Interactions of microorganisms within a urinary catheter polymicrobial biofilm model

Abstract

Biofilms are often polymicrobial in nature, which can impact their behavior and overall structure, often resulting in an increase in biomass and enhanced antimicrobial resistance. Using plate counts and locked nucleic acid/2′‐O‐methyl‐RNA fluorescence in situ hybridization (LNA/2′OMe‐FISH), we studied the interactions of four species commonly associated with catheter‐associated urinary tract infections (CAUTI): Enterococcus faecalis, Escherichia coli, Candida albicans, and Proteus mirabilis. Eleven combinations of biofilms were grown on silicone coupons placed in 24‐well plates for 24 h, 37°C, in artificial urine medium (AUM). Results showed that P. mirabilis was the dominant species and was able to inhibit both E. coli and C. albicans growth. In the absence of P. mirabilis, an antagonistic relationship between E. coli and C. albicans was observed, with the former being dominant. E. faecalis growth was not affected in any combination, showing a more mutualistic relationship with the other species. Imaging results correlated with the plate count data and provided visual verification of species undetected using the viable plate count. Moreover, the three bacterial species showed overall good repeatability SD (Sr) values (0.1–0.54) in all combinations tested, whereas C. albicans had higher repeatability Sr values (0.36–1.18). The study showed the complexity of early‐stage interactions in polymicrobial biofilms. These interactions could serve as a starting point when considering targets for preventing or treating CAUTI biofilms containing these species.

Horizontal Gene Transfer of Antibiotic Resistance Genes in Biofilms

“…As mixed-species biofilms and polymicrobial infections are much more common in nature [37,146,147], biofilm studies with multiple species should be performed. Clinical isolates from medical devices have shown that biofilm infections are not only due to single species but also to polymicrobial colonization including even copresence of fungi and bacteria [39].…”

Imaging biofilms using fluorescence in situ hybridization: seeing is believing

“…There are three main patterns of microbial spatial organization on biofilms that can be observed when FISH is applied ( Figure 1 -II): a) single cell microcolonies, where each species is in separated microcolonies, showing a non-commensal or neutral interactions ( Nielsen et al., 2000 ); b) co-aggregation, where the different species are all mixed and can be found together throughout the biofilm with a cooperative behavior ( Azevedo et al., 2016 ; Allkja et al., 2022 ); and c) the layered organization, where one species can be found in the lower layer of biofilm and the other in the upper layer, which might be related with both cooperative or competitive relations ( Habimana et al., 2010 ; Almeida et al., 2011 ).…”

“…Concernig the LNA and 2’OMe probes, there are just a few studies regarding their application to biofilms ( Azevedo et al., 2015 ; Azevedo et al., 2016 ; Allkja et al., 2022 ), and even these are limited for the in situ detection and localization of biofilm populations to assess the spatial organization. Despite of negative charge of LNA and 2’OMe probes, all these studies have shown a complete staining of the biofilm sample and a strong fluorescence signal, even in thicker biofilms ( Azevedo et al., 2015 ); it might be possible that the other characteristics (e.g., higher water solubility, more efficient hybridization) of LNA and 2’OMe ( Braasch and Corey, 2001 ; Elayadi et al., 2002 ; Robertson and Thach, 2009 ) are also determinant, assuring an efficient spatial characterization of biofilm populations.…”

“…For example, Azevedo et al., using LNA/2’OMe-FISH combined with CLSM, observed that E. coli and atypical bacteria ( Delftia tsuruhatensis and Achromobacter xylosoxidans ) are well mixed and aggregated to each other, resulting in a cooperative bahaviour after antibiotic treatment ( Figure 1 -II) ( Azevedo et al., 2016 ). Still, a recent study from Allkja et al., applying LNA/2’OMe-FISH, observed that Enterococcus faecalis , E. coli , Candida albicans , and Proteus mirabilis (species commonly associated with catheter-associated urinary tract infections), formed a co‐aggregated structures typical of a cooperative relationship ( Allkja et al., 2022 ). Yet, another application of LNA-FISH, was demonstrated by Vilas Boas et al., for the in situ analysis of bacteriophage-bacteria interactions in biofilm structures; these authors have easly detected and discriminated phage-infected and noninfected cells in biofilms formed by Pseudomonas aeruginosa and Acinetobacter baumannii , applying LNA probes to target a conserved gene highly expressed during multiplication of bacteriophage inside the bacterial cells ( Vilas Boas et al., 2016 ).…”