Extensive Reduction of Cell Viability and Enhanced Matrix Production in Pseudomonas aeruginosa PAO1 Flow Biofilms Treated with a d -Amino Acid Mixture

Abstract

bTreatment of Pseudomonas aeruginosa PAO1 flow biofilms with a D-amino acid mixture caused significant reductions in cell biomass by 75% and cell viability by 71%. No biofilm disassembly occurred, and matrix production increased by 30%, thereby providing a thick protective cover for remaining viable or persister cells.T he multifactorial tolerance of surface-attached complex microbial communities known as biofilms apparently renders conventional treatment strategies ineffective. Biofilms persist and are hard to eradicate because of mechanisms that involve restricted penetration of antimicrobials, differential physiological activity, presence of phenotypic variants and persisters, efflux systems, and enhanced repair systems (1, 2). Unless anti-biofilm strategies are able to block these tolerance mechanisms, biofilms will continue to exert detrimental effects, especially in the hospital and industrial settings.Since D-amino acids are synthesized and released by many bacterial species, including the opportunistic human pathogen Pseudomonas aeruginosa, and have been shown to lack significant toxicity, the idea of using them to combat biofilm-associated infections is highly attractive (3). Recent findings reveal that D-amino acids regulate bacterial cell wall remodeling in stationary phase and cause biofilm dispersal in aging bacterial communities (3, 4). Incorporation of a D-amino acid mixture (D-leucine, D-methionine, D-tryptophan, and D-tyrosine) into the peptidoglycan has also been reported to induce biofilm disassembly in Bacillus subtilis (5). In standing cultures, exogenous D-amino acids prevent biofilm formation in some bacterial strains (5). To date, there are no reports on the susceptibility of flow chamber-grown biofilms to D-amino acids. Flow-chamber biofilm systems not only simulate more physiologically relevant biofilm environments but also allow nondestructive, real-time, high-resolution characterization and quantification of live biofilm structures and development. Hence, in the present study, we investigated whether a Damino acid mixture could affect P. aeruginosa PAO1 biofilms grown under flow conditions using a multichannel microdevice flow system. With the use of confocal laser scanning microscopy (CLSM) and COMSTAT (6), we determined the effect of exogenous D-amino acid mixture on the structure, development, and viability of P. aeruginosa PAO1 flow biofilms.Pseudomonas aeruginosa strain PAO1 with the green fluorescent protein (GFP)-expressing plasmid pMRP9-1 (7) was used to enable detection of cells by CLSM. Cultures for inoculation of the microdevice were prepared in Luria-Bertani (LB) medium supplemented with 300 g ml Ϫ1 carbenicillin for plasmid maintenance as described previously (8) but at 30°C. Biofilms were grown in the microdevice at 30°C supplied with 1/3-diluted LB medium at an 11-ml h Ϫ1 flow rate. The multichannel microdevice flow system was assembled and prepared as described previously (8). In brief, this autoclavable, once-through, continuous-culture system consists of a stainless...

Impact of Environmental Conditions on the Form and Function of Candida albicans Biofilms

“…The uniformly vertical orientation of hyphae in most of the upper region of a/␣ biofilms formed in RPMI medium versus the less uniformly oriented hyphae in a/␣ biofilms formed in Spider medium, and the spaces in calcofluor white-stained biofilms formed in Spider medium that were absent from biofilm formed in RPMI medium, suggested to us that a difference may exist in matrix deposition between the two biofilms. To assess matrix density, we stained fixed preparations of a/␣ biofilms formed in the alternative media with Sypro Ruby, a general protein stain (35), which has previously been used to stain a variety of biofilm matrices (36)(37)(38)(39). This stain was not differentially sequestered at the top of biofilms, as was calcofluor white.…”

“…Sypro Ruby is a general protein dye (35) that has been extensively used for staining the matrix of fixed bacterial biofilms (36)(37)(38)(39). A comparison of matrix staining with calcofluor white, a chitin stain (25), was less effective because of the trapping effect at the top of both types of biofilms during staining.…”

What's on the Outside Matters: The Role of the Extracellular Polymeric Substance of Gram-negative Biofilms in Evading Host Immunity and as a Target for Therapeutic Intervention

“…Others found that specific D-amino acids disrupted Pa biofilms and were particularly effective when combined with antibiotics (86). When used alone, however, treatment of Pa biofilms with a mixture of D-amino acids induced ϳ30% increase in matrix production, thereby suggesting the potential to inadvertently provide protection for any remaining viable or persister cells (86). Recently, Leiman et al (87) indicated that D-tyrosine actually inhibited bacterial growth, and others have not found Damino acids to be effective (88).…”

The effects of d-Tyrosine combined with amikacin on the biofilms of Pseudomonas aeruginosa

“…In contrast to their planktonic counterparts, biofilm-derived bacteria have a distinctive phenotype regarding metabolic activity and gene expression, conferring an inherent resistance to antimicrobial agents as well as mechanisms of host clearance and making the treatment of biofilm-associated infections extremely difficult [2]. Biofilms persist and are hard to eradicate because of mechanisms that involve the restricted penetration of antimicrobials, differential physiological activity, and the presence of phenotypic variants and persisters, efflux systems, and enhanced repair systems [3]. Resistance is often genetically inherited and therefore transmitted progeny of bacterial colonies, or it can be acquired through horizontal gene transfer [4].…”

“…Since D-amino acids are synthesized and released by many bacterial species, including the opportunistic human pathogen P. aeruginosa, and have been shown to lack significant toxicity, the idea of using them to combat biofilm-associated infections is highly attractive [3]. Peptidoglycan (PG) is the major component of the bacterial cell wall and the most commonly cited source of D-amino acids in bacteria.…”