Hege Smith Tunsjø

Atlantic salmon bath challenged with Moritella viscosa - Pathogen invasion and host response

The Gram-negative bacterium Moritella viscosa is considered to be the main causative agent of winter ulcer, a disease that primarily affects salmonid fish in sea water during cold periods. The disease is initially characterised by localised swelling of the skin followed by development of lesions. To gain more knowledge of the role of M. viscosa in the pathogenesis of winter ulcer, 159 Atlantic salmon (80-110 g) were exposed to a bath challenge dose of 7 x 10(5) cfu ml(-1) for 1 h at 8.9 degrees C. The first mortalities were registered two days post-challenge and the mortality rate increased rapidly. Multi-organ samples were taken throughout the challenge for culture, immunohistochemistry and PCR analysis. Using real-time PCR, M. viscosa DNA was first detected in the gills of all fish examined 2, 6 and 12 h after challenge. From day 2, the bacterium was detected in the muscle/skin, head kidney, spleen and liver. This was in correlation with positive cultured samples and confirmed systemic infection. The early and consistent detection of M. viscosa DNA in gill samples, and less or not in muscle/skin or intestine, could suggest gills as a port of entry for the bacterium. Immunohistochemical analysis using a polyclonal antiserum against M. viscosa demonstrated generalised staining in the lumen of blood vessels and some positive mononuclear cells. The antigens recognised by the antiserum may have originated from extracellular bacterial products and be part of a bacterial invasion strategy. To better understand the immune response in salmon to M. viscosa infection, the expression profiles of the immune genes IL1 beta, C3, ISG15 and CD83 were studied. Increased expression of IL1 beta and C3 was not induced until day 7, which may suggest that M. viscosa might utilize escape mechanisms to evade the hosts immune system by suppressing relevant immune responses.

A Novel lux Operon in the Cryptically Bioluminescent Fish Pathogen Vibrio salmonicida Is Associated with Virulence

ABSTRACT The cold-water-fish pathogen Vibrio salmonicida expresses a functional bacterial luciferase but produces insufficient levels of its aliphatic-aldehyde substrate to be detectably luminous in culture. Our goals were to (i) better explain this cryptic bioluminescence phenotype through molecular characterization of the lux operon and (ii) test whether the bioluminescence gene cluster is associated with virulence. Cloning and sequencing of the V. salmonicida lux operon revealed that homologs of all of the genes required for luminescence are present: luxAB (luciferase) and luxCDE (aliphatic-aldehyde synthesis). The arrangement and sequence of these structural lux genes are conserved compared to those in related species of luminous bacteria. However, V. salmonicida strains have a novel arrangement and number of homologs of the luxR and luxI quorum-sensing regulatory genes. Reverse transcriptase PCR analysis suggests that this novel arrangement of quorum-sensing genes generates antisense transcripts that may be responsible for the reduced production of bioluminescence. In addition, infection with a strain in which the luxA gene was mutated resulted in a marked delay in mortality among Atlantic salmon relative to infection with the wild-type parent in single-strain challenge experiments. In mixed-strain competition between the luxA mutant and the wild type, the mutant was attenuated up to 50-fold. It remains unclear whether the attenuation results from a direct loss of luciferase or a polar disturbance elsewhere in the lux operon. Nevertheless, these findings document for the first time an association between a mutation in a structural lux gene and virulence, as well as provide a new molecular system to study Vibrio pathogenesis in a natural host.

Shiga Toxin 2a in Escherichia albertii

E scherichia albertii is an emerging human enteric pathogen ([1][1]). It belongs to the attaching and effacing group of bacteria, which also includes enteropathogenic and Shiga toxin-producing Escherichia coli (EPEC and STEC, respectively). Shiga toxin-producing E. albertii has been described,

Motility and flagellin gene expression in the fish pathogen Vibrio salmonicida: effects of salinity and temperature.

The success of several Vibrio species, including Vibrio cholerae, Vibrio anguillarum and Vibrio fischeri in colonizing their symbiont, or causing infection is linked to flagella-based motility. It is during early colonization or the initial phase of infection that motility appears to be critical. In this study we used Vibrio salmonicida, a psychrophilic and moderate halophilic bacterium that causes cold-water vibriosis in seawater-farmed Atlantic salmon (Salmo salar), to study motility and expression of flagellins under salt conditions mimicking the initial and later phases of an infection. Our results, which are based on motility in semi-solid agar, membrane protein proteomics, quantitation of flagellin gene expression, challenge infection of fish, and microscopy, show that V. salmonicida is highly motile, expresses elevated levels of flagellins, and typically contains several polar flagella under salt conditions that are seawater-like. In contrast, V. salmonicida cells are non-motile and express significantly lower levels of flagellins under physiological-like salt conditions.

Anaerobic blood culture isolates in a Norwegian university hospital: identification by MALDI‐TOF MS vs 16S rRNA sequencing and antimicrobial susceptibility profiles

We investigated 197 anaerobic isolates recovered from blood cultures in the period 2009–2013. The isolates included were Bacteroides spp., Clostridium spp., Prevotella spp., Fusobacterium spp. and Gram‐positive anaerobic cocci (GPAC). Identification results by MALDI‐TOF MS were compared to those obtained by 16S rRNA sequencing, and the MICs of benzylpenicillin, clindamycin, piperacillin‐tazobactam, meropenem and metronidazole were determined by Etests. The MALDI‐TOF MS correctly identified 94.9% of the anaerobes to the genus level, and 86.8% to the species level, with errors mainly among the non‐fragilis Bacteroides spp. and GPAC. About 73.3% of the isolates were non‐susceptible to penicillin, mainly due to high resistance rates in the Bacteroides spp. (99.2%) and Prevotella spp. (69.2%). About 18.5% of the isolates were clindamycin resistant. Piperacillin‐tazobactam had an excellent activity against all anaerobes except the non‐fragilis Bacteroides spp., of which 43.8% were non‐susceptible. The clinical significance of such a high resistance rate is unclear. Meropenem and metronidazole were the most active antibiotics, with 96.9% and 97.9% of the isolates being susceptible.

Putative virulence genes in Moritella viscosa: activity during in vitro inoculation and in vivo infection.

Moritella viscosa is considered to be the main aetiological agent of winter ulcer disease, primarily affecting farmed salmonid fish in cold marine waters. Transcription profiles of twelve M. viscosa genes, potentially involved in the pathogenesis, were studied during the course of an in vitro cell culture infection assay. Transcription of the same genes was compared in vivo, in head kidney and ulcer tissues of Atlantic salmon challenged with M. viscosa. During the in vitro infection, three putative toxins: a putative repeats in toxin gene (rtxA), a putative cytotoxic necrotizing factor (cnf) and a putative hemolysin increased their transcription significantly with time and coincident with cell rounding. Furthermore, the majority of the genes were stimulated by presence of fish cells and showed higher activity when adhered to fish cells compared to their planktonic counterpart. In vivo gene transcription studies revealed an up-regulation of a putative lateral flagellin in ulcer compared to head kidney tissues in the same individual. A similar trend was seen for cnf and a gene encoding a putative protease, indicating a role for these factors in colonization and tissue damage.

Comparison of nasopharyngeal aspirate with flocked swab for PCR-detection of respiratory viruses in children

Fast‐ and high‐throughput molecular workflows require sample matrices to be suitable for automation. Respiratory swabs are better suited for this purpose compared to the more viscous nasopharyngeal aspirates. Samples collected by nasopharyngeal aspiration and nasopharyngeal flocked swab from 81 children were compared for detection and recovery of respiratory viruses. Using real‐time RT‐PCR, no statistically significant differences in virus detection between the two sample types were found, supporting the use of flocked swabs in children aged one month to two years.

Suitability of stx‐PCR directly from fecal samples in clinical diagnostics of STEC

PCR‐based testing for Shiga toxin producing Escherichia coli (STEC) directly from fecal samples is increasingly being implemented in routine diagnostic laboratories. These methods aim to detect clinically relevant amounts of microbes and not stx‐carrying phages or low backgrounds of STEC. We present a diagnostic procedure and results from 1 year of stx‐targeted real‐time PCR of fecal samples from patients with gastrointestinal symptoms in Norway. A rapid stx2 subtyping strategy is described, which aims to quickly reveal the virulence potential of the microbe. stx was detected in 22 of 3320 samples, corresponding to a PCR positive rate of 0.66%. STEC were cultured from 72% of the PCR positive samples. Four stx1 isolates, eight stx2 isolates, and four isolates with both stx1 and stx2 were identified. With the method presented, stx‐carrying phages are not commonly detected. Our results support the use of molecular testing combined with classical culture techniques for routine diagnostic purposes.

A rapid, high-throughput screening method for carriage of methicillin-resistant Staphylococcus aureus

Rapid screening of methicillin‐resistant Staphylococcus aureus (MRSA) colonization prior to hospital admittance is important to reduce nosocomial infections and health care costs. Molecular detection of mecA and S. aureus specific target genes has become widely established for this purpose. However, there are still limitations in potential for high‐throughput screening in the methods described. We have compared the time aspects and workload of four different DNA preparation platforms, resulting in an automated and simple MRSA screening method which combines two liquid handling systems and a simple lysis buffer. We have further transferred our in‐house dual real‐time PCR to a fast‐PCR protocol, reducing the time and labour spent on these samples to a minimum.

Challenges in the identification of methicillin-resistant Staphylococcus argenteus by routine diagnostics

Current clinical diagnostic procedures have shortcomings in the differentiation of Staphylococcus argenteus from Staphylococcus aureus. This article presents three cases of Staphylococcus argenteus obtained from clinical samples. The initial results from biochemical and molecular methods led to an incorrect identification of the isolates as methicillin‐resistant Staphylococcus aureus. Whole genome sequencing and real‐time PCR targeting the nonribosomal peptide synthetase gene led to their correct identification as methicillin‐resistant Staphylococcus argenteus. The study shows that real‐time PCR can be used to differentiate the two species in routine diagnostics. For purposes of identification based on whole genome sequencing, the MinION portable sequencer is a simple and affordable approach which could be used by many laboratories.