Biology subjects

Sharon, B. M.

Publications and source records attributed to Sharon, B. M..

3 recordsLinked to original sources

bioRxiv2025

Factors affecting CRISPR-Cas defense against antibiotic resistance plasmids harbored by Enterococcus faecalis laboratory model strains and clinical isolates

Enterococcus faecalis is a Gram-positive bacterium and opportunistic pathogen that acquires resistance to a wide range of antibiotics by horizontal gene transfer (HGT). The rapid increase of multidrug-resistant (MDR) bacteria including MDR E. faecalis necessitates the development of alternative therapies and a deeper understanding of the factors that impact HGT. CRISPR-Cas systems provide sequence-specific defense against HGT. From previous studies, we know that E. faecalis CRISPR-Cas provides sequence-specific anti-plasmid defense during agar plate biofilm mating and in the murine intestine. Those studies were mainly conducted using laboratory model strains with a single, CRISPR-targeted plasmid in the donor. MDR E. faecalis typically possess multiple plasmids that are diverse in sequence and may interact with each other to impact plasmid transfer and CRISPR-Cas efficacy. Here, we altered multiple parameters of our standard in vitro conjugation assays to assess CRISPR-Cas efficacy, including the number and genotype of plasmids in the donor; and laboratory model strains as donor versus recent human isolates as donor during conjugation. We found that the plasmids pTEF2 and pCF10, which are not targeted by CRISPR-Cas in our recipient, enhance the conjugative transfer of the CRISPR-targeted plasmid pTEF1 into both wild-type and CRISPR-Cas-deficient (via deletion of cas9) recipient cells. However, the effect of pTEF2 on pTEF1 transfer is much more pronounced, with a striking 6-log increase in pTEF1 conjugation frequency when pTEF2 is also present in the donor and recipients are deficient for CRISPR-Cas (compared to 4-log for pCF10). Overall, this study provides insight about the interplay between plasmids and CRISPR-Cas defense, opening avenues for developing novel therapeutic strategies to curb HGT among bacterial pathogens, and highlighting pTEF2 as a plasmid for additional mechanistic study. IMPORTANCEThe emergence of MDR bacteria, including MDR E. faecalis, limits treatment options and necessitates development of alternative therapeutics. In these circumstances, bacterial CRISPR-Cas systems are being explored by the field to develop CRISPR-based antimicrobials. However, in many cases CRISPR-Cas efficacy has only been assessed using laboratory model strains. More studies are required that investigate clinical isolates, as those are the intended targets for CRISPR antimicrobials. Here, we demonstrated how the number of plasmids harbored by an E. faecalis donor strain can affect the apparent efficacy of CRISPR-Cas anti-plasmid defense in a recipient strain. Overall, our research is important to develop improved CRISPR-based antimicrobials to combat the spread and accumulation of antibiotic resistance determinants.

microbiology↗

bioRxiv2023

Functional and genetic adaptations contributing to Enterococcus faecalis persistence in the female urinary tract

Enterococcus faecalis is the leading Gram-positive bacterial species implicated in urinary tract infection (UTI). An opportunistic pathogen, E. faecalis is a commensal of the human gastrointestinal tract (GIT) and its presence in the GIT is a predisposing factor for UTI. The mechanisms by which E. faecalis colonizes and survives in the urinary tract (UT) are poorly understood, especially in uncomplicated or recurrent UTI. The UT is distinct from the GIT and is characterized by a sparse nutrient landscape and unique environmental stressors. In this study, we isolated and sequenced a collection of 37 clinical E. faecalis strains from the urine of primarily postmenopausal women. We generated 33 closed genome assemblies and four highly contiguous draft assemblies and conducted a comparative genomics to identify genetic features enriched in urinary E. faecalis with respect to E. faecalis isolated from the human GIT and blood. Phylogenetic analysis revealed high diversity among urinary strains and a closer relatedness between urine and gut isolates than blood isolates. Plasmid replicon (rep) typing further underscored possible UT-GIT interconnection identifying nine shared rep types between urine and gut E. faecalis. Both genotypic and phenotypic analysis of antimicrobial resistance among urinary E. faecalis revealed infrequent resistance to front-line UTI antibiotics nitrofurantoin and fluoroquinolones and no vancomycin resistance. Finally, we identified 19 candidate genes enriched among urinary strains that may play a role in adaptation to the UT. These genes are involved in the core processes of sugar transport, cobalamin import, glucose metabolism, and post-transcriptional regulation of gene expression. IMPORTANCEUrinary tract infection (UTI) is a global health issue that imposes substantial burden on healthcare systems. Women are disproportionately affected by UTI with >60% of women experiencing at least one UTI in their lifetime. UTIs can recur, particularly in postmenopausal women, leading to diminished quality of life and potentially life-threatening complications. Understanding how pathogens colonize and survive in the urinary tract is necessary to identify new therapeutic targets that are urgently needed due to rising rates of antimicrobial resistance. How Enterococcus faecalis, a bacterium commonly associated with UTI, adapts to the urinary tract remains understudied. Here, we generated a collection of high-quality closed genome assemblies of clinical urinary E. faecalis isolated from the urine of postmenopausal women that we used alongside detailed clinical metadata to perform a robust comparative genomic investigation of genetic factors that may mediate urinary E. faecalis adaptation to the female urinary tract.

microbiology↗

bioRxiv2021

Recurrent urinary tract infection and estrogen shape the taxonomic ecology and functional potential of the postmenopausal urobiome

Community-acquired urinary tract infection (UTI) is among the most common bacterial infections observed in humans. Postmenopausal women are a rapidly growing and underserved demographic group who are severely affected by recurrent UTI (rUTI) with a >50% recurrence rate. In this population, rUTI can persist for years, reducing quality of life and imposing a significant healthcare burden. rUTI is most often treated by antibiotics, but development of antibiotic resistance and allergy limit therapeutic options. The female urinary microbiome (urobiome) has been identified as a key component of the urogenital environment. However, compositional and functional changes in the urobiome underlying rUTI susceptibility in postmenopausal women are not well understood. Here, we used a controlled, cross-sectional cohort of postmenopausal women, to interrogate changes in urobiome structure and function linked to rUTI susceptibility by whole genome metagenomic sequencing (WGMS), advanced urine culture, estrogen metabolite profiling, and antibiotic sensitivity testing. Overall, we detected 276 bacterial, archaeal, and fungal species representing 106 genera. We find a putative commensal population consisting of species known to protect against bacterial vaginosis, such as Lactobacillus crispatus, within the urobiome of postmenopausal women who do not experience UTI. Integration of clinical metadata detected an almost exclusive enrichment of lactobacilli, including L. crispatus and L. vaginalis, in women taking estrogen hormone therapy (EHT). Integrating quantitative metabolite profiling of urinary estrogens with WGMS, we observed robust correlations between urobiome taxa, such as Bifidobacterium breve and L. crispatus, and urinary estrogen conjugate concentrations in women with no history of UTI that were absent in women with rUTI history. We further used functional metagenomic profiling and patient-derived isolate phenotyping to identify microbial metabolic pathways, antimicrobial resistance genes (ARGs), and clinically relevant antimicrobial resistance phenotypes enriched between disease-states. Our data indicate that distinct urobiome metabolic and ARG signatures are associated with current rUTI status and history. Importantly, we observed that rUTI history leaves an imprint of enriched ARGs even in women not currently experiencing UTI. Taken together, our data suggests that rUTI history and estrogen strongly shape the functional and taxonomic composition of the urobiome in postmenopausal women.

microbiology↗