Phenotypic and Genetic Predictors of Pathogenicity and Virulence in Flavobacterium psychrophilum

Abstract

Flavobacterium psychrophilum causes bacterial cold-water disease (BCWD) in farmed rainbow trout ( Oncorhynchus mykiss ), with the multilocus sequence typing (MLST) clonal complex (CC) CC-ST10 accounting for the majority of outbreaks globally. The development of alternative strategies to antibiotic treatment of BCWD using bacteriophage-based control of F. psychrophilum , or virulence factors as targets for therapy, requires knowledge of the phage-sensitivity of outbreak strains and of universal traits contributing to their pathogenicity. To examine the association between virulence and both genetic (MLST sequence type (ST) and PCR-serotype) and phenotypic characteristics (adherence, antibiotic resistance, colony spreading motility, hemolytic and proteolytic activity), the median lethal dose (LD 50 ) of 26 geographically disparate F. psychrophilum isolates was determined in rainbow trout. Furthermore, the in vitro sensitivity of the isolates against five bacteriophages was determined by the efficiency of plating (EOP). The tested F. psychrophilum isolates were mainly represented by CC-ST10 genotypes (22 out of 26) and showed up to 3-log differences in LD 50 (8.9 × 10 3 to 3.1 × 10 6 CFU). No association between MLST ST and virulence was found because of a high variation in LD 50 within STs. All identified serotypes (0, 1, and 2) were pathogenic, but ten most virulent isolates belonged to serotype 1 or 2. Isolates of high (LD 50 < 10 5 CFU), moderate (LD 50 = 10 5 –10 6 CFU), and weak (LD 50 > 10 6 CFU) virulence were similar in phenotypic characteristics in vitro . However, the only non-virulent CC-ST10 isolate was deficient in spreading motility and proteolytic activity, indicating that the characteristics are required for pathogenicity in F. psychrophilum . Univariate correlation studies found only non-significant associations between LD 50 and the measured phenotypic characteristics, and the multivariable analysis did neither reveal any significant predictors of virulence. The majority of isolates (16 out of 26) were sensitive to at least four bacteriophages, with up to a 6-log variation in the EOP. Most CC-ST10 isolates (16 out of 22) were sensitive to the examined phages, including 5 out of the 7 most virulent isolates represented by prevalent and antibiotic-resistant STs. Our findings suggest that control of BCWD using lytic phages or interventions targeting shared characteristics of pathogenic F. psychrophilum strains should be further explored.

Bacteriophages as Biocontrol Agents for Flavobacterium psychrophilum Biofilms and Rainbow Trout Infections

“…The therapeutic potential of phages FpV4 and FPSV-D22 was further emphasized by the fact that their lytic potential against isolate 950106-1/1 were relatively low (>1000-fold less efficient), compared with their infectivity against the FPS-S6 and 160401-1/5N isolates used in the biofilm experiments. 8 Previous studies have indicated that an initial PHR of at least 10 is required for rapid and effective control of F. psychrophilum infections in fish. 7 However, in liquid cultures, such high PHRs select for phage resistance in the bacterial host population, whereas phage-sensitive clones dominate (>99.8%) the regrowing population at lower (≤0.5) PHRs.…”

“…Also, several virulent F. psychrophilum strains resistant to several broad-range lytic phages, such as FPS-R9 used in this study, have been isolated from BCWD outbreaks. 8 For control of F. psychrophilum in fish farms, the development of phage resistance is maybe an easier issue to overcome since phages are evolving with their hosts in the environment and thus new ones can be isolated, whereas phage-resistant F. psychrophilum clones show significant decrease in fitness and pathogenicity. 24 …”

“…11 In our previous study, three of the isolates (FPS-S6, 160401-1/5N, and 950106-1/1) were susceptible to both FPSV-D22 and FpV4 phages in vitro , whereas one of the isolates (FPS-R9) was found to be intrinsically resistant to the phages in question and was included in the biofilm testing as a negative control of the methodology. 8 …”

Phage-Mediated Control of Flavobacterium psychrophilum in Aquaculture: In vivo Experiments to Compare Delivery Methods

“…Flavobacterium psychrophilum 950106-1/1, a well-characterized Danish strain isolated in 1995 from rainbow trout in a freshwater farm, was selected for the experiments (serotype Fd, virulent) ( Madsen and Dalsgaard, 1999 , 2000 ; Dalsgaard and Madsen, 2000 ; Sundell et al, 2019 ). Flavobacterium psychrophilum FPS-S6, a Swedish strain isolated in 2017 from rainbow trout (serotype Th, virulent), was utilized for the propagation of phage FPSV-D22 since it was the most efficient host for producing high phage titers ( Sundell et al, 2019 ). The bacteria were stored at −80°C in tryptone yeast extract salts medium [TYES: 0.4% tryptone, 0.04% yeast extract, 0.05% CaCl 2 × 2H 2 O, 0.05% MgSO 4 × 7H 2 O (pH 7.2)] ( Holt et al, 1993 ) and glycerol (15–20%).…”

“…Bacteriophages FpV4 and FPSV-D22 were used in these experiments ( Supplementary Table 1 ). FpV4 (lytic phage belonging to Podoviridae family, 90 kb genome) was isolated in 2005 from water with feces samples ( Stenholm et al, 2008 ; Castillo and Middelboe, 2016 ) and FPSV-D22 (lytic phage belonging to Siphoviridae family), isolated in 2017 from fish tissue samples collected at Danish freshwater farms of rainbow trout ( Sundell et al, 2019 ). Both phages were characterized to have a broad host range among F. psychrophilum strains [( Stenholm et al, 2008 ; Castillo et al, 2014 ) and unpublished data].…”