Increased Levels of (p)ppGpp Correlate with Virulence and Biofilm Formation, but Not with Growth, in Strains of Uropathogenic Escherichia coli

Abstract

Urinary tract infections are one of the most frequent bacterial diseases worldwide. UPECs are the most prominent group of bacterial strains among pathogens responsible for prompting such infections. As a group, these extra-intestinal infection-causing bacteria have developed specific features that allow them to sustain and develop in their inhabited niche of the urinary tract. In this study, we examined 118 UPEC isolates to determine their genetic background and antibiotic resistance. Moreover, we investigated correlations of these characteristics with the ability to form biofilm and to induce a general stress response. We showed that this strain collection expressed unique UPEC attributes, with the highest representation of FimH, SitA, Aer, and Sfa factors (100%, 92.5%, 75%, and 70%, respectively). According to CRA (Congo red agar) analysis, the strains particularly predisposed to biofilm formation represented 32.5% of the isolates. Those biofilm forming strains presented a significant ability to accumulate multi-resistance traits. Most notably, these strains presented a puzzling metabolic phenotype-they showed elevated basal levels of (p)ppGpp in the planktonic phase and simultaneously exhibited a shorter generation time when compared to non-biofilm-forming strains. Moreover, our virulence analysis showed these phenotypes to be crucial for the development of severe infections in the Galleria mellonella model.

Keywords: E. coli; Galleria mellonella; UPEC; UTI; antibiotic resistance; biofilm; infection; pathogenicity; phenotype; ppGpp; stringent response; virulence.

Figure 1

Genetic characteristics of the UPEC collection. (a) Percentage distribution of virulence factors identified by PCR in UPEC isolates. (b) Percentage distribution of phylogroups determined by phylogenetic typing using the quadruplex PCR method [17].

Figure 2

Co-occurrence matrix of virulence factors. Statistically significant results (Fisher’s exact test, p < 0.05) are shown in green, while non-significant correlations are shown in gray. The direction of correlations was determined using Pearson’s test, where “+” indicates a positive correlation and “-” a negative correlation (p < 0.05). The matrix presents horizontal values vs. vertical values.

Figure 3

Genetic relationships among UPEC strains. The dendrogram was created by the UPGMA method using the DendroUPGAMA server [36]. Colors mark the different phylogenetic groups identified, and strains that form biofilm on Congo red agar are underlined. The dashed lines divide the dendrogram into four clusters. The first cluster brings together all strains encoding the cnf gene.

Figure 4

Phenotypic evaluation of the UPEC strains. Non-biofilm forming (n = 24) and biofilm-forming (n = 23) strains were grown aerobically in MOPS minimal medium (0.2% glucose), with shaking. (a) The relative accumulation of the (p)ppGpp alarmones was assessed by the ³²P labeling of nucleotides and revealed chromatographically on PEI cellulose plates by TLC in 1.5M KH₂PO₄, pH 3.4. The presented data were collected at an exponential phase of growth (A600 = 0.3) (b) Doubling time was determined spectrophotometrically with a plate reader. (c) Linear regression plot obtained by combining both parameters (generation time and alarmone accumulation), with R2 values indicated. Results are obtained from at least three independent experiments and are expressed as the mean with SEM. Statistical significance was determined by unpaired Student’s t-test.

Figure 5

Evaluation of virulence of CRA+/− UPEC strains. Evaluation of virulence of biofilm-forming (gray part of panel) and non-biofilm-forming (colorless part of panel) UPEC strains in a surrogate infection model of G. mellonella. The 5 biofilm producers chosen were selected based on the greatest ability to form biofilm, and the 5 non-biofilm forming samples were randomly selected. (a) Kaplan–Meier survival curves of G. mellonella after injection of 10⁴–10⁷ cells/larva. (b) LD50 values calculated between 24 and 72 h. Percentage changes in LD50 between the 1st and 3rd days are indicated by arrows, with their value above them. The values of growth rate (min) and alarmone relative concentration (%) in particular strains are indicated under the plot.

Figure 5

Evaluation of virulence of CRA+/− UPEC strains. Evaluation of virulence of biofilm-forming (gray part of panel) and non-biofilm-forming (colorless part of panel) UPEC strains in a surrogate infection model of G. mellonella. The 5 biofilm producers chosen were selected based on the greatest ability to form biofilm, and the 5 non-biofilm forming samples were randomly selected. (a) Kaplan–Meier survival curves of G. mellonella after injection of 10⁴–10⁷ cells/larva. (b) LD50 values calculated between 24 and 72 h. Percentage changes in LD50 between the 1st and 3rd days are indicated by arrows, with their value above them. The values of growth rate (min) and alarmone relative concentration (%) in particular strains are indicated under the plot.