Biology subjects

Tedim, A. P.

Publications and source records attributed to Tedim, A. P..

5 recordsLinked to original sources

bioRxiv2023

Bacteriocin Distribution Patterns in Enterococcus faecium and Enterococcus lactis: Bioinformatic Analysis Using a Tailored Genomics Framework

Multidrug-resistant Enterococcus faecium represent a major concern due to their ability to thrive in diverse environments and cause life-threatening infections. While antimicrobial resistance and virulence mechanisms have been extensively studied, the contribution of bacteriocins to E. faeciums adaptability remains poorly explored. E. faecium, within the Bacillota phylum, is a prominent bacteriocin producer. Here, we developed a tailored database of 76 Bacillota bacteriocins (217 sequences, including 40 novel bacteriocins) and applied it to uncover bacteriocin distribution patterns in 997 quality-filtered E. faecium and Enterococcus lactis (former E. faecium clade B) genomes. Curated using computational pipelines and literature mining, our database demonstrates superior precision versus leading public tools in identifying diverse bacteriocins. Distinct bacteriocin profiles emerged between E. faecium and E. lactis, highlighting species-specific adaptations. E. faecium strains from hospitalized patients were significantly enriched for bacteriocins as entA, bac43, bacAS5 and bacAS11. These bacteriocins strongly associated with antibiotic resistance, particularly vancomycin and ampicillin, and Inc18 rep2_pRE25-derivative plasmids, classically associated with vancomycin resistance transposons. Our integrated genomic and epidemiological analysis elucidates meaningful connections between bacteriocin determinants, antimicrobial resistance, mobile genetic elements, and ecological origins in E. faecium. This work significantly expands the knowledge on the understudied bacteriocin diversity in opportunistic enterococci, revealing their contribution to environmental adaptation. Further characterization of strain-level bacteriocin landscapes could inform strategies to combat high-risk clones. Overall, these insights provide a framework for unravelling bacteriocins therapeutic and biotechnological potential.

genomics↗

bioRxiv2022

Quantification of bacterial DNA in blood using droplet digital PCR: a pilot study

AimTo use genus/species-specific genes droplet digital PCR (ddPCR) assays to detect/quantify bacterial DNA from Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and Enterococcus spp in blood samples. Methods and ResultsBacterial DNA from clinical strains (4<n<12) was extracted, quantified and diluted (10-0.0001ng/L) and ddPCR assays were performed in triplicate. These ddPCR assays showed low replication variability, low detection limit (1-0.1pg/L) and high genus/species specificity. ddPCR assays were also used to quantify bacterial DNA obtained from spiked blood (1x104-1CFU/mL) of each bacterial genus/species. Comparison between ddPCR assays and bacterial culture was performed by Pearson correlation. There was an almost perfect correlation (r[≥]0.997, p[≤]0.001) between the number of CFU/mL from bacterial culture and the number of gene copies/mL detected by ddPCR. The time from sample preparation to results was determined to be 3.5-4h. ConclusionsThe results demonstrated the quantification capacity and specificity of the ddPCR assays to detect/quantify four of the most important bloodstream infection (BSI) bacterial pathogens directly from blood. Significance and ImpactThis pilot study results reinforce the potential of ddPCR for the diagnosis and/or severity stratification of BSI. Applied to patients blood samples it can improve diagnosis and diminish sample-to-results time, improving patient care.

microbiology↗

bioRxiv2021

Fitness cost of vancomycin-resistant Enterococcus faecium plasmids associated with hospital infection outbreaks

BackgroundVancomycin resistance is mostly associated with Enterococcus faecium due to Tn1546-vanA located on narrow- and broad-host plasmids of various families. The studys aim was to analyse the effects of acquiring Tn1546-plasmids with proven epidemicity in different bacterial host backgrounds. MethodsWidespread Tn1546-plasmids of different families RepA_N (n=5), Inc18 (n=4) and/or pHT{beta} (n=1), and prototype plasmids RepA_N (pRUM) and Inc18 (pRE25, pIP501) were analysed. Plasmid transferability and fitness cost were assessed using E. faecium (GE1, 64/3) and Enterococcus faecalis (JH2-2/FA202/UV202) recipient strains. Growth curves (Bioscreen C) and Relative Growth Rates were obtained in presence/absence of vancomycin. Plasmid stability was analysed (300 generations). Whole genome sequencing (Illumina-MiSeq) of non-evolved and evolved strains (GE1/64/3 transconjugants, n=49) was performed. SNP calling (breseq software) of non-evolved strains was used for comparison. ResultsAll plasmids were successfully transferred to different E. faecium clonal backgrounds. Most Tn1546-plasmids and Inc18 and RepA_N prototypes reduced host fitness (-2%-18%) while the cost of Tn1546 expression varied according to the Tn1546-variant and the recipient strain (9-49%). Stability of Tn1546-plasmids was documented in all cases, often with loss of phenotypic resistance and/or partial plasmid deletions. Point mutations and/or indels associated with essential bacterial functions were observed on the chromosome of evolved strains, some of them linked to increased fitness. ConclusionsThe stability of E. faecium Tn1546-plasmids in the absence of selective pressure and the high intra-species conjugation rates might explain the persistence of vancomycin resistance in E. faecium populations despite the significant burden they might impose on bacterial host strains.

microbiology↗

bioRxiv2020

CoronaHiT: large scale multiplexing of SARS-CoV-2 genomes using Nanopore sequencing

The COVID-19 pandemic has spread to almost every country in the world since it started in China in late 2019. Controlling the pandemic requires a multifaceted approach including whole genome sequencing to support public health interventions at local and national levels. One of the most widely used methods for sequencing is the ARTIC protocol, a tiling PCR approach followed by Oxford Nanopore sequencing (ONT) of up to 96 samples at a time. There is a need, however, for a flexible, platform agnostic, method that can provide multiple throughput options depending on changing requirements as the pandemic peaks and troughs. Here we present CoronaHiT, a method capable of multiplexing up to 96 small genomes on a single MinION flowcell or >384 genomes on Illumina NextSeq, using transposase mediated addition of adapters and PCR based addition of barcodes to ARTIC PCR products. We demonstrate the method by sequencing 95 and 59 SARS-CoV-2 genomes for routine and rapid outbreak response runs, respectively, on Nanopore and Illumina platforms and compare to the standard ARTIC LoCost nanopore method. Of the 154 samples sequenced using the three approaches, genomes with [≥] 90% coverage (GISAID criteria) were generated for 64.3% of samples for ARTIC LoCost, 71.4% for CoronaHiT-ONT, and 76.6% for CoronaHiT-Illumina and have almost identical clustering on a maximum likelihood tree. In conclusion, we demonstrate that CoronaHiT can multiplex up to 96 SARS-CoV-2 genomes per MinION flowcell and that Illumina sequencing can be performed on the same libraries, which will allow significantly higher throughput. CoronaHiT provides increased coverage for higher Ct samples, thereby increasing the number of high quality genomes that pass the GISAID QC threshold. This protocol will aid the rapid expansion of SARS-CoV-2 genome sequencing globally, to help control the pandemic.

genomics↗

bioRxiv2019

Comparative genomics of global optrA-carrying Enterococcus faecalis uncovers common genetic features and a chromosomal hotspot for optrA acquisition

Linezolid-resistant Enterococcus faecalis (LREfs) carrying optrA are increasingly reported globally from multiple sources, but we still lack a comprehensive analysis of human and animal optrA-LREfs strains. We investigated the phylogenetic structure, genetic content [antimicrobial resistance (AMR), virulence, prophages, plasmidome] and optrA-containing platforms of 28 publicly available optrA-positive E. faecalis genomes from different hosts in 7 countries. In the genome-level analysis, in house databases with 57 virulence and 391 plasmid replication genes were tested for the first time. Our analysis showed a diversity of clones and adaptive gene sequences related to a wide range of genera, mainly but not exclusive from Firmicutes. The content in AMR and virulence genes was highly identical in contrast to the diversity of phages and plasmids observed. Epidemiologically unrelated clones (ST476-like and ST21-like) obtained from human clinical and animal hosts in different continents over 5 years (2012-2017) were phylogenetically related (3-122 SNPs difference). They also exhibited identical AMR and virulence profiles, highlighting a global spread of optrA-positive strains with relevant adaptive traits in livestock and that they might originate from an animal reservoir. optrA was located on the chromosome within a Tn6674-like element (n=9) or on medium-size plasmids (30-60 kb; n=14) belonging to main plasmid families (RepA_N/Inc18/Rep_3). In most cases, the immediate gene vicinity of optrA was identical in chromosomal (Tn6674) and plasmid (impB-fexA-optrA) backbones. Tn6674 was always inserted in the same {Delta}radC integration site and embedded in a 32 kb chromosomal platform common to diverse strains from different origins (patients, healthy humans, and animals) in Europe, Africa, and Asia during 2012-2018. This platform is conserved among hundreds of E. faecalis genomes and we here propose a conserved chromosomal hotspot for optrA integration. The finding of optrA in strains sharing identical adaptive features and genetic backgrounds across different hosts and countries suggest the occurrence of common and independent genetic events occurring in distant regions, and might explain the easy de novo generation of optrA-positive strains. It also anticipates a dramatic increase of optrA carriage and spread with a serious impact in the efficacy of linezolid for the treatment of Gram-positive infections.

genomics↗