Futoshi Aranishi

Relationship between gyrA Mutations and Quinolone Resistance in Flavobacterium psychrophilum Isolates

ABSTRACT Flavobacterium psychrophilum is the causative agent of the fish diseases called bacterial cold-water disease and rainbow trout fry syndrome. It has been reported that some isolates of F. psychrophilum are resistant to quinolones; however, the mechanism of this quinolone resistance has been unexplained. In this study, we examined the quinolone susceptibility patterns of 27 F. psychrophilum strains isolated in Japan and the United States. Out of 27 isolates, 14 were resistant to both nalidixic acid (NA) and oxolinic acid (OXA), and the others were susceptible to NA and OXA. When amino acid sequences deduced from gyrA nucleotide sequences of all isolates tested were analyzed, two amino acid substitutions (a threonine residue replaced by an alanine or isoleucine residue in position 83 of GyrA [Escherichia coli numbering] and an aspartic acid residue replaced by a tyrosine residue in position 87) were observed in the 14 quinolone-resistant isolates. These results strongly suggest that, as in other gram-negative bacteria, DNA gyrase is an important target for quinolones in F. psychrophilum.

Genetic differences among three species of the genus Trichiurus (Perciformes: Trichiuridae) based on mitochondrial DNA analysis

The mitochondrial DNA segment encoding the 16S ribosomal RNA (16S rRNA) gene sequence (ca. 600u2009bp) was compared among Trichiurus sp. 2 (sensu Nakabo, 2002) (obtained from various areas of Japan), T. japonicus Temminck and Schlegel (collected from various localities within Japan), and true T. lepturus Linnaeus (caught off the Atlantic coast of the United States and Brazil) of the family Trichiuridae using 10, 10, and 15 specimens, respectively. Based on phylogenetic analysis using a neighbor-joining (NJ) algorithm, the haplotypes of Trichiurus sp. 2, T. japonicus, and T. lepturus indicated three distinct monophyletic lineages, being supported by 100% bootstrap values with no haplotypes overlapping or sharing among the lineages. Trichiurus sp. 2, T. japonicus, and T. lepturus are genetically different from each other, suggesting that they are three distinct species.

PCR-based detection of allergenic mackerel ingredients in seafood.

Fish is one of the most common causes of food allergy, especially in coastal countries such as Japan. An investigation conducted by the Japanese Government in 1997 reported that fish was ranked second among various causes of food allergy for adults and that approximately 20% of food allergic adults in Japan were sensitive to fish. Several fish species and seafood products are known to cause allergy, and were classified into four groups consisting of (i) salmon, sardine and mackerel, (ii) cod and tuna, (iii) octopus and squid, and (iv) crab and shrimp, by a cluster analysis of immunoglobulin reactivities of children (Tanaka et al. 2000). Scomber mackerel belonging to the family Scombridae are one of the most commercially important seafoods in Japan, where the annual consumption of Scomber mackerel is much larger than that of salmon, sardine, cod and tuna, all of which are also allergenic fish. Three species – Japanese mackerel (Scomber japonicus), Pacific mackerel (Scomber australasicus) and Atlantic mackerel (Scomber scombrus) – constitute a significant part of the absolute mackerel consumption, and are considered to be most frequently involved in incidents of fish allergy in Japan. Since Scomber mackerel possess unique characteristics in the skin, they are easily differentiated from other whole fish, but processed foods containing Scomber mackerel with the skin removed are difficult to identify. In addition, foods containing even a trace of Scomber mackerel sometimes cause serious allergenic episodes (Sackesen and Adalioglu 2003). In this study, we report a rapid and reliable method to detect and differentiate Scomber mackerel fish from other Perciformes marine fish by a simple PCR technique using new Scomber genus specific primers. Nucleotide sequences of the nuclear 5S ribosomal RNA gene (5S rDNA) were compared among 15 marine fish species belonging to 13 genera in 6 families of Perciformes. Alignment of their sequences allowed the selection of a pair of oligonucleotide primers specific for Scomber mackerel. These primers enabled PCR amplification of the flanking nontranscribed spacer region of the 5S rDNA from S. japonicus, S. australasicus and S. scombrus, but no amplification from other fish including closely related Scombridae fish. This simple and robust method can serve in routine detection and authentication of allergenic Scomber mackerel ingredients in seafood in food inspection laboratories.

Polymerase chain reaction–restriction fragment length polymorphism analysis for species identification of hairtail fish fillets from supermarkets in Japan

Hairtail fillets are marketed as both fresh and frozen forms in retail outlets within Japan. The present study was undertaken to develop a rapid and reliable method for identification of the hairtail species in commercial fillets. A total of 64 fillet samples, caught from various localities within Japan and purchased from various supermarkets, were analyzed. Polymerase chain reaction (PCR) amplification of a portion of the mitochondrial DNA encoding 16S ribosomal RNA gene and subsequent restriction digestion of the amplified products, using Vspl endonuclease, generated different restriction patterns indicating two different species of hairtails in the fillet samples. The results indicated that the commercial hairtail fillets, labeled and marketed as ‘Tachiuo’ in Japan, were comprised of two species of hairtails with Trichiurus japonicus (commonly called Tachiuo) accounting for 47% and Trichiurus sp. 2 (commonly called Tenjikutachi) accounting for 53% of the analyzed samples. This simple and inexpensive PCR-restriction fragment length polymorphism analysis can be routinely applied to determine species composition of hairtails in commercial fillets.

A novel mitochondrial intergenic spacer reflecting population structure of Pacific oyster

Nucleotide sequence divergence in a novel major mitochondrial DNA intergenic spacer (IGS) of Pacific oysterCrassostrea gigas was analyzed for 29 cultured individuals within the Goseong population (Korea). A total of 7 variable sites were detected within the IGS, and the relative frequency of nucleotide alteration was determined to be 1.16%. All alterations were due to a single nucleotide substitution, and 5 transitions and 2 transversions were observed. Among 29 specimens, only 8 haplotypes could be identified, and 6 of the haplotypes were unique to particular specimens. Pairwise genetic diversity of all 8 haplotypes was calculated to be 0.412 ± 0.134 from multiple sequence substitutions based on the two-parameter model. The phylogenetic tree obtained for these haplotypes according to the neighbor-joining method illustrated a single cluster of linkages, which comprised 5 haplotypes associated with 23 specimens, while the other 3 haplotypes associated with 6 specimens were scattered. The results indicate that the IGS is higher polymorphic and thus more suitable as a genetic marker for population structure analysis of Pacific oyster than the mtDNA coding regions, such as cytochromec oxidase I and 16S ribosomal RNA genes.

Plasmid Profiling of Japanese Flavobacterium psychrophilum Isolates

Abstract Flavobacterium psychrophilum is the causative agent of bacterial coldwater disease and rainbow trout fry syndrome in salmonids, cyprinids, and ayu Plecoglossus altivelis. To investigate genetic variability of this bacterium, plasmid profiling analysis was undertaken in 104 isolates of F. psychrophilum from Japan (n = 92), Europe (n = 9) and the USA (n = 3). Plasmids were isolated by a simple and inexpensive procedure using silica particles and then analyzed by agarose gel electrophoresis. Seventy-two of 104 isolates (69.2%) possessed plasmids, which were distributed at 2.8 kilobases (kb) (n = 33), 3.4 kb (n = 15), 4.1 kb (n = 23), and 5.2 kb (n = 1). The type strain of F. psychrophilum possessed a 2.8-kb plasmid, and a 3.4-kb plasmid was found mainly in isolates from rainbow trout O. mykiss. The 4.1-kb and 5.2-kb plasmids were found in Japanese isolates. Although the functions of these plasmids remain cryptic, no plasmids were associated with antibiotic resistance to ampicillin, sodium nifurstyre...

EngineeredXcmI cassette-containing vector for PCR-based phylogenetic analyses

A simple and general method is described to construct a new vector bearing a synthetic XcmI cassette for direct cloning of PCR-amplified genes of interest. Cleavage of the vector with XcmI generates a linearized molecule with a single thymidine (T) overhang at the 3′ ends (T-vector) that facilitates TA cloning of PCR products carrying complementary 3′ adenosine (A) residue, without modification. Once constructed, this newly designed circularized vector (pEMX) is reproducible in any molecular biology laboratory, and is a high performance and cost effective alternative to commercially available T-vectors. PCR-based DNA analysis has become a widely used tool in investigating genetic relationships in naturally occurring and aquaculture populations of marine organisms. In general, genes of interest are amplified by PCR using thermostable DNA polymerase, and subsequently cloned into adequate vectors for sequencing and other manipulations. Taq polymerase, the most widely used PCR enzyme, adds a single adenosine at the 3′ ends of PCR products due to the terminal transferase activity (Clark 1988). PCR products derived using such polymerases can be directly cloned into vectors with complementary 3′ thymidine overhangs (T-vectors). T-vectors are routinely utilized for sequence analysis of 5S ribosomal RNA genes (5S rDNA) as a genetic marker to assess phylogenetic relationships in marine organisms (Martins and Galetti 2001). Multiple products encoding various sizes of multilocus 5S rDNA are amplified from a single fish species by PCR, and each product is separately cloned into a T-vector. This requires large numbers of T-vectors for constructing 5S rDNA library of even a single species. Moreover, commercially available T-vectors, such as pGEM-T (Promega) and pT7Blue (Novagen), are expensive and cannot be regenerated in the laboratory for further use. We describe here the development of a simple and general method for constructing T-vectors bearing an oligonucleotide cassette that produces complementary 3′ thymidine overhangs by restriction enzyme digestion.