Article Figures & Data
- FIG 1
Retargeting diffocins with a prophage RBP from C. difficile strain R20291. (A) Schematic representation of gene clusters coding for diffocin-4 (green) and modified diffocins Av-CD291.1 and Av-CD291.2 and including the tail structure genes of the phi027 prophage (blue). Genes are color coded according to source. For the phi027 prophage, the lysis cassette present only in the phi027 prophage is depicted in light blue and structural genes with no homology in the diffocin gene cluster are depicted in dark blue. The percentages of similarity between the diffocin-4 and phi027 genes are given (blue). (B) In vitro spot bioassays for bactericidal activity are shown for several strains. Preparations of diffocin-4, Av-CD291.1, and Av-CD291.2 were serially diluted and spotted on a soft agar lawn containing the indicated target strain. Dark zones of clearance indicate killing. Overlapping but distinct killing specificities for each diffocin preparation, which were all produced from a genetically identical B. subtilis host cell and by the same method, indicate killing is specific to the diffocin and not due to any nonspecific, contaminating B. subtilis protein. (C) The strain coverage for diffocin-4, Av-CD291.1, and Av-CD291.2 for ribotypes 001, 015, 017, 027, 053, and 087 is shown. White indicates no killing, and maroon indicates killing—with intensity of maroon reflecting robustness of killing. Strain designations in green and blue indicate known FQR1 and FQR2 phylogenies, respectively. An additional 20 strains representing 13 ribotypes were also tested and found not to be sensitive to Av-CD291.2 (data not shown).
- FIG 2
Enteric pharmacokinetics of diffocin-4 and Av-CD291.2 orally administered to mice. R-type bacteriocins that survive transit through the GI tract intact are detectable in feces by in vitro spot bioassays for bactericidal activity. Briefly, groups of 3 mice were administered diffocin-4 or Av-CD291.2 in 1% NaHCO3 (100 µg/dose [5 × 1011 KU]) via oral gavage (A) or in drinking water (60 µg/ml [3 × 1011 KU/ml]) containing 4% sucrose and 1% NaHCO3 for 28 h (B). Drinking water consumption averaged ~0.5 ml/h. Fecal pellets were collected and homogenized at the times indicated after oral gavage (A) or after introduction of Av-CD291.2 in drinking water (B). Fecal pellets collected at the indicated times were then filtered to remove microbial contaminants. The filtered homogenates were serially diluted and spotted on a soft agar lawn of the appropriately sensitive C. difficile strain R20291 (RT027). Dark zones of clearance indicate killing. Results from a representative mouse are shown for each condition.
- FIG 3
Oral administration of Av-CD291.2 in drinking water does not disturb the indigenous gut microbiota of healthy mice. (A) Analysis of the variance between microbial communities from the feces of healthy mice pretreatment (all) and posttreatment for each of the groups. The variance was assessed by the average relative abundance (weighted UniFrac distances) of OTU using principal component analysis. Samples are color coded according to treatment. Circled pretreatment samples indicate pretreatment outliers (one from each pretreatment cohort) that were removed from the microbiota analyses. Zone of inclusion are given with percentages. PCo1, principal component 1; PCo2, principal component 2. (B) Same as in panel A, except the average relative incidence (unweighted UniFrac distances) for OTU was assessed. (C) Bar charts depict the average family level composition (top 9) of OTU detected for each treatment group pre- and posttreatment by relative abundance. Significant differences between pre- and posttreatment are indicated by asterisks (*, 0.01 < P < 0.05; **, 0.001 < P < 0.01). Green asterisks indicate families elevated posttreatment, red asterisks indicate families reduced posttreatment, orange asterisks indicate families reduced posttreatment relative to all pretreatments, and purple indicates a family lower in one pretreatment cohort relative to most or all other pre- and posttreatment cohorts. (D) Same as in panel C, except the average family-level compositions (top 9) pre- and posttreatment were detected by relative incidence. Significant differences between pre- and posttreatment are indicated by asterisks as in panel C (***, P < 0.001). Circled asterisks indicate significant differences that remained after a false discovery rate correction was applied.
- FIG 4
Oral administration of Av-CD291.2 in drinking water does not disturb colonization resistance to C. difficile (A) or vancomycin-resistant E. faecium (VREF) (B) in mice. Mice were administered Av-CD291.2 (2× the equivalent daily dose [mg/kg] in the microbiota study), vancomycin (37.5 mg/kg/day), or the placebo control for 4 days in the drinking water. Following a washout period, mice were inoculated with the BI/NAP1/027-type C. difficile strain or VREF C68 strain (104 CFU) by oral gavage. Fecal samples were collected on days 1, 3, 5, and 10 postinoculation and assayed for C. difficile or VREF CFU. If pathogens were not detected in stool, the lower limit of detection (2 log10 CFU/g) was assigned. The standard errors of the means (SEM) for vancomycin-treated mice are shown.
- TABLE 1
Modified diffocin Av-CD291.2 prevents colonization in mice exposed to spores from C. difficile isolate BI/NAP1/027
Cohort Results fora: Day 4 Day 7b Infected (n) Total (n) % Infected (n) Total (n) % Placebo Female 8 10 80 10 10 100 Male 10 10 100 10 10 100 Total 18 20 90 20 20 100 Av-CD291.2 Female 0* 10 0* 9 10 90 Male 0** 10 0** 10 10 100 Total 0** 20 0** 19 20 95
Supplemental Material
Figure S1
Schematic of a diffocin and its mechanism of action. Diffocin is shown in the unbound and bound states on the C. difficile cell surface. RBP, receptor binding protein; BPAR, baseplate attachment region. Download Figure S1, PDF file, 0.2 MB.
Copyright © 2015 Gebhart et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.
Figure S2
Stability of R-type bacteriocins targeting RT027 strains. In vitro spot killing bioassays on strain R20291 (RT027) are shown. Preparations of diffocin-43593, Av-CD291.1, and Av-CD291.2 were isolated from B. subtilis production strains and then immediately spotted (day 0) or stored for 3 days before spotting on a soft agar lawn containing the target strain. Dark zones of clearance indicate killing. Download Figure S2, PDF file, 0.1 MB.
Copyright © 2015 Gebhart et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.
Figure S3
Spores are not killed by Avidocin-CDs in vitro. Approximately 50,000 CFU of R20291 spores were incubated with a 100-fold excess of Av-CD291.2 (5 × 106 KU) to CFU for 60 min in triplicate. After incubation, the samples were serially diluted and plated on brucella agar plates containing 0.1% taurocholate. A mock incubation with PBS for 60 min (and then heat treatment at 65°C to kill nonspores) was used as a control. No difference in viable CFU counts was observed. Similar experiments with R20291 vegetative cells (using up to 109 cells) and a 100-fold of excess Av-CD291.2 (up to 1011 KU) resulted in no CFU being recovered. Download Figure S3, PDF file, 0.001 MB.
Copyright © 2015 Gebhart et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.
Dataset S1
Total C. difficile shedding (CFU/g feces) during the prevention-of-colonization study. Download Dataset S1, XLSX file, 0.01 MB.
Copyright © 2015 Gebhart et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.
Dataset S2
Presence and absence of detected OTU in the fecal microbiota study. Download Dataset S2, XLSX file, 0.9 MB.
Copyright © 2015 Gebhart et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.
Figure S4
Additional microbiota analyses. (A) Alpha diversity pre- and posttreatment. The mean numbers of OTU observed for each cohort pre- and posttreatment were calculated. No significant differences were observed pre- and posttreatment for any cohort. Error bars indicate standard deviation for each condition (B and C). Comparison of microbiota change within each mouse after treatment shows no significant difference between treatment with Av-CD291.2 and treatment with the placebo control. (B) The variance between microbial communities posttreatment relative to pretreatment within each mouse was assessed by averaging the weighted UniFrac distances (relative change in OTU abundances) for each cohort. The mean for each treatment is indicated by a red bar and labeled. Error bars indicate standard deviations. Student's t tests were used to compare results. P values of <0.05 are indicated. Abundance variation after LD-fidaxomicin treatment was significantly different from Av-CD291.2 or placebo control treatments. (C) The plot is the same as in panel B, except the average unweighted UniFrac distance for each cohort was assessed. Incidence variation after LD-fidaxomicin treatment was significantly different from that of Av-CD291.2 or placebo control treatments. Download Figure S4, PDF file, 0.1 MB.
Copyright © 2015 Gebhart et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.
Table S1
Summary of P value results for Adonis tests. Table S1, PDF file, 0.2 MB.
Copyright © 2015 Gebhart et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.
Figure S5
Timeline schematics for prevention of colonization and microbiota studies in mice. (A) Av-CD291.2 efficacy study in mice exposed to spores from the BI-7 strain. Spore exposure and fecal sampling time points are depicted by arrows. The durations of the Av-CD291.2, placebo, and clindamycin treatments are indicated by parentheses. (B) Microbiota study in naive mice. Download Figure S5, PDF file, 0.1 MB.
Copyright © 2015 Gebhart et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.
Table S2
Strain list. Table S2, PDF file, 0.2 MB.
Copyright © 2015 Gebhart et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.
Table S3
Oligonucleotide and plasmid lists. Table S3, PDF file, 0.2 MB.
Copyright © 2015 Gebhart et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.
Additional Files
Supplementary Data
Supplementary Data
Files in this Data Supplement:
- Figure sf1, PDF - Figure sf1, PDF
- Figure sf2, PDF - Figure sf2, PDF
- Figure sf3, PDF - Figure sf3, PDF
- Figure sf4, PDF - Figure sf4, PDF
- Figure sf5, PDF - Figure sf5, PDF
- Table st1, PDF - Table st1, PDF
- Table st2, PDF - Table st2, PDF
- Table st3, PDF - Table st3, PDF
- Dataset sd1, XLSX - Dataset sd1, XLSX
- Dataset sd2, XLSX - Dataset sd2, XLSX
