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Increasing tolerance of hospital Enterococcus faecium to handwash alcohols

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Science Translational Medicine  01 Aug 2018:
Vol. 10, Issue 452, eaar6115
DOI: 10.1126/scitranslmed.aar6115
  • Fig. 1 Isopropanol tolerance variation among E. faecium isolates.

    Tolerance to isopropanol of 139 E. faecium isolates collected over 19 years at two hospitals in Melbourne, Australia. (A) The changing tolerance to isopropanol of E. faecium isolates collected between 1997 and 2015 is shown. The mean log10 CFU reduction values for each E. faecium isolate after exposure for 5 min to 23% (v/v) isopropanol are plotted against specimen collection date and clustered in 5- or 6-year windows. Two-tailed, unpaired Mann-Whitney test, ***P < 0.0001. (B to D) Mean log10 CFU reduction for the three dominant E. faecium clones ST17, ST203, and ST796 with the range (at least biological duplicates) displayed. The black arrows indicate the two isolates used in a previous handwash volunteer study (24).

  • Fig. 2 Isopropanol-tolerant E. faecium resists disinfection.

    A mouse gastrointestinal colonization assay was used to assess transmission of E. faecium. (A) The CD50 (gray dashed lines) for two vancomycin-resistant E. faecium isolates (Ef_aus0004 and Ef_aus0233) and two vancomycin-sensitive isolates (Ef_aus0026 and Ef_aus0099) was established (table S6). (B) The results of the contaminated cage floor mouse gut colonization experiment to quantify transmission of E. faecium are shown. The concentration of E. faecium before (the inoculum) and after standardized cage floor cleaning with 70% (v/v) isopropanol versus cleaning with water is plotted (table S7). The symbols show E. faecium CFU for the floor of three cages. (C) Percentage of mice with gut colonization by vancomycin-resistant or VSE after standardized cage floor cleaning with either 70% (v/v) isopropanol or sterile water. The results of at least three independent experiments based on six mice per experiment are shown (*P < 0.01; table S8). The null hypothesis (no difference between E. faecium that is sensitive versus tolerant to isopropanol) was rejected for P < 0.05, unpaired t test with Welch’s correction.

  • Fig. 3 Population structure of E. faecium isolates.

    (A) One hundred twenty-nine E. faecium isolates were subjected to whole-genome sequencing and alcohol tolerance testing, and their population structure was determined. The phylogeny was inferred using maximum likelihood with RAxML and was based on pairwise alignments of 18,292 core genome SNPs against the E. faecium Ef_aus0233 reference genome (filtered to remove recombination). Previous MLST designations are indicated by sequence type. A heat map summary of the fold difference in log10 kill for each selected pair of taxa is shown, with blue being the smallest fold difference in tolerance and red being the largest fold difference in tolerance. The prefix “A” or “M” before each pair number indicates pairs of strains from the Austin Hospital or Monash Medical Centre, respectively. (B) Analysis of convergent SNP differences among phylogenetically matched pairs of E. faecium isolates. The circular map represents the 2,888,087–base pair (bp) chromosome of the E. faecium Ef_aus0233 reference genome, showing the location of convergent core genome SNPs for each E. faecium pair. Track IDs are indicated by the color-coded key. The three homoplasic mutations (at chromosome positions 519,608, 2,396,698, and 2,838,889) that were conserved in the direction of nucleotide sequence change and present among three or more E. faecium pairs are highlighted and annotated. (C) Probabilistic separation of alcohol-sensitive (blue) and alcohol-tolerant (red) isolates according to a DAPC model built using accessory genome variation (table S3).

  • Fig. 4 Functional confirmation of genes associated with isopropanol tolerance in E. faecium.

    (A) Map of the 197-kb E. faecium plasmid showing the 70-kb region associated with isopropanol tolerance (blue) and the two carbohydrate PTS loci, including the 6.3-kb PTS-2 locus (purple) deleted by allelic exchange in the E. faecium reference strain Ef_aus0233. (B) Layout of the region around Ef_aus0233 chromosome position 519,608 showing the region deleted by allelic exchange in the gene 00501 encoding a putative galactoside symporter. Dark blue arrows indicate position of primers used to generate the recombination substrate for mutant construction. Primer positions: (a) 521,394 to 521,371; (b) 520,396 to 520,420; (c) 518,946 to 518,918; (d) 517,961 to 517,985. (C) Impact of the rpoBH486Y mutation, 197-kb plasmid ΔPTS-2, and Δ00501 galactoside symporter mutation on the ability of Ef_aus0233 to survive exposure to isopropanol. The means and SDs for biological triplicate experiments, with no differences between any of the mutants and wild-type (WT) E. faecium (table S9), are shown. (D) Growth phenotypes of the same rpoBH486Y mutation, 197-kb plasmid ΔPTS-2, and Δ00501 galactoside symporter mutation in the presence of 3% (v/v) isopropanol. Fold-change difference in doubling time for each mutant compared to wild-type E. faecium is shown. The phenotypes of the repaired mutants relative to wild-type E. faecium are also depicted. The null hypothesis (no difference between mean doubling time of mutant and repaired mutant or wild-type E. faecium) was rejected for P < 0.01, unpaired Mann-Whitney test (**P < 0.01, ***P < 0.0001; table S10). Error bars depict SDs. All data points are shown for at least three biological replicates and three technical replicates for each condition.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/452/eaar6115/DC1

    Fig. S1. Tolerance of E. faecium to ethanol exposure.

    Fig. S2. Isopropanol tolerance phenotype summary for 129 genome-sequenced E. faecium isolates.

    Fig. S3. Core and pan-genome analysis of 129 E. faecium genomes.

    Fig. S4. Growth curves of mutants.

    Table S1. Summary of E. faecium strains used in this study, with associated isopropanol tolerance data.

    Table S2. Pairwise comparisons of high-low alcohol-tolerant E. faecium.

    Table S3. DAPC analysis based on ortholog comparisons versus alcohol tolerance.

    Table S4. Oligonucleotides used in this study.

    Table S5. Bacterial strains and plasmids used for E. faecium mutagenesis.

    Table S6. Data for E. faecium CD50 experiments.

    Table S7. Data for E. faecium density (CFU/cm2) on the cage floor pre- and posttreatments.

    Table S8. Data for mice colonized by E. faecium after either water or alcohol cage floor disinfection.

    Table S9. Data for E. faecium (CFU/ml) remaining over time after exposure to isopropanol.

    Table S10. Data for change in doubling times of E. faecium mutants grown in the presence of isopropanol.

  • The PDF file includes:

    • Fig. S1. Tolerance of E. faecium to ethanol exposure.
    • Fig. S2. Isopropanol tolerance phenotype summary for 129 genome-sequenced E. faecium isolates.
    • Fig. S3. Core and pan-genome analysis of 129 E. faecium genomes.
    • Fig. S4. Growth curves of mutants.
    • Legends for tables S1 to S3
    • Table S4. Oligonucleotides used in this study.
    • Table S5. Bacterial strains and plasmids used for E. faecium mutagenesis.
    • Table S6. Data for E. faecium CD50 experiments.
    • Table S7. Data for E. faecium density (CFU/cm2) on the cage floor pre- and posttreatments.
    • Table S8. Data for mice colonized by E. faecium after either water or alcohol cage floor disinfection.
    • Table S9. Data for E. faecium (CFU/ml) remaining over time after exposure to isopropanol.
    • Table S10. Data for change in doubling times of E. faecium mutants grown in the presence of isopropanol.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Summary of E. faecium strains used in this study, with associated isopropanol tolerance data.
    • Table S2 (Microsoft Excel format). Pairwise comparisons of high-low alcohol-tolerant E. faecium.
    • Table S3 (Microsoft Excel format). DAPC analysis based on ortholog comparisons versus alcohol tolerance.

    [Download Tables S1 to S3]

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