Project description:<p>The study of antimicrobial resistance (AMR) in infectious diarrhea has generally been limited to cultivation, antimicrobial susceptibility testing and targeted PCR assays. When individual strains of significance are identified, whole genome shotgun (WGS) sequencing of important clones and clades is performed. Genes that encode resistance to antibiotics have been detected in environmental, insect, human and animal metagenomes and are known as "resistomes". While metagenomic datasets have been mined to characterize the healthy human gut resistome in the Human Microbiome Project and MetaHIT and in a Yanomani Amerindian cohort, directed metagenomic sequencing has not been used to examine the epidemiology of AMR. Especially in developing countries where sanitation is poor, diarrhea and enteric pathogens likely serve to disseminate antibiotic resistance elements of clinical significance. Unregulated use of antibiotics further exacerbates the problem by selection for acquisition of resistance. This is exemplified by recent reports of multiple antibiotic resistance in Shigella strains in India, in Escherichia coli in India and Pakistan, and in nontyphoidal Salmonella (NTS) in South-East Asia. We propose to use deep metagenomic sequencing and genome level assembly to study the epidemiology of AMR in stools of children suffering from diarrhea. Here the epidemiology component will be surveillance and analysis of the microbial composition (to the bacterial species/strain level where possible) and its constituent antimicrobial resistance genetic elements (such as plasmids, integrons, transposons and other mobile genetic elements, or MGEs) in samples from a cohort where diarrhea is prevalent and antibiotic exposure is endemic. The goal will be to assess whether consortia of specific mobile antimicrobial resistance elements associate with species/strains and whether their presence is enhanced or amplified in diarrheal microbiomes and in the presence of antibiotic exposure. This work could potentially identify clonal complexes of organisms and MGEs with enhanced resistance and the potential to transfer this resistance to other enteric pathogens.</p> <p>We have performed WGS, metagenomic assembly and gene/protein mapping to examine and characterize the types of AMR genes and transfer elements (transposons, integrons, bacteriophage, plasmids) and their distribution in bacterial species and strains assembled from DNA isolated from diarrheal and non-diarrheal stools. The samples were acquired from a cohort of pediatric patients and controls from Colombia, South America where antibiotic use is prevalent. As a control, the distribution and abundance of AMR genes can be compared to published studies where resistome gene lists from healthy cohort sequences were compiled. Our approach is more epidemiologic in nature, as we plan to identify and catalogue antimicrobial elements on MGEs capable of spread through a local population and further we will, where possible, link mobile antimicrobial resistance elements with specific strains within the population.</p>

Project description:Antimicrobial resistance poses a global threat. Natural-origin compounds represent a valuable source of antimicrobial agents used in both human and veterinary medicine. However, understanding their mechanisms of action at the molecular level is essential to support their safe and effective application. In this study, we evaluated the antimicrobial potential of trans-cinnamaldehyde (CNMA), a major constituent of cinnamon bark oil, which can account for up to 80% of the oil content in species such as Cinnamomum zeylanicum and cassia. Although CNMA exhibits broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria, its precise mode of action remains incompletely understood. To elucidate CNMA's molecular effects, we performed transcriptomic profiling of Escherichia coli MG1655 wild-type (GSE252441) and its ∆relA mutant upon treatment with a sub-inhibitory concentration (0.25×MIC) of CNMA. Total RNA was isolated and assessed using the Agilent Bioanalyzer 2100, and high-throughput sequencing was conducted on the Illumina NovaSeq6000 platform, generating ~30 million paired-end 101 bp reads per sample. The reference genome and annotations of E. coli MG1655 were obtained from GenBank. RNA-seq data were analyzed to identify differentially expressed genes (DEGs) compared to untreated controls at 30 and 60 min post-treatment, using thresholds of p ≤ 0.05 and |log₂FC| ≥ 2. Transcriptomic analysis revealed profound transcriptional remodeling. The most significantly enriched functional categories included genes involved in the tricarboxylic acid (TCA) cycle, flagellar biosynthesis, amino acid transport, and oxidoreductase activity. These findings indicate that CNMA-treated E. coli undergoes a marked metabolic downshift and initiates stress responses. These transcriptomic results were supported by complementary assays showing reduced growth kinetics, cytoplasmic shrinkage, NAD/NADH imbalance, and induction of the stringent response via elevated (p)ppGpp levels. Together, our findings suggest that CNMA disrupts bacterial fitness by impairing core metabolic and regulatory pathways, ultimately leading to loss of viability. Funding: This research was funded by the National Science Center, Poland (grant SONATA UMO-2018/31/D/NZ7/02258 to D.N.)

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