Synergistic interactions in mixed-species biofilms of pathogenic bacteria from the respiratory tract

Biofilms are groups of bacteria encased in a self-produced extracellular polymeric matrix¹, and which consist of superficial microbial colonies that attach to solid surfaces. Biofilms cause a variety of persistent infections, including chronic middle ear infections, chronic sinusitis, chronic otitis, and lung infections in people with the inherited disease, cystic fibrosis (CF). The microcolonies that constitute the biofilm can be composed of single-species populations or multimember communities of bacteria, depending on the environmental parameters under which they are formed². Different bacteria inside a biofilm structure are in close contact with each other; they display multiple phenotypes and have multiple genotypes, which arise by horizontal gene transfer. They also have quorum-sensing-specific effects on each other³.

Pseudomonas aeruginosa, Acinetobacter baumannii, and Stenotrophomonas maltophilia are among the most important causative agents in nosocomial respiratory diseases in Tehran hospitals. Treatment of these infections is difficult, as they show high levels of intrinsic or acquired resistance to different antimicrobial agents, markedly reducing the antibiotic options available for treatment^4,5. Bacterial species isolated from cases with respiratory tract infections usually show strong resistance to antimicrobial agents; this could be related to the potential for biofilm formation in respiratory tract. According to Starner et al.⁶, bacterial biofilms are increasingly recognized as the cause of persistence and disease pathogenesis in respiratory infections, such as CF⁶.

We have previously reported the occurrence of hospital-acquired pneumonia with different organisms in Tehran hospitals. Polymicrobial infections have been observed in such patients⁴. The aim of this study was to evaluate the occurrence of synergistic interactions during dual-species biofilm formation by the bacteria isolated from respiratory tract of these patients.

Bacterial strains, isolated from patients who were hospitalized in Tehran University Hospitals (Tehran, Iran), were screened for their biofilm production potential. The screening process was performed in 96-well microtiter plates using the crystal violet method^7,8. Six bacterial strains, including P. aeruginosa (n = 2), A. baumannii (n = 2), and S. maltophilia(n = 2) were selected for single- and dual-species biofilm formation. These organisms were isolated from patients with poly-microbial infections in the lower respiratory tract.

Dual-species biofilm formation was assessed on Foley catheter pieces^9,10. Briefly, discs of catheter material (surface area: 0.5cm²) were cut from catheters, with sterile cutters under aseptic conditions, and placed in 24-well Nunclon (Thermo Fisher Scientific, Waltham, MO) tissue culture plates. A standardized cell suspension (80µL) was applied to the surface of each disc, for dual species biofilms 40µL of each bacterial suspension were added and the discs were incubated for 1h at 37°C (adhesion period). Non-adherent organisms were removed by gentle washing with 0.15M PBS (5mL), and the discs were submerged in 1mL of brain heart infusion broth (BHIB), then incubated for 24h at 37°C to allow biofilm formation. In control experiments, discs lacking cells were incubated in medium containing 1mL BHIB. All biofilm and control assays were carried out three times, in triplicate. Quantitation of biofilm growth was measured using a tetrazolium reduction assay with tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (methylthiazol tetrazolium [MTT]; Sigma-Aldrich; St Louis, MO)¹¹.

After biofilm formation, 1mL MTT solution (0.3% in phosphate-buffered saline) was added to each well and subsequently incubated for 5h at 37°C. MTT was then removed, and the wells were washed three times with 0.15M PBS (2mL) to remove all traces of MTT. Dimethyl sulfoxide (1mL) was then added to solubilize the MTT formazan product. Then, dimethyl sulfoxide was transferred to 96-well microtiter plates and MTT formazan formation was measured at 550nm using an enzyme-linked immunosorbent assay (ELISA) reader (Anthos, Australia). Control wells containing medium plus MTT were used to determine background formazan values. All assays were carried out three times in triplicate.

Each of these six isolates was grown as single-species biofilms as well as in all possible combinations of two different species. Synergistic interactions were observed in the majority of combinations. With 18 combinations, biofilm formation by two species was significantly greater than biofilm production by any of the single species. According to Table 1, more than 100% increase in biofilm formation was observed in 14 out of 24 combinations. These included combination of isolates with low activity in making biofilm (Table 2). The results show that the biomass of a multi-species biofilm is not necessarily the sums of the biomass of each single species. Although P. aeruginosa 31 and A. baumannii 62 did not strongly produce biofilm (as clearly seen Figure 1), a considerable amount of biofilm (20-fold increase) was formed when these strains were grown in the presence of S. maltophilia (Figure 1). The strongest synergism was observed between P. aeruginosa 31 and S. maltophilia 106: the biofilm biomass of P31 was increased by more than 100-fold. The total number of cells remained constant during inoculation for both single- and dual-species biofilm and the optical density values indicated the biofilm biomass⁸.

Polymicrobial infections constitute a high percentage of nosocomial respiratory infections and cause serious problems in therapeutic procedures. In the present study, using 12 different combinations, the biofilm formation when two species were present was significantly greater than biofilm production by any of the species alone. Synergy was observed even between those who were not able to produce strong biofilm on their own. Therefore, the infections caused by mixed bacterial populations can increase the disease intensity via strong biofilm production. For instance, we demonstrated that S. maltophilia is able to induce biofilm production in P. aeruginosa 31, which was not able to produce biofilms by itself.

Since the total number of cells was kept constant during inoculation for both single- and dual-species biofilms, it became obvious that diversification is more effective than the number of cells involved in the primary stages of biofilm formation. Thus, in a niche with a specified number of cells, the presence of different species of bacteria can result in a greater quantity of biofilm, as has been reported previously^12,13.

Synergy in biofilm formation by bacteria involved in a mixed-species structure could be related to the co-aggregation process. Biofilm formation of some non-co-aggregating bacteria can be promoted by other strains in an environment with bacterial diversity. In an investigation of dental plaques, it was observed that Actinomyces species could induce biofilm formation of other non-co-aggregating bacteria; thus, the presence of Actinomyces is critical in dental plaque formation¹⁴. One probable explanation for the marked increase in biofilm formation by P31 and A62 isolates may be the enhanced attachment of these cells that occurs after the attachment of S. maltophilia to the surface.

Expression of some features only in mixed-species biofilms is another remarkable characteristic of bacterial cells in such structures. In an investigation on chronic rhinosinusitis, it was observed that Haemophilus influenzae expresses its virulence factor type IV pili (pilA) only in mixed-species biofilms. In the same niche, H. influenzaeand Streptococcus pneumoniae supported each other in adhering to the host epithelia and enhanced the eventual establishment of a recalcitrant polymicrobial community¹⁵.

In this study, bacteria from different genera were used together and inter-species communication between the strains, including the secreted factors (quorum-sensing molecules, secondary metabolites, carbohydrates, and proteins), which influence gene expression, metabolic co-operation and competition, physical contact, and the production of antimicrobial exoproducts, may lead to enhanced biofilm formation. The exact mechanism of synergism between these three bacteria is not known at present and one or more of the interactions mentioned above may have occurred during this experiment.

These results may suggest that studying the interactions between pathogens in respiratory tract infections could facilitate the more suitable and effective in future. A future challenge in biofilm research will be the dissection of these interactions.