High frequency of occupied attB regions in Norwegian Staphylococcus aureus isolates supports a two-step MRSA screening algorithm.

Rapid nucleic acid amplification tests for methicillin-resistant Staphylococcus aureus (MRSA) diagnostics commonly target the mec resistance gene, genes specific for S. aureus, and the integration site for the SCCmec resistance cassette, orfX. Due to poor specificity when these target genes are used individually, additional culture is required to verify positive results. The combination of these targets is useful, but the optimal algorithm may depend on the presence of the genetic markers in S. aureus isolates, as well as the prevalence of MRSA in a population. The aim of the present study was to identify a rapid, low-cost, and functional screening algorithm in order to reduce the response time for MRSA diagnostics. An in-house orfX-SCCmec polymerase chain reaction (PCR) assay was established and evaluated. The results were compared with an existing mec/nuc PCR assay and traditional culture. Methicillin-sensitive S. aureus (MSSA) that tested false-positive in the orfX-SCCmec PCR assay were further investigated with full genome sequencing using the Ion PGM™ System to verify results and causality. Based on these data, a two-step screening algorithm with initial mec/nuc PCR followed by orfX-SCCmec PCR on positive samples was suggested and tested on 1443 patient samples. 22.5 % of MSSA isolates tested false-positive with the orfX-SCCmec PCR. Full genome sequencing of these isolates identified genetic variation in the attB region of S. aureus, including empty cassette variants and non-mec SCC. The suggested two-step MRSA screening algorithm allowed us to report MRSA results for 95.6 % of all samples and 99 % of MRSA-negative samples after one day.

Purification of DNA eluates from the ProbeTec GC Q(x) assay on BD Viper™ XTR allows for further analysis and confirmation of gonorrhea.

Low positive predictive values of Neisseria gonorrhoeae nucleic acid amplification tests in low-prevalence populations are a major challenge for accurate diagnostics. It is therefore necessary to verify all positive N. gonorrhoeae results with a different assay and target gene, preferably using the same sample. The BD ProbeTec™ Q(x) Collection Kit for Endocervical or Lesion Specimens, which is recommended for BD Viper™ XTR, is incompatible with other commercial platforms. Therefore, a confirmatory PCR has not been available for samples received on this transport medium. To be able to verify results from these samples with another assay, our objective was to establish a procedure for using the DNA eluates from BD Viper™ XTR for further analysis. DNA eluates from BD Viper™ XTR were collected and analyzed in two in-house confirmatory real-time PCRs targeting the porA pseudogene and the opa multicopy gene. BD Viper™ XTR DNA eluates were analyzed directly and also after purification with the nucleic acid extraction system NucliSENS® easyMag®. Purification of BD Viper™ XTR DNA eluates with the nucleic acid extraction system NucliSENS® easyMag® provided a sensitivity of the in-house PCR comparable to BD Viper™. With the inclusion of two target genes in the confirmatory PCR, specific and reliable verification of results were obtained. This study presents a simple, inexpensive procedure which allows for rapid verification of gonorrhea from samples received on the BD ProbeTec™ Q(x) Collection Kit for Endocervical or Lesion Specimens.

Previously unrecognised division within Moritella viscosa isolated from fish farmed in the North Atlantic.

Previously undocumented phenotypical and genetic variation was identified amongst isolates of Moritella viscosa collected from various geographical locations and from different fish species. The studied isolates could be split into 2 major phenotypically and genetically different clusters, one of which was consistent with the species type strain (NCIMB 13548). Isolates consistent with the type strain originated exclusively from Atlantic salmon farmed in Norway, Scotland and the Faroe Isles, although a single isolate from farmed Norwegian cod clustered closely with this group. The 'variant' cluster comprised isolates originating from Norwegian farmed rainbow trout, Icelandic farmed rainbow trout and salmon, Canadian farmed (Atlantic) salmon, Icelandic lumpsucker and only exceptionally from Norwegian salmon. With the exception of the single aforementioned cod isolate, all isolates from Norwegian farmed cod belonged to the variant cluster. Phenotypically, the clusters could be absolutely separated only by elevated haemolytic activity in the variant strain, although approximately half of these isolates also produced acid from mannose, in contrast to the typical (type) strain. While 16S rRNA gene sequencing was unable to separate the 2 clusters, Western blot analyses, plasmid profile analysis, pulsed field gel electrophoresis and gyrB gene sequence analysis produced clusters consistent with the phenotypic data. Macroscopically and histologically the disease in rainbow trout caused by the variant strain was consistent with that previously described in Atlantic salmon. The results of the present study may indicate a degree of host specificity of the typical strain for Atlantic salmon.

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 host's immune system by suppressing relevant immune responses.