The use of a multilocus sequence typing (MLST) technique has identified the intraspecific genetic diversity of U.S. Flavobacterium psychrophilum, an important pathogen of salmonids worldwide. Prior to this analysis, little U.S. F. psychrophilum genetic information was known; this is of importance when considering targeted control strategies, including vaccine development. Herein, MLST was used to investigate the genetic diversity of 96 F. psychrophilum isolates recovered from rainbow trout (Oncorhynchus mykiss), coho salmon (Oncorhynchus kisutch), and Chinook salmon (Oncorhynchus tshawytscha) that originated from nine U.S. states. The isolates fell into 34 distinct sequence types (STs) that clustered in 5 clonal complexes (CCs) (n ‫؍‬ 63) or were singletons (n ‫؍‬ 33). The distribution of STs varied spatially, by host species, and in association with mortality events. Several STs (i.e., ST9, ST10, ST30, and ST78) were found in multiple states, whereas the remaining STs were localized to single states. With the exception of ST256, which was recovered from rainbow trout and Chinook salmon, all STs were found to infect a single host species. Isolates that were collected during bacterial cold water disease outbreaks most frequently belonged to CC-ST10 (e.g., ST10 and ST78). Collectively, the results of this study clearly demonstrate the genetic diversity of F. psychrophilum within the United States and identify STs of clinical significance. Although the majority of STs described herein were novel, some (e.g., ST9, ST10, ST13, ST30, and ST31) were previously recovered on other continents, which demonstrates the transcontinental distribution of F. psychrophilum genotypes. IMPORTANCEFlavobacterium psychrophilum is the causative agent of bacterial cold water disease (BCWD) and rainbow trout fry syndrome (RTFS) and is an important bacterial pathogen of wild and farmed salmonids worldwide. These infections are responsible for large economic losses globally, yet the genetic diversity of this pathogen remains to be fully investigated. Previous studies have identified the genetic diversity of this pathogen in other main aquaculture regions; however, little effort has been focused on the United States. In this context, this study aims to examine the genetic diversity of F. psychrophilum from the United States, as this region remains important in salmonid aquaculture.T he causative agent of bacterial cold water disease (BCWD) and rainbow trout fry syndrome (RTFS), Flavobacterium psychrophilum, is an important bacterial pathogen of wild and farmed salmonids worldwide (1). In addition to horizontal transmission, F. psychrophilum is suspected of being vertically transmitted (2-5) and appears to resist standard povidone-iodine treatment during egg disinfection (4, 6, 7), which make efforts to control this bacterium particularly problematic. Since the initial isolation of F. psychrophilum in North America (8), F. psychrophilum infections have been reported in Europe, South America, Asia, and Australia (9-11) and from all of...

Flavobacterium psychrophilum, the etiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), causes significant economic losses in salmonid aquaculture, particularly in rainbow trout (Oncorhynchus mykiss). Prior studies have used multilocus sequence typing (MLST) to examine genetic heterogeneity withinF. psychrophilum. At present, however, its population structure in North America is incompletely understood, as only 107 isolates have been genotyped. Herein, MLST was used to investigate the genetic diversity of an additional 314 North AmericanF. psychrophilumisolates that were recovered from ten fish host species from 20 U.S. states and 1 Canadian province over nearly four decades. These isolates were placed into 66 sequence types (STs), 47 of which were novel, increasing the number of clonal complexes (CCs) in North America from 7 to 12. Newly identified CCs were diverse in terms of host association, distribution, and association with disease. The largestF. psychrophilumCC identified was CC-ST10, within which 10 novel genotypes were discovered, most of which came fromO. mykissexperiencing BCWD. This discovery, among others, provides evidence for the hypothesis that ST10 (i.e., the founding ST of CC-ST10) originated in North America. Furthermore, ST275 (in CC-ST10) was recovered from wild/feral adult steelhead and marks the first recovery of CC-ST10 from wild/feral fish in North America. Analyses also revealed that at the allele level, the diversification ofF. psychrophilumin North America is driven three times more frequently by recombination than random nucleic acid mutation, possibly indicating how new phenotypes emerge within this species.IMPORTANCEFlavobacterium psychrophilumis the causative agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), both of which cause substantial losses in farmed fish populations worldwide. To better prevent and control BCWD and RTFS outbreaks, we sought to characterize the genetic diversity of several hundredF. psychrophilumisolates that were recovered from diseased fish across North America. Results highlighted multipleF. psychrophilumgenetic strains that appear to play an important role in disease events in North American aquaculture facilities and suggest that the practice of trading fish eggs has led to the continental and transcontinental spread of this bacterium. The knowledge generated herein will be invaluable toward guiding the development of future disease prevention techniques.

Flavobacterium psychrophilum is a serious pathogen of salmonids worldwide, a matter that is compounded by the lack of effective vaccine preparations. As a result, biosecurity measures and antimicrobial agents remain the only available methods to control diseases caused by F. psychrophilum. It is feared that antimicrobial use may have led to the development of F. psychrophilum strains with reduced susceptibility. Therefore, the primary objective of this study was to determine the antimicrobial susceptibility profiles of 50 F. psychrophilum isolates from Michigan in response to 10 antimicrobial compounds. As recommended by the Clinical and Laboratory Standard Institute and the World Organization of Animal Health, a standardized microdilution broth assay was employed to determine the minimum inhibitory concentrations (MICs) for ampicillin (AMP), gentamicin (GEN), enrofloxacin (ENRO), oxolinic acid (OXO), flumequine (FLUQ), trimethoprim-sulphamethoxazole (SXT), ormetoprim-sulphadimethoxine (PRI), erythromycin (ERY), florfenicol (FFN), and oxytetracycline (OXY). Epidemiological cutoff values were calculated using the normalized resistance interpretation (CO) and the ECOFFinder analysis methods (CO). The MIC distributions in response to OXY exhibited bimodality, indicating the presence of isolates with reduced susceptibility in addition to the wild-type isolates. The OXY epidemiological cutoff values (CO <0.06 μg/ml; CO <0.12 μg/ml) demonstrated that 24% of Michigan isolates exhibited reduced susceptibility to this commonly used drug. No other antimicrobial exhibited a bimodal distribution of MICs. This study represents the first antimicrobial susceptibility assessment of F. psychrophilum strains recovered from Michigan and contributes valuable data to the worldwide validation efforts to determine universal epidemiological cutoff values of this deadly fish pathogen.