Christian Michel

Complete genome sequence of the fish pathogen Flavobacterium psychrophilum

We report here the complete genome sequence of the virulent strain JIP02/86 (ATCC 49511) of Flavobacterium psychrophilum, a widely distributed pathogen of wild and cultured salmonid fish. The genome consists of a 2,861,988–base pair (bp) circular chromosome with 2,432 predicted protein-coding genes. Among these predicted proteins, stress response mediators, gliding motility proteins, adhesins and many putative secreted proteases are probably involved in colonization, invasion and destruction of the host tissues. The genome sequence provides the basis for explaining the relationships of the pathogen to the host and opens new perspectives for the development of more efficient disease control strategies. It also allows for a better understanding of the physiology and evolution of a significant representative of the family Flavobacteriaceae, whose members are associated with an interesting diversity of lifestyles and habitats.

Production of viable cultures of Flavobacterium psychrophilum: approach and control

Although the fish pathogen Flavobacterium psychrophilum is a major source of concern in salmonid hatcheries, few studies have been conducted on its pathogenicity. Difficulties are often experienced when trying to control or quantify standard procedures for in vitro culture of the bacterium. Plate enumeration and counting chamber enumeration combined with epifluorescent microscopy with fluorescent dyes determined that no more than 25% of the bacterial cells present in the cultures were able to produce colonies on agar media. This was strongly dependent upon different medium components. Tryptone-enriched Anacker and Ordal medium proved more suitable than tryptone-yeast extract-salts with skimmed milk. Adding horse serum and trace elements in controlled proportions offered the most reproducible results. Viable but nonculturable forms were apparently not responsible for the difficulties in production of F. psychrophilum, but the cells were highly susceptible to osmotic conditions. Improvements in the media and careful handling of the bacteria in isotonic suspension media resulted in predictable production of viable bacteria and allowed an absorbance/colony-forming-units relation curve to be established.

Diversity of Lactic Acid Bacteria Associated with Fish and the Fish Farm Environment, Established by Amplified rRNA Gene Restriction Analysis

ABSTRACT Lactic acid bacteria have become a major source of concern for aquaculture in recent decades. In addition to true pathogenic species of worldwide significance, such as Streptococcus iniae and Lactococcus garvieae, several species have been reported to produce occasional fish mortalities in limited geographic areas, and many unidentifiable or ill-defined isolates are regularly isolated from fish or fish products. To clarify the nature and prevalence of different fish-associated bacteria belonging to the lactic acid bacterium group, a collection of 57 isolates of different origins was studied and compared with a set of 22 type strains, using amplified rRNA gene restriction analysis (ARDRA). Twelve distinct clusters were delineated on the basis of ARDRA profiles and were confirmed by sequencing of sodA and 16S rRNA genes. These clusters included the following: Lactococcus raffinolactis, L. garvieae, Lactococcus l., S. iniae, S. dysgalactiae, S. parauberis, S. agalactiae, Carnobacterium spp., the Enterococcus “faecium” group, a heterogeneous Enterococcus-like cluster comprising indiscernible representatives of Vagococcus fluvialis or the recently recognized V. carniphilus, V. salmoninarum, and Aerococcus spp. Interestingly, the L. lactis and L. raffinolactis clusters appeared to include many commensals of fish, so opportunistic infections caused by these species cannot be disregarded. The significance for fish populations and fish food processing of three or four genetic clusters of uncertain or complex definition, namely, Aerococcus and Enterococcus clusters, should be established more accurately.

Transcriptional Responses of Resistant and Susceptible Fish Clones to the Bacterial Pathogen Flavobacterium psychrophilum

Flavobacterium psychrophilum is a bacterial species that represents one of the most important pathogens for aquaculture worldwide, especially for salmonids. To gain insights into the genetic basis of the natural resistance to F. psychrophilum, we selected homozygous clones of rainbow trout with contrasted susceptibility to the infection. We compared the transcriptional response to the bacteria in the pronephros of a susceptible and a resistant line by micro-array analysis five days after infection. While the basal transcriptome of healthy fish was significantly different in the resistant and susceptible lines, the transcriptome modifications induced by the bacteria involved essentially the same genes and pathways. The response to F. psychrophilum involved antimicrobial peptides, complement, and a number of enzymes and chemokines. The matrix metalloproteases mmp9 and mmp13 were among the most highly induced genes in both genetic backgrounds. Key genes of both pro- and anti-inflammatory response such as IL1 and IL10, were up-regulated with a greater magnitude in susceptible animals where the bacterial load was also much higher. While higher resistance to F. psychrophilum does not seem to be based on extensive differences in the orientation of the immune response, several genes including complement C3 showed stronger induction in the resistant fish. They may be important for the variation of susceptibility to the infection.

Genetic diversity and population structure of Mycobacterium marinum: New insights into host and environmental specificities

ABSTRACT Mycobacterium marinum causes a systemic tuberculosis-like disease in fish and skin infections in humans that can spread to deeper structures, resulting in tenosynovitis, arthritis, and osteomyelitis. However, little information is available concerning (i) the intraspecific genetic diversity of M. marinum isolated from humans and animals; (ii) M. marinum genotype circulation in the different ecosystems, and (iii) the link between M. marinum genetic diversity and hosts (humans and fish). Here, we conducted a genetic study on 89 M. marinum isolates from humans (n = 68) and fish (n = 21) by using mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing. The results show that the M. marinum population is genetically structured not only according to the host but also according to the ecosystem as well as to tissue tropism in humans. This suggests the existence of different genetic pools in the function of the biological and ecological compartments. Moreover, the presence of only certain M. marinum genotypes in humans suggests a different zoonotic potential of the M. marinum genotypes. Considering that the infection is linked to aquarium activity, a significant genetic difference was also detected when the human tissue tropism of M. marinum was taken into consideration, with a higher genetic polymorphism in strains isolated from patients with cutaneous forms than from individuals with deeper-structure infection. It appears that only few genotypes can produce deeper infections in humans, suggesting that the immune system might play a filtering role.

Minimal inhibitory concentration methodology in aquaculture: the temperature effect

Although antimicrobial drugs testing and minimal inhibitory concentration (MIC) assessment have become of common use for guiding bacterial disease treatments, it soon appeared that temperature conditions under which tests were performed could result in strong variations and cause misinterpretations. This is specially inconvenient with fish pathogenic bacteria, many of which require low temperature incubation and do not fit easily with the standardized procedures generally accepted in medical laboratories. Temperature may affect both antimicrobial drugs and bacterial physiology through a variety of mechanisms such as stability and diffusion kinetics of the drug species, growth rate, membrane properties, enzymatic activity and genetic regulations of the bacterial cells. Interference and combination of so many effects make any prediction unrealistic. In MIC studies, however, dilution methods appear much less susceptible than diffusion methods to temperature-induced fluctuations. Provided interpretation is done carefully they should always be preferred to perform routinely antimicrobial tests.