Coexistence of Pseudomonas aeruginosa With Candida albicans Enhances Biofilm Thickness Through Alginate-Related Extracellular Matrix but Is Attenuated by N-acetyl-l-cysteine

Abstract

Bacteria and Candidaalbicans are prominent gut microbiota, and the translocation of these organisms into blood circulation might induce mixed-organism biofilms, which warrants the exploration of mixed- versus single-organism biofilms in vitro and in vivo. In single-organism biofilms, Acinetobacter baumannii and Pseudomonas aeruginosa (PA) produced the least and the most prominent biofilms, respectively. C. albicans with P. aeruginosa (PA+CA) induced the highest biofilms among mixed-organism groups as determined by crystal violet straining. The sessile form of PA+CA induced higher macrophage responses than sessile PA, which supports enhanced immune activation toward mixed-organism biofilms. In addition, Candida incubated in pre-formed Pseudomonas biofilms (PA>CA) produced even higher biofilms than PA+CA (simultaneous incubation of both organisms) as determined by fluorescent staining on biofilm matrix (AF647 color). Despite the initially lower bacteria during preparation, bacterial burdens by culture in mixed-organism biofilms (PA+CA and PA>CA) were not different from biofilms of PA alone, supporting Candida-enhanced Pseudomonas growth. Moreover, proteomic analysis in PA>CA biofilms demonstrated high AlgU and mucA with low mucB when compared with PA alone or PA+CA, implying an alginate-related mucoid phenotype in PA>CA biofilms. Furthermore, mice with PA>CA biofilms demonstrated higher bacteremia with more severe sepsis compared with mice with PA+CA biofilms. This is possibly due to the different structures. Interestingly, l-cysteine, a biofilm matrix inhibitor, attenuated mixed-organism biofilms both in vitro and in mice. In conclusion, Candida enhanced Pseudomonas alginate–related biofilm production, and Candida presentation in pre-formed Pseudomonas biofilms might alter biofilm structures that affect clinical manifestations but was attenuated by l-cysteine.

Biofilm-Forming Potential of Ocular Fluid Staphylococcus aureus and Staphylococcus epidermidis on Ex Vivo Human Corneas from Attachment to Dispersal Phase

“…The CV method is a quantitative method which monitors biofilm mass. CV is a positively charged molecule which binds to negatively charged bacteria and polysaccharides of EPS which could be quantitated [ 67 , 68 , 69 ]. In this method an overnight culture was diluted 10,000 times ( v / v ) and then 100 µL of this suspension was added to a 96 well polystyrene plate (Nunclon™, Thermo scientific, Roskilde, Denmark) containing 100 μL of BHI medium.…”

“…Excess crystal violet was discarded and each well was washed twice with 200 µL of PBS and dried at RT. CV associated with the bacteria was extracted with 200 μL of absolute ethanol and quantified using a Spectrophotometer [SpectraMax M3, with a cuvette adaptor (Molecular Devices, San Jose, CA, USA)] set at 595 nm [ 67 , 68 , 69 ]. Wells without cells served as the control (OD was < 0.1 at 595 nm) and the OD value was deducted from the biofilm positive (OD > 0.3 at 595 nm) and biofilm negative strains (OD < 0.3 at 595 nm) [ 62 , 63 ].…”

Polymicrobial Interactions in the Cystic Fibrosis Airway Microbiome Impact the Antimicrobial Susceptibility of Pseudomonas aeruginosa

“…C. albicans enhanced P. aeruginosa ECM production, consisting of proteins, polysaccharides, and nucleic acids, through increased expression of the alginate genes AlgU and mucA [171]. Alam et al (2019) showed C. albicans can enhance meropenem tolerance of P. aeruginosa in a dual-species biofilm [172].…”

Microbial Interkingdom Biofilms and the Quest for Novel Therapeutic Strategies

“…In P. aeruginosa-C. albicans mixed biofilms, C. albicans enhances P. aeruginosa EPS production by increasing alginate-producing genes, AlgU and mucA. Thus, the synergy between these two microorganisms leads to thicker biofilms with an easier bacterial dissemination [53]. However, this effect seems to be attenuated in the presence of N-acetyl-L-cysteine both in vitro and in an in vivo catheter-subcutaneous implantation model.…”

“…This thiol-containing cysteine derivative is commonly known to disrupt disulfide bonds and cysteine utilization, exhibiting antimicrobial activity [ 54 ]. Hence, N -acetyl- l -cysteine may be a promising agent to prevent biofilm formation and to attenuate catheter-related sepsis due to its ability to inhibit matrix production on P. aeruginosa – C. albicans biofilms [ 53 ].…”