Antimicrobial susceptibility pattern of Flavobacterium columnare isolates collected worldwide from 17 fish species

Flavobacterium columnare is the causative agent of columnaris disease in diverse fish species worldwide. Although columnaris is an important disease, the antimicrobial susceptibility pattern of F. columnare is not well studied. Thus, the purpose of this study was to test the in vitro antimicrobial susceptibility of 97 F. columnare isolates collected worldwide between 1987 and 2011 from 17 fish species. The broth microdilution technique was utilized for reliable testing of these fastidious organisms. None of the isolates displayed acquired resistance to florfenicol, gentamicin, ormetoprim‐sulfadimethoxine and trimethoprim‐sulfamethoxazole. Acquired resistance to chloramphenicol was detected in 1%, to nitrofuran in 5%, to oxytetracycline in 11% and to enrofloxacin, flumequine and oxolinic acid in 10%, 16% and 16% of the isolates, respectively, as reflected by a bimodal or trimodal distribution of their minimum inhibitory concentrations (MICs). One isolate showed acquired resistance towards several antimicrobial agents including erythromycin. Another isolate revealed acquired resistance towards – amongst others – ampicillin. The isolates displaying acquired resistance originated from ornamental fish species or Vietnamese catfish, except for two isolates coming from wild channel catfish in which acquired resistance was encountered towards oxytetracycline only. Fifty per cent of the resistant isolates from ornamental fish were shown to have acquired resistance against three classes of antimicrobial agents, assigning these isolates as multiple resistant. These data might indicate less prudent use of antimicrobials especially in ornamental fish species.     

Antimicrobial susceptibility of Flavobacterium psychrophilum isolates from the United Kingdom

Routine application of antimicrobials is the current treatment of choice for rainbow trout fry syndrome (RTFS) or bacterial coldwater disease (BCWD) caused by Flavobacterium psychrophilum. In this study, the antimicrobial susceptibilities of 133 F. psychrophilum isolates, 118 of which were from the UK, were evaluated by broth microdilution and disc diffusion methods following VET04‐A2 and VET03‐A guidelines of Clinical and Laboratory Standards Institute (CLSI), respectively. Isolates were categorized as wild type (fully susceptible, WT) or non‐wild type (NWT) using normalized resistance interpretation (NRI)‐determined cut‐off values (CO_WT). Broth microdilution testing showed that only 12% of UK isolates were WT to oxolinic acid (MIC CO_WT ≤ 0.25 mg/L) and 42% were WT for oxytetracycline (MIC CO_WT ≤ 0.25 mg/L). In contrast, all the isolates tested were WT (MIC CO_WT ≤ 2 mg/L) for florfenicol, the main antimicrobial for RTFS control in the UK. Disc diffusion‐based CO_WT values were ≥51 mm for 10 μg amoxicillin, ≥44 mm for 30 μg florfenicol, ≥30 mm for 2 μg oxolinic acid and ≥51 mm for 30 μg oxytetracycline. There was a high categorical agreement between the classifications of the isolates by two testing methods for florfenicol (100%), oxytetracycline (93%) and oxolinic acid (99%).     

Epidemiological cut‐off values for Flavobacterium psychrophilum MIC data generated by a standard test protocol

Epidemiological cut‐off values were developed for application to antibiotic susceptibility data for Flavobacterium psychrophilum generated by standard CLSI test protocols. The MIC values for ten antibiotic agents against Flavobacterium psychrophilum were determined in two laboratories. For five antibiotics, the data sets were of sufficient quality and quantity to allow the setting of valid epidemiological cut‐off values. For these agents, the cut‐off values, calculated by the application of the statistically based normalized resistance interpretation method, were ≤16 mg L^?1 for erythromycin, ≤2 mg L^?1 for florfenicol, ≤0.025 mg L^?1 for oxolinic acid (OXO), ≤0.125 mg L^?1 for oxytetracycline and ≤20 (1/19) mg L^?1 for trimethoprim/sulphamethoxazole. For ampicillin and amoxicillin, the majority of putative wild‐type observations were ‘off scale’, and therefore, statistically valid cut‐off values could not be calculated. For ormetoprim/sulphadimethoxine, the data were excessively diverse and a valid cut‐off could not be determined. For flumequine, the putative wild‐type data were extremely skewed, and for enrofloxacin, there was inadequate separation in the MIC values for putative wild‐type and non‐wild‐type strains. It is argued that the adoption of OXO as a class representative for the quinolone group would be a valid method of determining susceptibilities to these agents.     

Comprehensive antibiotic susceptibility profiling of Chilean Piscirickettsia salmonis field isolates

Antibiotics have been extensively used against infections produced by Piscirickettsia salmonis, a fish pathogen and causative agent of piscirickettsiosis and one of the major concerns for the Chilean salmon industry. Therefore, the emergence of resistant phenotypes is to be expected. With the aim of obtaining a landscape of the antimicrobial resistance of P. salmonis in Chile, the susceptibility profiles for quinolones, florfenicol and oxytetracycline (OTC) of 292 field isolates derived from main rearing areas, different hosts and collected over 5 years were assessed. The results allowed for the determination of epidemiological cut‐off values that were used to characterize the pathogen population. This work represents the first large‐scale field study addressing the antimicrobial susceptibility of P. salmonis, providing evidence of the existence of resistant types with a high incidence of resistance to quinolones. Remarkably, despite the amounts and frequency of therapies, our results disclosed that the issue of resistance to florfenicol and OTC is still in the onset.     

Sickeningly Sweet: L‐rhamnose stimulates Flavobacterium columnare biofilm formation and virulence

Flavobacterium columnare, the causative agent of columnaris disease, causes substantial mortality worldwide in numerous freshwater finfish species. Due to its global significance and impact on the aquaculture industry continual efforts to better understand basic mechanisms that contribute to disease are urgently needed. The current work sought to evaluate the effect of L‐rhamnose on the growth characteristics of F. columnare. While we initially did not observe any key changes during the total growth of F. columnare isolates tested when treated with L‐rhamnose, it soon became apparent that the difference lies in the ability of this carbohydrate to facilitate the formation of biofilms. The addition of different concentrations of L‐rhamnose consistently promoted the development of biofilms among different F. columnare isolates; however, it does not appear to be sufficient as a sole carbon source for biofilm growth. Our data also suggest that iron acquisition machinery is required for biofilm development. Finally, the addition of different concentrations of L‐rhamnose to F. columnare prior to a laboratory challenge increased mortality rates in channel catfish (Ictalurus punctatus) as compared to controls. These results provide further evidence that biofilm formation is an integral virulence factor in the initiation of disease in fish.     

Isolation and characterization of Flavobacterium columnare strains infecting fishes inhabiting the Laurentian Great Lakes basin

Flavobacterium columnare, the aetiological agent of columnaris disease, causes significant losses in fish worldwide. In this study, the prevalence of F. columnare infection was assessed in representative Great Lakes fish species. Over 2000 wild, feral and hatchery‐propagated salmonids, percids, centrarchids, esocids and cyprinids were examined for systemic F. columnare infections. Logistic regression analyses showed that the prevalence of F. columnare infection varied temporally and by the sex of the fish, whereby females had significantly higher prevalence of infection. A total of 305 isolates of F. columnare were recovered. Amplification of the near complete 16S rRNA gene from 34 representative isolates and subsequent restriction fragment length polymorphism analyses demonstrated that all belonged to F. columnare genomovar I. Phylogenetic analysis of near complete 16S rRNA gene sequences also placed the isolates in genomovar I, but revealed some intragenomovar heterogeneity. Together, these results suggest that F. columnare genomovar I is widespread in the Great Lakes Basin, where its presence may lead to mortality.     

Evaluation of a 4‐h static copper sulphate treatment against experimental infection of Flavobacterium columnare in channel catfish (Ictalurus punctatus)

The efficacy of copper sulphate (CuSO₄) against the early stages of an experimental acute infection of Flavobacterium columnare in channel catfish (Ictalurus punctatus) was evaluated. Fish were challenged, by waterborne exposure to F. columnare in an ultra‐low flow‐through water delivery system, and treated with CuSO₄ at 4.2 and 2.1 mg L^?1 at 5.5 h post challenge for 4 h. Bacterial challenged non‐treated fish acted as a positive control and non‐challenged non‐treated fish acted as a negative control. Fish challenged with F. columnare and treated with CuSO₄ at 4.2 and 2.1 mg L^?1 post challenge had mortalities of 6.4% and 18.3%, respectively, compared with the positive control, which had 90.2% mortality. The mortality of challenged fish treated with CuSO₄ at 4.2 and 2.1 mg L^?1 was significantly different from the positive control. There was no significant difference between the mortalities of the 4.2 and 2.1 mg L^?1 treatments. The results suggest that CuSO₄ has clear therapeutic value against columnaris infections.     

Protective efficacy of Nigella sativa seeds and oil against columnaris disease in fishes

Columnaris disease, caused by the bacterium Flavobacterium columnare, is currently the most frequently reported bacterial disease affecting farm‐raised channel catfish in the USA. Common treatments against the disease include the use of medicated feed that has led to emergent antibiotic resistant strains of F. columnare. Nigella sativa (Black cumin) is a medicinal herb commonly used by many cultures as a natural remedy for numerous disorders. Recently, we have discovered the antibacterial activity of N. sativa and its oil extract against F. columnare. In this study, we showed N. sativa oil (NSO) strongly inhibited the growth of all of the strains of F. columnare tested and yielded significantly larger zones of inhibition than those produced by oxytetracyclin. We tested the protective effect against columnaris disease in vivo by incorporating NSO (5%) or N. sativa seeds (NSS) (5%) into fish feeds. Fishes (Ictalurus punctatus and Danio rerio) fed amended diets displayed significantly lower mortality than those fed control diets. Per cent mortalities in control groups ranged from 77% to 44% and from 70% to 18% in zebrafish and channel catfish, respectively. A dose study using different NSS concentrations showed that 5% NSS offered the most protection against columnaris disease in channel catfish.     

In vitro comparisons of the inhibitory activity of florfenicol,copper sulphate and potassium permanganate towards Aeromonas hydrophila and Flavobacterium columnare

Aeromonas hydrophila and Flavobacterium columnare, the aetiological agents of motile aeromonas septicaemia (MAS) and columnaris disease respectively, have been recently causing crippling mortalities to the sunshine bass, Morone chrysops female ×Morone saxatilis male (Percichthyidae), industry in the United States. Isolates of A. hydrophila and F. columnare obtained from fish that died during farm outbreaks were subjected to in vitro evaluation of florfenicol (FFC), copper sulphate (CuSO₄) and potassium permanganate (KMnO₄). Florfenicol inhibited the growth of A. hydrophila and F. columnare more than CuSO₄ and KMnO₄. The minimum inhibition concentration (MIC) of FFC was 0.04 ± 0 and 0.2 ± 0.1 mg L^?1 for A. hydrophila and F. columnare respectively, while the 50% inhibition concentration (IC50) for A. hydrophila and F. columnare was 0.23 ± 0.01 and 0.4 ± 0.2 mg L^?1 respectively. Copper sulphate was more effective against A. hydrophila than KMnO₄; CuSO4 had a MIC of 83.2 ± 0 mg L^?1 compared to 158.0 ± 0 mg L^?1 for KMnO₄. Copper sulphate was also more effective against F. columnare than KMnO₄. The IC50 values of CuSO₄ and KMnO₄ towards F. columnare were 4.8 ± 0.3 and 8.7 ± 1.6 mg L^?1 respectively, and the minimum bactericidal concentration values of CuSO₄ and KMnO₄ towards F. columnare were 25.0 ± 0 and > 158.0 mg L^?1 respectively. In addition, F. columnare was more sensitive to CuSO₄ and KMnO₄ than A. hydrophila.     

Identification,annotation of Mucin genes in channel catfish (Ictalurus punctatus) and their expression after bacterial infections revealed by RNA‐Seq analysis

Mucins are large glycoproteins that cover epithelial surfaces of the body and play important roles in prevention of inflammatory and various infectious diseases. In this study, five membrane‐bound and seven secreted mucin genes in the channel catfish were identified. All these identified mucin genes possess at least one PTS, von Willebrand D (VWD) or SEA domains. The expression of the 12 mucin genes in channel catfish was first studied with infection of Edwardsiella ictaluri and Flavobacterium columnare. Expression difference in MUC13a, MUC13, MUC2 and MUC5b was found in the intestine after E. ictaluri infection. Eight mucin gene expressions (except MUC3a, MUC2, MUC4 and MUC5f) were up‐regulated at 4 hr and down‐regulated after 24 hr in the gill with F. columnare infection. Expression level of MUC2 gene was up‐regulated in the intestine with E. ictaluri infection without no significant change in the gill under the F. columnare infection, which indicate that MUC2 is tissue‐specific gene expression and has different immune respond to two bacterial challenge. Taken together, the study showed mucin from the gill by F. columnare challenge induced an obvious response than mucin from the intestine with E. ictaluri infection.     

The effect of piscidin antimicrobial peptides on the formation of Gram-negative bacterial biofilms

Fish-derived antimicrobial peptides are an important part of the innate immune system due to their potent antimicrobial properties. Piscidins are a class of antimicrobial peptides first described in hybrid striped bass (Morone chrysops x Morone saxatilis) but have also been identified in many other fish species. Previous work demonstrated the broad antimicrobial activity of piscidins against Gram-negative and Gram-positive bacterial species. This study sought to determine the extent to which class I (striped bass piscidin 1, white bass piscidin 1 and striped bass/white bass piscidin 3) and class II (striped bass piscidin 4 and white bass piscidin 5) piscidins inhibit biofilm formation of different Gram-negative bacteria. In general, the class I and II piscidins demonstrate potent activity against Escherichia coli and Flavobacterium columnare biofilms. The class II piscidins showed more activity against E. coli and F. columnare isolates than did the class I piscidins. The piscidins in general were much less effective against inhibiting Aeromonas hydrophila and A. veronii biofilm growth. Only the class I piscidins showed significant growth inhibition among the Aeromonas spp. examined.     

Prevalence and antimicrobial resistance of vibrios of human health significance in inland saline aquaculture areas

This study was conducted to evaluate the prevalence, potential pathogenicity and antimicrobial resistance of Vibrio isolates from 65 soil/water/fish samples collected from inland saline aquaculture areas. Depending on the sample type, presumptive Vibrio counts ranged from 2.50 to 6.16 log₁₀ CFU/ml (or/g). Among the 119 confirmed Vibrio isolates, Vibrio cholerae was found to most dominant (91.6%) and it was detected in all the samples from inland saline areas. Seven other Vibrio spp. including Vibrio parahaemolyticus and Vibrio vulnificus were also detected. Except one O139 serotype, rest of the V. cholerae isolates were found belonging to non‐O1/non‐O139 serogroups. None of the V. cholerae isolate was found positive for ctx gene. Antimicrobial susceptibility testing against 7 commonly used antibiotics revealed highest resistance (50.4%) against ampicillin. Very high intermediate resistance (87.4%) was also observed against erythromycin. Contrary to previous studies, high susceptibility (>70%) to chloramphenicol, nalidixic acid, tetracycline and trimethoprim was observed in Vibrio isolates obtained in present study. Almost 20% of Vibrio isolates were resistant to two or more antibiotic classes with multiple antibiotic resistance (MAR) index value of ≥0.28. Presence of V. cholerae isolates with very high MAR index value of 0.85 also suggested that these multidrug‐resistant environment isolates could serve as reservoir of antibiotic‐resistant genes in aquatic systems. The presence of multiple drug resistance vibrios in emerging inland saline aquaculture systems emphasizes the need for their routine monitoring for developing the risk assessment and mitigation strategies.     

Lack of association between Flavobacterium columnare genomovar and virulence in hybrid tilapia Oreochromis niloticus (L.) × Oreochromis aureus (Steindachner)

Columnaris disease can be problematic in tilapia (Oreochromis spp.) production. An understanding of the pathogenesis and virulence of Flavobacterium columnare is needed to develop prevention strategies. The objective of this study was to determine the virulence of genetically defined isolates of F. columnare in sex‐reversed hybrid tilapia, Oreochromis niloticus (L.) × O. aureus (Steindachner). A series of immersion challenge trials were performed using isolates of the five established genomovars of F. columnare: I, II, II‐B, III and I/II. The mean per cent mortality of fish challenged with genomovar I, II and III isolates ranged from 0 to 100, 3.3–78 and 3.3–75%, respectively. The mean per cent mortality of fish challenged with genomovar II‐B ranged from 35 to 96.7%, and the only genomovar I/II isolate tested caused no mortality. Contrary to previous work in other fish species, there did not appear to be an association between F. columnare genomovar and virulence in tilapia. The challenge model used resulted in acute mortality. An alternative challenge model was tested by cohabitating healthy fish with dead fish infected with F. columnare. This method resulted in rapid appearance of clinical signs and mortality, suggesting the potential for F. columnare to increase in virulence upon growth on/in a fish host.     

Genetic diversity and antimicrobial resistance of Flavobacterium psychrophilum isolated from cultured rainbow trout,Onchorynchus mykiss (Walbaum), in Spain

Flavobacterium psychrophilum is the causative agent of bacterial cold‐water disease (CWD) and rainbow trout fry syndrome (RTFS) in salmonids. These diseases are a major problem in the aquaculture industry in Spain, and a better understanding of the epidemiology of F. psychrophilum isolates is necessary to improve management strategies. In this study, to investigate genetic variability of this bacterium, pulsed‐field gel electrophoresis after DNA digestion with endonuclease StuI, plasmid profiling analysis and antimicrobial susceptibility testing were undertaken with 25 isolates of F. psychrophilum from Spain. These isolates were classified into 17 patterns by PFGE analysis, which were grouped into four clusters and seven independent branches. Twenty isolates (80%) possessed plasmids of 3.5 kb (n = 13) or 5.5 kb (n = 7). No plasmids were associated with antibiotic resistance to oxytetracycline (OTC) or florfenicol (FLO). Twenty isolates (80%) had minimum inhibitory concentrations (MICs) to OTC of between 2.4 and 9.7 μg mL^?1, and all isolates were susceptible to FLO. A relationship between the origin of the isolates and PFGE genotypes was found. Plasmid profile typing correlated with PFGE profile typing, whereas no correlation was found between antimicrobial susceptibility testing and PFGE profiles. These results suggest that the population of F. psychrophilum with pathogenic potential in northern Spain is quite heterogeneous.     

Virulence assay of rhizoid and non‐rhizoid morphotypes of Flavobacterium columnare in red tilapia,Oreochromis sp., fry

Numerous isolates of Flavobacterium columnare were previously recovered from red tilapia, Oreochromis sp., exhibiting columnaris‐like disease in Thai farms, and the phenotypic and genetic characteristics were described. The objective of this study was to determine the virulence of two morphotypes (rhizoid and non‐rhizoid colonies) of F. columnare and to determine their ability to adhere to and persist in red tilapia fry. The results showed that the typical rhizoid isolate (CUVET1214) was a highly virulent isolate and caused 100% mortality within 24 h following bath challenge of red tilapia with three different doses. The non‐rhizoid isolate (CUVET1201) was avirulent to red tilapia fry. Both morphotypes adhered to and persisted in tilapia similarly at 0.5 and 6 h post‐challenge as determined by whole fish bacterial loads. At 24 and 48 h post‐challenge, fry challenged with the rhizoid morphotype exhibited significantly higher bacterial loads than the non‐rhizoid morphotype. The results suggested that an inability of the non‐rhizoid morphotype to persist in tilapia fry may explain lack of virulence.     

Influence of native catfish mucus on Flavobacterium columnare growth and proteolytic activity

Flavobacterium columnare causes columnaris disease of farmed and wild freshwater fish. Skin mucus is an important factor in early stages of columnaris pathogenesis, albeit little studied. Our objectives were to (a) characterize the terminal glycosylation pattern (TGP) of catfish mucus, (b) determine the growth of F. columnare in formulated water (FW)‐containing channel catfish (Ictalurus punctatus) or hybrid catfish (Ictalurus punctatus X Ictalurus furcatus) mucus and (c) examine extracellular protease activity of two F. columnare isolates differing in virulence. The TGP of catfish mucus by lectin binding was as follows: alpha‐D‐mannose/alpha‐D‐glucose >N‐acetyl‐beta‐D‐glucosamine >N‐acetyl‐beta‐D‐glucosamine/N‐acetylneuraminic acid >N‐acetyl‐D‐galactosamine >alpha‐D‐galactose/N‐acetyl‐alpha‐D‐galactosamine >beta‐D‐galactose = alpha‐L‐fucose. Virulence studies demonstrated isolate AL‐02‐36 was highly virulent in channel catfish fry (0.1 g) with cumulative mortality of 90%‐100% versus 60% for isolate ALG‐00‐530 at equivalent doses (~3 × 10⁶ CFU/ml); a similar result was observed in larger (0.7 g) catfish. In multiple experiments, F. columnare replicated (2‐3 logs) and survived (28 days) in formulated water‐containing catfish mucus. Highly virulent isolate AL‐02‐36 possessed at least 2.5‐ to fivefold higher protease activity following growth in mucus than the less virulent ALG‐00‐530. Flavobacterium columnare utilized catfish mucus as a nutrient source and mucus presence modulated extracellular protease production.     

Multilocus sequence typing (MLST) analysis of California Flavobacterium psychrophilum reveals novel genotypes and predominance of CC‐ST10 in California salmonid hatcheries

The Gram‐negative bacterium, Flavobacterium psychrophilum, is endemic to California, USA, where it is an important pathogen in salmonid aquaculture, especially in rainbow trout (Oncorhynchus mykiss). Disease outbreaks caused by F. psychrophilum in rainbow trout fingerlings can approach 90% mortality, resulting in millions of dollars of economic losses annually. The focus of this study was to investigate the genetic diversity of 49 F. psychrophilum isolates collected from disease outbreaks in 17 salmonid hatcheries in California, USA, from 2015 to 2018 using multilocus sequence typing. Results suggest California F. psychrophilum isolates are diverse, representing 11 distinct sequence types (STs), three of which were previously undescribed. Still, the majority of genotyped isolates (n = 41) belonged to a single clonal complex (CC), CC‐ST10, which is the largest CC worldwide and has been linked to disease outbreaks on several continents. Results of this study provide evidence of marked intraspecific genetic diversity of F. psychrophilum from California. The biological significance of this genetic variability is unclear but could have implications for future vaccine development and treatments. Further studies investigating the virulence, antigenic, and antimicrobial susceptibility profiles of F. psychrophilum are warranted to better understand the epizootiology of this pathogen in the Western United States.     

Vertebrate mucus stimulates biofilm development and upregulates iron acquisition genes in Flavobacterium columnare

Columnaris disease is responsible for substantial losses throughout the production of many freshwater fish species. One of the ways in which the bacterium Flavobacterium columnare is so effective in initiating disease is through the formation of biofilms on fish skin and gills. To further explore the interaction between host factors and bacterial cells, we assayed the ability of vertebrate mucus to enhance F. columnare biofilm development. Different concentrations of catfish, tilapia and pig mucus (5–60 µg/ml) increased biofilm growth at varying degrees among F. columnare isolates. Our data suggest that vertebrate mucus acts as a signalling molecule for the development of F. columnare biofilms; however, there are clear disparities in how individual isolates respond to different mucus fractions to stimulate biofilms. The expression of iron acquisition genes among two genomovar II isolates showed that ferroxidase, TonB receptor and the siderophore synthetase gene were all significantly upregulated among F. columnare biofilms. Interestingly, the siderophore acetyltransferase gene was only shown to be significantly upregulated in one of the genomovar II isolates. This work provides insight into our understanding of the interaction between F. columnare and vertebrate mucus, which likely contributes to the growth of planktonic cells and the transition into biofilms.