Out of the 36 isolates in this study, 35 showed strong biofilm formation, and 1 showed moderate biofilm formation. It demonstrates that the formation of biofilms is a crucial virulence factor¹⁴ for both Pseudomonas aeruginosa and the species of Candida. Because of biofilms, these infections can adhere to a variety of surfaces, shielding them from the host immune system and various environmental factors such as dehydration and violence, as well as natural killer cells, phagocytes, complement, and ROS-mediated damage. These interspecies interactions are typically more resistant to antimicrobial agents than other microorganisms.⁸

Together, the fungi and bacteria can be found in a range of settings, but biofilms are particularly common. In these structures, the attached species communicate with one another via a variety of signaling pathways. Due to the formation of biofilms, interspecies interactions can influence drug intolerance, virulence factors, and the outcome of polymicrobial infections.¹⁵ Pseudomonas aeruginosa caused pneumonia may be predisposed to by Candida albicans colonization, according to a clinical study done on ventilated patients.¹

According to this study, Pseudomonas aeruginosa exposed to Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis showed an increase in OD. One significant effect of these phenotypic modifications is modified antibiotic tolerance. Antimicrobial concentrations needed to destroy biofilms are many times greater than those needed to treat planktonic bacterial infections, which are more common. As a result, for biofilm-associated infections, microbiological indices of antimicrobial susceptibility like minimal inhibitory concentration (MIC) that direct the selection of treatment for common infections are unreliable. Furthermore, the patient’s symptoms are frequently exacerbated by an inflammatory response, as the immune system is unable to effectively eradicate the infection. Lastly, live bacteria in the form of persistence or small colony variants can also be found within the biofilm structure. Consequently, biofilm-related infections are thought to be challenging to treat. In certain cases, such as infections related to catheters, patients’ biofilms can be effectively treated by removing the infected foreign body; however, this is not always the case, necessitating prolonged use of high doses of antibiotics and frequent surgery.¹⁵

In this study, it was found that there was a difference in OD between Pseudomonas aeruginosa and the OD of Pseudomonas aeruginosa with exposure to Candida albicans, where there was an increase in OD which indicated a synergistic interaction. Candida albicans produces ethanol due to the induction by Phenazine from Pseudomonas aeruginosa and the ethanol which stimulates adhesion and biofilm formation of Pseudomonas aeruginosa. There is a positive feedback mechanism where Candida albicans ethanol production can increase 5-methyl-phenazine-1-carboxylic acid (5-MPCA) production in Pseudomonas aeruginosa and increase biofilm formation. Then, 5-MPCA stimulates ethanol production in Candida albicans.¹⁶

Similar findings were also found in a study conducted in 2021 by Kasetty et al., which showed that Pseudomonas aeruginosa biofilms containing Candida albicans had higher OD levels than Pseudomonas aeruginosa biofilms alone.¹⁴ It also bore similarities to Phuengmaung’s research from 2022, which discovered that prominent interkingdom biofilms with more severe infections were induced by Pseudomonas and Candida in the lungs of patients suffering from cystic fibrosis and ventilation-associated pneumonia (VAP). Compared to Pseudomonas and single Candida,⁹ the biofilm production between Pseudomonas added with Candida was more increased. However, this is not the same as the results of research by Fourie et al. in 2019, which showed a decrease in OD indicating an antagonistic interaction.⁸

Pseudomonas aeruginosa and Candida albicans both produce more biofilms than a single Pseudomonas aeruginosa, most likely as a result of their distinct biofilm properties and the significance of multiple genes for Pseudomonas biofilm production, especially for biofilms from the polysaccharide synthesis locus (Psl) as opposed to the alginate-mediated pathway. Pseudomonas virulence factors that were significant were the genes (alginate, psl, and pel) that produce the pseudomonas biofilm. Alginate was a negatively charged acetylated polymer that was used to increase reactive oxygen species in host cells and inhibit phagocytosis. It contained nonrepetitive b-1,4-linked L -guluronic and D -mannuronic acids. Psl is a neutral branched pentasaccharide that mediates attachment to lung epithelial cells, promoting pro-inflammatory responses and host cell damage. It has a 1:1:3 ratio of D-glucose, D-rhamnose, and D-mannose. Pel, on the other hand, was a positively charged polysaccharide that stabilized the biofilm structure by acetylating one to four glycosidic branches from N-acetyl galactosamine and N-acetylglucosamine. We can therefore conclude that the psl gene plays a role in the increase in OD in mixed biofilms.¹⁰

A number of publications have demonstrated that Pseudomonas aeruginosa and Candida albicans interact antagonistically. One such publication, by Fourie et al., demonstrates that quorum sensing molecules and phenazines mediate antagonistic interactions between Pseudomonas aeruginosa and Candida albicans. However, the presence of biofilm heterogeneity between in vitro and in vivo studies can lead to different results.⁹

Additional research demonstrates that interactions between the fungus Candida albicans and the bacteria Pseudomonas aeruginosa led to more severe infections in the human host. Pseudomonas aeruginosa’s production of biofilms is linked to more challenging-to-treat infections. Pseudomonas aeruginosa is stimulated by ethanol to colonize respiratory tract cells and plastic surfaces. The fungus also produces ethanol, which modifies the toxic spectrum of Pseudomonas aeruginosa’s phenazine, which is linked to worsening lung function in cystic fibrosis patients.⁸ A positive feedback loop between Candida albicans and Pseudomonas aeruginosa occurs as a result of phenazine’s interaction with Candida albicans to promote ethanol production, which exacerbates the disease. When exposed to low concentrations of Candida albicans, phenazine increased the production of fermentation products like ethanol by three to five times. However, at high concentrations, phenazine is toxic to Candida albicans.¹⁷

In this investigation, it was discovered that Pseudomonas aeruginosa’s OD differed from that of the bacteria when exposed to Candida glabrata, with the latter exhibiting an increase in OD that suggested a synergistic interaction. One of the typical flora commonly found in the gastrointestinal tract, vagina, and oral cavity is Candida glabrata, an organism that is widely distributed. But it can also be harmful and lead to severe infections, which are more likely to happen in patients with compromised immune systems.¹⁸ One of Candida glabrata’s worrisome virulence factors is its capacity to form biofilms.¹⁹ These days, the relationship between Pseudomonas aeruginosa and Candida glabrata in biofilm formation is still largely unexplored in polymicrobial research. Only two published studies have indicated that Pseudomonas aeruginosa and Candida glabrata have an antagonistic interaction. These studies also demonstrate a decrease in the OD of the mixture of Pseudomonas aeruginosa and Candida glabrata when compared to the OD of Pseudomonas aeruginosa alone.^9,20

Phenazines are among the molecules produced by Pseudomonas that induce ethanol from Candida. Candida glabrata also produces ethanol, and this ethanol can help Pseudomonas through the psl operon facilitate its extracellular matrix as a positive feedback to adapt to the microenvironment and increase the formation of biofilm.¹⁷ As a result, when Pseudomonas aeruginosa is exposed with Candida glabrata, the amount of OD increases. It has been demonstrated by a study that, in contrast to the other three species, C. glabrata can form biofilms in low glucose concentrations and under unfavorable nutritional conditions. This is most likely because C. glabrata can acclimate to these circumstances and selectively colonize certain human tissues.²¹

The results of this study showed that Pseudomonas aeruginosa and those exposed to Candida krusei differed in their OD values, with the latter showing an increase in OD that suggested a synergistic interaction. One of Candida krusei’s virulence factors is its capacity to form biofilms.²² Pseudomonas aeruginosa can stimulate Candida krusei’s ethanol production, which thickens Pseudomonas aeruginosa’s extracellular matrix and raises OD. This mechanism is similar to that of Candida albicans and Candida glabrata.¹⁷

Interactions between various species alter the host response, the effectiveness of antibiotics, the pathogenesis and virulence of the bacteria, and generally make infections worse and make them more resistant to traditional treatment.²³ Polymicrobial research on the role of Candida krusei and Pseudomonas aeruginosa in biofilm formation is still lacking at this time. Only one published study, which demonstrated a drop in the optical density (OD) of the mixture of Pseudomonas aeruginosa and Candida krusei relative to the OD of single Pseudomonas aeruginosa, suggested that there was an antagonistic interaction between the two species.⁸

Similar to other Candida, Pseudomonas aeruginosa can stimulate ethanol production in Candida parapsilosis which causes thickening of the extracellular matrix in Pseudomonas aeruginosa¹⁵ so that it causes an increase in OD. In this study, it was found that there was a difference in OD between Pseudomonas aeruginosa and the OD of Pseudomonas aeruginosa exposed to Candida parapsilosis, in which there was an increase in OD indicating a synergistic interaction.

Candida parapsilosis forms thinner, less complex biofilms than Candida albicans. On implanted plastic medical devices, however, C. parapsilosis biofilms continue to be a major source of infection.²⁴ Polymicrobial research on the role of Candida parapsilosis and Pseudomonas aeruginosa in biofilm formation is still lacking. The antagonistic interaction between Pseudomonas aeruginosa and Candida parapsilosis, indicated by a decrease in the optical density (OD) of the mixture of Pseudomonas aeruginosa and Candida parapsilosis relative to the OD of single Pseudomonas aeruginosa, has only been reported in two published studies.^9,20

The results of this investigation showed that Pseudomonas aeruginosa and those exposed to Candida tropicalis differed in terms of their OD, with the former showing an increase in OD that suggested a synergistic interaction. Like other Candida, Pseudomonas aeruginosa can induce Candida tropicalis to produce ethanol, which thickens Pseudomonas aeruginosa’s extracellular matrix and raises the organism’s optical density (OD).¹⁷ In most studies, Candida tropicalis outperforms Candida albicans in its ability to produce biofilm.²⁵

In a previous investigation of the antagonistic relationship between Candida tropicalis and Pseudomonas aeruginosa, Wahyuning et al.²⁶ discovered that Pseudomonas aeruginosa inhibited the formation of biofilms in Candida tropicalis. Pseudomonas aeruginosa and Candida tropicalis biofilm former isolates were also used in this investigation. Because the strain used in this study is capable of killing C. tropicalis hyphae and biofilms, which are caused by phenazine compounds that compromise the integrity of cell walls, antagonistic relationships arise in P. aeruginosa mixed species. This work, however, is in line with earlier in vitro biofilm studies that demonstrated P. aeruginosa can inhibit the growth of Candida biofilms that are not albicans, such as C. tropicalis.

This is due to the fact that Gram negative bacteria contain N-acyl homoserine lactone (AHL) and that these two microbes have the ability to release quorum sensing molecules. In the meantime, AHL in P. aeruginosa can be decreased in vitro to prevent Candida spp. biofilms.²⁵ Comparable to studies by Fourie et al. and Bandara et al. that reported an antagonistic interaction between Pseudomonas aeruginosa and Candida tropicalis and demonstrated a drop in the optical density (OD) of a mixture of the two species relative to the OD of Pseudomonas aeruginosa alone.^9,20 Pseudomonas aeruginosa and Candida spp. both form biofilms, according to a study by Bandara. Each isolate used in the study was the ATCC strain, which can result in various interactions between Pseudomonas aeruginosa and Candida spp.²⁰