee endorsed the CBDE and CAT-10 methods for colistin testing of Enterobacterales and P. aeruginosa.

Two-Site Evaluation of the Colistin Broth Disk Elution Test To Determine Colistin In Vitro Activity against Gram-Negative Bacilli.

Limited methods for colistin MIC determination are available to clinical microbiology laboratories. The purpose of this study was to evaluate the accuracy of the colistin broth disk elution (CBDE) test compared to that of broth microdilution (BMD) for identifying colistin MICs. CBDE was compared to colistin BMD using a collection of Gram-negative bacilli tested at two U.S. microbiology laboratories. The isolates tested included 121 retrospective clinical isolates, 45 prospective clinical isolates, and 6 mcr-1-positive Escherichia coli isolates. CBDE was performed with four 10-ml cation-adjusted Mueller-Hinton broth tubes per isolate, to which 0, 1, 2, and 4 colistin 10-µg disks were added, generating final concentrations in the tubes of 0 (growth control), 1, 2, and 4 µg/ml, respectively. MICs were evaluated visually and interpreted using Clinical and Laboratory Standards Institute breakpoints. Site 2 also compared CBDE to the reference broth macrodilution (BMAD) method (n = 110 isolates). Overall, CBDE yielded a categorical agreement (CA) and essential agreement (EA) of 98% and 99%, respectively, compared to the results of colistin BMD. Very major errors occurred for mcr-1-producing strains, with MICs fluctuating from 2 to 4 µg/ml on repeat testing. The results for all other isolates were in CA with those of BMD. CBDE versus BMAD had an EA of 100% and a CA of 100%. Compared to currently used techniques, CBDE is an easy and practical method to perform colistin MIC testing. Some mcr-1-producing isolates yielded MICs of 2 µg/ml by CBDE and 4 µg/ml by BMD. As such, the results for isolates with colistin MICs of 2 µg/ml by CBDE should be confirmed by the reference BMD method, and isolates with MICs of ≥2 µg/ml should be evaluated for the presence of mcr genes.