2. Materials and Methods
Anonymized antimicrobial susceptibility data for bacterial isolates from farmed finfish species were provided by the Animal Health Centre (AHC), BC Ministry of Agriculture and Food. The data included antimicrobial susceptibilities for bacterial isolates from finfish for FLOR, OXY, SXT, and triple sulphonamide combination (TRI: sulphamerazine, sulphathiazole, and sulfadiazine) for the years 2007 to 2018, as well as sulfa-ormetoprim for 2007 to 2009. This study represents a descriptive analysis as the sample size did not allow for formal trend analysis.
Samples for bacterial culture were plated on blood agar and TSA agar (supplemented with NaCl) and incubated at 15 °C for up to 48 h. Any colonies that grew were initially identified by colony morphology and basic biochemical testing, including Gram staining, Oxidase, Indole, and O129 tests. PCR and Biolog Microstation System were used for species-level identification when necessary. Antimicrobial susceptibility testing (AST) and quality control were performed using the Kirby–Bauer disc diffusion method following Clinical and Laboratory Standards Institute (CLSI) guidelines with results interpreted as sensitive or resistant based on the guidelines that were current when testing was performed [
18]. For
A. salmonicida, results were interpreted as sensitive or resistant based on clinical breakpoints for zone diameters correlated to minimum inhibitory concentrations (MICs) [
18]. Other organisms lacked CLSI interpretive criteria; categorizations were extrapolated from breakpoints established for
A. salmonicida or from values reported in the published literature or drug manufacturers. Specific details regarding these interpretations (e.g., breakpoint zone diameters) were not available due to the historical nature of the dataset.
The isolates came from different finfish species, including Atlantic salmon, Pacific salmon (including Chinook, Coho, and Pink salmon), Rainbow trout (including freshwater and marine “Steelhead” trout), Sablefish, Tilapia, and White sturgeon. Rainbow trout were kept as a separate category because it was not possible to determine if they were from marine (often called Steelhead trout) or freshwater sources.
A large variety of organisms were isolated and tested for antimicrobial susceptibility from salmon submissions to the AHC. Susceptibility for isolates of the genera
Aeromonas,
Vibrio,
Aliivibrio,
Yersinia,
Photobacterium,
Pseudoalteromonas,
Pseudomonas,
Psychrobacter, and
Serratia spp. was reported to provide a broad overview of antimicrobial susceptibility in commonly isolated finfish bacteria. Of these genera, the following species were reported separately:
A. salmonicida,
Aeromonas sobria,
Aliivibrio wodanis,
V. anguillarum,
Vibrio ordalii,
Vibrio splendidus,
Vibrio tapetis, and
Y. ruckeri. The BC AHC also highlighted
Tenacibaculum maritimum as a species of interest. Descriptive statistics were prepared in Excel
® (Microsoft Corporation, Redmond, WA, USA). A visual assessment was performed to compare isolate numbers and AST from 2007 to 2014 and 2015 to 2018 to look for any differences in results when submissions were voluntary compared to the mandatory requirement from the Department of Fisheries and Oceans Canada that came into effect starting in 2015 [
19]. The same assessment considered the addition of MALDI-TOF to the AHC in 2018 for bacterial species identification compared to traditional biochemical identification prior to that year.
4. Discussion
Our study describes the antimicrobial susceptibilities of a historical collection of bacterial isolates from farmed salmonids in BC, Canada, that were submitted to the provincial diagnostic laboratory. Overall, the prevalence of AMR to Canadian-approved antimicrobials for bacterial species of concern was low, with only A. salmonicida having any resistance to tested antimicrobials that were more than a single isolate over the study period. Among the bacterial species of interest, isolates were dominated by A. salmonicida, particularly from Atlantic salmon, followed by A. wodanis, A. sobria, Y. ruckeri, and Vibrio species. These susceptibility results will support future Canadian AMR surveillance in farmed aquaculture by providing baseline data. Future estimates of the prevalence of AMR can be compared carefully to these, with the caveat that the sampling design may be different than these results of isolates from diagnostic submissions. These data represent prevalence among isolates from submitted cases, which may not be directly comparable to systematic sampling from surveillance. They provide value in that no other data exist for BC for the time period reported. For A. salmonicida, the only species with enough isolates to warrant an annual AMR, the apparent peaks of resistance to all four antimicrobials in 2010, 2012, and 2013 should not be over-interpreted, given the low numbers of isolates in other years around these.
Although little work on AMR and aquaculture has been undertaken in Canada, three additional studies were identified. A recent AMR surveillance study of isolates from farmed salmonids in Canada’s Atlantic region found some similar and different patterns of isolates and AMR [
13]. The study investigated 26 genera of bacteria from 2291 samples from farmed salmonids over a similar time-period (2000–2021). Over 90% of the samples in the study were gathered from Atlantic salmon. Of the 2291 samples, 515 resulted in species-level identification, of which 336 were tested for AMR. The authors stratified
A. salmonicida isolates into furunculosis-causing (typical) and non-furunculosis-causing (atypical) subspecies. In terms of cases,
Y. ruckeri was the most common, followed by
A. salmonicida (when grouping typical and atypical strains together), and
R. salmoninarum. Comparatively, when considering isolates tested for susceptibility, the three most common pathogens were
Y. ruckeri,
A. salmonicida (grouped), and
Pseudomonas fluorescens. This partly coincides with the results of our study, where the top three pathogens by isolate were
A. salmonicida,
A. wodanis, and
Y. ruckeri. Our study was limited to historical isolates from the BC AHC and the methods they used for bacterial isolation at the time. Laboratory methodology at the BC AHC could not detect
R. salmoninarum as their protocol relied on PCR for detection. It is not known how methodology may have impacted the ability to detect
T. maritimum, which typically requires specialized culture medium to increase the ability for isolation.
The Atlantic bacterial isolates were tested for susceptibility to FLOR, OXY, sulfa-ormetoprim, a potentiated sulfonamide related to SXT—trimethoprim-sulfamethoxazole, and enrofloxacin [
13]. When considering isolates from all fish species, results varied for some isolates and were similar for others. In the Atlantic
A. salmonicida isolates, resistance to FLOR was 11.7% (
n = 13/111) (atypical) and 27.6% (
n = 8/29) (typical), roughly similar to 18% in our
A. salmonicida isolates. Resistance to OXY in
A. salmonicida in their study (95.5% (
n = 106/111) (atypical) and 58.6% (
n = 17/29) (typical) was higher than what was detected (22%) in our study. Resistance to SXT was 12.6% (
n = 14/111) (atypical) and 24.1% (
n = 7/29) (typical) in
A. salmonicida, compared to 23% in our study. Resistance to FLOR, OXY, and SXT was not detected or negligible in
Y. ruckeri isolates from both studies.
In addition to this study, an older Canadian study included data on 17 isolates from finfish in Atlantic Canada (Nova Scotia and New Brunswick) from 2002 to 2004, reporting high levels of resistance in
A. salmonicida to FLOR (76%;
n = 13/17) and OXY (94%;
n = 16/17) [
16]. Another older source for finfish from Newfoundland found slightly higher levels of resistance to OXY (37%;
n = 42/113) and sulfa-ormetoprim (34.6%;
n = 36/104) in
A. salmonicida isolates from 1990 to 1995 [
15].
Published studies including AMR data from salmonid isolates in other countries reported almost exclusively on
A. salmonicida, except for one Chilean study that tested
Piscirickettsia salmonis [
20], one Chilean study on a variety of finfish bacterial isolates of a variety of species [
21], two Norwegian studies that included
Moritella viscosa [
22] and
V. anguillarum [
23], and a recent whole genome sequencing (WGS) analysis from the USA, further described below [
24]. In the USA, a six-year historical study on wild Chinook salmon (
n = 806), Coho salmon (
n = 623), Atlantic salmon (
n = 301), and Steelhead trout (
n = 385) in Michigan isolated
A. salmonicida from 234 samples (11%) and found moderate resistance to OXY (22% of isolates), which is the same level of resistance found in our study (22%;
n = 38/174) [
25]. Compared to BC,
A. salmonicida isolates from farmed salmon in China (2012–2016) had higher resistance to FLOR (52%,
n = 31/60) and OXY (40%,
n = 24/60) [
26]. The Chilean study identified 5018 isolates from salmon fecal and intestinal samples that spanned
Aeromonas spp.,
Pseudomonas spp.,
Serratia spp.,
Shewanella spp., and
Psychrobacter spp., among others, of which 47 isolates were resistant to FLOR and 44 isolates were resistant to OXY [
21].
Resistance to FLOR was 0% in Spanish isolates from 2001 to 2004 [
27] and in Scottish isolates in 1993 [
28]. A study from Spain on Atlantic salmon and brown trout (sample sizes for each species not available) found 90% of
A. salmonicida isolates displayed resistance or intermediate susceptibility to OXY, while 0% of isolates displayed resistance to FLOR [
27]. They tested a further nine antimicrobials, including amoxicillin, doxycycline, erythromycin, nalidixic acid, cotrimoxazole, flumequine, chloramphenicol, enrofloxacin, and novobiocin, and found resistance to at least one of these antimicrobials in 56% (
n = 190/341) of isolates.
Our study identified that 4.3% of all bacterial isolates from Atlantic salmon were resistant to all three antimicrobial classes tested. While we only tested a small number of drug classes for susceptibility, this does create some cause for concern about treatment effectiveness, given that these include the only drugs approved for use in finfish in Canada [
11]. Resistance to additional antimicrobials that were not included in the BC panel (e.g., erythromycin, oxolinic acid, or flumequine) is common internationally, but could not be compared to our results in BC due to a lack of inclusion in the testing panel for our isolates [
27,
28,
29]. The Atlantic Canada study identified that 2.5% of all isolates were resistant to all antimicrobials tested, but this is not directly comparable as this included five antimicrobials across four antimicrobial classes: FLOR, OXY, sulfa-ormetoprim, SXT, and enrofloxacin. Similarly, the study of Chilean isolates by Higuera-Llantén et al. (2018) found a high degree of cross-resistance between FLOR, OXY, and other antimicrobials [
21]. A Scottish study investigated multidrug resistance in isolates already resistant to OXY and found that all isolates were resistant to at least one alternative antimicrobial, 63% to three or more antimicrobials, and 95% of OXY-resistant isolates were also resistant to trimethoprim [
28]. The WGS study of 61 isolates—including
Aeromonas spp.,
Flavobacterium spp.,
Edwardsiella spp.,
Yersinia spp.,
Vibrio spp.,
Shewanella spp.,
Photobacterium spp.,
Pseudomonas spp.,
Acinetobacter spp., and
Streptococcus spp. sourced from a variety of saltwater, freshwater, and ornamental fish from across the USA—found five
Edwardsiella isolates with ARGs to FLOR, OXY, and sulfa-ormetoprim located on a single plasmid [
24]. Due to the high degree of potential for horizontal gene transfer in marine settings, the prevalence of multi-drug resistance and rate of ARG dissemination in marine organisms may be high [
30]. Genetic and molecular testing of future BC isolates is an important area of future research to investigate the potential AMR genes responsible for underlying phenotypic resistance, and to look for horizontal genetic elements potentially responsible for co-selection of AMR to these approved and used antimicrobials in finfish.
The US study showed that WGS was well suited for the detection of resistance in the marine environment due to its ability to simultaneously analyze numerous ARGs that often coexist in fish [
24]. The authors also demonstrated that WGS is superior for isolate detection to other analytic methods, such as MALDI-TOF MS, due to better specificity [
24]. Indeed, WGS is becoming increasingly popular as a method of identifying AMR genes in bacterial isolates from aquaculture samples. For example, a genomic analysis of
Tenacibaculum spp. from farmed Atlantic salmon in BC in 2017–2022 by Nowlan et al. [
31] found acquired ARGs for OXY, while in Chile, Suarez et al. (2021) found ARGs in
Mycobacterium spp. from farmed Atlantic and Coho salmon for beta-lactams, tetracyclines, gentamycin, macrolides, and rifampin [
31,
32]. Similarly, Domínguez et al. (2019) found multiple transferable ARGs against sulfonamides and trimethoprim in
Pseudomonas spp. isolates against a backdrop of high multidrug resistance to FLOR, erythromycin, furazolidone, and amoxicillin [
33]. In Turkey, Saticioglu et al. (2021) found ARGs for OXY, FLOR, and sulfamethoxazole-trimethoprim in
Chryseobacterium (a member of the family Flavobacteriaceae) isolates [
34]. These studies reinforce the future need to utilize WGS methods for AMR surveillance in farmed salmonid sampling in BC.
There were differences in the predominant bacterial species between submissions from Atlantic and Pacific salmon in this isolate collection. Atlantic salmon submissions were dominated by
A. salmonicida, compared to
V. anguillarum and
V. ordalii in Pacific salmon submissions. These two
Vibrio spp. can infect both species of salmon [
35]. While
A. salmonicida is a known clinical pathogen for Atlantic salmon, its clinical significance in Pacific salmon is not known [
36]. The reason for these differences in this BC collection is not known and should continue to be monitored. However, in consultation with the BC finfish industry, the relative clinical importance of these organisms, particularly
A. salmonicida in Atlantic salmon, remains secondary to yellowmouth caused by
T. maritimum, which represents the primary reason for antimicrobial use in salmonid production in BC [
37].
T. maritimum was not isolated in this collection. It is possible that culture methods may not have been optimized for its detection, given the specific salinity requirements for its growth in culture media [
38]. While
A. salmonicida represents the primary reservoir for FLOR resistance in this isolate collection, its relative importance as a reservoir remains uncertain compared to
T. maritimum. Until recently, there were no MIC interpretive criteria for FLOR in
T. maritimum. However, a recent study reported the development of epidemiological cutoff values based on data from farmed Atlantic salmon in BC that did not find evidence of reduced FLOR efficacy for the treatment of
T. maritimum in this population [
39].
The apparent low levels of resistance to FLOR in this isolate collection from farmed finfish in BC bode well compared to other salmon-producing regions of the globe. This antimicrobial remains an important tool for finfish aquaculture in BC, given its importance for treating yellowmouth caused by
T. maritimum [
37]. Future monitoring of this organism is important for the BC finfish industry.
This study on historical data regarding resistance in BC finfish aquaculture had some limitations. This dataset is biased towards bacterial isolates from farmed Atlantic salmon, with the sparse data limiting the generalizability to farmed Pacific salmon, Rainbow trout, and other farmed finfish species in BC. However, given that Atlantic salmon makes up the vast bulk of production in BC [
40], these data are useful for current salmonid production. We were reliant on historical antimicrobial susceptibility data from the AHC with no control over bacterial isolation, speciation, and antimicrobial susceptibility testing methods or interpretation. Antimicrobial susceptibility testing was completed exclusively using disc diffusion and subsequent MICs from zone diameters were not available. We also acknowledge that the prevalence estimates provided are based on a dichotomous categorization of isolates as susceptible or resistant. We did not have the raw zone diameter data to assess the potential for impact of isolates near resistance breakpoints, shifts in MIC over time, or the ability to assess isolates with intermediate susceptibility. Past work on
Vibrio spp. from seafood products suggests that there is little ambiguity between susceptibility and resistance for OTC and FLOR [
41], but future work is required. While these things could result in subtle differences in prevalence estimates, they are unlikely to result in dramatic change prevalence and findings based on the low levels of AMR or for most species or to have little impact over time for
A. salmonicida for which there were more isolates. Having MIC data for future surveillance would provide the ability to assess this more closely.
Interpretive criteria for all organisms did not exist and breakpoints for similar organism and drug combinations were applied by the AHC. These caveats are consistent with approaches taken and reported for eastern Canadian AMR surveillance data for finfish submissions to a diagnostic laboratory and allow for this regional comparison [
13]. However, comparisons of AMR prevalence between these and other studies should be conducted with caution and attention to these differences. We also did not have access to isolates for further investigation by WGS. This, and the lack of specific knowledge of intrinsic resistance in the large number of species identified, precluded the ability to clearly understand the impact of the detection of multi-drug resistance. However, given the paucity of historical data for farmed salmonids in BC, these data still represent an important baseline for bacteria from diagnostic submissions for future surveillance. Future systematic surveillance in BC that includes
T. maritimum, standardized MIC-based AST, harmonized interpretive criteria, and routine WGS of key pathogens at the farm-level, linked to farm-level AMU data, will be important to track trends and support antimicrobial stewardship for the industry. Adding in additional antimicrobials, such as erythromycin or oxolinic acid, will allow for comparison to other countries that use these drugs and test for their susceptibility.
Changes in Canadian finfish surveillance policy may have impacted annual levels of AMR in this dataset. The federal Department of Fisheries and Oceans Canada changed their surveillance regulations in 2015 for BC finfish [
19]. Finfish submissions prior to this point were voluntary, while submissions after 2015 were mandated through the new auditing programme [
19]. It is not known how this influenced annual resistance levels. However, given the relatively low numbers of isolates by pathogen species over time, it was not possible to formally assess the relative annual impact on AMR. Farm-specific AMU data directly linked to isolate submissions were also not available, precluding the ability to assess direct selection pressures at the farm level. However, the low levels of resistance to some commonly used antimicrobials, such as FLOR, create interesting questions about any links between its use and AMR. This is an important area for future research.