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

Introduction: Mixed-species biofi lms are involved in a wide variety of infections. We studied the synergistic interactions during dual-species biofi lm formation among isolates of Pseudomonas aeruginosa, Acinetobacter baumannii, and Stenotrophomonas maltophilia. Methods: Isolates were cultured as single-species and all possible combinations of dual-species biofi lms. Results: The 61 A. baumannii biofi lms increased by 26-fold when cultured with S. maltophilia isolates; 62 A. baumannii biofi lms increased by 20-fold when cultured with S. maltophilia isolates; and 31 P. aeruginosa biofi lms increased by 102-fold when cultured with S. maltophilia 106. Conclusions: Synergy was observed between two isolates, including those that inherently lacked biofi lm formation ability.

Biofi lms are groups of bacteria encased in a self-produced extracellular polymeric matrix 1 , and which consist of superfi cial microbial colonies that attach to solid surfaces.Biofi lms 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 fi brosis (CF).The microcolonies that constitute the biofi lm can be composed of singlespecies populations or multimember communities of bacteria, depending on the environmental parameters under which they are formed 2 .Different bacteria inside a biofi lm 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-specifi c effects on each other 3 .
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 biofi lm formation in respiratory tract.According to Starner et al. 6 , bacterial biofi lms are increasingly recognized as the cause of persistence and disease pathogenesis in respiratory infections, such as CF 6 .
We have previously reported the occurrence of hospitalacquired pneumonia with different organisms in Tehran hospitals.Polymicrobial infections have been observed in such patients 4 .The aim of this study was to evaluate the occurrence of synergistic interactions during dual-species biofi lm 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 biofi lm formation.These organisms were isolated from patients with poly-microbial infections in the lower respiratory tract.
Dual-species biofi lm formation was assessed on Foley catheter pieces 9,10 .Briefl y, discs of catheter material (surface area: 0.5cm 2 ) 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 biofi lms 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 biofi lm growth was measured using a tetrazolium reduction assay with tetrazolium salt 3-[4,5-dimethylthiazol-2yl]-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.

Short Communication
Each of these six isolates was grown as single-species biofi lms as well as in all possible combinations of two different species.Synergistic interactions were observed in the majority of combinations.With 18 combinations, biofi lm formation by two species was signifi cantly greater than biofi lm production by any of the single species.According to Table 1, more than 100% increase in biofi lm formation was observed in 14 out of 24 combinations.These included combination of isolates with low activity in making biofi lm (Table 2).The results show that the biomass of a multi-species biofi lm is not necessarily the sums of the biomass of each single species.Although P. aeruginosa 31 and A. baumannii 62 did not strongly produce biofi lm (as clearly seen Figure 1), a considerable amount of biofi lm (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 biofi lm 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 biofi lm and the optical density values indicated the biofi lm biomass 8 .
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 biofi lm formation when two species were present was signifi cantly greater than biofi lm production by any of the species alone.Synergy was observed even between those who were not able to produce strong biofi lm on their own.Therefore, the infections caused by mixed bacterial populations can increase the disease intensity via strong biofi lm production.For instance, we demonstrated that S. maltophilia is able to induce biofi lm production in P. aeruginosa 31, which was not able to produce biofi lms by itself.
Since the total number of cells was kept constant during inoculation for both single-and dual-species biofi lms, it became obvious that diversifi cation is more effective than the number of cells involved in the primary stages of biofi lm formation.Thus, in a niche with a specifi ed number of cells, the presence of different species of bacteria can result in a greater quantity of biofi lm, as has been reported previously 12,13 .
Synergy in biofi lm formation by bacteria involved in a mixed-species structure could be related to the co-aggregation process.Biofi lm 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 biofi lm formation of other non-co-aggregating bacteria; thus, the presence of Actinomyces is critical in dental plaque formation 14 .One probable explanation for the marked increase in biofi lm 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 biofi lms is another remarkable characteristic of bacterial cells in such structures.In an investigation on chronic rhinosinusitis, it was observed that Haemophilus infl uenzae expresses its virulence factor type IV pili (pilA) only in mixed-species biofi lms.In the same niche, H. infl uenzae and Streptococcus pneumoniae supported each other in adhering to the host epithelia and enhanced the eventual establishment of a recalcitrant polymicrobial community 15 .
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 infl uence gene expression, metabolic co-operation and competition, physical contact, and the production of antimicrobial exoproducts, may lead to enhanced biofi lm 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 biofi lm research will be the dissection of these interactions.

Varposhti M et al. -
Mixed-species biofi lms of respiratory tract

TABLE 1 -Percentage of increase in the amount of biofi lm formation of individual bacterial strains when combined with the other bacterial strains.
Stenotrophomonas maltophilia 107.We did not combine the same bacterial strains; thus, some places in the table show no data.