Dataset Information

Selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes in third-generation cephalosporin resistant Enterobacteriaceae.

ABSTRACT: Metagenomic sequencing of fecal DNA can usefully characterise an individual's intestinal resistome but is limited by its inability to detect important pathogens that may be present at low abundance, such as carbapenemase or extended-spectrum beta-lactamase producing Enterobacteriaceae. Here we aimed to develop a hybrid protocol to improve detection of resistance genes in Enterobacteriaceae by using a short period of culture enrichment prior to sequencing of DNA extracted directly from the enriched sample. Volunteer feces were spiked with carbapenemase-producing Enterobacteriaceae and incubated in selective broth culture for 6 hours before sequencing. Different DNA extraction methods were compared, including a plasmid extraction protocol to increase the detection of plasmid-associated resistance genes. Although enrichment prior to sequencing increased the detection of carbapenemase genes, the differing growth characteristics of the spike organisms precluded accurate quantification of their concentration prior to culture. Plasmid extraction increased detection of resistance genes present on plasmids, but the effects were heterogeneous and dependent on plasmid size. Our results demonstrate methods of improving the limit of detection of selected resistance mechanisms in a fecal resistome assay, but they also highlight the difficulties in using these techniques for accurate quantification and should inform future efforts to achieve this goal.

SUBMITTER: Peto L

PROVIDER: S-EPMC6839868 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes in third-generation cephalosporin resistant Enterobacteriaceae.

Peto Leon L Fawcett Nicola J NJ Crook Derrick W DW Peto Tim E A TEA Llewelyn Martin J MJ Walker A Sarah AS

PloS one 20191108 11

Metagenomic sequencing of fecal DNA can usefully characterise an individual's intestinal resistome but is limited by its inability to detect important pathogens that may be present at low abundance, such as carbapenemase or extended-spectrum beta-lactamase producing Enterobacteriaceae. Here we aimed to develop a hybrid protocol to improve detection of resistance genes in Enterobacteriaceae by using a short period of culture enrichment prior to sequencing of DNA extracted directly from the enrich ...[more]

PMID: 31703058

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Project description:Background:The epidemiology of antibiotic-resistant Enterobacteriaceae intestinal carriage in healthy US children has not been well characterized. Methods:Children between 14 days and 14 years of age were enrolled during well-child visits in Oakland, California, Kansas City, Kansas, and Nashville, Tennessee, between December 2013 and March 2015. Data on recent antibiotic use by the child and travel and hospitalization history of all members of each child's household were obtained with a risk-factor survey. Stool specimens collected from the subjects were screened for extended-spectrum ?-lactamase-producing (ESBL-P) bacteria using CHROMagar ESBL medium. Putative ESBL-P Escherichia coli and Klebsiella colonies underwent phenotypic confirmation by double-disk synergy testing; confirmed third-generation cephalosporin-resistant (3GCR) isolates underwent additional antibiotic-susceptibility testing. Results:In 519 subjects, the overall 3GCR Enterobacteriaceae carriage rate was 4.4% (n = 23) and ranged from 3.4% to 5.1% among the study sites. The ESBL-P Enterobacteriaceae carriage rate was 3.5% (n = 18). The rates of 3GCR Enterobacteriaceae carriage was highest in 1 to <2 year olds at 6.5%, and was 5.2% in <5 year-olds vs 1.7% in ?5-year-olds (P = .11). 3GCR and ESBL-P Enterobacteriaceae carriage was associated with international travel within the previous year; 11.1% of ESBL-P Enterobacteriaceae carriers reported this history compared with 1.6% of noncarriers (P = .004). No other queried factor was found to increase risk. Of the 24 analyzed 3GCR isolates, 58% were multidrug resistant. Conclusions:The 3GCR Enterobacteriaceae carriage rate exceeds 5% in healthy US children <5 years of age. International travel within the previous year increased the risk of 3GCR and ESBL-P Enterobacteriaceae carriage. In contrast, we found no differences in the rates of hospitalization or recent antibiotic exposure between carriers and noncarriers. Young children, who have the highest prevalence of colonization, might be a sentinel population to study to gain a better understanding of community sources of antibiotic-resistant Enterobacteriaceae.

Project description:BackgroundMany patients admitted to a hospital are already colonized with multi-drug resistant organisms (MDRO) including third-generation cephalosporin-resistant Enterobacteriaceae (3GCREB). The aim of our study was to determine the prevalence of rectal 3GCREB colonization at admission to a large German university hospital and to estimate infection incidences. In addition, risk factors for 3GCREB colonization were identified.Materials/methodsIn 2014 and 2015, patients were screened for rectal colonization with 3GCREB and filled out a questionnaire on potential risk factors at admission to a non-intensive care unit (non-ICU). All patients were retrospectively monitored for bacterial infections. Descriptive, univariable and multivariable logistic regression analyses were conducted to identify risk factors for 3GCREB colonization at admission.ResultsOf 4,013 patients included, 10.3% (n = 415) were rectally colonized with 3GCREB at admission. Incidence of nosocomial infections was 3.5 (95% CI 2.0-6.1) per 100 patients rectally colonized with 3GCREB compared to 2.3 (95% CI 1.8-3.0, P = 0.213) per 100 3GCREB negative patients. Independent risk factors for 3GCREB colonization were prior colonization / infection with MDRO (OR 2.30, 95% CI 1.59-3.32), prior antimicrobial treatment (OR 1.97, 95% CI 1.59-2.45), male sex (OR 1.38, 95% CI 1.12-1.70), prior travelling outside Europe (OR 2.39, 95% CI 1.77-3.22) and places of residence in the Berlin districts Charlottenburg-Wilmersdorf (OR 1.52, 95% CI 1.06-2.18), Friedrichshain-Kreuzberg (OR 2.32, 95% CI 1.44-3.74) and Mitte (OR 1.73, 95% CI 1.26-2.36).ConclusionsAdmission prevalence of rectal colonization with 3GCREB was high, while infection incidence did not significantly differ between patients rectally colonized or not with 3GCREB at hospital admission. In consequence, hospitals should prioritize improvement of standard precautions including hand hygiene to prevent infections among all patients irrespective of their 3GCREB status at hospital admission.

Dataset Information

Selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes in third-generation cephalosporin resistant Enterobacteriaceae.

Publications

Selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes in third-generation cephalosporin resistant Enterobacteriaceae.

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