The Effect of the Gallbladder Environment during Chronic Infection on Salmonella Persister Cell Formation

Abstract

Typhoid fever is caused by Salmonella enterica serovar Typhi (S. Typhi). Around 3-5% of individuals infected become chronic carriers, with the gallbladder (GB) as the predominant site of persistence. Gallstones (GS) aid in the development and maintenance of GB carriage, serving as a substrate to which Salmonellae attach and form a biofilm. This biofilm matrix protects bacteria from the host immune system and environmental stress. This shielded environment is an ideal place for the development of persister cells, a transient phenotype of a subset of cells within a population that allows survival after antibiotic treatment. Persisters can also arise in response to harsh environments such as the GB. Here we investigate if GB conditions affect the number of persisters in a Salmonella population. To simulate the chronic GB environment, we cultured biofilms in cholesterol-coated 96-well plates in the presence of ox or human bile. We then treated planktonic or biofilm Salmonella cultures with high concentrations of different antibiotics. This study suggests that biofilms provide a niche for persister cells, but GB conditions either play no role or have a negative influence on persister formation, especially after kanamycin treatment. The antibiotic target was important, as antimicrobials directed against DNA replication or the cell wall had no effect on persister cell formation. Interestingly, repeated treatment with ciprofloxacin increased the percentage of S. Typhimurium persisters in a biofilm, but this increase was abolished by GB conditions. On the other hand, repeated ciprofloxacin treatment of S. Typhi biofilms in GB conditions slightly increased the fraction of persisters. Thus, while the harsh conditions in the GB would be thought to give rise to increased persisters, therefore contributing to the development of chronic carriage, these data suggest persister cell formation is dampened in this environment.

Keywords: Salmonella; bile; biofilm; persisters; typhoid.

Figure 1

Persister cells recovered in S. Typhimurium 14028 and S. Typhi Ty2 laboratory strains after treatment with ciprofloxacin (red circles) or kanamycin (blue circles) in planktonic cells growing in ox bile (OB) or human bile (HB) versus total cells (strains not treated with antibiotics are represented by black circles). Representative charts are shown from one experiment performed with four or more biological replicates and repeated at least three times with similar results. A one-way ANOVA followed by Tukey’s multiple-comparison test were used to compare the different conditions (**, p ≤ 0.01; ****, p ≤ 0.0001).

Figure 2

Persister cells recovered in S. Typhimurium 14028 and S. Typhi Ty2 laboratory strains after treatment with ciprofloxacin (red circles) or kanamycin (blue circles) in biofilms growing in ox bile (A) or human bile (B) versus total cells (strains not treated with antibiotics are represented by black circles). Representative charts are shown from one experiment performed with four or more biological replicates and repeated at least three times with similar results. A one-way ANOVA followed by Tukey’s multiple-comparison test were used to compare the different conditions (ns, not significant).

Figure 3

Persister cells recovered in S. Typhi clinical strains after treatment with ciprofloxacin (red circles) or kanamycin (blue circles) in biofilms on human bile versus total cells (strains not treated with antibiotics are represented by black circles). Representative charts are shown from one experiment performed with four or more biological replicates and repeated at least three times with similar results. A one-way ANOVA followed by Tukey’s multiple-comparison test were used to compare the different conditions (ns, not significant; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001).

Figure 4

Persister cells recovered in S. Typhimurium 14028 and S. Typhi Ty2 laboratory strains after treatment with ampicillin (purple circles) or cefepime (orange circles) in biofilms growing in human bile (HB) versus total cells (strains not treated with antibiotics are represented by black circles). Representative charts are shown from one experiment performed with four or more biological replicates and repeated at least three times with similar results. A one-way ANOVA followed by Tukey’s multiple-comparison test were used to compare the different conditions (ns, not significant; *, p ≤ 0.05).

Figure 5

Multiple rounds of ciprofloxacin treatment on biofilms. (A) Experimental set-up; growth, treatment, and CFU counts for rounds 2–5 were performed the same as for round 1. (B) Colony forming units recovered for S. Typhimurium 14028 and S. Typhi Ty2 laboratory strains in different growth conditions after every round of ciprofloxacin treatment (red symbols) vs. control total cell numbers (black and green symbols). Representative charts are shown from one experiment performed with four or more biological replicates and repeated at least three times with similar results.