Proteus mirabilis outcompetes Klebsiella pneumoniae in artificial urine medium through secretion of ammonia and other volatile compounds

Abstract

Klebsiella pneumoniae and Proteus mirabilis form mixed biofilms in catheter-associated urinary tract infections. However, co-inoculation of P. mirabilis with K. pneumoniae in artificial urine medium (AUM) resulted in a drastic reduction of K. pneumoniae cells in both biofilm and planktonic growth. Here, the mechanism behind this competitive interaction was studied. Both pH and aqueous ammonia (NH_3aq) increased in mixed cultures (to 9.3 and 150 mM, respectively), while K. pneumoniae viable cells dramatically diminished over time (>6-log reduction, p < 0.05). Mixed cultures developed in either 2-(N-morpholino) ethanesulfonic acid (MES)-buffered AUM (pH 6.5) or AUM without urea did not show bacterial competition, evidencing that the increase in pH and/or NH_3aq concentration play a role in the competitive interaction. Viability of K. pneumoniae single-species cultures decreased 1.5-log in alkaline AUM containing 150 mM NH_3aq after 24 h inoculation, suggesting that ammonia is involved in this inter-species competition. Besides NH_3aq, additional antimicrobials should be present to get the whole competitive effect. Supernatants from P. mirabilis-containing cultures significantly diminished K. pneumoniae viability in planktonic cultures and affected biofilm biomass (p < 0.05). When subjected to evaporation, these supernatants lost their antimicrobial activity suggesting the volatile nature of the antimicrobial compounds. Exposure of K. pneumoniae to volatile compounds released by P. mirabilis significantly decreased cell viability in both planktonic and biofilm cultures (p < 0.05). The current investigation also evidenced a similar bactericidal effect of P. mirabilis volatiles over Escherichia coli and Morganella morganii. Altogether, these results evidence the secretion of ammonia and other volatile compounds by P. mirabilis, with antimicrobial activity against gram-negative uropathogens including K. pneumoniae. This investigation provides novel insight into competitive inter-species interactions that are mediated by production of volatile molecules.

Keywords: Artificial urine medium; Bacteria; Biofilms; Dual-species competition; Klebsiella pneumoniae; Metabolite; Microbiology; Proteus mirabilis; Volatile compounds.

Figure 1

Time-course of medium pH, aqueous ammonia concentration, and bacterial cell number of P. mirabilis and K. pneumoniae mixed and single-species planktonic cultures in AUM. P. mirabilis (Pm) and K. pneumoniae (Kp) were inoculated, alone or mixed, in AUM and incubated at 37 °C. At different time-points both pH and NH_3aq levels were measured (A-C) and the number of cultivable cells was assessed (D-F). Each point represents the mean ± SDs of at least three independent experiments.

Figure 2

Effect of AUM modifications on the development of mixed P. mirabilis and K. pneumoniae planktonic cultures. MES-buffered AUM (pH 6.5) (A, C) or AUM without urea (B, D) were co-inoculated with P. mirabilis (Pm) and K. pneumoniae (Kp) and incubated at 37 °C. At different time-points both pH and NH_3aq levels were measured (A-B) and the number of cultivable cells was assessed (C-D). Each point represents the mean ± SDs of at least three independent experiments.

Figure 3

Survival of K. pneumoniae in AUM modified by alkalinization and increased aqueous ammonia concentration. K. pneumoniae was inoculated in AUM and modified-AUMs and the number of viable cells was assessed at 24 h post-inoculation. Modified AUMs having alkaline pH (8.0, 9.0 and 9.3) and increasing NH_3aq concentrations (25–150 mM at pH 9.1) were prepared as described in Material and Methods. Each bar represents the mean ± SDs of at least three independent experiments. (*) p < 0.01 compared to control AUM by ANOVA with Dunnett's post test.

Figure 4

Effect of bacterial supernatants on K. pneumoniae planktonic and biofilm growth. Cell-free supernatants (SN) from 16-h-old single-species K. pneumoniae (Kp), P. mirabilis (Pm), and mixed (Mix) planktonic cultures were analyzed. (A) The number of cultivable K. pneumoniae cells per ml was determined after planktonic growth in AUM containing SN at a ratio 1:1, for 24 h. (B) K. pneumoniae pre-formed biofilms (4-days-old) were challenged with AUM containing SN at a ratio 1:1. After 24 h, biofilm biomass was assessed by crystal violet staining (A_595nm). In both panels, each bar represents the mean ± SDs of three independent experiments. (*) p < 0.05 and (**) p < 0.001 compared to control AUM by ANOVA with Dunnett's post test.

Figure 5

Bactericidal activity of P. mirabilis volatiles over K. pneumoniae cultures. (A) Effect of evaporation on the antimicrobial activity of supernatants from mixed cultures (SN Mix; see legend of Figure 4). SN without any treatment (untreated), after evaporation (evaporated), or adjusted to pH 9.3 after evaporation were evaluated as described in legend of Figure 4. Each bar represents the mean ± SDs of three independent experiments. (*) p < 0.05 and (**) p < 0.001 compared to control AUM by ANOVA with Dunnett's post test. (B) Effect of P. mirabilis (Pm) volatiles on the viability of K. pneumoniae (Kp) in planktonic cultures in AUM. Kp was inoculated in a small open tube placed inside a larger tube containing Pm (Kp in/Pm out). Cell numbers in both compartments were quantified after 24 h at 37 °C. Kp in/AUM out was performed ss control. (C-D) Pm volatile mediated antimicrobial activity determined by 2-Petri-dish assays. (C) A small lidless Petri dish was placed inside a larger one, closed by its lid. Kp were spotted on the central small AUM-agar Petri dish (left panels) and Pm, on the external AUM-agar ring (upper left panel), and incubated for 48 h at 37 °C. As control, no Pm were spotted on the external AUM-agar ring (lower left panel). Quantification of bacterial viability in each colony by CFU counts (right panel). VCs: volatile compounds. (D) 2-Petri-dish assays performed as described in (C) to test Pm VCs over Escherichia coli (Ec), Morganella morganii (Mm), and Enterococcus faecalis (Ef). In panels B–D, each bar represents the mean ± SDs of three independent experiments. (*) p < 0.05 and (**) p < 0.001 by Student's t test.

Figure 6

Detrimental effect of P. mirabilis volatiles over K. pneumoniae pre-formed biofilms. (A) K. pneumoniae (Kp) biofilms were developed in 16 wells placed in the middle of a 96-well plate and, at day 3, P. mirabilis (Pm) was inoculated in the 48 wells surrounding Kp biofilms. This condition corresponds to biofilms exposed to Pm volatile compounds (VCs). As control, AUM was placed in the wells surrounding Kp biofilms (control biofilms). The plates were then incubated for additional 24 h at 37 °C. Kp biofilms were evaluated for both cell viability by CFU counts after mechanically disruption (B) and biofilm biomass by crystal violet staining (C). Each bar represents the mean ± SDs of three independent experiments. (**) p < 0.001 by Student's t test.