Fig 1.
A frameshift mutation is present in rpoS from CFT073 rpoSam and strain derivatives thereof.
(A) The rpoS allele from CFT073 (WAM4505), CFT073 gyrAS83L (WAM2266), and CFT073 rpoSam (WAM2267) was sequenced via Sanger sequencing using the “rpoS sequencing F” and “rpoS sequencing R” primers listed in Table 1 and an alignment of the rpoS coding regions from these strains was performed using the ClustalW function in MacVector 9.0.2. (B) Dendrogram showing relevant strains used in this study (Also listed in Table 1) and their applicable characteristics. Strains mentioned in Panel A are outlined with a double black box.
Fig 2.
Inactivation of dsdA does not engender a competitive advantage in CBA/J mice during experimental UTI.
CFT073 ΔlacZYA rpoSam and CFT073 ΔdsdA rpoSam or CFT073 dsdAΔ445bp gyrAS83L or CFT073 gyrAS83L were co-inoculated at a 1:1 ratio into CBA/J mice (n = 16, 8, and 18, respectively). Mice were sacrificed at 48hpi. Bacteria from (A) bladder and (B) kidney homogenates were enumerated on MacConkey’s medium plus lactose. Several mice from the co-infections had no detectable bacteria in their kidneys (4 of 16, 5 of 8, and 3 of 18, respectively). Lines are drawn at the geometric mean relative competitive index (RCI). Statistical Significance was assessed by a Wilcoxon signed-rank test relative to a hypothetical RCI of 1.
Fig 3.
CFT073 ΔdsdA colonizes the murine urinary tract indistinguishably from wild type.
(A) CFT073 ΔdsdA and CFT073 ΔlacZYA were co-inoculated at a 1:1 ratio into CBA/J mice (n = 17). Bacteria from bladder and kidney homogenates were enumerated on MacConkey’s medium plus lactose. Lines are drawn at the geometric mean relative competitive index (RCI). Nine mice had no detectable bacteria in their kidneys. Statistical Significance was assessed by a Wilcoxon signed-rank test relative to a hypothetical RCI of 1. (B) Manipulations were carried out as described for panel A except that single strains were used (n = 16 for each). Two mice infected with CFT073 had no detectable bacteria in their kidneys and three mice infected with CFT073 ΔdsdA had no detectable bacteria in their kidneys. Lines are drawn at the geometric mean CFU/organ. Statistical significance was assessed by the Mann-Whitney U test.
Fig 4.
dsdA- strains have a growth defect in human urine.
Bacteria were grown overnight in pooled, filter sterilized human urine. Bacteria were washed 2x in phosphate buffered saline and the OD600 of each cell suspension was normalized to OD600 = 1.5. Bacteria were then inoculated into fresh, pre-warmed urine to OD600 = 0.03. Bacteria were allowed to grow for 12 hours. OD600 (Panel A) and viable counts (Panel B) were measured at the time points indicated above. dsdA+ strains used in this analysis are CFT073, CFT073 gyrAS83L, and CFT073 rpoSam, and dsdA- strains are CFT073 ΔdsdA, CFT073 dsdAΔ445bp gyrAS83L, and CFT073 ΔdsdA rpoSam. Data points represent mean OD600 and mean viable counts for each strain type where applicable and error bars are drawn to represent standard error of the mean (±SEM).
Table 1.
Strains, plasmids, and oligonucleotides used in this study.