Dataset Information

Constitutive expression of the cryptic vanGCd operon promotes vancomycin resistance in Clostridioides difficile clinical isolates.

ABSTRACT:

Objectives

To describe, for the first time (to the best of our knowledge), the genetic mechanisms of vancomycin resistance in clinical isolates of Clostridioides difficile ribotype 027.

Methods

Clinical isolates and laboratory mutants were analysed: genomically to identify resistance mutations; by transcriptional analysis of vanGCd, the vancomycin resistance operon encoding lipid II d-alanine-d-serine that is less bound by vancomycin than native lipid II d-alanine-d-alanine; by imaging of vancomycin binding to cell walls; and for changes in vancomycin bactericidal activity and autolysis.

Results

Vancomycin-resistant laboratory mutants and clinical isolates acquired mutations to the vanSR two-component system that regulates vanGCd. The substitutions impaired VanSR's function, resulting in constitutive transcription of vanGCd. Resistance was reversed by silencing vanG, encoding d-alanine-d-serine ligase in the vanGCd operon. In resistant cells, vancomycin was less bound to the cell wall septum, the site where vancomycin interacts with lipid II. Vancomycin's bactericidal activity was reduced against clinical isolates and laboratory mutants (64 and ?1024?mg/L, respectively) compared with WT strains (4?mg/L). Truncation of the potassium transporter TrkA occurred in laboratory mutants, which were refractory to autolysis, accounting for their survival in high drug concentrations.

Conclusions

Ribotype 027 evolved first-step resistance to vancomycin by constitutively expressing vanGCd, which is otherwise silent. Experimental evolutions and bactericidal assays show that ribotype 027 can acquire mutations to drastically enhance its tolerance to vancomycin. Thus, further epidemiological studies are warranted to examine the extent to which vancomycin resistance impacts clinical outcomes and the potential for these strains to evolve higher-level resistance, which would be devastating.

SUBMITTER: Shen WJ

PROVIDER: S-EPMC7069472 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Publications

Constitutive expression of the cryptic vanGCd operon promotes vancomycin resistance in Clostridioides difficile clinical isolates.

Shen Wan-Jou WJ Deshpande Aditi A Hevener Kirk E KE Endres Bradley T BT Garey Kevin W KW Palmer Kelli L KL Hurdle Julian G JG

The Journal of antimicrobial chemotherapy 20200401 4

<h4>Objectives</h4>To describe, for the first time (to the best of our knowledge), the genetic mechanisms of vancomycin resistance in clinical isolates of Clostridioides difficile ribotype 027.<h4>Methods</h4>Clinical isolates and laboratory mutants were analysed: genomically to identify resistance mutations; by transcriptional analysis of vanGCd, the vancomycin resistance operon encoding lipid II d-alanine-d-serine that is less bound by vancomycin than native lipid II d-alanine-d-alanine; by im ...[more]

PMID: 31873741

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Project description:Clostridioides difficile infection (CDI) is most commonly diagnosed using nucleic acid amplification tests (NAAT); the low positive predictive value of these assays results in patients colonized with C. difficile unnecessarily receiving CDI treatment antibiotics. The risks and benefits of antibiotic treatment in individuals with such cases are unknown. Fecal samples of NAAT-positive, toxin enzyme immunoassay (EIA)-negative patients were collected before, during, and after randomization to vancomycin (n = 8) or placebo (n = 7). C. difficile and antibiotic-resistant organisms (AROs) were selectively cultured from fecal and environmental samples. Shotgun metagenomics and comparative isolate genomics were used to understand the impact of oral vancomycin on the microbiome and environmental contamination. Overall, 80% of placebo patients and 71% of vancomycin patients were colonized with C. difficile posttreatment. One person randomized to placebo subsequently received treatment for CDI. In the vancomycin-treated group, beta-diversity (P = 0.0059) and macrolide-lincosamide-streptogramin (MLS) resistance genes (P = 0.037) increased after treatment; C. difficile and vancomycin-resistant enterococci (VRE) environmental contamination was found in 53% of patients and 26% of patients, respectively. We found that vancomycin alters the gut microbiota, does not permanently clear C. difficile, and is associated with VRE colonization/environmental contamination. (This study has been registered at ClinicalTrials.gov under registration no. NCT03388268.)IMPORTANCE A gold standard diagnostic for Clostridioides difficile infection (CDI) does not exist. An area of controversy is how to manage patients whose stool tests positive by nucleic acid amplification tests but negative by toxin enzyme immunoassay. Existing data suggest most of these patients do not have CDI, but most are treated with oral vancomycin. Potential benefits to treatment include a decreased risk for adverse outcomes if the patient does have CDI and the potential to decrease C. difficile shedding/transmission. However, oral vancomycin perturbs the intestinal microbiota and promotes antibiotic-resistant organism colonization/transmission. We conducted a double-blinded randomized controlled trial to assess the risk-benefit of oral vancomycin treatment in this population. Oral vancomycin did not result in long-term clearance of C. difficile, perturbed the microbiota, and was associated with colonization/shedding of vancomycin-resistant enterococci. This work underscores the need to better understand this population of patients in the context of C. difficile/ARO-related outcomes and transmission.

Dataset Information

Constitutive expression of the cryptic vanGCd operon promotes vancomycin resistance in Clostridioides difficile clinical isolates.

Objectives

Methods

Results

Conclusions

Publications

Constitutive expression of the cryptic vanGCd operon promotes vancomycin resistance in Clostridioides difficile clinical isolates.

Similar Datasets

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