Tenacibaculum maritimum CRISPR loci analysis and evaluation of isolate spoligotyping.

AIMS: We performed in silico analysis of CRISPRcas loci from Tenacibaculum maritimum, evaluated spoligotyping as a subtyping method and genotyped uncharacterized Turkish isolates from European sea bass by multilocus sequence typing (MLST). METHODS AND RESULTS: Spoligotyping was performed with primers designed to allow amplification and sequencing of whole CRISPR-arrays from 23 T. maritimum isolates. Twenty-three completed/draft genomes were also downloaded from the NCBI database and analysed. MLST of Turkish isolates was achieved with a well-established 7-gene scheme. Tenacibaculum maritimum genomes carry a structurally complete but partially defective class II CRISPRcas locus due to known amino acid substitutions in encoded Cas9 proteins. Our spacer identification suggests that the host range of bacteriophage P2559Y and Vibrio phage nt-1 include T. maritimum and that the most recurrent infection recorded by isolates has been with Tenacibaculum phage PTm5. Thirty-eight isolates with this CRISPRcas locus belonged to 25 spoligotypes and to 24 sequence types by MLST, respectively. According to MLST, T. maritimum isolates from Turkey are most related to previously defined sequence types ST3, ST40 and ST41 isolates from Spain, Malta and France. CONCLUSIONS: The evaluated spoligotyping offers discriminatory power comparable to MLST. SIGNIFICANCE AND IMPACT OF THE STUDY: Spoligotyping has potential as a quick, easy and cheap tool for subtyping of T. maritimum isolates.

Environmental biofilm communities associated with early-stage common dentex (Dentex dentex) culture.

AIMS: To describe the biofilm microbiota associated with various feeding phases during larval common dentex (Dentex dentex) culture. METHODS AND RESULTS: A targeted metagenomic (metagenetic) study was performed by means of 16S rRNA gene-based PCR and NextGen pyrosequencing. The resulting dataset was scrutinized with microbial community analysis software (r packages) using r/Rstudio. While median observed and estimated alpha-diversities were 171 ± 38 and 207 ± 27 taxa, respectively, 72·1-85·8% of individual biofilm communities comprised only 27-46 taxa. Members of the genus Methylobacterium and family Rhodobacteraceae dominated biofilms formed during all feeding phases while genera Nannochloropsis and Tetraselmis microalgae were major constituents of biofilms during rotifer live feeding. Both potential fish pathogenic genera, for example, Vibrio and putatively probiotic taxa, for example, Phaeobacter gallaeciensis were identified. CONCLUSIONS: Relatively stable biofilm communities were identified during each feeding phase but varied significantly between feeding phases, most likely in response to the introduction of live feed/microalgae-associated bacteria into rearing tanks. SIGNIFICANCE AND IMPACT OF THE STUDY: The structure of the bacterial communities identified represents a 'template' for successful larval dentex culture and provides a foundation for future investigations into failed production cycles.

Real time PCR detection of Piscirickettsia salmonis from formalin-fixed paraffin-embedded tissues.

Piscirickettsia salmonis is the causative agent of piscirickettsiosis, a transmissible disease of salmonid fish. Diagnosis of piscirickettsiosis has traditionally been based upon identification of typical pathological changes by histological investigation, with confirmation by immunohistochemistry on formalin-fixed, paraffin-embedded tissues. However, implementation of more rapid confirmatory techniques, preferably with higher levels of sensitivity and possibilities for quantification, is desirable. A real-time polymerase chain reaction (PCR) assay was designed for specific detection of P. salmonis and tested on samples extracted from formalin-fixed paraffin-embedded material. Construction of a PCR-target mimic allowed determination of detection limits, linearity of the real-time PCR and quantitative detection of P. salmonis. The present study demonstrates the capability of the described real time PCR assay for detection of P. salmonis from paraffin-embedded material with a high degree of sensitivity and specificity. Implementation of this assay constitutes an important development for a rapid and secure diagnosis of piscirickettsiosis.

Organization of the antiseptic resistance gene qacA and Tn552-related beta-lactamase genes in multidrug- resistant Staphylococcus haemolyticus strains of animal and human origins.

A part (12 kb) of a plasmid containing the beta-lactamase genes of Tn552, the disinfectant resistance gene qacA, and flanking DNA has been cloned from a Staphylococcus haemolyticus isolate and sequenced. This region was used to map the corresponding regions in six other multiresistant S. haemolyticus isolates of human and animal origin. The organizations of the genetic structures were almost identical in all isolates studied. The beta-lactamase and qacA genes from S. haemolyticus have >99.9% identities at the nucleotide level with the same genes from S. aureus, demonstrating that various staphylococcal species able to colonize animal and human hosts can exchange the genetic elements involved in resistance to antibiotics and disinfectants. The use of antibiotics and disinfectants in veterinary practice and animal husbandry may also contribute to the selection and maintenance of resistance factors among the staphylococcal species. Different parts of the 12-kb section analyzed had high degrees of nucleotide identity with regions from several other different Staphylococcus aureus plasmids. This suggests the contribution of interplasmid recombination in the evolutionary makeup of this 12-kb section involving plasmids that can intermingle between various staphylococcal species. The lateral spread of resistance genes between various staphylococcal species is probably facilitated by the generation of large multiresistance plasmids and the subsequent interspecies exchange of them.

Stability of barley aleurone transcripts: Dependence on protein synthesis, influence of the starchy endosperm and destabilization by GA3.

We have studied the stability of Barley aleurone and embryo expressed (Balem) transcripts in aleurone layers. The Per1, Ole1 and Ole2 transcripts are abundant during desiccation and in dry resting seeds, while B12D and B22E transcripts are expressed mainly during seed maturation and germination. From 21 to 40 days post anthesis (DPA) incubation of aleurone layers resulted in a substantial, but differential reduction in the levels of these transcripts. In contrast, Balem transcript levels in aleurone layers of incubated embryoless grains were (except for B22E) similar to those of freshly dissected layers. Cycloheximide lowered transcript levels significantly. This indicates that a protein-synthesis-dependent mRNA-stabilizing mechanism is active in the aleurone cells when attached to the starchy endosperm. At the onset of seed desiccation (40 DPA), half-lives of transcripts to be stored in the dry seed were up to several days longer than the half-life of B22E, which decreases during seed maturation. While the Per1, Ole1 and Ole2 transcript levels decline rapidly in the aleurone layers of mature, germinating seeds, the genes are actively transcribed and their transcripts highly stable in the aleurone of incubated embryoless seeds. The expression of Ole1 and Ole2, as well as Per1, can be repressed 100-1 000-fold by gibberellic acid (GA3) in a dose-dependent manner. Abscisic acid can counteract the GA3 repression. Incubations with transcriptional and translational inhibitors indicate that GA3 inhibits the transcription of these genes and at the same time induces a protein-synthesis-dependent mechanism destabilizing their mRNA molecules present.

Horizontal transfer of a multi-drug resistance plasmid between coliform bacteria of human and bovine origin in a farm environment.

Multi-drug-resistant coliform bacteria were isolated from feces of cattle exposed to antimicrobial agents and humans associated with the animals. Isolates from both cattle and humans harbored an R plasmid of 65 kb (pTMS1) that may have been transferred between them due to selective antibiotic pressure in the farm environment.