We coupled multiplex PCR and a DNA microarray to construct an assay suitable for the simultaneous detection of five important marine fish pathogens ( Vibrio vulnificus , Listonella anguillarum , Photobacterium damselae subsp. damselae , Aeromonas salmonicida subsp. salmonicida , and Vibrio parahaemolyticus ). The array was composed of nine short oligonucleotide probes (25-mer) complementary to seven chromosomal loci ( cyt , rpoN , gyrB , toxR , ureC , dly , and vapA ) and two plasmid-borne loci ( fatA and A.sal). Nine primer sets were designed to amplify short fragments of these loci (100 to 177 bp) in a multiplex PCR. PCR products were subsequently labeled by nick translation and hybridized to the microarray. All strains of the five target species ( n = 1 to 21) hybridized to at least one species-specific probe. Assay sensitivities ranged from 100% for seven probes to 83 and 67% for the two remaining probes. Multiplex PCR did not produce any nonspecific amplification products when tested against 23 related species of bacteria ( n = 40 strains; 100% specificity). Using purified genomic DNA, we were able to detect PCR products with <20 fg of genomic DNA per reaction (equivalent to four or five cells), and the array was at least fourfold more sensitive than agarose gel electrophoresis for detecting PCR products. In addition, our method allowed the tentative identification of virulent strains of L. anguillarum serotype O1 based on the presence of the fatA gene (67% sensitivity and 100% specificity). This assay is a sensitive and specific tool for the simultaneous detection of multiple pathogenic bacteria that cause disease in fish and humans.

Two Gram-negative, rod-shaped, gliding bacterial strains, designated strain LL04 11.1.1T and strain A37.1T, were isolated from a diseased sole (Solea senegalensis) and from seawater from a holding tank for turbot (Psetta maxima), respectively. The strains grew on solid media as bright yellow colonies with uneven edges; the colonies did not adhere to the agar. The bacteria were able to grow at temperatures in the range 14 to 38 °C and from pH 6.0 to pH 8.0. The DNA G+C contents of strains LL04 11.1.1T and A37.1T were 32.1 and 32.7 mol%, respectively. Analysis of the 16S rRNA gene sequences indicated that strains LL04 11.1.1T and A37.1T were members of the genus Tenacibaculum in the family Flavobacteriaceae. The sequence similarities of the two isolates with respect to the type strains of recognized members of the genus ranged from 94.2 to 99.4 %. DNA–DNA hybridization studies revealed that the strains studied represent two distinct novel species of the genus Tenacibaculum, for which the names Tenacibaculum discolor sp. nov. [type strain LL04 11.1.1T (=NCIMB 14278T=DSM 18842T)] and Tenacibaculum gallaicum sp. nov. [type strain A37.1T (=NCIMB 14147T=DSM 18841T)] are proposed.

A new series of amino/nitro-substituted 3-arylcoumarins were synthesized and their antibacterial activity against clinical isolates of Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative) was evaluated. Some of these molecules exhibited antibacterial activity against S. aureus comparable to the standards used (oxolinic acid and ampicillin). The preliminary structure-activity relationship (SAR) study showed that the antibacterial activity against S. aureus depends on the position of the 3-arylcoumarin substitution pattern. With the aim of finding the structural features for the antibacterial activity and selectivity, in the present manuscript different positions of nitro, methyl, methoxy, amino and bromo substituents on the 3-arylcoumarin scaffold were reported.

The pathobiological activities in vivo and in vitro of live cells and extracellular products (ECP) of eleven Pasteurella piscicida strains of different origin were examined. Infectivity trials showed that P. piscicida did not possess strict host specificity since the majority of the isolates were virulent for gilthead seabream, rainbow trout and turbot, with LD50 values ranging between 10(3) and 10(6) live cells. However, none of the strains tested were pathogenic for mice (LD50 > 10(8) cells)). In addition, the ECP were strongly toxic for fish (LD50 ranging from 1.0 to 4.6 micrograms protein per g fish), which clearly demonstrates their important role in the pathogenesis of pasteurellosis. All the ECP samples were cytotoxic for fish and homoiothermic cell lines, possessed notable phospholipase activity and displayed haemolytic activity for sheep, salmon and turbot erythrocytes (but not for trout erythrocytes). However, the production of proteolytic enzymes differed among the P. piscicida strains. Although no strain displayed elastase activity, five isolates (the Japanese and Italian strains) hydrolysed casein and gelatin. All these biological activities in vivo and in vitro were lost after heat treatment (100 degrees C for 10 min). The general enzymic patterns of both live cells and ECP evaluated by the API-ZYM system also revealed some variation among the P. piscicida isolates. Generally, whole cells showed a wider range of enzymic activities than ECP. The results presented here are important for the selection of strains in the development of effective polyvalent pasteurellosis vaccines containing both whole cells and ECP.