P-glycoprotein Research Articles

Growth of microbes in competitive lifestyles promotes increased ARGs in soil microbiota: insights based on genetic traits

BackgroundThe widespread selective pressure of antibiotics in the environment has led to the propagation of antibiotic resistance genes (ARGs). However, the mechanisms by which microbes balance population growth with the enrichment of ARGs remain poorly understood. To address this, we employed microcosm cultivation at different antibiotic (i.e., Oxytetracycline, OTC) stresses across the concentrations from the environmental to the clinical. Paired with shot-gun metagenomics analysis and quantification of bacterial growth, trait-based assessment of soil microbiota was applied to reveal the association between key ARG subtypes, representative bacterial taxa, and functional-gene features that drive the growth of ARGs.ResultsOur results illuminate that resistome variation is closely associated with bacterial growth. A non-monotonic change in ARG abundance and richness was observed over a concentration gradient from none to 10 mg/l. Soil microbiota exposed to intermediate OTC concentrations (i.e., 0.1 and 0.5 mg/l) showed greater increases in the total abundance of ARGs. Community compositionally, the growth of representative taxa, i.e., Pseudomonadaceae was considered to boost the increase of ARGs. It has chromosomally carried kinds of multidrug resistance genes such as mexAB-oprM and mexCD-oprJ could mediate the intrinsic resistance to OTC. Streptomycetaceae has shown a better adaptive ability than other microbes at the clinical OTC concentrations. However, it contributed less to the ARGs growth as it represents a stress-tolerant lifestyle that grows slowly and carries fewer ARGs. In terms of community genetic features, the community aggregated traits analysis further indicates the enhancement in traits of resource acquisition and growth yield is driving the increase of ARGs abundance. Moreover, optimizations in energy production and conversion, alongside a streamlining of bypass metabolic pathways, further boost the growth of ARGs in sub-inhibitory antibiotic conditions.ConclusionThe results of this study suggest that microbes with competitive lifestyles are selected under the stress of environmental sub-inhibitory concentrations of antibiotics and nutrient scarcity. They possess greater substrate utilization capacity and carry more ARGs, due to this they were faster growing and leading to a greater increase in the abundance of ARGs. This study has expanded the application of trait-based assessments in understanding the ecology of ARGs propagation. And the finding illustrated changes in soil resistome are accompanied by the lifestyle switching of the microbiome, which theoretically supports the ARGs control approach based on the principle of species competitive exclusion.4ZqjNsN9dRS1iWJDwru5hLVideo

Effect of Fecal Microbiota Transplant on Antibiotic Resistance Genes Among Patients with Chronic Pouchitis.

Pouchitis is common among patients with ulcerative colitis (UC) who have had colectomy with ileal pouch-anal anastomosis. Antibiotics are first-line therapy for pouch inflammation, increasing the potential for gut colonization with multi-drug resistant organisms (MDRO). Fecal microbial transplant (FMT) is being studied in the treatment of pouchitis and in the eradication of MDRO. Prior work using aerobic antibiotic culture disks suggests that some patients with chronic pouchitis may regain fluoroquinolone sensitivity after FMT. However, gut MDRO include anaerobic, fastidious organisms that are difficult to culture using traditional methods. We aimed to assess whether FMT reduced the abundance of antibiotic resistance genes (ARG) or affected resistome diversity, evenness, or richness in patients with chronic pouchitis. We collected clinical characteristics regarding infections and antibiotic exposures for 18 patients who had previously been enrolled in an observational study investigating FMT as a treatment for pouchitis. Twenty-six pre- and post-FMT stool samples were analyzed using FLASH (Finding Low Abundance Sequences by Hybridization), a CRISPR/Cas9-based shotgun metagenomic sequence enrichment technique that detects acquired and chromosomal bacterial ARGs. Wilcoxon rank sum tests were used to assess differences in clinical characteristics, ARG counts, resistome diversity and ARG richness, pre- and post-FMT. All 13 of the patients with sufficient stool samples for analysis had recently received antibiotics for pouchitis prior to a single endoscopic FMT. Fecal microbiomes of all patients had evidence of multi-drug resistance genes and ESBL resistance genes at baseline; 62% encoded fluoroquinolone resistance genes. A numerical decrease in overall ARG counts was noted post-FMT, but no statistically significant differences were noted (P = 0.19). Richness and diversity were not significantly altered. Three patients developed infections during the 5-year follow-up period, none of which were associated with MDRO. Antibiotic resistance genes are prevalent among antibiotic-exposed patients with chronic pouchitis. FMT led to a numerical decrease, but no statistically significant change in ARG, nor were there significant changes in the diversity, richness, or evenness of ARGs. Further investigations to improve FMT engraftment and to optimize FMT delivery in patients with inflammatory pouch disorders are warranted.

A family with gallstone disease: defining inherited risk in the era of clinical genetic testing.

Gallstones are among the most frequent hepatobiliary conditions. Although in most cases, they remain asymptomatic, they can cause complications and, in such cases, invasive treatments like endoscopic retrograde cholangiography (ERC) or cholecystectomy are required. Here, we present the results of genetic testing of a single family with a high incidence of symptomatic gallstones and cholestatic liver phenotypes. Gallstone disease was detected among seven family members spanning three generations, and DNA samples were available from five of them. Genotyping was performed using TaqMan assays for known, selected genetic risk factors for gallstones and cholestasis, as well as next generation sequencing (NGS) of three genes involved in hepatobiliary transport. In all genotyped patients, we detected at least one copy of the gallstone-predisposing p.D19H variant in the hepatobiliary sterol transporter ABCG5/8, and in three cases, this variant was found in the rare homozygous state. In addition, the patients were all homozygous carriers of two intronic variants (c.2211+16C >T and c.3508-16T>C) and two common polymorphisms (c.504C>T and c.711A>T) in the ABCB4 gene, as well as the ATP8B1 gene variant c.696T>C. All genotyped patients also carried the predisposing variants c.1331C>T and c.3084A>G of the hepatobiliary bile salt export pump ABCB11 in either heterozygous or homozygous form. Hence, we propose that these variants taken together may have contributed to the high frequency of gallstone disease in this family, although functional studies for some variants are still lacking. In this report, we present these findings and discuss the challenges associated with interpreting sequencing data.

Influence of ABCB1 polymorphisms on aripiprazole and dehydroaripiprazole plasma concentrations

Aripiprazole (ARI) is an atypical antipsychotic which is a substrate of P-glycoprotein (P-gp), a transmembrane glycoprotein that plays a crucial role in eliminating potentially harmful compounds from the organism. ARI once-monthly (AOM) is a long-acting injectable form which improves treatment compliance. Genetic polymorphisms in ABCB1 may lead to changes in P-gp function, leading to individual differences in drug disposition. The present study aims to determine how the different variants of the three most prevalent SNPs of the ABCB1 gene affect plasma concentrations of ARI, of its active metabolite dehydroaripiprazole (DHA) and ARI/DHA ratio in patients under AOM treatment. The metabolizing state of the two main aripiprazole metabolizing enzymes (CYP2D6 and CYP3A4) were considered to specifically study the effect of P-gp on plasma concentrations of the parent compound and its active metabolite. The study found a clear relationship between the genotypes found for the different ABCB1 SNPs and the ARI/DHA ratio. Specifically, patients with GG genotype in G2677T have almost twice the ratio compared to TT genotype. Similarly, this increase is also found in C3435T with 1.4-fold and in C1236T with 1.6-fold for the same genotypes. Regarding haplotypes, significant differences were obtained between CC-GG-CC and TT-TT-TT patients, with an 87.9% higher ratio in patients with the CC-GG-CC haplotype. There was a clear trend towards lower ARI concentrations and higher DHA concentrations when the presence of mutated T alleles increases. The ABCB1 gene could be a good partner along with CYP2D6 and CYP3A4 genotyping in conjunction with monitoring ARI plasma concentrations.

Genomic Analysis of Virulent, Multidrug Resistant Klebsiella pneumoniae and Klebsiella oxytoca from Bloodstream Infections, South Africa.

A total of 46 suspected Klebsiella species (spp.) were collected from blood cultures within the uMgungundlovu District in the KwaZulu-Natal Province. Antibiotic susceptibility was determined against a panel of 19 antibiotics using the disk diffusion test. A subset of 14 MDR K. pneumoniae (n=10) and K. oxytoca (n=4) isolates were selected based on their antibiograms and subjected to whole genome sequencing (WGS). The sequence types (STs), resistome, virulome, mobilome, capsule loci (KLs) were analysed using relevant WGS and bioinformatics tools. Of the 10 K. pneumoniae sequence types (ST) identified, the most common were ST25 (n=3), ST101 (n=3), and 4 K. oxytoca belonged to ST450 (n=3). The two high-risk K. pneumoniae clones ST15, and ST17 were identified. O and K capsule types were identified, with predominance of KL2, KL17, KL29, O1/O2v2, O1/O2v1, and OL104 respectively. The majority of isolates displayed multidrug resistance predominantly carrying β-lactamase genes, including blaCTX-M-15, blaTEM-1B, blaSHV, and blaOXA-1, and blaOXY including the carbapenemase blaOXA-181 in two (14.3%) study isolates. Other resistance genes included: aac(6')-lb-cr, aac(3), aac, aph, aad, dfr, tet(A), and tet(D), mph(A), sul1, sul2, oqx, qnr, acrR, ramR, parC, gyrA, arr-3, cat, fosA, qacE genes conferring resistance to aminoglycosides, trimethoprim, tetracycline, macrolide, sulfonamides, fluoroquinolones, rifampicin phenicols, fosfomycin, and quaternary ammonium compound disinfectant. Virulence factors related to hypervirulence: encoding aerobactin (iuc, iutA), salmochelin (iro), yersiniabactin (ybt), enterobactin (ent), type 1 and 3 (mrk and fim), and capsule synthesis (rcsA and rcsB) were identified. IncF, IncR, and Col plasmid replicon types and class I integrons were detected, with IncFIB(K) predominance. The blaCTX-M-15 and blaTEM-1 genes were bracketed by Tn3 transposons, ISEc9, recombinase and IS91 insertion sequences. The convergence of multidrug resistance and hypervirulence genes in Klebsiella strains is a potential clinical concern. Carbapenemase, ESBL screening and genomic surveillance are urgently required in hospital environments.

Plasmid hybrids as vectors for antibiotic resistance in environmental Escherichia coli.

This study investigated the potential role of phages in the dissemination of antimicrobial resistance genes (ARGs) and virulence factor genes (VFGs) in Escherichia coli (E. coli). A comprehensive in silico analysis of 18,410 phage sequences retrieved from the National Center for Biotechnology Information database (NCBI) revealed distinct carriage patterns for ARGs and VFGs between lytic, temperate, and chronic phage types. Notably, 57 temperate phages carried ARGs, particularly associated with multidrug and aminoglycoside resistance. Temperate phages (8.97%, 635/7081) and chronic phages (8.09%, 14/173) exhibited a significantly higher prevalence of VFGs (Chi-Square, p≤0.05), particularly associated with exotoxin-related genes, compared to lytic phages (0.05%, 6/11,156). This underscores the role phages play as reservoirs and potential vectors for the dissemination of ARGs and VFGs in bacteria. Our environmental E. coli isolates (n=60) were found to carry 179 intact prophages containing polymyxin, macrolide, tetracycline, and multidrug resistance genes as well as various VFGs. This study documents the presence of phage-plasmids (P-Ps) in environmental E. coli isolates, offering new insights into horizontal gene transfer (HGT) mechanisms. Notably, the blaCTX-M-15 gene, associated with beta-lactam resistance, was identified in two P-Ps, suggesting a potentially novel route for the dissemination of beta-lactam resistance. The diverse replicon types observed in P-Ps suggest a broader integration capacity compared to traditional plasmids, potentially enabling the blaCTX-M-15 gene dissemination across diverse bacterial species. This study provides valuable insights into the multifaceted role of phages in shaping the antimicrobial resistance landscape. Further research is necessary to fully understand the intricate mechanisms underlying phage-mediated ARG and VFG dissemination.

Effect of microplastics concentration and size on pollutants removal and antibiotic resistance genes (ARGs) generation in constructed wetlands: a metagenomics insight

The accrual of microplastics (MPs) and antibiotics poses synergistic threats to the environment. This study systemically examined the effect of environmental-level (μg/L) MPs (90–110 μm) and nanoplastics (NPs, 700nm) on constructed wetlands (CWs) treating oxytetracycline-contaminated wastewater via metagenomics analysis. Polystyrene (PS) MPs notably hindered the removal of nitrogen, phosphorus, and oxytetracycline, particularly at high level (1000μg/L), with removal rates of 73.34%, 59.59%, and 99.34%, respectively. Among them, the removal of NH4+-N decreased the most in comparison to CK, at 15.26%. Antibiotic resistance genes (ARGs) copies/16S rRNA ranged from 0.26 to 0.42 in CWs, exceeding that found in rivers by a factor of 1.5 to 2.5 times. The relative abundance of multidrug resistance genes (mdtB, acrB, mexF, mdtC, and mexT) and tetracycline resistance genes (txtA, tetG, and tetP) exhibited a pronounced increase under MPs exposure, ranging from 0.06 to 0.14 and 0.01 to 0.08 copies/16S rRNA, respectively. Redundancy and network analyses emphasized robust associations among contaminant reduction, ARG abundance, and microbial community. Partial least squares path modeling indicated MPs exerted a more profound influence on pollutant removal (coefficient = 0.8194), microbial community (coefficient = 0.3358) and ARGs dissemination (coefficient = 0.6566) compared to NPs. MPs concentrations significantly affects pollutants removal and ARGs proliferation, and MPs with larger sizes amplified ARG dissemination. This research highlights the influence of MPs on CW-mediated wastewater treatment and ARGs accumulation, offering valuable insights for developing ecological wastewater treatment strategies tailored to multi-pollutant scenarios. These insights are fundamental in developing sustainable solutions to the adverse impacts of MPs on ecosystems.

Molecular genetic characteristic of pulmonary tuberculosis associated with ABCB1 gene expression of multidrugresistance protein P-gp

Background: Tuberculous inflammation is mediated by a complex molecular signaling pathway, the analysis of which makes it possible to identify promising biomarkers and targets for the development of new diagnostic, prognostic and pharmacological approaches in order to improve the effectiveness of anti-tuberculosis chemotherapy. Determining the relationship between key inflammatory cytokines, the multidrug-resistant protein P-gp and the activity of specific inflammation in the surgical material of patients with pulmonary tuberculosis may prove to be a novel tool in the development of pathogenetic therapy and personalized medicine. Aims: to characterize molecular and genetic profiles of tuberculomas and identify genes that correlate with the expression of the ABCB1 gene of the P-gp protein in the surgical material of patients with pulmonary tuberculosis. Research objectives: 1) to obtain molecular and genetic characteristics of tuberculosis by real-time PCR and compare it with the activity of tuberculous inflammation; 2) to carry out a correlation analysis between the expression of the ABCB1 gene and key cytokines of the tuberculosis process: IL-6, IL-10, IFN-γ, TGF-β, TNF-α, IL-1β. Materials and methods: A prospective cohort study was conducted on the basis of the FSBI CTRI. The object of the study was the surgical material of 35 patients diagnosed with multiple pulmonary tuberculomas. Histological examination methods were used for the morphological assessment of the surgical material. A real-time quantitative PCR method was used to analyze gene expression. Statistical processing was performed using the GraphPad Prism Version 7.04 software package (GraphPad Software, USA). The data is presented as a median with an interquartile range. The nonparametric Mann-Whitney U-test was used to compare the two groups. All p-values were two-sided and p 0.05 was considered statistically significant. The correlation between the variables was estimated using the Spearman correlation coefficient. The correlation analysis was carried out in the Microsoft Office Excel 2010 software. Results: The study revealed that the highest level of expression of ABCB1 gene of the P-gp protein is observed in tuberculomas with high activity of tuberculous inflammation, and its expression is correlated with the expression of the IL6 gene (p0.001) and the expression of the IL10 gene (p0.01). Tuberculomas of this group are also characterized by higher expression of the TGFB1, TNF and IL1B genes, compared with the group of moderate activity of specific inflammation. Conclusions: The data obtained indicate that in addition to pro-/anti-inflammatory cytokines, the P-gp protein plays an important role in the pathogenesis of tuberculous inflammation, especially with its high activity. Further clarification of the P-gp role in tuberculous inflammation may be an important step for the development of new approaches to treat tuberculosis using methods of HDT and personalized medicine.

Identification and validation of KIF20A for predicting prognosis and treatment outcomes in patients with breast cancer

Breast cancer is a leading cause of cancer-related deaths among women globally. It is imperative to explore novel biomarkers to predict breast cancer treatment response as well as progression. Here, we collected six breast cancer samples and paired normal tissues for high-throughput sequencing. By differential expression analysis, we found 1687 DEGs and identified the top 10 hub genes, including TOP2A, CDK1, BUB1B, KIF11, CCNA2, BUB1, CCNB1, KIF20A, DLGAP5 and CDC20. Univariate and multivariate Cox analyses on the METABRIC database and GSE96058 dataset demonstrated that KIF20A was an independent prognostic predictor for overall survival. KIF20A was positively correlated with cell cycle phases, including the cell cycle process, cycle G2 M phase transition and cell cycle DNA replication initiation. Single-cell analyses revealed that KIF20A was enriched in fibroblasts and endothelial within breast cancer stroma. Meanwhile, multidrug resistance (MDR) genes ABCB1, ABCC1 and ABCG2 were co-expressed with KIF20A in fibroblasts and endothelial cells within the stroma. MTABRIC database confirmed that high expression of KIF20A was positively correlated with treatment efficacy in patients with breast cancer. In conclusion, KIF20A could be served as a predictive biomarker for breast cancer prognosis and treatment outcomes. KIF20A may play a significant role by regulating cell cycle progression and modulating stromal progression in breast cancer. Our findings provided novel molecular insights that can guide personalized treatment strategies in breast cancer.

ATP-Binding Cassette Transporter Genes and microRNAs in Pediatric B-Cell ALL: Expression Insights.

Acute lymphocytic leukemia (ALL), characterized by uncontrolled growth of abnormal lymphocytes, predominantly affects children. Genetic analysis focusing on genes and microRNAs reveals important information about the biology of ALL, enabling accurate diagnosis and treatment. This study examines gene and microRNA expression in B cell ALL to improve early diagnosis and personalized treatment. Bone marrow samples were collected from patients both before and after the induction phase of chemotherapy. Comprehensive diagnostic techniques including flow cytometry, molecular assays, real-time PCR for common translocations, karyotyping, and complete blood count (CBC) analysis were employed. These methods were utilized to determine the type and risk assessment of ALL, identify specific gene and microRNA expressions, and measure blood cell counts. The study comprised 12 patients, all under the age of 18. Post-treatment RT-PCR analysis revealed significant reductions in the expression of the ABCB1 gene, miR-129-5p, and miR-9-5p following the induction phase of chemotherapy. Karyotype analysis indicated that two patients were hypodiploid; unfortunately, both of these patients did not survive. MicroRNAs and ABC genes serve as predictive and prognostic biomarkers in Acute Lymphoblastic Leukemia (ALL) and should be carefully reconsidered. It is more accurate to state that while microRNAs and ABC genes may potentially influence treatment response in ALL, further research is crucial to fully understand their roles in determining treatment outcomes.

Dissemination mechanisms of unique antibiotic resistance genes from flowback water to soil revealed by combined Illumina and Nanopore sequencing.

As a byproduct of shale gas extraction, flowback water (FW) is produced in large quantities globally. Due to the unique interactions between pollutants and microorganisms, FW always harbor multiple antibiotic resistance genes (ARGs) that have been confirmed in our previous findings, potentially serving as a point source for ARGs released into the environment. However, whether ARGs in FW can disseminate or integrate into the environmental resistome remains unclear. In this study, unique ARGs from FW were identified, and the ARG profiles in soil and FW-spiked soil were compared using a combination of Illumina and Nanopore sequencing. The results indicated that the total abundance of the soil resistome increased by 30.8 % in soil contaminated with FW. Of this increase, 11.1 % was attributable to the integration of exogenous ARGs from FW into the soil resistome. Sequence alignment at the gene level further confirmed the successful integration of 20 unique ARG sequences classified as multidrug and vancomycin resistance genes into the soil resistome. These 20 ARG sequences were detected only in the FW. Multiple lines of evidence indicated that horizontal gene transfer dominated ARG dissemination in soil contaminated by FW. This conclusion is supported by the discrepancy between changes in mobile ARGs and host abundance, the upregulation of oxidative stress-related genes (SOD1 and SOD2) and the SOS response (lexA and recA), as well as the upregulation of genes related to quorum sensing (virD4, virB9, and virB3) and naked DNA uptake (pilD, pilT, and pilQ).

Transcriptional responses to in vitro macrocyclic lactone exposure in Toxocara canis larvae using RNA-seq.

Toxocara canis, the causative agent of zoonotic toxocariasis in humans, is a parasitic roundworm of canids with a complex lifecycle. While macrocyclic lactones (MLs) are successful at treating adult T. canis infections when used at FDA-approved doses in dogs, they fail to kill somatic third-stage larvae. In this study, we profiled the transcriptome of third-stage larvae derived from larvated eggs and treated in vitro with 10 μM of the MLs - ivermectin and moxidectin with Illumina sequencing. We analyzed transcriptional changes in comparison with untreated control larvae. In ivermectin-treated larvae, we identified 608 differentially expressed genes (DEGs), of which 453 were upregulated and 155 were downregulated. In moxidectin-treated larvae, we identified 1,413 DEGs, of which 902 were upregulated and 511 were downregulated. Notably, many DEGs were involved in critical biological processes and pathways including transcriptional regulation, energy metabolism, neuronal structure and function, physiological processes such as reproduction, excretory/secretory molecule production, host-parasite response mechanisms, and parasite elimination. We also assessed the expression of known ML targets and transporters, including glutamate-gated chloride channels (GluCls), and ATP-binding cassette (ABC) transporters, subfamily B, with a particular focus on P-glycoproteins (P-gps). We present gene names for previously uncharacterized T. canis GluCl genes using phylogenetic analysis of nematode orthologs to provide uniform gene nomenclature. Our study revealed that the expression of Tca-glc-3 and six ABCB genes, particularly four P-gps, were significantly altered in response to ML treatment. Compared to controls, Tca-glc-3, Tca-Pgp-11.2, and Tca-Pgp-13.2 were downregulated in ivermectin-treated larvae, while Tca-abcb1, Tca-abcb7, Tca-Pgp-11.2, and Tca-Pgp-13.2 were downregulated in moxidectin-treated larvae. Conversely, Tca-abcb9.1 and Tca-Pgp-11.3 were upregulated in moxidectin-treated larvae. These findings suggest that MLs broadly impact transcriptional regulation in T. canis larvae.

Isolation method of brain microvessels from small frozen human brain tissue for blood-brain barrier protein expression analysis

BackgroundProtein expression analysis of isolated brain microvessels provides valuable insights into the function of the blood-brain barrier (BBB). However, isolation of brain microvessels from human brain tissue, particularly in small quantities, poses significant challenges. This study presents a method for isolating brain microvessels from a small amount of frozen human brain tissue, adapting techniques from an established mouse brain capillary isolation method.MethodsBrain microvessel fractions were obtained from approximately 0.3 g of frozen human brain tissue (frontal cortex) using a bead homogenizer for homogenization, followed by purification with a combination of cell strainers and glass beads. Protein expression in the isolated human microvessel fractions and whole-brain lysates was analyzed by western blot and proteomic analysis.ResultsMicroscopic imaging confirmed the successful isolation of brain microvessels from frozen human brain tissue. Protein quantification assays demonstrated that the microvessel fraction yielded sufficient protein for detailed expression analysis. Western blot analysis revealed an enrichment of BBB-selective proteins including multidrug resistance 1 (MDR1)/ATP-binding cassette sub-family B member 1 (ABCB1), glucose transporter protein type 1 (GLUT1)/solute carrier family 2 member 1 (SLC2A1), and claudin 5 (CLDN5), in the brain microvessel fraction compared to whole-brain lysates. Multiple reaction monitoring quantification of six BBB-selective proteins—MDR1, breast cancer resistance protein (BCRP)/ATP binding cassette subfamily G member 2 (ABCG2), GLUT1, monocarboxylate transporter 1 (MCT1)/solute carrier family 16 member 1 (SLC16A1), transferrin receptor, and CLDN5—revealed expression levels consistent with those observed in larger human brain samples. Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS)-based quantitative proteomics further demonstrated significant enrichment of human microvascular endothelial cells in the isolated fraction, corroborating the findings from mouse models.ConclusionsWe successfully developed a method for isolation of brain microvessels from a small amount of frozen human brain tissue, facilitating detailed study of BBB proteome in aging or pathological conditions. This technique provides valuable insights into BBB dysfunction in central nervous system disorders and holds potential for improving brain-targeted drug delivery strategies.

Fecal carriage of ESBL-producing E. coli and genetic characterization in rural children and livestock in the Somali region, Ethiopia: a one health approach

BackgroundThe emergence and spread of Extended-Spectrum Beta-Lactamase (ESBL)-producing Escherichia coli pose significant challenges for treatment of infections globally. This challenge is exacerbated in sub-Saharan African countries, where the prevalence of ESBL-producing E. coli is high. This, combined with the lack of a strong and supportive healthcare system, leads to increased morbidity and mortality due to treatment failures. Notably, studies in Ethiopia have primarily focused on hospital settings, leaving a gap in understanding ESBL prevalence in rural communities, where human-animal proximity may facilitate microbial exchange.MethodsWe conducted a community-based study in the rural Somali region of Ethiopia, simultaneously examining the fecal carriage of ESBL-producing E. coli in children aged 2–5 years and their livestock (cattle, camel, goat). Fecal samples from 366 children and 243 animals underwent phenotypic screening for ESBL-producing E. coli. Following phenotypic confirmation, ESBL resistance genes were identified via conventional PCR. Whole-genome sequencing (WGS) was performed on a subset of isolates from human feces.ResultsWe found that 43% (159/366) of children and 3.7% (9/244) of livestock harbored ESBL-producing E. coli. The ESBL gene blaCTX-M-15 was predominant in human (82.7%, 120/145) and livestock (100%) isolates. In the 48 human E. coli isolates subjected to WGS, a high diversity resulting in 40 sequence types (STs) was observed. Among these, ST-2353 was the most prevalent (5/48), followed by ST-10 and ST-48 (3/48) and ST-38, ST-450, and ST-4750 (2/48). These STs were associated with multiple resistance genes, such as blaCTX-M-15, blaTEM-1B, blaOXA-1, blaCTX-M-14 and blaTEM-35.ConclusionWe report a high prevalence of ESBL E. coli in rural children, which outnumbers its prevalence in livestock. These isolates displayed a high diversity of sequence types (STs) with ST-2353 being the dominant ST. Our study is the first to report the association of ST-2353 with multi-drug resistance genes in Ethiopia. Further research using an integrated approach including other domains such as water and food products is needed to truly understand and combat AMR transmission and acquisition in this region.

Suppressed Protein Translation Caused by MSP-8 Deficiency Determines Fungal Multidrug Resistance with Fitness Cost.

Antifungal resistance, particularly the rise of multidrug-resistance strains, poses a significant public health threat. In this study, the study identifies a novel multidrug-resistance gene, msp-8, encoding a helicase, through experimental evolution with Neurospora crassa as a model. Deletion of msp-8 conferred multidrug resistance in N. crassa, Aspergillus fumigatus, and Fusarium verticillioides. However, the transcript levels of genes encoding known drug targets or efflux pumps remain unaltered with msp-8 deletion. Interestingly, MSP-8 interacted with ribosomal proteins, and this mutant displays compromised ribosomal function, causing translational disturbance. Notably, inhibition of protein translation enhances resistance to azoles, amphotericin B, and polyoxin B. Furthermore, MSP-8 deficiency or inhibition of translation reduces intracellular ketoconazole accumulation and membrane-bound amphotericin B content, directly causing antifungal resistance. Additionaly, MSP-8 deficiency induces cell wall remodeling, and decreases intracellular ROS levels, further contributing to resistance. The findings reveal a novel multidrug resistance mechanism independent of changes in drug target or efflux pump, while MSP-8 deficiency suppresses protein translation, thereby facilitating the development of resistance with fitness cost. This study provides the first evidence that MSP-8 participates in protein translation and that translation suppression can cause multidrug resistance in fungi, offering new insights into resistance mechanisms in clinical and environmental fungal strains.

Integrated Analysis of Transcriptome and Metabolome in the Brain After Cold Stress of Red Tilapia During Overwintering.

Cold stress during overwintering is considered a bottleneck problem limiting the development of the red tilapia (Oreochromis spp.) industry, and the regulation mechanism is currently not well understood. In this study, the fish (initial weight: 72.71 ± 1.32 g) were divided into the cold stress group (cold) and the control (normal) group. In the control group, the water temperature was maintained at 20 °C, which is basically consistent with the overwintering water temperature in greenhouses of local areas. In the cold group, the water temperature decreased from 20 °C to 8 °C by 2 °C per day during the experiment. At the end of the experiment, the levels of fish serum urea nitrogen, glucose, norepinephrine, alkaline phosphatase, total bilirubin, and total cholesterol in the cold group changed significantly compared with that in the control group (P < 0.05). Then transcriptome sequencing and LC-MS metabolome of brain tissue were further employed to obtain the mRNA and metabolite datasets. We found that the FoxO signaling pathway and ABC transporters played an important role by transcriptome-metabolome association analysis. In the FoxO signaling pathway, the differentially expressed genes were related to cell cycle regulation, apoptosis and immune-regulation, and oxidative stress resistance and DNA repair. In the ABC transporters pathway, the ATP-binding cassette (ABC) subfamily abca, abcb, and abcc gene expression levels, and the deoxycytidine, L-lysine, L-glutamic acid, L-threonine, ornithine, and uridine metabolite contents changed. Our results suggested that the cold stress may promote apoptosis through regulation of the FoxO signaling pathway. The ABC transporters may respond to cold stress by regulating amino acid metabolism. The results provided a comprehensive understanding of fish cold stress during overwintering, which will facilitate the breeding of new cold-resistant varieties of red tilapia in the future.

Pharmacogenetics of Neoadjuvant MAP Chemotherapy in Localized Osteosarcoma: A Study Based on Data from the GEIS-33 Protocol.

Background: Osteosarcoma is a rare disease, but it is the most frequent malignant bone tumor. Primary treatment consists of preoperative MAP (methotrexate (MTX), doxorubicin and cisplatin) chemotherapy followed by surgery and adjuvant chemotherapy. Pathological response to preoperative chemotherapy is one of the most important prognostic factors, but molecular biomarkers are lacking. Additionally, chemotherapy-induced toxicity might jeopardize treatment completion. We evaluated variants in genes involved in DNA repair and drug metabolism pathways as predictors of response to MAP-based treatment. Material and Methods: Germline polymorphisms in MTHFR, SLC19A1, ABCB1, ABCC2, ABCC3, ERCC1, ERCC2 and GSTP1 genes were determined for association studies in 69 patients diagnosed with localized osteosarcoma who enrolled in the prospective GEIS-33 trial. P-glycoprotein expression in tumor tissue was also analyzed. Results: In the multivariate analysis, the ABCC2 rs2273697 (odds ratio [OR] 12.3, 95% CI 2.3-66.2; p = 0.003) and ERCC2 rs1799793 (OR 9.6, 95% CI 2.1-43.2; p = 0.003) variants were associated with poor pathological response. P-glycoprotein expression did not correlate with pathological response. The ABCB1 rs1128503 (OR 11.4, 95% CI 2.2-58.0; p = 0.003) and ABCC3 rs4793665 (OR 12.0, 95% CI 2.1-70.2; p = 0.006) variants were associated with MTX grade 3-4 hepatotoxicity. Conclusions: Our findings add to the evidence that genetic variants in the ABC transporters and DNA-repair genes may serve as predictive biomarkers for MAP chemotherapy and contribute to treatment personalization.

Genome Insights into Beneficial Microbial Strains Composing SIMBA Microbial Consortia Applied as Biofertilizers for Maize, Wheat and Tomato.

For the safe use of microbiome-based solutions in agriculture, the genome sequencing of strains composing the inoculum is mandatory to avoid the spread of virulence and multidrug resistance genes carried by them through horizontal gene transfer to other bacteria in the environment. Moreover, the annotated genomes can enable the design of specific primers to trace the inoculum into the soil and provide insights into the molecular and genetic mechanisms of plant growth promotion and biocontrol activity. In the present work, the genome sequences of some members of beneficial microbial consortia that have previously been tested in greenhouse and field trials as promising biofertilizers for maize, tomato and wheat crops have been determined. Strains belong to well-known plant-growth-promoting bacterial genera such as Bacillus, Burkholderia, Pseudomonas and Rahnella. The genome size of strains ranged from 4.5 to 7.5 Mbp, carrying many genes spanning from 4402 to 6697, and a GC content of 0.04% to 3.3%. The annotation of the genomes revealed the presence of genes that are implicated in functions related to antagonism, pathogenesis and other secondary metabolites possibly involved in plant growth promotion and gene clusters for protection against oxidative damage, confirming the plant-growth-promoting (PGP) activity of selected strains. All the target genomes were found to possess at least 3000 different PGP traits, belonging to the categories of nitrogen acquisition, colonization for plant-derived substrate usage, quorum sensing response for biofilm formation and, to a lesser extent, bacterial fitness and root colonization. No genes putatively involved in pathogenesis were identified. Overall, our study suggests the safe application of selected strains as "plant probiotics" for sustainable agriculture.

Resistome in the indoor dust samples from workplaces and households: a pilot study.

The antibiotic resistance genes (ARGs) limit the susceptibility of bacteria to antimicrobials, representing a problem of high importance. Current research on the presence of ARGs in microorganisms focuses mainly on humans, livestock, hospitals, or wastewater. However, the spectrum of ARGs in the dust resistome in workplaces and households has gone relatively unexplored. This pilot study aimed to analyze resistome in indoor dust samples from participants' workplaces (a pediatric hospital, a maternity hospital, and a research center) and households and compare two different approaches to the ARGs analysis; high-throughput quantitative PCR (HT-qPCR) and whole metagenome shotgun sequencing (WMGS). In total, 143 ARGs were detected using HT-qPCR, with ARGs associated with the macrolides, lincosamides, and streptogramin B (MLSB) phenotype being the most abundant, followed by MDR (multi-drug resistance) genes, and genes conferring resistance to aminoglycosides. A higher overall relative quantity of ARGs was observed in indoor dust samples from workplaces than from households, with the pediatric hospital being associated with the highest relative quantity of ARGs. WMGS analysis revealed 36 ARGs, of which five were detected by both HT-qPCR and WMGS techniques. Accordingly, the efficacy of the WMGS approach to detect ARGs was lower than that of HT-qPCR. In summary, our pilot data revealed that indoor dust in buildings where people spend most of their time (workplaces, households) can be a significant source of antimicrobial-resistant microorganisms, which may potentially pose a health risk to both humans and animals.

Pharmacogenetic Testing in Treatment-resistant Panic Disorder: a Preliminary Analysis

Background Many pharmacological treatments are considered effective in the treatment of panic disorder (PD), however, about 20 to 40% of the patients have treatment-resistant PD. Pharmacogenetics could explain why some patients are treatment-resistant. Objective Our objective was to gather preliminary data on the clinical usefulness of pharmacogenetic testing in this disorder. Methods Twenty patients with treatment-resistant PD were included in this observational study and submitted to commercial pharmacogenetic testing. Testing panel included gene polymorphisms related to CYP, genes EPHX1, UGT1A4, UGT2B15, ABCB1, ADRA2A, ANKK1, COMT, DRD2, FKBP5, GRIK4, GSK3B, HTR1A, HTR2A, HTR2C, MC4R, OPRM1, SCN1A, SLC6A4 and MTHFR. Participants received treatment-as-usual for PD before being enrolled in this study, including first-line and second-line medications for PD. Results In 30% of the patients, the tests indicated reduced chance of response to the prescribed drug, while they indicated very low serum levels of the prescribed drug in 20% of the subjects. The pharmacogenetic tests predicted reduction of MTHFR enzyme activity in 74% of the patients. ABCB1 gene alleles associated to drug resistance were found in 90% of the samples. Conclusion Commercial pharmacogenetic testing failed to predict negative treatment outcome in most patients with PD. The association between treatment-resistance in PD and the genes CYP2C19, MTHFR and ABCB1 deserves further study.