Who arrived first? Priority effects on Candida albicans and Pseudomonas aeruginosa dual biofilms

doi:10.21203/rs.3.rs-4672806/v1

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Who arrived first? Priority effects on Candida albicans and Pseudomonas aeruginosa dual biofilms

https://doi.org/10.21203/rs.3.rs-4672806/v1

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Historical processes in community assembly, such as the arrival order of species, strongly influence their interactions causing priority effects. Candida albicans and Pseudomonas aeruginosa are frequently co-isolated from biofilm-based infections of the skin, lungs, and medical devices. Their relationship, predominantly antagonistic, involves physical and chemical interactions. However, the presence and implications of priority effects among these microorganisms remain largely unexplored. Here, we developed dual biofilms with differing inoculation times for each species and assessed the resulting microbial communities' in vitro virulence and susceptibility. Our findings showed that the inoculation order impacts biofilm composition, structure, virulence, and antimicrobial susceptibility. The first colonizer had an advantage for surface colonization. Consecutive colonization increased biofilm virulence and negated C. albicans’ protective effect on P. aeruginosa PAET1 against meropenem treatment. Finally, we propose N-acetylcysteine as an adjuvant for treating C. albicans and P. aeruginosa interkingdom infections, working independently of priority effects.

Biological sciences/Microbiology/Biofilms

Biological sciences/Microbiology/Microbial communities/Clinical microbiology

Biological sciences/Microbiology/Applied microbiology

Health sciences/Pathogenesis/Infection

polymicrobial interactions

first colonizer

co-infections

antifungal treatment

N-acetylcysteine

interkingdom biofilms

fungi-bacteria

Fungi and bacteria colonization of various body microenvironments leads to interkingdom polymicrobial infections, which are common in the oral cavity, gastrointestinal system, skin, respiratory tract, and urinary tract ^1,2. The most prevalent fungal pathogen in constant interaction with a diverse array of bacteria is Candida albicans ². Moreover, most human infectious diseases are caused by biofilm-forming pathogens, which can colonize biotic surfaces and medical devices ¹. C. albicans is particularly known for its propensity to form biofilms independently or in conjunction with other microorganisms ³.

C. albicans and Pseudomonas aeruginosa are frequently co-isolated from biofilm-based infections of the skin, lungs, and medical devices ⁴. The relationship between these microorganisms involves physical and chemical interactions and is predominantly classified as antagonistic ¹. However, their polymicrobial biofilms have reported virulence and susceptibility advantages ¹, highlighting that interactions within microbial communities are complex and dynamic ⁵. In pulmonary co-infections of C. albicans and P. aeruginosa, the clinical impact of the fungal species remains incompletely understood ⁶. Experimental and clinical observations suggest that the presence of Candida may contribute to respiratory diseases, albeit indirectly ⁷. However, there is currently insufficient clinical data to support antifungal treatment following Candida identification ⁷.

Mixed-species biofilms pose a significant clinical challenge due to their difficulty in diagnosis and treatment ³. Interspecies interactions within polymicrobial biofilms are relevant for host-response, drug resistance, and disease progression ³. For example, in polymicrobial biofilms, Candida acts as a physical scaffold for bacteria attachment and growth, creates hypoxic microenvironments that protect anaerobic bacteria, and produces an extracellular matrix that shields bacteria from antibiotics ^1,8,9. However, most biofilm studies focus on single species.

Studies in microbial ecology have revealed that historical processes in community assembly, such as the order and time of species arrival to a habitat, strongly influence species interaction ^10,11. These interactions, termed priority effects, have been observed in mammalian gut and skin microbiomes, as well as in fungal-bacterial biofilms ^5,11. In vitro and ex-vivo biofilm experiments with C. albicans and bacteria found in chronic wound infections have shown that priority effects increase the relative abundance of early colonizers at the expense of late colonizers ⁵. However, the presence of priority effects on dual biofilms of C. albicans and P. aeruginosa and their implications remain largely unexplored.

Therefore, this study aimed to evaluate whether priority effects influence the virulence of C. albicans and P. aeruginosa polymicrobial biofilms and explore therapeutic strategies for their treatment. We developed dual biofilms with differing inoculation times for each species and studied the resulting microbial communities. We found that priority effects influence biofilm structure and composition. In addition, successive colonization increased biofilm virulence and negated C. albicans’ protective effect on P. aeruginosa PAET1 against meropenem treatment. Our findings shows that priority effects impact significantly the virulence and susceptibility of resulting biofilms, underscoring the importance of C. albicans presence in interkingdom biofilms.

Composition and structure of C. albicans and P. aeruginosa biofilms with different inoculation times

We devised a protocol to obtain polymicrobial biofilms of C. albicans and P. aeruginosa PAET1 with different inoculation orders (Fig. 1a). The CFUs count revealed differences in polymicrobial communities composition. P. aeruginosa PAET1 biomass (CFUs per mL) is significantly higher when it is the early colonizer (1Pa2Ca) compared to the other polymicrobial conditions (1Ca2Pa and CaPa) and the monomicrobial control (Pa) (Fig. 1a and Supplementary Table 1). This translates into a higher relative abundance (97.75%) of P. aeruginosa PAET1 in the 1Pa2Ca condition, compared to the 86–89% reported in 1Ca2Pa and CaPa conditions (Fig. 1b). Notably, no significant differences in the total number of CFUs per mL (sum of both P. aeruginosa PAET1 and C. albicans CFUs per mL) were observed within the different polymicrobial biofilms (Fig. 1b and Suplemmentary Table 1).

However, it is important to consider that C. albicans cells are larger than bacteria cells, resulting in biofilms where fungal growth accounts for a significant portion of biofilm structure (Fig. 2). Notably, C. albicans was primarily found in blastospore morphology under all conditions. The differences in biomass composition reported by CFUs counting were also observed in the microscopy images, with more bacteria cells present in the 1Pa2Ca condition (Fig. 2a). Overall, bacteria size allowed P. aeruginosa PAET1 to be located within Candida cells, reaching the surface regardless of the inoculation order (Fig. 2b). Notably, variations in bacteria distribution along biofilm layers were observed among conditions.

When P. aeruginosa PAET1 was added earlier (1Pa2Ca), bacterial cells formed a thick bottom layer (Fig. 2b), covering almost all the abiotic surface and reducing the space for C. albicans attachment (Fig. 2c). In contrast, in the condition where C. albicans was added earlier (1Ca2Pa), the bacteria had less abiotic surface available to attach, resulting in a bottom layer that was less thick and dense (Fig. 2b). This caused that P. aeruginosa PAET1 to use C. albicans as a biotic surface for attachment, leading to a more pornounced cell-to-cell interaction between the interkingdom species (Fig. 2c) and a greater distribution of the bacteria along all biofilm layers (Fig. 2b). Conversely, when both microorganisms were inoculated simultaneously (CaPa), the species competed for atachment to the abiotic surface, favoring intra-species aggregation, observed as clusters of cells, over inter-species interaction (Fig. 2c). This resulted in an overlap of C. albicans and P. aeruginosa PAET1 throughout biofilm layers (Fig. 2b). Moreover, the higher competitive behaviour among the microorganisms in this polymicrobial condition (CaPa )represented a higher mortality for C. albicans compared to its monomicrobial biofilm (Ca) (Supplementary Fig. 1), resulting in polymicrobial biofilms that were less thick than the others (Fig. 2b).

Virulence assessment of C. albicans and P. aeruginosa biofilms with different inoculation times

To evaluate whether the resulting priority effects induce virulence variations of the polymicrobial biofilms, early staged biofilms (20–24 h) were formed on top of A549 alveolar cells (Fig. 3). In these experiments, polymicrobial biofilms were generated from an inoculum of ~ 10⁵ CFUs per mL, while the monomicrobial controls were formed from inoculum of ~ 10⁵ CFUs per mL (Ca 1x and Pa 1x) and ~ 10⁶ CFUs per mL (Ca 2x and Pa 2x). In this way, we could evaluate the impact of inoculum size on the final biomass of each microorganism by comparing the monomicrobial biofilms 1x to 2x (Fig. 3a). We found that halving the inoculum size did not affect the obtained number of CFUs per mL from P. aeruginosa PAET1 biofilms (Supplementary Table 1). However, the reduction of the inoculum had a statistically significant effect on C. albicans biofilms, causing a decrease of ~ 1 Log₁₀ CFUs per mL in Ca 1x compared to Ca 2x (Supplementary Table 1).

Next, we assessed whether the inclusion of eukaryotic cells in the in vitro model induced alterations in the biomass of each species. We compared the biomass of Ca and Pa monomicrobial biofilms from Fig. 1a protocol to Ca 2x and Pa 2x monomicrobial biofilms from the Fig. 3a protocol. No statistically significant differences were observed in the final number of CFUs per mL for either microorganism under both conditions (Supplementary Table 1). These protocols used the same inoculum size for bacteria and fungi; however, the experiments that included eukaryotic cells (Fig. 3) were incubated for half the time compared to those that did not (Fig. 1) (24 h vs 48 h for C. albicans and 20 h vs 42 h for P. aeruginosa PAET1). This indicates that the inclusion of eukaryotic cells stimulates microorganism growth in monomicrobial biofilms of C. albicans and P. aeruginosa PAET1.

Figure 3 showed that the 1Pa2Ca condition formed in the presence of eukaryotic cells retained a significantly higher biomass of P. aeruginosa PAET1 compared to the other polymicrobial biofilms (Fig. 3a and Supplementary Table 1). In terms of total CFUs per mL, no statistically significant differences were reported among the three polymicrobial biofilms (Fig. 3b and Supplementary Table 1). However, the composition of the polymicrobial communities was different when eukaryotic cells were included in the experiment. The relative abundance of P. aeruginosa PAET1 in the 1Ca2Pa condition decreases to 75% but reaches almost 100% in CaPa and 1Pa2Ca, compared to biofilms formed directly on the plate (Fig. 3b vs Fig. 1b).

After biofilm characterization, we focused on the viability evaluation of the A549 cells (Fig. 4). Observing the morphologies of the infected eukaryotic cells compared to the control, we noticed different alterations that led to cell death in some cases (dark cells in Fig. 4a). In general, infected cells appeared to be expanded, particularly in the conditions Pa 1x, Pa 2x, and 1Pa2Ca. The quantification of the viability of A549 cells by trypan blue (Fig. 4b) showed that all monomicrobial biofilms decreased cell viability in ~ 0.1–0.4 log₁₀, with the highest values reported for Ca 2x but without statistical significance (Supplementary Table 1). Regarding the polymicrobial biofilms, those formed by simultaneous colonization (CaPa) showed a viability reduction similar to the monomicrobial controls (0.35 log₁₀ cells per mL). However, the scenario changes when biofilms were formed by successive colonization. When P. aeruginosa was added first (1Pa2Ca), the log₁₀ reduction in cell viability reached 0.63, indicating that these biofilms are statistically significantly more virulent than the monomicrobial biofilms (Supplementary Table 1). But, when C. albicans was the early colonizer (1Ca2Pa), the reduction in cell viability increases to 1.07 log₁₀ cells per mL, making this condition the most virulent compared to all the others (Supplementary Table 1).

Finally, we created a multiple linear regression model to establish if viability differences were associated with the quantity of fungal cells per bacteria cells present in the polymicrobial community. We found a high goodness of fit of the model (r² = 0.9918), in which each parameter was statistically significant: CFUs per mL of P. aeruginosa PAET1 (p-value = 0.0281), CFUs per mL of C. albicans (p-value = 0.0127), and the interaction of CFUs per mL of both microorganisms (p-value = 0.0003). This can be clearly seen in Fig. 4c, where an increase in the number of CFUs per mL in both Ca (y-axis) and Pa (x-axis) led to higher mortality of A549 cells (higher viability reduction). Likewise, in polymicrobial biofilms, the interaction of the quantity of both microorganism also accounts for higher virulence of the interkingdom communities than the monomicrobial counterparts.

Treatment response of C. albicans and P. aeruginosa biofilms with different inoculation times

Considering that priority effects of C. albicans and P. aeruginosa dual biofilms involved virulence differences, we decided to explore the response of the polymicrobial biofilms against a 15 h treatment with conventional antimicrobials (Fig. 5a). Each biofilm condition, monomicrobial and polymicrobial, had an untreated control that allowed us to calculate the change in the Log₁₀ CFUs per mL of the treated samples. Thus, a higher change represents a more significant reduction in the Log₁₀ CFUs per mL and, therefore, higher susceptibility of the evaluated microorganism to the treatment.

We found that meropenem (MER) at 5 µg per mL has the same effect on P. aeruginosa PAET1 when grown in the polymicrobial biofilms 1Ca2Pa and 1Pa2Ca, compared to Pa monomicrobial control (Fig. 5b and Supplementary Table 1). However, a decrease in susceptibility to this antibiotic was found in the condition CaPa. On the other hand, C. albicans susceptibility to caspofungin (CAS) at 5 µg per mL was reduced in all polymicrobial biofilms in comparison to Ca (Fig. 5c and Supplementary Table 1). When both antimicrobial agents were given simultaneously (Fig. 5d), we observed that the variation in susceptibility to MER found in P. aeruginosa PAET1 in the CaPa biofilm was lost, but the decreased response to CAS in C. albicans polymicrobial biofilms remained (Fig. 5d and Supplementary Table 1).

We went further and added the mucolytic agent N-acetylcysteine (NAC) at 1 mg per mL to the previous combined treatment (Fig. 5e). As a result, no differences in susceptibility of P. aeruginosa PAET1 and C. albicans were longer observed between the polymicrobial biofilms and their monomicrobial counterparts (Supplementary Table 1). In the case of P. aeruginosa PAET1, NAC acted as a potent antimicrobial by itself (Supplementary Fig. 2a) or as an adjuvant on the antibiotic treatment (Supplementary Fig. 2b) of monomicrobial and polymicrobial biofilms. On the contrary, the concentration used of NAC did not have a relevant antifungal activity by itself (Supplementary Fig. 2a and Supplementary Table 1). Moreover, the adjuvant effect of NAC observed in the combined therapies MER + CAS + NAC (Fig. 5d) and NAC + CAS (Supplementary Fig. 2c) was restricted to the polymicrobial biofilms (Supplementary Table 1). This suggests that the decrease in susceptibility to CAS is associated with the protection that the P. aeruginosa matrix confers to C. albicans. As can be observed in the Supplementary Fig. 3, most ECM polysaccharides in the polymicrobial biofilms appear to be secreted by P. aeruginosa PAET1.

The coexistence of multiple pathogenic species in the same host can lead to higher virulence, increasing disease risk and severity, compared to monomicrobial infections ^12,13. The order in which microorganisms infect the host strongly influences their interactions ¹². Depending on the system, the first or second arriver could have advantages that favor survival ¹². However, the priority effects of pathogen coexistence have been little explored.

In this study, we studied polymicrobial biofilms of C. albicans and P. aeruginosa PAET1 with different inoculation times. CFUs counts and microscopy images showed that being the first colonizer confers an advantage for surface adhesion and further biofilm development in polymicrobial biofilms. This is clearly observed in the 1Pa2Ca condition, where P. aeruginosa PAET1 arrival reduces the potential adhesion sites for C. albicans, resulting in the domination of the community composition by the bacteria (Fig. 1–3). This exclusion effect occurs naturally in the gastrointestinal and vaginal tracts, where local microbiota restricts C. albicans colonization ¹⁴. It has also been reported in vitro with bacteria from burn wounds ⁵.

When fungi arrive first (1Ca2Pa), the percentage of C. albicans in the community composition increased in comparison with the other polymicrobial biofilms (Fig. 1, and more markedly in Fig. 3), but P. aeruginosa PAET1 continued dominating in number. We explain this bacterial dampening of the priority effects by their small size and ability to colonize fungal structures ^5,15, allowing them to attach between and on top of C. albicans cells (Fig. 2). This reaffirms the scaffold role of C. albicans in polymicrobial biofilms by increasing the attachment surface, and the importance of including fungi in the study of microbial communities associated with disease ⁵.

Lastly, in the situation of the synchronous arrival of bacteria and yeast (CaPa), both species compete for attachment to the underlying substrate, as previously reported for other C. albicans-bacteria interactions ^5,16. This competition led to a patchy distribution of both microorganisms on the surface, with intra-species clumps (Fig. 2). Interestingly, in the CaPa condition, higher C. albicans death was observed, followed by 1Ca2Pa (Supplementary Fig. 1). This could indicate that P. aeruginosa PAET1 utilizes mechanisms that compromise fungal integrity to gain access to colonization space, supported by the antagonistic relationship described elsewhere among this duo ^17–27.

However, the clinical consequences of the association of C. albicans and P. aeruginosa remain unclear. Contradictory conclusions from in vivo studies range from reduction of virulence to synergy and enhanced mortality of co-infections compared to mono-infections ⁶. P. aeruginosa and C. albicans are frequently co-isolated from tracheobronchial samples, being more common in patients with cystic fibrosis (CF) and ventilator-associated pneumonia (VAP) ^6,27–29. In both conditions, co-infection with this pair leads to reduced lung function, prolonged hospital stays, and higher intensive care unit mortality ²⁷. For those reasons, we decided to include pulmonary epithelial cells in the model and evaluate virulence variations of the polymicrobial biofilms associated with priority effects.

The simultaneous inoculation of bacteria and fungi (CaPa) showed an increase in virulence compared to Ca or Pa biofilms (Fig. 4b), but it was not statistically significant. This disagrees with previous in vivo studies in pulmonary models that found higher biofilm biomass, virulence, and inflammation in polymicrobial biofilms of C. albicans and P. aeruginosa compared to their monomicrobial infections ^30–34. We believe the discrepancy is due to the short duration of the experiment (20 h of inter-species interaction); prolonged exposure of the epithelial cells to biofilm growth would likely increase the magnitude of virulence differences. However, it is interesting to note that CaPa was the condition with more inter-species competition (Supplementary Fig. 1) but less virulence (Fig. 4b).

On the other hand, biofilms formed by sequential inclusion of microorganisms produced a significant reduction in epithelial cell viability compared to the monomicrobial control, being 1Ca2Pa the most virulent group (Fig. 4b). This result is highly significant because co-infections in the real context are unlikely to be simultaneous ³⁵. Previous work showed that mouse mortality is higher when preformed P. aeruginosa biofilms are inoculated with C. albicans than when they are not ^32,36. Moreover, the addition of C. albicans to preformed biofilms of P. aeruginosa induces the expression of the bacterial genes mucA and psl, and higher inflammatory responses than mixed biofilms formed by simultaneous inoculation ³². In contrast, the effect of P. aeruginosa after C. albicans colonization is not that clear. Previous instillation of C. albicans protected mice against P. aeruginosa virulence (bacterial burden and lung injury), presumably by a priming effect ^28,37,38. Conversely, Roux et al. reported that previous instillation of C. albicans increases P. aeruginosa colonization, pneumonia development, and cytokine production in rats ^39,40. In patients, C. albicans colonization of the respiratory tract was identified as a risk factor for Pseudomonas VAP ^41,42.

Our model showed that not only does the individual burden of each specie account for the virulence of polymicrobial biofilms, but also the community composition (interaction of both microorganisms’ biomass) (Fig. 4c). However, we do not know which type of priority effects are responsible for the increase in P. aeruginosa PAET1 virulence in the epithelial pulmonary cells after C. albicans colonization (1Ca2Pa) compared to its monomicrobial infection (Pa) (Fig. 4b). Described interactions between both microorganisms could play a role, such as C. albicans stimulation of P. aeruginosa expression of virulence factors (pyoverdine, phenazines and rhamnolipids) and biofilm formation ^27,33,34,43.

Our results support the hypothesis of C. albicans as a co-conspirator in pulmonary infections, agreeing with previous studies that associate its presence in the respiratory tract with higher morbidity or mortality ^44,45. However, the co-isolation of C. albicans and P. aeruginosa from respiratory samples does not consider antifungal administration unless there is histological evidence of fungal infection ^27,28,46. Mainly due to the lack of agreement on the significance of C. albicans presence and inconclusive evidence on the implementation of antifungal treatment in humans ^{6,28,33,44,46,47}. Therefore, C. albicans detection is usually assumed as colonization ⁴⁶. Hoping to contribute to elucidating these questions, we tested whether priority effects influence microbial response to antibiotic and antifungal treatment, alone and in combination.

We found that P. aeruginosa PAET1 susceptibility to MER decreases when its biofilm is formed concomitantly with C. albicans (CaPa) compared to when it is formed alone (Pa) (Fig. 5b). This effect has been reported before and was associated with the mannan and glucan polysaccharides of the extracellular matrix (ECM) ⁴⁸. However, we found that the protective effect was abolished after the mixed antibiotic and antifungal treatment MER + CAS (Fig. 5b), suggesting some contribution of fungal activity. Surprisingly, this synergistic effect was not present in polymicrobial biofilms formed from the sequential addition of colonizers (1Ca2Pa and 1Pa2Ca) (Fig. 5b), showing that community priority effects can impact microbial antibiotic susceptibility.

We also saw that in polymicrobial biofilms, P. aeruginosa PAET1 protects C. albicans from CAS treatment, regardless of the arrival time of colonizers (Fig. 5c). Combined antibiotic and antifungal treatment MER + CAS did not alter this effect (Fig. 5d), but the inclusion of NAC did (Fig. 5e), suggesting involvement of P. aeruginosa PAET1 ECM rather than bacterial activity (Supplementary Fig. 3). More studies need to confirm these findings, but susceptibility changes derived from polymicrobial interactions are frequently associated with the extended protection of one species’ ECM for another ^48–50.

Nevertheless, as the history of colonization events can be patient-specific, treatments not affected by priority effects should be preferred in the management of polymicrobial biofilms. For these reasons, we reaffirm the inclusion of N-acetylcysteine as an exciting option in the treatment of C. albicans and P. aeruginosa co-infections, as previously reported ³². We believe that the inclusion of biofilm disrupting agents could help elucidate whether antifungals are required to eradicate interkingdom biofilms. Our study showed that MER + CAS combination did not significantly improve the reduction of both microorganisms in any polymicrobial condition compared to MER treatment alone (Fig. 5b vs Fig. 5d). However, the comparison of MER treatment with NAC + MER and MER + CAS + NAC resulted in an increased reduction of at least 1 Log₁₀ of CFUs per mL of P. aeruginosa, and C. albicans and P. aeruginosa, respectively, in all polymicrobial conditions (Fig. 5b vs Fig. 5e and Supplementary Fig. 2b). Whether the reduction in C. albicans biomass has clinical relevance in disease outcomes remains a question that needs to be answered.

Our study confirms that priority effects modify community structure and function ^51,52, making it necessary to consider them in modeling in vitro systems. We recognize that the protocol used to assess biofilm virulence can be tested to extend the duration of biofilm growth and to evaluate the treatment effectiveness. But it allowed us to identify that sequential colonization favors microbial interactions that enhance virulence against alveolar epithelial cells, being more marked in the 1Ca2Pa condition. Contrary, the successive addition of species abolishes the protective effect of C. albicans to P. aeruginosa against MER. Finally, N-acetylcysteine could enhance the treatment efficacy of C. albicans and P. aeruginosa interkingdom infections, independently of the priority effects.

Bacterial strains and growing conditions

This study used two clinical isolates: Candida albicans 10045727 obtained from a patient with fungaemia ⁵³ and Pseudomonas aeruginosa PAET1 from a cystic fibrosis patient with persistent infection ⁵⁴. Additionally, P. aeruginosa PAET1 expressing the GFP plasmid pETS-PA was employed in imaging experiments ⁵⁵. Bacteria were cultured at 37 ºC in Luria Bertani (LB) medium (Scharlab S.L.), while fungi were grown at 30 ºC in Yeast Petone Dextrose (YPD) medium consisting of 1% Yeast Extract (Gibco), 2% Meat Peptone (Scharlab S.L.), 2% D-glucose (Fisher Scientific S.L.).

Biofilm formation

Overnight cultures (16 h at 200 rpm) of C. albicans (30 ºC) and P. aeruginosa PAET1 (37 ºC) were centrifuged at 4000 rpm (Labnet Spectrafuge™ 6C) for 5 min and washed twice with Phosphate Buffer Saline 1X (PBS) (Fisher Scientific S.L.). Suspensions with a final optical density of λ = 550 nm (OD₅₅₀) of 0.1 for yeast and 0.005 for bacteria, which corresponds to ~ 10⁶ Colony Forming Units per mL (CFUs per mL) of both microorganisms, were prepared in RPMI-1640 with L-glutamine without sodium bicarbonate (Sigma-Aldrich) supplemented with 0.2% D-glucose (referred to as RPMId). Then, suspensions were added to 96-well polystyrene plates with a flat bottom (Corning, USA).

Three different protocols for polymicrobial biofilm formation were established based on the colonization order: 1) Initial inoculum of C. albicans followed by addition of P. aeruginosa PAET1 6 h later, referred to as 1Ca2Pa. 2) Initial inoculum of P. aeruginosa PAET1 followed by C. albicans addition 6 h later, referred to as 1Pa2Ca. 3) Initial inoculum with both species, referred to as CaPa. Monomicrobial biofilms of C. albicans and P. aeruginosa were included as controls and denoted as Ca and Pa, respectively (Fig. 1A). To enhance adhesion to the surface, RPMId used for the initial inoculation (t₀) was supplemented with 10% Fetal Bovine Serum (FBS) (Gibco). A PBS wash was performed before the second inoculation (t₁). Biofilms were incubated at 37 ºC for a total time of 48 h, with an intermediate step of media renewal at 24 h.

To quantify the resulting biomass from each biofilm protocol, wells underwent a PBS wash before the removal of biofilms using a pipette tip. The biofilm suspensions were then subjected to sonication in an ultrasonic bath (Branson 200 ultrasonic cleaner) for 5 min, followed by 30 s vortexing to aid cell disaggregation. Serial dilutions were plated on LB and YPD agar for P. aeruginosa and C. albicans growth, respectively. CFUs were counted after 24 h.

Microscopic characterization

To observe the microscopic structure of biofilms, P. aeruginosa PAET1-GFP and C. albicans biofilms were formed on 8-well cell culture slides (SPL Life Sciences) following the protocol described above. The cell culture slides underwent an overnight pretreatment with FBS at 37°C to enhance microorganism adhesion to the surface. The wells were washed with PBS before t₀ to remove the excess of FBS.

The cell culture slides were then incubated, and matured biofilms were visualized using a LSM 800 confocal scanning laser microscope (Zeiss). P. aeruginosa PAET1 was visualized by the GFP plasmid, while C. albicans was stained with the chitin dye Calcofluor White (CW) (Thermo Fisher Scientific) at 10 µM. Propidium Iodide from the LIVE/DEAD BacLight Bacterial Viability Kit (Thermo Scientific) was used to stain dead cells (compromised membranes) and Concanavalin A-Alexa Fluor 647 (Invitrogen) to stain polysaccharides of the extracellular matrix (ECM), according to manufacturer’s instructions. Images were processed using Zeiss software.

Virulence assessment

Differences in the virulence of early staged biofilms of C. albicans and P. aeruginosa PAET1 with distinct inoculation times were evaluated in vitro using the adenocarcinomic alveolar A549 cell line (ATCC CCL-185). Cells were cultured in T-75 tissue culture flasks (Thermo Fisher Scientific) and grown in Dulbecco’s Modified Eagle Medium: Nutrient mixture F12 (DMEM/F12; Thermo Fisher Scientific), supplemented with 10% (v/v) FBS (Thermo Fisher Scientific), 100 Units per mL of penicillin/streptomycin (Thermo Fisher Scientific) and 2.5 µg per mL of amphotericin B (Thermo Fisher Scientific). Cells were passaged and maintained in a humidified incubator at 37 ºC and 5% CO₂ (ICOmed, Memmert).

For viability experiments, 2x10⁵ cells per well were seeded in a 24-well plate (SPL Life Sciences) in Dulbecco’s Modified Eagle Medium and incubated at 37 ºC and 5% CO₂ for 24 h. On day 2, the media was changed to antibiotic and antifungal-free DMEM/F12 (only with 10% FBS) to avoid any possible interferences with infection. On day 3, before cell infection, the media was replaced with DMEM/F12 supplemented with 10% FBS and 5% bovine serum albumin (BSA; Panreac Química S.L.U.). Cells were then infected with ~ 10⁵ CFUs per mL of each microorganism from an overnight culture previously washed with PBS to obtain the three different polymicrobial biofilms based on the inoculation time (Fig. 3A). Uninfected cells and cells infected with P. aeruginosa PAET1 (Pa 1x) and C. albicans (Ca 1x) alone served as controls. Additional controls of monomicrobial biofilms (Ca 2x and Pa 2x), where the concentration of the inoculum was doubled (~ 10⁶ CFUs per mL), were included to discard mortality associated with higher infection load in polymicrobial biofilms. After 4 hours of t₀, wells were washed with PBS, and t₁ was performed. Biofilms were allowed to grow for a total of 24 h since t₀.

Subsequently, wells were washed once with PBS, and cells were detached by the addition of 0.2 ml of trypsin-EDTA at 0.25% (Thermo Fisher Scientific) for 10 minutes at 37 ºC and 5% CO₂. Following trypsin inactivation with 0.2 ml of antibiotic and antifungal-free media, 10 µl of cell suspensions was mixed 1:1 with trypan blue and counted in a Neubauer chamber. Cell viability was calculated by averaging the count of live cells in 5 squares of the chamber, multiplied by 2 (dilution factor of the dye) and by 10⁴ to obtain the number of cells per mL. The reduction in cell viability after infection was calculated by subtracting the base ten logarithms of viable cells per mL in infected wells from the control well (ΔLog₁₀ Viable cells per mL = Log₁₀ Viable cells per mL_Control - Log₁₀ Viable cells per mL_infected). The same detached and stained eukaryotic cells were visualized in differential interference contrast (DIC) to see morphological changes after infection, using a Nikon inverted fluorescent microscope ECLIPSE Ti–SperL100 (Nikon, Japan) coupled with a DS-Qi2 Nikon camera (Nikon, Japan). Images were processed with Image J Fiji software.

In parallel, wells were processed to recover bacteria and fungi attached to cells. This involved washing wells thrice with PBS, before trypsin addition and inactivation. The suspensions were sonicated in an ultrasonic bath for 5 min and vortexed for 30 s. Serial dilutions were plated onto LB and YPD agar for colony counting of P. aeruginosa PAET1 and C. albicans, respectively.

Susceptibility Evaluation

The antimicrobial susceptibility of polymicrobial biofilms according to the inoculation time of each species was assessed using the antibiotic meropenem (MER) at 5 µg per mL (Sharlab, S.L.), the antifungal caspofungin (CAS) at 5 µg per mL (Merck Life Science), and the mucolytic N-acetylcysteine (NAC) at 1 mg per mL (Sigma-Aldrich), as well as their combinations (MER + CAS, NAC + MER, NAC + CAS, MER + CAS + NAC). Each biofilm condition included control wells treated with RPMId.

Biofilms were treated 24 h after t₁ inoculation (Fig. 5A). 15 h post-treatment, biofilms were washed and prepared for CFUs quantification as described in the “biofilm formation” section. The reduction in biomass after treatment was calculated by subtracting the base ten logarithms of CFUs per mL of treated samples from their respective control sample (ΔLog₁₀ CFUs per mL = Log₁₀ CFUs per mL_Control - Log₁₀ CFUs per mL_Treated).

Statistics and Reproducibility

Graphics and comparisons were conducted using GraphPad Prism v10. Statistical differences among the mean of conditions were evaluated by Unpaired t-test, One-way ANOVA or two-way ANOVA as appropriate and stated in each figure and Supplementary table 1. Correction for multiple comparisons was also included when required. In addition, a multiple linear regression model to explain variations in the viability reduction of the infected eukaryotic cells was created: A549 viability reduction (Δlog cells per mL) ~ Intercept + P. aeruginosa (CFUs per mL) + C. albicans (CFUs per mL) + P. aeruginosa (CFUs per mL): C. albicans (CFUs per mL). A significant level of p < 0.05 was established for all tests.

Conflict of Interest

All authors declare no conflict of interest.

Funding sources

were not involved in the research conduction or article preparation.

Author contributions

B.V.A.-J. contributed to the study design, performed the experiments, analyzed the data and wrote the manuscript. J.A. collaborated in the design and execution of experiments that involved eukaryotic cells and reviewed the manuscript. E. T. contributed to the design and supervision of the study and the revision of the manuscript. All authors read and approved the final version of the manuscript.

Acknowledgements

We thank Dr Jesus Guinea Ortega from the Department of Clinical Microbiology and Infectious Diseases, Hospital General Universitario Gregorio Marañón, Madrid, Spain, for the generous gift of the Candida albicans 10045727 used in this study. Likewise, A549 cells were a generous gift of Dr. Esther Julián from the Autonomous University of Barcelona, Bellaterra, Spain.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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Who arrived first? Priority effects on Candida albicans and Pseudomonas aeruginosa dual biofilms

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