Lone Madsen

Antimicrobial resistance patterns in Danish isolates of Flavobacterium psychrophilum

The resistance pattern of Flavobacterium psychrophilum to the antimicrobial agents used in fish farming in Denmark was assessed in vitro using an agar dilution method. After identification of 387 isolates from clinical outbreaks of rainbow trout fry syndrome (RTFS) and the environment, the isolates were tested and the resulting antibiograms were used to predict the theoretical therapeutic efficacy and to evaluate if resistance had changed as a course of time. Antimicrobial agents included in this investigation were oxolinic acid (OXA), amoxicillin (AMX), potentiated sulfadiazine, oxytetracycline (OTC) and florfenicol (FLO). We found that F. psychrophilum isolates divided in susceptible and resistant clusters reflecting the reduced efficacy in practice when using OTC and AMX. The most recent isolates were less susceptible to AMX and OXA, whereas resistance to OTC seemed stable over the last 5 years. Apparently, F. psychrophilum carries intrinsic resistance towards the potentiated sulfonamides, and in correlation with this, we found very few susceptible isolates. All isolates were susceptible to FLO. The method used for determining minimum inhibitory concentration (MIC) follows the American guideline from The National Committee for Clinical Laboratory Standards (NCCLS) with some modifications to comply with the growth demand of this fastidious bacterium. We suggest future investigations use these guidelines as a benchmark.

Detection of Flavobacterium psychrophilum from fish tissue and water samples by PCR amplification

Rainbow trout fry syndrome and cold‐water disease, caused by Flavobacterium psychrophilum, are important diseases in farmed salmonids. Some of the presently available techniques for the detection of Fl. psychrophilum are either time consuming or lack sufficient sensitivity. In the present investigation, the possible detection of Fl. psychrophilum from fish tissue and water samples was examined using nested PCR with DNA probes against a sequence of the 16S rRNA genes. The DNA was extracted using Chelex® 100 chelating resin. The primers, which were tested against strains isolated from diseased fish, healthy fish, fish farm environments and reference strains, proved to be specific for Fl. psychrophilum. The obtained detection limit of Fl. psychrophilum seeded into rainbow trout brain tissue was 0·4 cfu in the PCR tube, corresponding to 17 cfu mg−1 brain tissue. The PCR‐assay proved to be more sensitive than agar cultivation of tissue samples from the brain of rainbow trout injected with Fl. psychrophilum. In non‐sterile fresh water seeded with Fl. psychrophilum the detection limit of the PCR‐assay was 1·7 cfu in the PCR tube, corresponding to 110 cfu ml−1 water. The PCR‐assay detected Fl. psychrophilum in water samples taken from a rainbow trout farm, but Fl. psychrophilum could not be isolated using inoculation on selective agar. The method presented here has the potential to detect low levels of Fl. psychrophilum in fish tissue and in water samples, and the technique can be a useful tool for understanding the epidemiology of Fl. psychrophilum.

Porcine transcriptome analysis based on 97 non-normalized cDNA libraries and assembly of 1,021,891 expressed sequence tags

BackgroundKnowledge of the structure of gene expression is essential for mammalian transcriptomics research. We analyzed a collection of more than one million porcine expressed sequence tags (ESTs), of which two-thirds were generated in the Sino-Danish Pig Genome Project and one-third are from public databases. The Sino-Danish ESTs were generated from one normalized and 97 non-normalized cDNA libraries representing 35 different tissues and three developmental stages.ResultsUsing the Distiller package, the ESTs were assembled to roughly 48,000 contigs and 73,000 singletons, of which approximately 25% have a high confidence match to UniProt. Approximately 6,000 new porcine gene clusters were identified. Expression analysis based on the non-normalized libraries resulted in the following findings. The distribution of cluster sizes is scaling invariant. Brain and testes are among the tissues with the greatest number of different expressed genes, whereas tissues with more specialized function, such as developing liver, have fewer expressed genes. There are at least 65 high confidence housekeeping gene candidates and 876 cDNA library-specific gene candidates. We identified differential expression of genes between different tissues, in particular brain/spinal cord, and found patterns of correlation between genes that share expression in pairs of libraries. Finally, there was remarkable agreement in expression between specialized tissues according to Gene Ontology categories.ConclusionThis EST collection, the largest to date in pig, represents an essential resource for annotation, comparative genomics, assembly of the pig genome sequence, and further porcine transcription studies.

Vertebral column deformities in farmed rainbow trout ( Oncorhynchus mykiss )

Abstract Farmed rainbow trout ( Oncorhynchus mykiss ) were fed diets with either different levels of vitamin C, or diets enriched with glucan or chitin, from feeding start and 6 months onwards. At an average weight of 100 g, the trout were X-rayed to determine the deformity level. The investigations showed deformity levels from 4.8% to 12.5% among the different diet groups. Fish fed the chitin-enriched diet, the low vitamin C diet, the high vitamin C diet, and the control diet had the highest deformity levels, ranging from 8.9% to 12.5%, while the group fed the glucan-enriched diet had the lowest level of deformities (4.8%). In all groups examined, the deformities were spread over the whole vertebral column. The deformities in the group fed the low vitamin C diet were more severe than those found in the other groups. An outbreak of the disease rainbow trout fry syndrome (RTFS) caused by the bacterium Flavobacterium psychrophilum was observed in all groups during the investigation. The findings of deformities in all examined groups indicate a possible role of F. psychrophilum in the aetiology of the observed deformities. The lower level of deformities in the group fed the glucan-enriched diet might be caused by the ability of glucan to stimulate the immune response and increase the disease resistance of fish.

Involvement of a sialic acid-binding lectin with hemagglutination and hydrophobicity of Flavobacterium psychrophilum.

ABSTRACT Strains of Flavobacterium psychrophilum were studied for their ability to adhere and cause agglutination of erythrocytes and yeast cells. Strains of the serotype Th showed low or no hemagglutinating (HA) properties toward human, avian, bovine, and rainbow trout erythrocytes, whereas strains of serotype Fd and FpT exhibited distinct HA properties. None of the strains was able to cause agglutination of yeast cells. Greater adherence specificity toward rainbow trout blood cells was seen for the HA-positive strains. Growth at 5°C, compared to that at 15°C, induced an increase in the hemagglutination of some strains. HA activities of F. psychrophilum were inhibited only by sialic acid (N-acetyl-neuraminic acid), heat treatment at 65°C, and proteinase K treatment and not by any of seven other carbohydrates, periodate oxidation, or treatment with trypsin. The supernatant from washed bacterial cells also showed some HA properties. All strains were shown to be highly hydrophobic by the hydrophobic interaction chromatography test, although some contradictions to the results of the salt aggregation test (showing some strains as less hydrophobic) were seen. These results indicate that the aggregation of F. psychrophilum and erythrocytes depend on a lectin present on the surface of HA-positive F. psychrophilum strains and absent on HA-negative strains. This lectin reacts specifically with sialic acid. The adhesion differences observed for F. psychrophilum strains do not appear to correlate with the virulence but still provide insights into the interaction of F. psychrophilum and rainbow trout.

Efficiency of oxytetracycline treatment in rainbow trout experimentally infected with Flavobacterium psychrophilum strains having different in vitro antibiotic susceptibilities

Abstract The medication effect of oxytetracycline on groups of rainbow trout fry experimentally infected with three strains of Flavobacterium psychrophilum was investigated. The infection model was based on intraperitoneal injection of the pathogen and treatment was done using medicated feed resulting in 100 mg oxytetracycline/kg fish for 10 days. The three F. psychrophilum strains had different antimicrobial susceptibilities and successful treatment was only obtained in the trial using a strain with a MIC OTC of 0.25 μg/ml. No effect of treatment was seen in the group infected with a strain having MIC OTC of 8.0 μg/ml and only little effect was seen when the strain MIC OTC was 4.0 μg/ml. This shows that it is valid to predict the treatment efficiency of OTC from in vitro data facing an outbreak of rainbow trout fry syndrome. The importance of doing susceptibility testing is emphasized, and as shown the selection of media for antimicrobial susceptibility testing of F. psychrophilum is important.

Diversity of Flavobacterium psychrophilum and the potential use of its phages for protection against bacterial cold water disease in salmonids

Flavobacterium psychrophilum causes rainbow trout fry syndrome (RTFS) and cold water disease (CWD) in salmonid aquaculture. We report characterization of F. psychrophilum strains and their bacteriophages isolated in Chilean salmonid aquaculture. Results suggest that under laboratory conditions phages can decrease mortality of salmonids from infection by their F. psychrophilum host strain. Twelve F. psychrophilum isolates were characterized, with DNA restriction patterns showing low diversity between strains despite their being obtained from different salmonid production sites and from different tissues. We isolated 15 bacteriophages able to infect some of the F. psychrophilum isolates and characterized six of them in detail. DNA genome sizes were close to 50 Kbp and corresponded to the Siphoviridae and Podoviridae families. One isolate, 6H, probably contains lipids as an essential virion component, based on its chloroform sensitivity and low buoyant density in CsCl. Each phage isolate rarely infected F. psychrophilum strains other than the strain used for its enrichment and isolation. Some bacteriophages could decrease mortality from intraperitoneal injection of its host strain when added together with the bacteria in a ratio of 10 plaque-forming units per colony-forming unit. While we recognize the artificial laboratory conditions used for these protection assays, this work is the first to demonstrate that phages might be able protect salmonids from RTFS or CWD.

SNP mining porcine ESTs with MAVIANT, a novel tool for SNP evaluation and annotation

MOTIVATION Single nucleotide polymorphisms (SNPs) analysis is an important means to study genetic variation. A fast and cost-efficient approach to identify large numbers of novel candidates is the SNP mining of large scale sequencing projects. The increasing availability of sequence trace data in public repositories makes it feasible to evaluate SNP predictions on the DNA chromatogram level. MAVIANT, a platform-independent Multipurpose Alignment VIewing and Annotation Tool, provides DNA chromatogram and alignment views and facilitates evaluation of predictions. In addition, it supports direct manual annotation, which is immediately accessible and can be easily shared with external collaborators. RESULTS Large-scale SNP mining of polymorphisms bases on porcine EST sequences yielded more than 7900 candidate SNPs in coding regions (cSNPs), which were annotated relative to the human genome. Non-synonymous SNPs were analyzed for their potential effect on the protein structure/function using the PolyPhen and SIFT prediction programs. Predicted SNPs and annotations are stored in a web-based database. Using MAVIANT SNPs can visually be verified based on the DNA sequencing traces. A subset of candidate SNPs was selected for experimental validation by resequencing and genotyping. This study provides a web-based DNA chromatogram and contig browser that facilitates the evaluation and selection of candidate SNPs, which can be applied as genetic markers for genome wide genetic studies. AVAILABILITY The stand-alone version of MAVIANT program for local use is freely available under GPL license terms at http://snp.agrsci.dk/maviant. SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Multilocus Sequence Typing Identifies Epidemic Clones of Flavobacterium psychrophilum in Nordic Countries

ABSTRACT Flavobacterium psychrophilum is the causative agent of bacterial cold water disease (BCWD), which affects a variety of freshwater-reared salmonid species. A large-scale study was performed to investigate the genetic diversity of F. psychrophilum in the four Nordic countries: Denmark, Finland, Norway, and Sweden. Multilocus sequence typing of 560 geographically and temporally disparate F. psychrophilum isolates collected from various sources between 1983 and 2012 revealed 81 different sequence types (STs) belonging to 12 clonal complexes (CCs) and 30 singleton STs. The largest CC, CC-ST10, which represented almost exclusively isolates from rainbow trout and included the most predominant genotype, ST2, comprised 65% of all isolates examined. In Norway, with a shorter history (<10 years) of BCWD in rainbow trout, ST2 was the only isolated CC-ST10 genotype, suggesting a recent introduction of an epidemic clone. The study identified five additional CCs shared between countries and five country-specific CCs, some with apparent host specificity. Almost 80% of the singleton STs were isolated from non-rainbow trout species or the environment. The present study reveals a simultaneous presence of genetically distinct CCs in the Nordic countries and points out specific F. psychrophilum STs posing a threat to the salmonid production. The study provides a significant contribution toward mapping the genetic diversity of F. psychrophilum globally and support for the existence of an epidemic population structure where recombination is a significant driver in F. psychrophilum evolution. Evidence indicating dissemination of a putatively virulent clonal complex (CC-ST10) with commercial movement of fish or fish products is strengthened.

Bacteriophage Resistance Mechanisms in the Fish Pathogen Flavobacterium psychrophilum: Linking Genomic Mutations to Changes in Bacterial Virulence Factors

ABSTRACT Flavobacterium psychrophilum is an important fish pathogen in salmonid aquaculture worldwide. Due to increased antibiotic resistance, pathogen control using bacteriophages has been explored as a possible alternative treatment. However, the effective use of bacteriophages in pathogen control requires overcoming the selection for phage resistance in the bacterial populations. Here, we analyzed resistance mechanisms in F. psychrophilum after phage exposure using whole-genome sequencing of the ancestral phage-sensitive strain 950106-1/1 and six phage-resistant isolates. The phage-resistant strains had all obtained unique insertions and/or deletions and point mutations distributed among intergenic and genic regions. Mutations in genes related to cell surface properties, gliding motility, and biosynthesis of lipopolysaccharides and cell wall were found. The observed links between phage resistance and the genetic modifications were supported by direct measurements of bacteriophage adsorption rates, biofilm formation, and secretion of extracellular enzymes, which were all impaired in the resistant strains, probably due to superficial structural changes. The clustered regularly interspaced short palindromic repeat (CRISPR) region was unaffected in the resistant isolates and thus did not play a role as a resistance mechanism for F. psychrophilum under the current conditions. All together, the results suggest that resistance in F. psychrophilum was driven by spontaneous mutations, which were associated with a number of derived effects on the physiological properties of the pathogen, including reduced virulence under in vitro conditions. Consequently, phage-driven physiological changes associated with resistance may have implications for the impact of the pathogen in aquaculture, and these effects of phage resistance on host properties are therefore important for the ongoing exploration of phage-based control of F. psychrophilum.