OBJECTIVES/GOALS: In this study, we aim to report the role of porins and bla CTX-M β-lactamases among Escherichia coli and Klebsiella pneumoniae, focusing on emerging carbapenem resistant Enterobacterales (CRE) subtypes, including non-carbapenemase producing Enterobacterales (NCPE) and ertapenem-resistant but meropenem-susceptible (ErMs) strains. METHODS/STUDY POPULATION: Whole genome sequencing was conducted on 76 carbapenem-resistant isolates across 5 hospitals in San Antonio, U.S. Among these, NCP isolates accounted for the majority of CRE (41/76). Identification and antimicrobial susceptibility testing (AST) results were collected from the clinical charts. Repeat speciation was determined through whole genome sequencing (WGS) analysis and repeat AST, performed with microdilution or ETEST®. Minimum inhibitory concentrations (MIC) were consistent with Clinical and Laboratory Standards Institute (CLSI M100, ED33). WGS and qPCR were used to characterize the resistome of all clinical CRE subtypes, while western blotting and liquid chromatography with tandem mass spectrometry (LC-MS-MS) were used to determine porin expression and carbapenem hydrolysis, respectively. RESULTS/ANTICIPATED RESULTS: bla CTX-M was found to be most prevalent among NCP isolates (p = 0.02). LC-MS/MS analysis of carbapenem hydrolysis revealed that bla CTX-M -mediated carbapenem hydrolysis, indicating the need to reappraise the term, “non-carbapenemase (NCP)®” for quantitatively uncharacterized CRE strains harboring bla CTX-M . Susceptibility results showed that 56% of all NCPE isolates had an ErMs phenotype (NCPE vs. CPE, p < 0.001), with E. coli driving the phenotype (E. coli vs. K. pneumoniae, p < 0.001). ErMs strains carrying bla CTX-M , had 4-fold more copies of bla CTX-M than ceftriaxone-resistant but ertapenem-susceptible isolates (3.7 v. 0.9, p < 0.001). Immunoblot analysis demonstrated the absence of OmpC expression in NCP-ErMs E. coli, with 92% of strains lacking full contig coverage ofompC. DISCUSSION/SIGNIFICANCE: Overall, this work provides evidence of a collaborative effort between bla CTX-M and OmpC in NCP strains that confer resistance to ertapenem but not meropenem. Clinically, CRE subtypes are not readily appreciated, potentially leading to mismanagement of CRE infected patients. A greater focus on optimal treatments for CRE subtypes is needed.
Metformin may potentially reverse various age-related conditions; however, it is unclear whether metformin can also mitigate or delay the deterioration of immunological resilience that occurs in the context of infections that are commonly observed in older persons. We examined whether metformin promotes the preservation of immunological resilience in an acute S. pneumoniae (SPN) infection challenge in young adult mice. Mice were fed metformin (MET-alone) or standard chow (controls-alone) for 10 weeks prior to receiving intratracheal inoculation of SPN. A subset of each diet group received pneumococcal conjugate vaccine at week 6 (MET + PCV and control + PCV). Compared to controls-alone, MET-alone had significantly less infection-associated morbidity and attenuated inflammatory responses during acute SPN infection. Metformin lowered the expression of genes in the lungs related to inflammation as well as shorter lifespan in humans. This was accompanied by significantly lower levels of pro-inflammatory cytokines (e.g., IL6). MET + PCV vs. control + PCV manifested enhanced SPN anticapsular IgM and IgG levels. The levels of SPN IgM production negatively correlated with expression levels of genes linked to intestinal epithelial structure among MET + PCV vs. control + PCV groups. Correspondingly, the gut microbial composition of metformin-fed mice had a significantly higher abundance in the Verrucomicrobia, Akkermansia muciniphila, a species previously associated with beneficial effects on intestinal integrity and longevity. Together, these findings indicate metformin’s immunoprotective potential to protect against infection-associated declines in immunologic resilience.
The risk of severe coronavirus disease 2019 (COVID-19) varies significantly among persons of similar age and is higher in males. Age-independent, sex-biased differences in susceptibility to severe COVID-19 may be ascribable to deficits in a sexually dimorphic protective attribute that we termed immunologic resilience (IR).
This study investigates the emergence of ertapenem-resistant, meropenem-susceptible (ErMs) among non-carbapenemase producing (NCP) and carbapenemase producing (CP) Escherichia coli and Klebsiella pneumoniae. As mutations for ertapenem resistance establish the genetic background for non-carbapenemase meropenem resistance, there is a great need for antibiotic stewards and researchers to understand the determinants of a strain’s propensity to become resistant. Whole genome sequencing was conducted on clinical carbapenem-resistant E. coli (CREC) and K. pneumoniae (CRKP) across 5 hospitals in San Antonio, U.S. from 2012-2018. The majority of carbapenem resistant Enterobacterales (CRE) were NCP (54%; 41/76). The blaCTX-M was found to be most prevalent among NCP isolates (p = 0.02). LC-MS/MS analysis of carbapenem hydrolysis revealed that blaCTX-M-mediated carbapenem hydrolysis, indicating the need to reappraise the term, “non-carbapenemase (NCP)” for quantitatively uncharacterized CRE strains harboring blaCTX-M. Antimicrobial susceptibility results showed that 56% of all NCPE isolates had an ErMs phenotype (NCPE vs. CPE, p &lt; 0.001), with E. coli driving the phenotype (E. coli vs. K. pneumoniae, p &lt; 0.001). ErMs strains carrying blaCTX-M, had approximately 4-fold more copies of blaCTX-M than ceftriaxone-resistant but ertapenem and meropenem susceptible (EsMs) isolates (3.7 v. 0.9, p &lt; 0.001). ErMs also carried more mobile genetic elements (MGEs), particularly IS26 composite transposons, than EsMs (37 vs. 0.2, p &lt; 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, this work provides evidence of a collaborative effort between blaCTX-M and OmpC in NCP strains that confer resistance to ertapenem but not meropenem. To thwart potential mismanagement of CRE infected patients, future efforts should focus on understanding the mechanism(s) underlying OmpC loss, developing rapid methods to detect blaCTX-M copy number variants, and targeted antimicrobials for NCPE and ErMs strains.
Abstract Background Carbapenem-resistant Enterobacterales are a growing threat globally. Early detection of CRE is necessary for appropriate treatment and infection control measures. Many hospital labs can test for carbapenemase production; however, in some regions, including South Texas, the majority of CRE are non-carbapenemase producing (NCPE). This study had two interrelated aims to develop decision rules tailored to a region with high prevalence of NCPE to predict 1) antimicrobial resistance (AMR) from whole genome sequencing (WGS) data and 2) CRE treatment outcomes. Methods To better understand links between resistome, phenotypic AMR, and prediction of outcomes for CRE, we developed decision rules to build machine learning prediction models. We conducted WGS and antibiotic susceptibility testing (21 antibiotics) on CRE isolates from unique patients across 5 hospitals in the South Texas region between 2013 and 2020. Day 30 outcomes were based on desirability of outcome ranking (DOOR). The overall classification accuracies of the models are reported. Results Overall 146 CRE isolates were included, 97 were used to train each model, and 49 were used for validation. Among the K. pneumoniae and E. coli CRE isolates that were available with susceptibility data, the majority (62%) were NCPE. For the clinical recovery model (DOOR), the combination of admission ICU status, piperacillin-tazobactam (PT) MIC > 16, presence of sul gene, and polymyxin-sparring regimens associated with an overall accuracy of 95% for having a worse DOOR. Majority (60%) of patients were empirically treated with piperacillin-tazobactam; notably, less than 33% isolates had PT MIC ≤ 16. Interestingly, combined effects of isolates that did not harbor carbapenemases, blaOXA-1, blaCTX-M-15, blaCMY, or aac(6’)ib-cr genes resulted in a decision rule with a 95.7% overall accuracy for susceptibility to PT (MIC < 16 ug/mL). Conclusion Herein, we used machine learning approaches to predict AMR and treatment-based outcomes linked with WGS data in a region with predominantly NCPE infections. Machine learning can obtain a model that can make repeatable predictions, further data can improve the accuracy and guide tailored clinical decision-making. Disclosures Grace Lee, PharmD, PhD, BCPS, Merck Co. (Grant/Research Support)NIA/NIH (Research Grant or Support)
Abstract Some of the most effective drugs used in the treatment of breast cancer are microtubule stabilizers. However, there are limitations to their clinical efficacy, including inherent and acquired drug resistance. All microtubule stabilizers that are currently approved for clinical use bind within the taxane pocket on β-tubulin in a reversible manner. The taccalonolides are a novel class of microtubule stabilizers that have a similar profile of microtubule stabilization as the taxanes, but circumvent drug resistance mediated by expression of drug efflux pumps, mutations in the taxane binding site, or overexpression of the βIII isotype of tubulin. We have shown that one important difference between the taccalonolides and clinically approved microtubule stabilizers is that the taccalonolides form a covalent bond to β-tubulin. This distinct interaction allows for irreversible binding, which explains their ability to avoid drug efflux mechanisms and likely belies their exquisite potency in in vivo antitumor models which allows for delivery in aqueous solvents. Serum stability and binding studies, microsomal clearance and pharmacokinetic analysis were performed with both taccalonolides AF and AJ to more fully understand the properties of this class of compounds. We found that both taccalonolides had low microsomal intrinsic clearance rates with no evidence of serum binding and had half-lives similar to paclitaxel in vivo. Like other microtubule targeted agents, taccalonolide AF has a narrow therapeutic window with antitumor effects accompanied by body weight loss. Interestingly, direct injection of taccalonolide AF into a xenograft tumor was highly effective with no associated toxicities at low doses, indicating that targeted delivery to the tumor would greatly increase the efficacy and decrease toxicities. To this end, efforts to promote the targeted delivery of taccalonolide AF to the tumor are being evaluated. Citation Format: Risinger AL, Li J, Benavides R, Kuhn JG, Mooberry SL. The taccalonolides are novel microtubule stabilizers that covalently bind tubulin and have in vivo efficacy in drug resistant tumors. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-13-07.
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.
Background Carbapenem-resistant Enterobacterales (CRE) pose a significant global public health threat. Resistance among CRE is particularly complex, owing to numerous possible resistance mechanisms and broad definitions. We aimed to characterize the clinical and molecular profiles of CRE in the South Texas region. Materials and methods We compared the clinical, genotypic, and phenotypic profiles of carbapenemase producing Enterobacterales (CPE) with those of non-carbapenemase producers (NCPE) isolated from South Texas, United States between 2011 and 2019. Molecular characteristics and resistance mechanisms were analyzed using whole-genome sequences. Results The majority (59%) of the CRE isolates were NCPE while 41% of isolates harbored carbapenemases, predmonantly bla KPC -type. The most common CPE was Klebsiella pneumoniae while majority of Enterobacter cloacae and Escherichia coli were NCPE Among K. pneumoniae , the clonal group 307 has emerged as a predmoninant group and was associated with as many CRE infections as the previous common clonal group 258. Patients with NCPE compared to CPE infections were associated with higher antimicrobial exposure prior to culture collection (days of therapy, 795 vs. 242; p < 0.001) and emergency department visits within past 90 days (22% vs. 4%; p = 0.011). The all cause 30-day mortality was 21%. Conclusions This study highlights the diversity of resistance mechanisms underlying CRE in South Texas, with 59% not harboring a carbapenemase. Individuals with NCPE infections were more likely to have had prior antimicrobial therapy and emergency department visits compared to those with CPE. Identification and distinction of these mechanisms by rapid identification of species and carbapenemase would allow for optimal treatment and infection control efforts.
The taccalonolides are microtubule stabilizers that covalently bind tubulin and circumvent clinically relevant forms of resistance to other drugs of this class. Efforts are under way to identify a taccalonolide with optimal properties for clinical development. The structurally similar taccalonolides AF and AJ have comparable microtubule-stabilizing activities in vitro, but taccalonolide AF has excellent in vivo antitumor efficacy when administered systemically, while taccalonolide AJ does not elicit this activity even at maximum tolerated dose. The hypothesis that pharmacokinetic differences underlie the differential efficacies of taccalonolides AF and AJ was tested. The effects of serum on their in vivo potency, metabolism by human liver microsomes and in vivo pharmacokinetic properties were evaluated. Taccalonolides AF and AJ were found to have elimination half-lives of 44 and 8.1 min, respectively. Furthermore, taccalonolide AJ was found to have excellent and highly persistent antitumor efficacy when administered directly to the tumor, suggesting that the lack of antitumor efficacy seen with systemic administration of AJ is likely due to its short half-life in vivo. These results help define why some, but not all, taccalonolides inhibit the growth of tumors at systemically tolerable doses and prompt studies to further improve their pharmacokinetic profile and antitumor efficacy.
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