Next Generation Sequencing (NGS) combined with powerful bioinformatic approaches are revolutionising food microbiology. Whole genome sequencing (WGS) of single isolates allows the most detailed comparison possible hitherto of individual strains. The two principle approaches for strain discrimination, single nucleotide polymorphism (SNP) analysis and genomic multi-locus sequence typing (MLST) are showing concordant results for phylogenetic clustering and are complementary to each other. Metabarcoding and metagenomics, applied to total DNA isolated from either food materials or the production environment, allows the identification of complete microbial populations. Metagenomics identifies the entire gene content and when coupled to transcriptomics or proteomics, allows the identification of functional capacity and biochemical activity of microbial populations. The focus of this review is on the recent use and future potential of NGS in food microbiology and on current challenges. Guidance is provided for new users, such as public health departments and the food industry, on the implementation of NGS and how to critically interpret results and place them in a broader context. The review aims to promote the broader application of NGS technologies within the food industry as well as highlight knowledge gaps and novel applications of NGS with the aim of driving future research and increasing food safety outputs from its wider use.

A total of 11 strains of moderately halophilic histamine-producing bacteria isolated from fermented squid liver sauce were studied phenotypically, genotypically, and phylogenetically. These strains are considered members of the genus Tetragenococcus based on their physiological, morphological, and chemotaxonomic characteristics. A 16s rRNA gene sequence analysis showed that these strains clustered with, but were separate from, Tetragenococcus halophilus. The results of DNA-DNA hybridization experiments indicated that the new isolates represent a new Tetragenococcus species, for which we propose the name Tetragenococcus muriaticus; strain X-1 (= JCM 10006) is the type strain of this species.Pediococcus halophilus was described as a halophilic lactic acid bacterium which requires sodium chloride (NaC1) for growth and is tolerant of a high NaCl concentration (more than 18%). According to Bergey's Manual of Systematic Bacteriology (6), tetrad cocci which are tolerant of 18% NaCl are members of P. haloThilus. Recently, however, Collins et al. (4) reported that P. halophilus is phylogenetically more closely related to enterococci and lactobacilli than to pediococci on the basis of 16s 16s rRNA sequence data, and thus they proposed that P. halophilus should be reclassified in a new genus, the genus Tetragenococcus, with Tetragenococcus halophilus as the single species recognized in the genus (2).Previous workers have isolated large numbers of T. halophilus as the predominant bacteria (7, 8) from Shottsuru, a Japanese local traditional fermented fish sauce, which contains more than 25% NaC1. In this study, we isolated histamineproducing lactic acid bacteria from Shottsuru-like squid liver sauce. Eleven strains were designated strains X-lT to X-11 and were different from authentic T. halophilus strains in the following characteristics: production of histamine, lack of L-arabinose fermentation, growth at 40°C, and 16s rRNA gene (rDNA) sequence. To precisely identify and phylogenetically place the new isolates, phenotypic characterization, analysis of fatty acid compositions, 16s rDNA sequencing, and DNA-DNA hybridization were performed. On the basis of our results we propose that our isolates should be placed in a new species, Tetragenococcus muriaticus. MATERIALS AND METHODSBacterial strains and growth conditions. A total of 11 strains (X-lT to X-11) were isolated from squid liver sauce, a Japanese local traditional fermented seafood. These strains were maintained on stab cultures in 10% NaCl-GYP agar (lo), which was supplemented with 10% sodium chloride, 1% magnesium sulfate, 0.1% potassium chloride, 0.5% calcium carbonate, and 0.3% agar. The pH was adjusted to 7.0 with 1.0 N NaOH. The cultures were incubated for about 2 days at 30°C. The strains were stored at 4°C and were transferred to fresh medium every third month. Unless indicated otherwise, the inocula used for phenotypic tests were prepared from 48-to 60-h-old cultures in 10% NaCl-GYP broth and incubated at 30°C.Morphology. Cell morphology was observed by using ...

The use of molecular tools for early and rapid detection of gram-negative histamine-producing bacteria is important for preventing the accumulation of histamine in fish products. To date, no molecular detection or identification system for gram-negative histamine-producing bacteria has been developed. A molecular method that allows the rapid detection of gram-negative histamine producers by PCR and simultaneous differentiation by single-strand conformation polymorphism (SSCP) analysis using the amplification product of the histidine decarboxylase genes (hdc) was developed. A collection of 37 strains of histamine-producing bacteria (8 reference strains from culture collections and 29 isolates from fish) and 470 strains of non-histamineproducing bacteria isolated from fish were tested. Histamine production of bacteria was determined by paper chromatography and confirmed by high-performance liquid chromatography. Among 37 strains of histamineproducing bacteria, all histidine-decarboxylating gram-negative bacteria produced a PCR product, except for a strain of Citrobacter braakii. In contrast, none of the non-histamine-producing strains (470 strains) produced an amplification product. Specificity of the amplification was further confirmed by sequencing the 0.7-kbp amplification product. A phylogenetic tree of the isolates constructed using newly determined sequences of partial hdc was similar to the phylogenetic tree generated from 16S ribosomal DNA sequences. Histamine accumulation occurred when PCR amplification of hdc was positive in all of fish samples tested and the presence of powerful histamine producers was confirmed by subsequent SSCP identification. The potential application of the PCR-SSCP method as a rapid monitoring tool is discussed.

We encountered two cases of fatal necrotizing fasciitis caused by Photobacterium damsela in Japan. Both cases occurred in fishermen who became sick after fishing. They developed multiple organ failure within 20 to 36 h from the onset of initial symptoms despite intensive chemotherapy and surgical treatments. CASE REPORTS Case 1.A 58-year-old fisherman with a history of diabetes mellitus noticed mild itching on his left wrist after fishing off the east coast of Okinawa on 16 December 1998. Complaints of developing hyperemia and swelling of the hand made the patient consult a local clinic. He received medications, but the symptoms were not improved. Twelve hours later, the patient consulted another hospital for a more precise diagnosis and further treatment. On initial physical examination, the patient showed almost clear consciousness, a blood pressure of 110 over 70 mmHg, a pulse rate of 127/min, a body temperature of 37.4°C, and hyperventilation. He felt exquisite pain in his left upper limb. Patchy purple-gray discoloration of the skin of the left arm was noted. His left three fingers (i.e., middle to little fingers) became cold and hypesthesic. Initial laboratory tests showed a leukocyte count of 21,000 cells/mm 3 and a platelet count of 174,000/mm 3 . The results of standard coagulation tests, prothrombin time, activated partial thromboplastin time, and bleeding time were within normal limits. The C-reactive protein concentration was 3.14 mg/dl. Hyperglycemia (343 mg/ dl), glycosuria, and ketonuria were noted. Ultrasonography of the left hand and forearm showed a low-echoic area. Compartment syndrome due to staphylococcal infection was strongly suspected, and 1 g of cefotiam per day was administered intravenously only for 1 day. Emergency surgical exploration was performed under general anesthesia. A skin incision was made to the level of the distal biceps. No bleeding or intravenous thrombi were observed in the purple-gray skin area. Two hours after anesthesia, the patient became anuric and hyperkalemic (6.6 mEq/liter). Emergency hemodialysis was prepared, but the patient went into cardiac arrest 20 h after the initial itchy feeling in his left wrist. Tubular necrosis without intravascular thrombosis was observed in the kidneys at autopsy, and myoglobin was not detected in the urine by immunohistochemical tests.Case 2. The second case was a 76-year-old fisherman with no special past medical history. He suffered a minor puncture wound to his right thumb from the fin of a sea bass on 18 September 2000 in Okayama. The wound was so slight that the patient did not seek any treatment. About 27 h later, he suffered from severe pain, swelling, and flaring of the palm, so he consulted a local clinic, where the wound was sanitized and an anti-inflammatory drug was prescribed. However, 36 h later, the severe pain and swelling persisted, and the symptoms had spread to the right shoulder. The patient was then transferred to an emergency medical center. On initial physical examination, he appeared to be sick and somnolen...

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