Autoinducer-2 of Streptococcus mitis as a Target Molecule to Inhibit Pathogenic Multi-Species Biofilm Formation In Vitro and in an Endotracheal Intubation Rat Model

Abstract

Streptococcus mitis (S. mitis) and Pseudomonas aeruginosa (P. aeruginosa) are typically found in the upper respiratory tract of infants. We previously found that P. aeruginosa and S. mitis were two of the most common bacteria in biofilms on newborns' endotracheal tubes (ETTs) and in their sputa and that S. mitis was able to produce autoinducer-2 (AI-2), whereas P. aeruginosa was not. Recently, we also found that exogenous AI-2 and S. mitis could influence the behaviors of P. aeruginosa. We hypothesized that S. mitis contributes to this interspecies interaction and that inhibition of AI-2 could result in inhibition of these effects. To test this hypothesis, we selected PAO1 as a representative model strain of P. aeruginosa and evaluated the effect of S. mitis as well as an AI-2 analog (D-ribose) on mono- and co-culture biofilms in both in vitro and in vivo models. In this context, S. mitis promoted PAO1 biofilm formation and pathogenicity. Dual-species (PAO1 and S. mitis) biofilms exhibited higher expression of quorum sensing genes than single-species (PAO1) biofilms did. Additionally, ETTs covered in dual-species biofilms increased the mortality rate and aggravated lung infection compared with ETTs covered in mono-species biofilms in an endotracheal intubation rat model, all of which was inhibited by D-ribose. Our results demonstrated that S. mitis AI-2 plays an important role in interspecies interactions with PAO1 and may be a target for inhibition of biofilm formation and infection in ventilator-associated pneumonia.

Keywords: AI-2; PAO1; Streptococcus mitis; biofilms; ventilator-associated pneumonia.

FIGURE 1

Scanning electron microscopy (SEM) analysis of biofilms obtained from PAO1 and Streptococcus mitis. (A) PAO1 mono-culture. (B) PAO1 and S. mitis co-culture. (C) PAO1 and S. mitis co-culture with 50 mM D-ribose. (D) S. mitis mono-culture. Red arrows: S. mitis; Blue arrows: PAO1.

FIGURE 2

Quantification of biofilm formation by crystal violet staining. Quantification of the biofilm biomass by spectrophotometry (OD 570). The error bars indicate standard deviations. P represents PAO1 group; PS represents PAO1&S. mitis group; PSD represents PAO1&S. mitis group co-culture with D-ribose (50 mM). (A) Effects of D-ribose on planktonic growth of PAO1 and S. mitis. Cells were grown in BHI medium, in the presence of 50 mM concentrations of D-ribose or dH₂O. The data represent mean values of three independent experiments. (B) The biomass of co-cultured PAO1 and S. mitis was significantly higher than that of co-cultured PAO1 and S. mitis with 50 mM D-ribose and mono-cultured PAO1 or S. mitis. (C) PAO1 was exposed to fresh spent supernatant (SPNT) or heat-treated supernatant (HI SPNT) from S. mitis. Both the SPNT and the HI SPNT from S. mitis could increase the biomass of the PAO1 biofilm. (D) Bacterial colony counts of mono- and dual-species biofilms. (E) No significant difference was found between co-cultures of PAO1 with dead S. mitis cells and co-cultures with dead PAO1 cells. ‘P-dead’ represents dead PAO1 cells, and ‘S-dead’ represents dead S. mitis cells. (F) Bioluminescencing activity of partially purified S. mitis AI-2 and BB170 (AB medium for V. harveyi, BHI for S. mitis and PAO1).

FIGURE 3

Survival rates of rats inoculated with different biofilm-covered ETTs. Fifteen rats in each group were inoculated with PAO1 (P), PAO1&S. mitis (PS), or S. mitis (S) biofilm-covered ETTs, and sterilized tubes were used as controls. The PSD represents PS group was treated with D-ribose, whereas the other four groups were treated with PBS. (A) The survival rate was estimated at the indicated times, and the results are displayed as a Kaplan–Meier plot. The survival time of the PAO1&S. mitis group was significantly shorter than the survival times of the other three groups (P < 0.05). (B) Number of bacteria in the lung tissue (log CFU per gram). (C) Total numbers of cells in the BALF of the different groups. (D–F) Protein levels of IL-4, IL-6, and IL-10 in the BALF of the different groups.

FIGURE 4

Histopathological analysis of the lungs of rats inoculated with different biofilm-covered ETTs. Sections of lungs stained with hematoxylin-eosin on day 7 post-infection are shown. (A–E) Depict the lungs of P group, PS group, PSD group, S group, and the control group, respectively. The destination area was selected using an inverted microscope at 400×. Both (A,B) show large numbers of inflammatory cells infiltrating the alveolar spaces and lung necrosis. Furthermore, the pulmonary tissues in the PAO1&S. mitis group presented significantly more inflammation than those in the PAO1 group.