Pathway Antagonists Research Articles

Pharmacology of Veratrum californicum Alkaloids as Hedgehog Pathway Antagonists.

Veratrum californicum contains steroidal alkaloids that function as inhibitors of hedgehog (Hh) signaling, a pathway involved in the growth and differentiation of cells and normal tissue development. This same Hh pathway is abnormally active for cell proliferation in more than 20 types of cancer. In this current study, alkaloids have been extracted from the root and rhizome of V. californicum, followed by their separation into five fractions using high performance liquid chromatography. Mass spectrometry was used to identify the presence of twenty-five alkaloids, nine more than are commonly cited in literature reports, and the Bruker Compass Data Analysis software was used to predict the molecular formula for every detected alkaloid. The Gli activity of the raw extract and each fraction were compared to 0.1 µM cyclopamine, and fractions 1, 2, and 4 showed increased bioactivity through suppression of the Hh signaling pathway. Fractions 2 and 4 had enhanced bioactivity, but fraction 1 was most effective in inhibiting Hh signaling. The composition of fraction 1 consisted of veratrosine, cycloposine, and potential isomers of each.

Distinct expression patterns of Hedgehog signaling components in mouse gustatory system during postnatal tongue development and adult homeostasis.

The Hedgehog (HH) pathway regulates embryonic development of anterior tongue taste fungiform papilla (FP) and the posterior circumvallate (CVP) and foliate (FOP) taste papillae. HH signaling also mediates taste organ maintenance and regeneration in adults. However, there are knowledge gaps in HH pathway component expression during postnatal taste organ differentiation and maturation. Importantly, the HH transcriptional effectors GLI1, GLI2 and GLI3 have not been investigated in early postnatal stages; the HH receptors PTCH1, GAS1, CDON and HHIP, required to either drive HH pathway activation or antagonism, also remain unexplored. Using lacZ reporter mouse models, we mapped expression of the HH ligand SHH, HH receptors, and GLI transcription factors in FP, CVP and FOP in early and late postnatal and adult stages. In adults we also studied the soft palate, and the geniculate and trigeminal ganglia, which extend afferent fibers to the anterior tongue. Shh and Gas1 are the only components that were consistently expressed within taste buds of all three papillae and the soft palate. In the first postnatal week, we observed broad expression of HH signaling components in FP and adjacent, non-taste filiform (FILIF) papillae in epithelium or stroma and tongue muscles. Notably, we observed elimination of Gli1 in FILIF and Gas1 in muscles, and downregulation of Ptch1 in lingual epithelium and of Cdon, Gas1 and Hhip in stroma from late postnatal stages. Further, HH receptor expression patterns in CVP and FOP epithelium differed from anterior FP. Among all the components, only known positive regulators of HH signaling, SHH, Ptch1, Gli1 and Gli2, were expressed in the ganglia. Our studies emphasize differential regulation of HH signaling in distinct postnatal developmental periods and in anterior versus posterior taste organs, and lay the foundation for functional studies to understand the roles of numerous HH signaling components in postnatal tongue development.

Soluble CD83 modulates human-monocyte-derived macrophages toward alternative phenotype, function, and metabolism.

Alterations in macrophage (Mφ) polarization, function, and metabolic signature can foster development of chronic diseases, such as autoimmunity or fibrotic tissue remodeling. Thus, identification of novel therapeutic agents that modulate human Mφ biology is crucial for treatment of such conditions. Herein, we demonstrate that the soluble CD83 (sCD83) protein induces pro-resolving features in human monocyte-derived Mφ biology. We show that sCD83 strikingly increases the expression of inhibitory molecules including ILT-2 (immunoglobulin-like transcript 2), ILT-4, ILT-5, and CD163, whereas activation markers, such as MHC-II and MSR-1, were significantly downregulated. This goes along with a decreased capacity to stimulate alloreactive T cells in mixed lymphocyte reaction (MLR) assays. Bulk RNA sequencing and pathway analyses revealed that sCD83 downregulates pathways associated with pro-inflammatory, classically activated Mφ (CAM) differentiation including HIF-1A, IL-6, and cytokine storm, whereas pathways related to alternative Mφ activation and liver X receptor were significantly induced. By using the LXR pathway antagonist GSK2033, we show that transcription of specific genes (e.g., PPARG, ABCA1, ABCG1, CD36) induced by sCD83 is dependent on LXR activation. In summary, we herein reveal for the first time mechanistic insights into the modulation of human Mφ biology by sCD83, which is a further crucial preclinical study for the establishment of sCD83 as a new therapeutical agent to treat inflammatory conditions.

Schisandrin B Alleviates Renal Tubular Cell Epithelial–Mesenchymal Transition and Mitochondrial Dysfunction by Kielin/Chordin-like Protein Upregulation via Akt Pathway Inactivation and Adenosine 5′-Monophosphate (AMP)-Activated Protein Kinase Pathway Activation in Diabetic Kidney Disease

Diabetic kidney disease is a common complication of diabetes and remains the primary cause of end-stage kidney disease in the general population. Schisandrin B (Sch B) is an active ingredient in Schisandra chinensis. Our study illustrates that Sch B can mitigate renal tubular cell (RTC) epithelial–mesenchymal transition (EMT) and mitochondrial dysfunction in db/db mice, accompanied by the downregulation of TGF-β1 and the upregulation of PGC-1α. Similarly, Sch B demonstrated a protective effect by reducing the expression of TGF-β1, α-SMA, fibronectin, and Col I, meanwhile enhancing the expression of E-cadherin in human RTCs (HK2 cells) stimulated with high glucose. Moreover, under high glucose conditions, Sch B effectively increased mitochondrial membrane potential, lowered ROS production, and increased the ATP content in HK2 cells, accompanied by the upregulation of PGC-1α, TFAM, MFN1, and MFN2. Mechanistically, the RNA-seq results showed a significant increase in KCP mRNA levels in HK2 cells treated with Sch B in a high glucose culture. The influence of Sch B on KCP mRNA levels was confirmed by real-time PCR in high glucose-treated HK2 cells. Depletion of the KCP gene reversed the impact of Sch B on TGF-β1 and PGC-1α in HK2 cells with high glucose level exposure, whereas overexpression of the KCP gene blocked EMT and mitochondrial dysfunction. Furthermore, the PI3K/Akt pathway was inhibited and the AMPK pathway was activated in HK2 cells exposed to a high concentration of glucose after the Sch B treatment. Treatment with the PI3K/Akt pathway agonist insulin and the AMPK pathway antagonist compound C attenuated the Sch B-induced KCP expression in HK2 cells exposed to a high level of glucose. Finally, molecular autodock experiments illustrated that Sch B could bind to Akt and AMPK. In summary, our findings suggested that Sch B could alleviate RTC EMT and mitochondrial dysfunction by upregulating KCP via inhibiting the Akt pathway and activating the AMPK pathway in DKD.

Characteristics of antibiotic resistance of non-typhoidal Salmonella circulating in the Russian Federation in the period from 2019 to 2022

Introduction. Non-typhoidal Salmonella make a significant contribution to the incidence of enteric infections and are characterized by an increasing proportion of strains resistant to antimicrobial agents (AMA), including the first choice antibiotics (cephalosporins III and fluoroquinolones).
 The purpose of the study is to assess the phenotypic resistance of Salmonella to various classes of AMAs and determine the relationship between the phenotypic resistance, serotype, source of isolation and nature of incidence.
 Materials and methods. We studied 752 representative strains of Salmonella of 2494 strains isolated from various sources (clinical samples, food products, environment) received from 59 regions of Russia in the period from 2019 to 2022. The phenotypic resistance to 22 antibiotics of 11 CLSI classes of AMAs was assessed by broth microdilution method (minimum inhibitory concentration). The diversity of resistance profiles of Salmonella serotypes was compared using the Shannon index.
 Results. The dominant position in terms of isolation frequency is occupied by the serotypes Salmonella Enteritidis, S. Infantis, S. Muenchen, S. Typhimurium, S. Bovismorbificans, which accounted for 64.4% of the studied strains. 543 (72.2%) strains showed resistance to at least one of the tested antibiotics; 193 (25.7%) strains were characterized by multidrug resistance phenotype (MDR). Resistance to AMA classes was characterized by the following distribution: quinolones (61.3%), tetracyclines (28.1%), penicillins (19.1%), β-lactam combination agents (18.6%), folate pathway antagonists (16, 5%), phenicols (10.1%), aminoglycosides (5.6%), cephems (4.7%), monobactams (4.4%), lipopeptides (3.9%). No penem-resistant strains have been identified. The features of Salmonella resistance by AMA classes are shown to depend on the sources of isolation, the Salmonella serotype and the nature of the incidence (outbreak and sporadic).
 Conclusions. Monitoring of phenotypic antibiotic resistance is an important tool for epidemiological surveillance in order to prevent the spread of bacterial resistance to AMAs.

A Comparative Study on Antimicrobial Resistance in Escherichia coli Isolated from Channel Catfish and Related Freshwater Fish Species

Antimicrobial resistance (AMR) trends in 114 generic Escherichia coli isolated from channel catfish and related fish species were investigated in this study. Of these, 45 isolates were from commercial-sized channel catfish harvested from fishponds in Alabama, while 69 isolates were from Siluriformes products, accessed from the U.S. Department of Agriculture Food Safety and Inspection Service’ (FSIS) National Antimicrobial Resistance Monitoring System (NARMS) program. Antibiotic susceptibility testing and whole genome sequencing were performed using the GenomeTrakr protocol. Upon analysis, the fishpond isolates showed resistance to ampicillin (44%), meropenem (7%) and azithromycin (4%). The FSIS NARMS isolates showed resistance to tetracycline (31.9%), chloramphenicol (20.3%), sulfisoxazole (17.4%), ampicillin (5.8%) and trimethoprim-sulfamethoxazole, nalidixic acid, amoxicillin-clavulanic acid, azithromycin and cefoxitin below 5% each. There was no correlation between genotypic and phenotypic resistance in the fishpond isolates, however, there was in NARMS isolates for folate pathway antagonists: Sulfisoxazole vs. sul1 and sul2 (p = 0.0042 and p < 0.0001, respectively) and trimethoprim-sulfamethoxazole vs. dfrA16 and sul1 (p = 0.0290 and p = 0.013, respectively). Furthermore, correlations were found for tetracyclines: Tetracycline vs. tet(A) and tet(B) (p < 0.0001 each), macrolides: Azithromycin vs. mph(E) and msr(E) (p = 0.0145 each), phenicols: Chloramphenicol vs. mdtM (p < 0.0001), quinolones: Nalidixic acid vs. gyrA_S83L=POINT (p = 0.0004), and β-lactams: Ampicillin vs. blaTEM-1 (p < 0.0001). Overall, we recorded differences in antimicrobial susceptibility testing profiles, phenotypic-genotypic concordance, and resistance to critically important antimicrobials, which may be a public health concern.

Association of health, nutrition, and socioeconomic variables with global antimicrobial resistance: a modelling study

Although antimicrobial use is a key selector for antimicrobial resistance, recent studies have suggested that the ecological context in which antimicrobials are used might provide important factors for the prediction of the emergence and spread of antimicrobial resistance. We used 1547 variables from the World Bank dataset consisting of socioeconomic, developmental, health, and nutritional indicators; data from a global sewage-based study on antimicrobial resistance (abundance of antimicrobial resistance genes [ARGs]); and data on antimicrobial usage computed from the ECDC database and the IQVIA database. We characterised and built models predicting the global resistome at an antimicrobial class level. We used a generalised linear mixed-effects model to estimate the association between antimicrobial usage and ARG abundance in the sewage samples; a multivariate random forest model to build predictive models for each antimicrobial resistance class and to select the most important variables for ARG abundance; logistic regression models to test the association between the predicted country-level antimicrobial resistance abundance and the country-level proportion of clinical resistant bacterial isolates; finite mixture models to investigate geographical heterogeneities in the abundance of ARGs; and multivariate finite mixture models with covariates to investigate the effect of heterogeneity in the association between the most important variables and the observed ARG abundance across the different country subgroups. We compared our predictions with available clinical phenotypic data from the SENTRY Antimicrobial Surveillance Program from eight antimicrobial classes and 12 genera from 56 countries. Using antimicrobial use data from between Jan 1, 2016, and Dec 31, 2019, we found that antimicrobial usage was not significantly associated with the global ARG abundance in sewage (p=0·72; incidence rate ratio 1·02 [95% CI 0·92-1·13]), whereas country-specific World Bank's variables explained a large amount of variation. The importance of the World Bank variables differed between antimicrobial classes and countries. Generally, the estimated global ARG abundance was positively associated with the prevalence of clinical phenotypic resistance, with a strong association for bacterial groups in the human gut. The associations between bacterial groups and ARG abundance were positive and significantly different from zero for the aminoglycosides (three of the four of the taxa tested), β-lactam (all the six microbial groups), fluoroquinolones (seven of nine of the microbial groups), glycopeptide (one microbial group tested), folate pathway antagonists (four of five microbial groups), and tetracycline (two of nine microbial groups). Metagenomic analysis of sewage is a robust approach for the surveillance of antimicrobial resistance in pathogens, especially for bacterial groups associated with the human gut. Additional studies on the associations between important socioeconomic, nutritional, and health factors and antimicrobial resistance should consider the variation in these associations between countries and antimicrobial classes. EU Horizon 2020 and Novo Nordisk Foundation.

Interrogating direct NLRP3 engagement and functional inflammasome inhibition using cellular assays

As a key regulator of the innate immune system, the NLRP3 inflammasome responds to a variety of environmental insults through activation of caspase-1 and release of the proinflammatory cytokines IL-1β and IL-18. Aberrant NLRP3 inflammasome function is implicated in numerous inflammatory diseases, spurring drug discovery efforts at NLRP3 as a therapeutic target. A diverse array of small molecules is undergoing preclinical/clinical evaluation with a reported mode of action involving direct modulation of the NLRP3 pathway. However, for a subset of these ligands the functional link between live-cell target engagement and pathway inhibition has yet to be fully established. Herein we present a cohort of mechanistic assays to both query direct NLRP3 engagement in cells, and functionally interrogate different nodes of NLRP3 pathway activity. This system enabled the stratification of potency for five confirmed NLRP3 inhibitors, and identification of two reported NLRP3 inhibitors that failed to demonstrate direct pathway antagonism.

Detection and genomic characterization of a multidrug-resistant Salmonella Newport co-harbouring blaCMY-2, qnrB19 and mcr-9 from the diarrheic faeces of a foal

ObjectivesThis study reports the genomic characterization of the multidrug resistant Salmonella Newport strain 195_20 recovered from the diarrheic faeces of a foal in Brazil and co-harbouring the mcr-9, blaCMY-2 and qnrB19 antibiotic resistance genes. MethodsBacterial isolate positive for mobile colistin resistance gene (mcr-9) was submitted to antimicrobial susceptibility testing by disk diffusion and broth microdilution for colistin and polymyxin B. The isolate was submitted to whole genome sequencing by Illumina technology and Nanopore Sequencing. Conjugation assays, plasmid sizes determined by S1-PFGE and plasmid content were investigated by hybrid assembly after MinIon long reads sequencing. ResultsIsolate 195_20 was identified as sequence type ST45, resistant to penicillin and cephalosporins (ampicillin, ceftazidime, ceftriaxone and cefotaxime), aminoglycosides (streptomycin and gentamicin), phenicol (chloramphenicol), quinolones and fluoroquinolones (nalidixic acid, ciprofloxacin, and pefloxacin), folate pathway antagonists (sulfonamides and trimethoprim-sulfamethoxazole), and tetracycline. A transferable IncHI2/IncHI2A plasmid sized ca. 262kb was found to carry the mcr-9 gene in a module consisting of IS903-mcr-9-wbuC-IS26. In addition, an 174kb IncC and a 48kb IncN plasmid were also identified in the 195_20 isolate, carrying blaCMY-2 and qnrB19, respectively. ConclusionsNot surprisingly, isolate 195_20 was susceptible to polymyxins, possibly due to absence of qseBC regulatory operon. Presence of mobile colistin resistance (mcr-9), third-generation cephalosporins (blaCMY-2) and quinolone (qnrB19) resistance determinants in zoonotic pathogens from animals in close contact with humans alerts for the possible route of transmission between these different reservoirs.

Establishment of a 96-well transwell system using primary human gut organoids to capture multiple quantitative pathway readouts

Disruptions in the gut epithelial barrier can lead to the development of chronic indications such as inflammatory bowel disease (IBD). Historically, barrier function has been assessed in cancer cell lines, which do not contain all human intestinal cell types, leading to poor translatability. To bridge this gap, we adapted human primary gut organoids grown as monolayers to quantify transcription factor phosphorylation, gene expression, cytokine production, and barrier function. In this work we describe and characterize a novel 96-well human gut organoid-derived monolayer system that enables quantitative assessment of candidate therapeutics. Normal human intestine differentiation patterns and barrier function were characterized and confirmed to recapitulate key aspects of in vivo biology. Next, cellular response to TNF-α (a central driver of IBD) was determined using a diverse cadre of quantitative readouts. We showed that TNF-α pathway antagonists rescued damage caused by TNF-α in a dose-dependent manner, indicating that this system is suitable for quantitative assessment of barrier modulating factors. Taken together, we have established a robust primary cell-based 96-well system capable of interrogating questions around mucosal response. This system is well suited to provide pivotal functional data to support translational target and drug discovery efforts.

Cross-species analysis of SHH medulloblastoma models reveals significant inhibitory effects of trametinib on tumor progression

Sonic Hedgehog (SHH) medulloblastomas (MBs) exhibit an intermediate prognosis and extensive intertumoral heterogeneity. While SHH pathway antagonists are effective in post-pubertal patients, younger patients exhibit significant side effects, and tumors that harbor mutations in downstream SHH pathway genes will be drug resistant. Thus, novel targeted therapies are needed. Here, we performed preclinical testing of the potent MEK inhibitor (MEKi) trametinib on tumor properties across 2 human and 3 mouse SHH MB models in vitro and in 3 orthotopic MB xenograft models in vivo. Trametinib significantly reduces tumorsphere size, stem/progenitor cell proliferation, viability, and migration. RNA-sequencing on human and mouse trametinib treated cells corroborated these findings with decreased expression of cell cycle, stem cell pathways and SHH-pathway related genes concomitant with increases in genes associated with cell death and ciliopathies. Importantly, trametinib also decreases tumor growth and increases survival in vivo. Cell cycle related E2F target gene sets are significantly enriched for genes that are commonly downregulated in both trametinib treated tumorspheres and primary xenografts. However, IL6/JAK STAT3 and TNFα/NFκB signaling gene sets are specifically upregulated following trametinib treatment in vivo indicative of compensatory molecular changes following long-term MEK inhibition. Our study reveals a novel role for trametinib in effectively attenuating SHH MB tumor progression and warrants further investigation of this potent MEK1/2 inhibitor either alone or in combination with other targeted therapies for the treatment of SHH MB exhibiting elevated MAPK pathway activity.

Extract of Chenopodium album lowers blood pressure in rats through endothelium-dependent and -independent vasorelaxation

To investigate the antihypertensive effect of crude extract of Chenopodium album (Ca.Cr), based on its medicinal use in hypertension. Ca.Cr and its fractions were tested in-vivo in normotensive anesthetized rats for blood pressure-lowering effect. In-vitro experiments were performed on isolated rat aortae to explore the vascular mechanism(s). In normotensive anesthetized rats, Ca.Cr produced a dose-dependent (1-300mg/kg) fall (30%mmHg) in mean arterial pressure (MAP). Among the fractions, nHexane was the most potent (46% fall). In rat aortic rings precontracted with phenylephrine (PE), Ca.Cr and its fractions (except Ca.Aq) produced endothelium-dependent vasorelaxation, which was partially reversed with endothelium removal and by pretreating intact aortic rings with L-NAME (10μM) and atropine (1μM). This relaxation to Ca.Cr and fractions (nHexane, ethylacetate and chloroform) was also eliminated with indomethacin pretreatment, however, it unmasked a vasoconstriction effect with Ca.Cr only. Surprisingly, the aqueous fraction produced a calcium sensitive strong vasoconstriction instead of vasorelaxation. The crude extract and its fractions (except Ca.Aq) also antagonized vasoconstriction induced with high K+ (80mM), suggesting calcium antagonistic effect. The aqueous fraction produced mild vasorelaxation against high K+. This effect was further confirmed when pretreatment of the aortic rings with different concentrations of crude extract and fractions suppressed CaCl2 concentration response curves, similar to verapamil. In acute toxicity test, Ca.Cr extract was found safe up to 5g/kg body weight in mice. These findings suggest that crude extract and fractions of C.album produced vasorelaxant effect through muscarinic receptors linked-NO pathway, prostaglandin (endothelium-dependent) and calcium antagonism (endothelium-independent), which explains the blood pressure lowering effect of C.album in rats.

An Overview of Antimicrobial Resistance Profiles of Publicly Available Salmonella Genomes with Sufficient Quality and Metadata.

Salmonella enterica (S. enterica) is a commensal organism or pathogen causing diseases in animals and humans, as well as widespread in the environment. Antimicrobial resistance (AMR) has increasingly affected both animal and human health and continues to raise public health concerns. A decade ago, it was estimated that the increased use of whole genome sequencing (WGS) combined with sharing of public data would drastically change and improve the surveillance and understanding of Salmonella epidemiology and AMR. This study aimed to evaluate the current usefulness of public WGS data for Salmonella surveillance and to investigate the associations between serovars, antibiotic resistance genes (ARGs), and metadata. Out of 191,306 Salmonella genomes deposited in European Nucleotide Archive and NCBI databases, 47,452 WGS with sufficient minimum metadata (country, year, and source) of S. enterica were retrieved from 116 countries and isolated between 1905 and 2020. For in silico analysis of the WGS data, KmerFinder, SISTR, and ResFinder were used for species, serovars, and AMR identification, respectively. The results showed that the five common isolation sources of S. enterica are human (29.10%), avian (22.50%), environment (11.89%), water (9.33%), and swine (6.62%). The most common ARG profiles for each class of antimicrobials are β-lactam (blaTEM-1B; 6.78%), fluoroquinolone [(parC[T57S], qnrB19); 0.87%], folate pathway antagonist (sul2; 8.35%), macrolide [mph(A); 0.39%], phenicol (floR; 5.94%), polymyxin B (mcr-1.1; 0.09%), and tetracycline [tet(A); 12.95%]. Our study reports the first overview of ARG profiles in publicly available Salmonella genomes from online databases. All data sets from this study can be searched at Microreact.

Smoothened antagonist sonidegib affects the development of D. melanogaster larvae via suppression of epidermis formation

Hedgehog (Hh) signaling is essential for the regulation of embryonic growth and development, the maintenance of stem cell autostasis, and tissue formation, whether in vertebrates or invertebrates. However, exploration into the Hh pathway antagonists in Drosophila or other pests of agricultural importance has been scant. In order to gain a better understanding of the potential utility of the antagonists in insect investigations, a conventional Hh antagonist, sonidegib, was used to evaluate the effects on the development of Drosophila larvae. The results showed that early instar larvae exposed to sonidegib exhibited new epidermal abnormalities and decreased motility after molting. Transcriptome analysis revealed that Sonidegib had a profound effect on chitin-based cuticle development throughout all stages of larvae. Physiological experiments revealed that sonidegib suppressed the epidermis formation and decreased the chitin content. The results of this study shed new light on the potential use of Hh antagonists in agricultural pest management.

Genome Sequence Analysis of a Wohlfahrtiimonas chitiniclastica Strain Isolated from a Septic Wound of a Hospitalized Patient in Uganda

We report a genome sequence of Wohlfahrtiimonas chitiniclastica strain MUWRP0946, isolated from a hospitalized patient in Uganda. The genome size was 2.08 million bases, and the genome completeness was 94.22%. The strain carries tetracycline, folate pathway antagonist, β-lactam, and aminoglycoside antibiotic resistance genes.

Antibiotic resistance genes, mobile elements, virulence genes, and phages in cultivated ESBL-producing Escherichia coli of poultry origin in Kwara State, North Central Nigeria

The paucity of information on the genomic diversity of drug-resistant bacteria in most food-producing animals, including poultry in Nigeria, has led to poor hazard characterization and the lack of critical control points to safeguard public health. Hence, this study used whole genome sequencing (WGS) to assess the presence and the diversity of antibiotic resistance genes, mobile genetic elements, virulence genes, and phages in Extended Spectrum Beta Lactamase producing Escherichia coli (ESBL – E. coli) isolates obtained from poultry via the EURL guideline of 2017 in Ilorin, Nigeria. The prevalence of ESBL – E. coli in poultry was 10.5 % (n = 37/354). The phenotypic antibiotic susceptibility testing showed that all the ESBL— E. coli isolates were multi-drug resistant (MDR). The in-silico analysis of the WGS raw-read data from 11 purposively selected isolates showed that the isolates had a wide array of ARGs that conferred resistance to beta-lactam antibiotics, and 8 other classes of antibiotics (fluoroquinolones, foliate pathway antagonists, aminoglycoside, phenicol, tetracycline, epoxide, macrolides, and rifamycin). All the ARGs were in the bacterial chromosome except in two isolates where plasmid-mediated quinolone resistance (PMQR) was detected. Two isolates carried the gyrAp.S83L mutation which confers resistance to certain fluoroquinolones. The mobilome consisted of several Col-plasmids and the predominant IncF plasmids belonged to the IncF64:A-:B27 sequence type. The virulome consisted of genes that function as adhesins, iron acquisition genes, toxins, and protectins. Intact phages were found in 8 of the 11 isolates and the phageome consisted of representatives of four families of viruses: Myoviridae (62.5 %, n = 5/8), Siphoviridae (37.5 %, n = 3/8), Inoviridae (12.5 %, n = 1), and Podoviridae (12.5 %, n = 1/8). ESBL - E. coli isolates harboured 1–5 intact phages and no ARGs were identified on any of the phages. Although five of the isolates belonged to phylogroup A, the isolates were diverse as they belonged to different serotype and sequence types. Our findings demonstrate the high genomic diversity of ESBL - E. coli of poultry origin in Ilorin, Nigeria. These diverse isolates harbor clinically relevant ARGs, mobile elements, virulence genes, and phages that may have detrimental zoonotic potentials on human health.

Frem1 activity is regulated by Sonic hedgehog signaling in the cranial neural crest mesenchyme during midfacial morphogenesis.

Frem1 has been linked to human face shape variation, dysmorphology, and malformation, but little is known about its regulation and biological role in facial development. During midfacial morphogenesis in mice, we observed Frem1 expression in the embryonic growth centers that form the median upper lip, nose, and palate. Expansive spatial gradients of Frem1 expression in the cranial neural crest cell (cNCC) mesenchyme of these tissues suggested transcriptional regulation by a secreted morphogen. Accordingly, Frem1 expression paralleled that of the conserved Sonic Hedgehog (Shh) target gene Gli1 in the cNCC mesenchyme. Suggesting direct transcriptional regulation by Shh signaling, we found that Frem1 expression is induced by SHH ligand stimulation or downstream pathway activation in cNCCs and observed GLI transcription factor binding at the Frem1 transcriptional start site during midfacial morphogenesis. Finally, we found that FREM1 is sufficient to induce cNCC proliferation in a concentration-dependent manner and that Shh pathway antagonism reduces Frem1 expression during pathogenesis of midfacial hypoplasia. By demonstrating that the Shh signaling pathway regulates Frem1 expression in cNCCs, these findings provide novel insight into the mechanisms underlying variation in midfacial morphogenesis.

2005. Direct Hospitalization Costs Associated with Resistant and Susceptible Salmonella and Shigella Infections in the United States — 2012–2019

Abstract Background Foodborne illnesses cost ∼$90 billion in the United States annually. Antibiotic-resistant infections may be more difficult to treat and can be associated with severe outcomes, leading to higher costs and longer length of stay (LOS). We described LOS and costs for hospital admissions for Salmonella and Shigella infections by resistance status. Methods We reviewed all inpatient admissions from ∼300 hospitals in the Premier Healthcare database during 2012–2019 and included records with culture-confirmed nontyphoidal Salmonella (NTS), typhoidal Salmonella (serotypes Typhi and Paratyphi A, B, and C), or Shigella. We classified infections as resistant if laboratory testing found resistance to ≥1 drug in the clinically important antibiotic categories (fluoroquinolones, macrolides, penicillins, folate pathway antagonists, or third-generation cephalosporins) or susceptible otherwise. We used the Wilcoxon rank sum test to assess difference in median LOS and cost of patient hospitalization for resistant vs. susceptible infections by pathogen type. Results Of 4,168 persons hospitalized, about half (n=2,129, 51%) were female; median age was 52 [IQR: 26‒68] years; 29% (n=1,217) of infections were resistant. For NTS infections, median LOS was 4 [IQR: 3‒7] days for resistant and 4 [IQR: 3‒6] (p=0.04) for susceptible; for typhoidal Salmonella, 6 [IQR: 5‒8] for resistant and 5 [IQR: 3‒8] for susceptible (p=0.15); and for Shigella, 3 [IQR: 2‒4] for both (p=0.09). Median costs were highest for typhoidal Salmonella ($12,577 resistant vs. $9,238 susceptible; p=0.21), followed by NTS ($7,179 vs $6,421, p=0.01) and Shigella ($4,975 vs $5,335; p=0.26). Conclusion Hospitalization costs were higher for patients with some resistant infections. Longer LOS may contribute to higher costs; however, it is not clear whether other pathogen or patient factors (e.g., comorbidities) also contributed. Additional data and adjusted analyses may provide insight into reasons for the cost difference. If resistant infections are shown to be the cause, our findings support that public health interventions to limit the spread of resistant infections could reduce costs. Disclosures All Authors: No reported disclosures.

A comprehensive description of the TolC effect on the antimicrobial susceptibility profile in Enterobacter bugandensis.

Enterobacter bugandensis is an emerging human pathogen in which multidrug resistant strains have been continuously isolated from various environments. Thus, this organism possesses the potential to pose challenges in human healthcare. However, the mechanisms, especially the efflux pumps, responsible for the multidrug resistance in E. bugandensis remain to be well elucidated. The Enterobacter strain CMCC(B) 45301 was specifically identified using whole genome sequencing. The specific CMCC(B) 45301 homologues of the TolC dependent efflux-pump genes characterized in Escherichia coli were identified. The tolC deletion mutant in CMCC(B) 45301 was constructed and subjected to susceptibility tests using 26 different antimicrobial agents, along with the wild type strain. The synergistic effects combining the Bacillus crude extract (BCE) and several other TolC-affected compounds against CMCC(B) 45301 were assayed. We reclassified the Enterobacter CMCC(B) 45301 strain from species cloacae to bugandensis, on the basis of its whole genome sequence. We found that the CMCC(B) 45301 TolC, AcrAB, AcrD, AcrEF, MdtABC, EmrAB, and MacAB exhibit high similarity with their respective homologues in E. coli and Enterobacter cloacae. Our results for the susceptibility tests revealed that lacking tolC causes 4- to 256-fold decrease in the minimal inhibitory concentrations of piperacillin, gentamicin, kanamycin, tetracycline, norfloxacin, ciprofloxacin, chloramphenicol, and erythromycin against CMCC(B) 45301. In addition, the inhibition zones formed by cefuroxime, cefoperazone, amikacin, streptomycin, minocycline, doxycycline, levofloxacin, florfenicol, trimethoprim-sulfamethoxazole, azithromycin, lincomycin, and clindamycin for the tolC mutant were larger or more obvious than that for the parent. Our data suggested the important role played by TolC in CMCC(B) 45301 susceptibility to common antibiotic families covering ß-lactam, aminoglycoside, tetracycline, fluoroquinolone, phenicol, folate pathway antagonist, macrolide, and lincosamide. Deletion for tolC also increased the susceptibility of CMCC(B) 45301 to berberine hydrochloride and BCE, two natural product-based agents. Finally, we found that erythromycin, norfloxacin, and ciprofloxacin can potentiate the antibacterial activity of BCE against CMCC(B) 45301. The present study elaborated the comprehensive TolC effect on the antimicrobial susceptibility profile in E. bugandensis, which might contribute to the development of more therapeutic options against this nosocomial pathogen.

PHF8 promotes osteogenic differentiation of BMSCs in old rat with osteoporosis by regulating Wnt/β-catenin pathway.

Osteoporosis is a progressive bone disorder with a higher incidence in the elderly and has become a major public health concern all over the world. Therefore, it is urgent to investigate the mechanisms underlying the pathogenesis of osteoporosis. In this study, the osteoporosis animal model was established, and then rat bone marrow mesenchymal stem cells (rBMSCs) were cultured. The results showed that PHF8 expression was decreased in osteoporosis rats compared to controls. Overexpression of PHF8 promoted BMSC osteogenic differentiation and the expression of osteogenesis-related genes. In addition, the Wnt/β-catenin signaling pathway in BMSCs was inhibited in osteoporosis rats, which was rescued by overexpression of PHF8. After treatment with the Wnt pathway antagonist, the improved osteogenic differentiation of BMSCs induced by overexpression of PHF8 was blocked. Collectively, our data revealed that the decreased expression of PHF8 in osteoporosis rats suppressed the osteogenic differentiation of BMSCs, which was then restored by PHF8 overexpression. Furthermore, the inhibition of the Wnt/β-catenin signaling pathway in BMSCs suppressed osteogenic differentiation. Thus, these findings indicated that PHF8 plays a role in osteogenic differentiation through the Wnt/β-catenin signaling pathway.