Inhibition Activity of Compounds and Bacteriophages against Flavobacterium psychrophilum Biofilms In Vitro

Abstract

Flavobacterium psychrophilum produces biofilms under laboratory conditions, and it has been inconclusively suggested that F. psychrophilum biofilms can be a potential reservoir for transmission of the pathogen to a fish population under fish farming conditions. Therefore, there is a need for anti-biofilm compounds. The main aim of this study was to determine the anti-biofilm properties of certain compounds and bacteriophages on F. psychrophilum biofilms under static conditions using a standard 96-well microtiter plate biofilm assay in vitro. Eight compounds (A-type proanthocyanidins, D-leucine, EDTA, emodin, fucoidan, L-alliin, parthenolide, and 2-aminoimidazole) at three sub-minimum inhibitory concentrations (sub-MICs), four bacteriophages (Fpv-3, Fpv-9, Fpv-10, and Fpv-21), and a phage combination (Fpv-9 + Fpv-10) were tested for inhibition of biofilm formation and reduction of the biomass of mature biofilms formed by two smooth isolates (P7-9/10 and P1-10B/10) and two rough isolates (P7-9/2R/10 and P1-10B/2R/10) of F. psychrophilum. The crystal violet staining method was used to stain the biofilms. Most of the compounds at sub-MICs inhibited the biofilm formation of mainly smooth isolates, attaining up to 80% inhibition. Additionally, the same reduction trend was also observed for 2-aminoimidazole, emodin, parthenolide, and D-leucine on the biomass of mature biofilms in a concentration-dependent manner. The anti-biofilm properties of the compounds are believed to lie in their ability to disturb the cellular interactions during biofilm formation and probably to cause cell dispersal in already formed biofilms. Lytic bacteriophages efficiently inhibited biofilm formation of F. psychrophilum, while they partially reduced the biomass of mature biofilms. However, the phage combination (Fpv-9 + Fpv-10) showed a successful reduction in the biomass of F. psychrophilum mature biofilms. We conclude that inhibiting compounds together with bacteriophages may supplement the use of disinfectants against bacterial biofilms (e.g., F. psychrophilum biofilms), leading to a reduced occurrence of bacterial coldwater disease outbreaks at fish farms.Biofilms are defined as clusters of bacterial cells attached to different surfaces and surrounded by a selfproduced matrix of extracellular polymeric substances (EPSs), consisting of polysaccharides, proteins, and environmental DNA, among others (Flemming and Wingender 2010). These cell clusters exist across a range of ecological niches, both natural and man-made. The development of bacterial biofilms seems to be a cyclic path that

Quantification and comparison of gene expression associated with iron regulation and metabolism in a virulent and attenuated strain of Flavobacterium psychrophilum

“…(2019) recently identified a novel elastase that may be involved with infectivity and establishment in host tissues. The ability of biofilm formation and adherence for F. psychrophilum has also been of interest with respect to virulence assessments, transcriptomic changes and targeted treatment options via chemical therapeutants and/or targeted phage treatments (Levipan et al., 2018; Papadopoulou et al., 2019). In F. columnare , the host's ability to acquire iron has also been tied to biofilm formation and manipulations in iron availability in growth media may modulate strain virulence.…”

“…This degree of diversification has placed an emphasis on the importance of identifying new strains via genomic sequencing and conversely the resistance via genomic analyses in host fish species (Fraslin et al, 2018;Söderlund et al, 2018). In addition to strain identification efforts, recent research efforts have also focused on discerning F. psychrophilum F. psychrophilum has also been of interest with respect to virulence assessments, transcriptomic changes and targeted treatment options via chemical therapeutants and/or targeted phage treatments (Levipan et al, 2018;Papadopoulou et al, 2019). In F. columnare, the host's ability to acquire iron has also been tied to biofilm formation and manipulations in iron availability in growth media may modulate strain virulence.…”

Phage‐Host Interactions in Flavobacterium Pathogens and the Potential of Phage‐Based Disease Control in Salmonid Aquaculture

“…Bacterial biofilms are challenging to control using traditional treatment strategies due to their increased tolerance to antimicrobials [227]. In laboratory experiments, F. psychrophilum biofilms were efficiently eradicated by phage exposure at an MOI ratio as low as 0.01 [100,105]. Moreover, when applied to RAS, F. columnare-specific phages preferentially clustered in biofilms attached to biofilter media, indicating that biofilms could serve to maintain phages in the RAS environment [228].…”

“…As particularly F. psychrophilum infections are most severe in the early life stages, vaccine development has been challenged by the undeveloped immune systems of juvenile fish [92,94]. Other disease prevention strategies include good management and high hygienic levels at farms [95], the use of antagonistic bacteria against F. columnare and F. psychrophilum [96,97], selective breeding to increase resistance against BCWD in rainbow trout [98,99] as well as inhibiting compounds against F. psychrophilum biofilms [100].…”

“…In F. psychrophilum , changes in colony morphologies in association with phage exposure have not been reported. Interestingly, while both smooth and rough isolates are associated with virulence to rainbow trout [188], rough isolates appear to show a higher level of resistance to phages [100].…”