Enterococcus faecalis Antagonizes Pseudomonas aeruginosa Growth in Mixed-Species Interactions

Abstract

Enterococcus faecalis is often coisolated with Pseudomonas aeruginosa in polymicrobial biofilm-associated infections of wounds and the urinary tract. As a defense strategy, the host innately restricts iron availability at infection sites. Despite their coprevalence, the polymicrobial interactions of these two species in biofilms and under iron-restricted conditions remain unexplored. Here, we show that E. faecalis inhibits P. aeruginosa growth within biofilms when iron is restricted. E. faecalis lactate dehydrogenase (ldh1) gives rise to l-lactate production during fermentative growth. We find that an E. faecalis ldh1 mutant fails to inhibit P. aeruginosa growth. Additionally, we demonstrate that ldh1 expression is induced under iron-restricted conditions, resulting in increased lactic acid exported and, consequently, a reduction in local environmental pH. Together, our results suggest that E. faecalis synergistically inhibits P. aeruginosa growth by decreasing environmental pH and l-lactate-mediated iron chelation. Overall, this study emphasizes the importance of the microenvironment in polymicrobial interactions and how manipulating the microenvironment can impact the growth trajectory of bacterial communities. IMPORTANCE Many infections are polymicrobial and biofilm-associated in nature. Iron is essential for many metabolic processes and plays an important role in controlling infections, where the host restricts iron as a defense mechanism against invading pathogens. However, polymicrobial interactions between pathogens are underexplored under iron-restricted conditions. Here, we explore the polymicrobial interactions between commonly coisolated E. faecalis and P. aeruginosa within biofilms. We find that E. faecalis modulates the microenvironment by exporting lactic acid which further chelates already limited iron and also lowers the environmental pH to antagonize P. aeruginosa growth under iron-restricted conditions. Our findings provide insights into polymicrobial interactions between bacteria and how manipulating the microenvironment can be taken advantage of to better control infections.

Keywords: Enterococcus faecalis; Pseudomonas aeruginosa; iron restriction; l-lactate; lactate dehydrogenase (LDH); mixed species; polymicrobial interactions.

FIG 1

E. faecalis inhibits P. aeruginosa growth under iron-restricted conditions. (A to D) Enumeration of PAO1 BAA-47 (A), E. faecalis clinical isolates (B), P. aeruginosa clinical isolates (C), and OG1RF (D) from 24-h biofilms with single or mixed inocula grown in TSBG medium supplemented with 1 mM 22D. Dotted lines represent inoculum of bacteria spotted. n ≥ 3 with 3 technical replicates; error bars represent standard deviation from the mean. Statistical analysis was performed using the Mann-Whitney U test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. For panel A, statistical significances are for all strains compared to PAO1 BAA-47. (E) Enumeration of PAO1 BAA-47 from 24-h cultures grown with fresh TSBG medium, water, or cell-free supernatant obtained from 24-h biofilms of PAO1 BAA-47, OG1RF, and PAO1 BAA-47 mixed with OG1RF. The water and respective supernatants were mixed at a 1:1 ratio with fresh TSBG medium supplemented with 1 mM 22D for PAO1 BAA-47 growth. n = 4 with 3 technical replicates; error bars represent standard deviation from the mean. Statistical analysis was performed using the Mann-Whitney U test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (F and G) Enumeration of PADP6 (F) and OG1RF (G) from 24-h macrocolonies with single or mixed inocula grown in TSBG medium supplemented without and with 2 mM 22D. Bacterial species were mixed at a 1:1 ratio for mixed-species macrocolonies. Dotted lines represent inoculum of bacteria spotted. n = 3 with 3 technical replicates; error bars represent standard deviation from the mean. Statistical analysis was performed using the Mann-Whitney U test: ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 2

l-Lactate produced by E. faecalis inhibits P. aeruginosa growth in iron-restricted media. (A and B) Enumeration of PADP6 (A) and OG1RF, Δldh1, and Δldh1::ldh1 (B) from 48-h macrocolonies with single or mixed inocula grown in 2 mM 22D-chelated TSBG medium. Bacterial species were mixed at a 1:1 ratio for mixed-species macrocolonies. Dotted lines represent inoculum of bacteria spotted. n = 4 with 3 technical replicates; error bars represent standard deviation from the mean. Statistical analysis was performed using the Mann-Whitney U test: ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (C) Quantification of l-lactate exported from 48-h single- and mixed-species macrocolonies grown in TSBG medium supplemented without and with 2 mM 22D. n = 3 with 2 technical replicates; error bars represent standard deviation from the mean. Statistical analysis was performed using two-way analysis of variance with Tukey’s test for multiple comparisons: ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (D) Quantification of iron when 200 μM iron(II) sulfate heptahydrate (Fe²⁺) and iron(III) chloride hexahydrate (Fe³⁺) were supplemented without and with various concentrations of 22D (0.5, 1, and 2 mM) or l-lactate (10 and 20 mM). n ≥ 3 with 2 technical replicates; error bars represent standard deviation from the mean. Statistical analysis was performed using the Mann-Whitney U test: ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (E and F) Enumeration of PADP6 (E) and OG1RF and Δldh1 (F) from 48-h macrocolonies with single or mixed inocula grown in 2 mM 22D-chelated TSBG medium without and with increasing lactic acid concentrations (2.5, 5, and 10 mM). Bacterial species were mixed at a 1:1 ratio for mixed-species macrocolonies. Dotted lines represent inoculum of bacteria spotted. n = 3 with 3 technical replicates; error bars represent standard deviation from the mean. Statistical analysis was performed using the Mann-Whitney U test: ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 3

P. aeruginosa growth inhibition is due to lowered environmental pH under iron-restricted conditions. (A) Enumeration of PADP6 from 48-h single-species macrocolonies grown in TSBG medium supplemented without and with increasing 22D concentrations (2, 3, and 4 mM), which is then further supplemented without and with lactic acid (10 and 20 mM). Dotted lines represent inoculum of bacteria spotted. n ≥ 3 with 3 technical replicates; error bars represent standard deviation from the mean. Statistical analysis was performed using the Mann-Whitney U test: ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (B) Enumeration of PADP6 from 48-h single-species macrocolonies grown in 2 mM 22D-chelated TSBG medium supplemented without and with 20 mM lactic acid (pH unadjusted and pH adjusted to pH 6.60) or 20 mM citric acid (pH unadjusted and pH adjusted to pH 6.61). Dotted lines represent inoculum of bacteria spotted, and dashed lines represent limit of detection. n = 3 with 3 technical replicates; error bars represent standard deviationl from the mean. Statistical analysis was performed using the Mann-Whitney U test: ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (C) Planktonic growth of PADP6 in pH-unadjusted (pH 7.29) and pH-adjusted (pH 6.52, 5.50, 4.52, and 3.54) TSBG medium supplemented with 2 mM 22D. n = 3 with 3 technical replicates; error bars represent standard deviation from the mean.

FIG 4

Proposed working model of E. faecalis and P. aeruginosa in vitro polymicrobial interactions. Interactions between E. faecalis and P. aeruginosa under unchelated (A) and iron-restricted (B) conditions. (A) In unchelated conditions, l-lactate produced in E. faecalis is exported with hydrogen ions via a symporter (purple) as lactic acid, which is then deprotonated in the environment into l-lactate and hydrogen ions (H⁺). This l-lactate then chelates iron in the environment. (B) In iron-restricted conditions, E. faecalis ldh1 expression is upregulated. Consequently, as E. faecalis grows, l-lactate production and lactic acid secretion increase. This further chelates iron under iron-restricted conditions and lowers the environmental pH to a point at which P. aeruginosa cannot grow. The figure was created with BioRender.com.