Fig 1.
The Kp terZ-F genes are sufficient for TeO3-2 resistance.
(A) The ter locus is organized in two operons, a putative biosynthetic cluster and a TeO3-2 resistance cluster. These sections are found on opposite DNA strands and are encoded bidirectionally. The representative ter locus from the hvKp strain NTUH-K2044 is shown. NTUH-K2044 containing the empty vector pACYC184, the isogenic ΔterC mutant (clone Kp2259) containing an empty vector, the pTerC, or the pTerZ-F plasmid (B), and the E. coli K12 strain MG1655 with or without the pTerZ-F plasmid (C) were grown on LB or LB containing 10 or 100 μM K2TeO3-2 to visualize inhibition of growth (dilution series 100−10−7 of overnight culture). Two representative clones (labeled #1 and #2) of NTUH-K2044ΔterC containing the pTerC or the pTerZ-F plasmid and MG1655 containing the pTerZ-F plasmid are shown.
Fig 2.
The Kp ter operon is not exclusive to hypervirulence plasmids.
ter+ plasmids from Martin et al. mSystems, 2018 [10] (A-C) and reference strains from the NCBI database (D-F) were analyzed. (A,D) Relative frequencies of sequence types (ST) of Kp strains containing ter+ plasmids. HvKp sequence types previously associated with the ter operon are outlined in a dashed line. (B,E) Heat map of ter+ plasmid sequence similarity to genes known to influence infection and antibiotic resistance genes. Each row represents an individual plasmid in the order of S2 Table (Martin et al. mSystems, 2018 [10] index 1–14, NCBI reference strains index 15–102). The pK2044 hvKp plasmid is highlighted by the red box, and hypervirulent Kp sequence types (hvST) previously associated with the ter operon are indicated. (C,F) To determine if any neighboring gene was consistently associated with ter, the gene neighborhood of ter plasmids encoding the ter operon from Martin et al. mSystems, 2018 [10] was visualized (C) and the frequency of ORFs adjacent to the ter operon encoded on reference plasmids from the NCBI database was calculated (F).
Fig 3.
TerC is a fitness factor during gut colonization.
(A) Three days prior to inoculation, male and female C57BL6/J mice sourced from barriers RB16 and RB07 were treated with 0.5 g/L ampicillin or regular drinking water. (B-E) NTUH-K2044 and the isogenic ΔterC mutant (clone Kp2259) were mixed 1:1 and approximately 5x106 CFU were orally gavaged into mice (n = 9–18 per group). A fresh fecal pellet was collected daily from each animal, CFUs were enumerated, and log competitive indices (mutant:WT) were calculated (median and IQR displayed, *P < 0.05, **P < 0.005, ***P < 0.0005, one-sample t test compared to a hypothetical value of 0). (F) NTUH-K2044 and the isogenic ΔterC mutant containing an empty vector or the pTerZ-F plasmid were mixed 1:1 and approximately 5x106 CFU were orally gavaged into mice sourced from barrier RB16 (n = 14–16). A fresh fecal pellet was collected 24 hours after inoculation, CFUs were enumerated, and log competitive indices (mutant:WT) were calculated (F, median and IQR displayed, ****P < 0.00005, one-sample t test compared to a hypothetical value of 0 or Student’s t test). Each data point represents an individual animal.
Fig 4.
The fecal microbiota in which terC is (RB16) and is not (RB07) a fitness factor are distinct.
Fecal pellets collected from male and female C57BL6/J mice sourced from barriers RB16 and RB07 (n = 9–20 mice per group) on the day of Kp inoculation were subjected to 16S rRNA sequencing. Pairwise community dissimilarity values between the fecal microbiota communities of barriers RB16 and RB07 with or without three days treatment with 0.5 g/L ampicillin were visualized by Principal coordinates analysis (A, AMOVA) and individually (B, **P < 0.005, ****P < 0.00005, one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test). (C) Diversity of the fecal microbiota was summarized by inverse Simpson index (blue points: RB16+Abx, orange points: RB07+Abx, **P < 0.005, one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test). LEfSe was used to determine if specific bacterial families (D) or OTUs (F) were differentially abundant between the fecal microbiota of RB16 and RB07 (D, LDA ≥ 3.5 and P < 0.05 are shown). Differential bacterial families (E) or OTUs (G) relative abundance values were plotted (E, *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.00005, Student’s t test).
Fig 5.
Exogenous treatment of mice with SCFAs results in a terC fitness defect.
Seven days prior to inoculation, male and female C57BL6/J mice sourced from barriers RB16 and RB07 were treated with a SCFA cocktail or regular drinking water (sham). NTUH-K2044 and the isogenic ΔterC mutant (clone Kp2257) were mixed 1:1 and approximately 5x106 CFU were orally gavaged into mice (n = 19 per group). A fresh fecal pellet was collected daily from each animal, CFUs were enumerated, and log competitive indices (mutant:WT) were calculated (median and IQR displayed, *P < 0.05, **P < 0.005, ***P < 0.0005, one-sample t test compared to a hypothetical value of 0 or Holm-Sidak multiple-comparison test following one-way ANOVA).