A number of nucleic acid amplification assays (NAAs) have been employed to detect tubercle bacilli in clinical specimens for tuberculosis (TB) diagnosis. Among these, loop-mediated isothermal amplification (LAMP) is an NAA possessing superior isothermal reaction characteristics. In the present study, a set of six specific primers targeting the Mycobacterium tuberculosis 16S rRNA gene with high sensitivity was selected and a LAMP system (MTB-LAMP) was developed. Using this system, a total of 200 sputum samples from Nepalese patients were investigated. The sensitivity of MTB-LAMP in culture-positive samples was 100 % (96/96), and the specificity in culture-negative samples was 94.2 % (98/104, 95 % confidence interval 90.5-97.9 %). The positive and negative predictive values of MTB-LAMP were 94.1 and 100 %, respectively. These results indicate that this MTB-LAMP method may prove to be a powerful tool for the early diagnosis of TB.

Clostridium perfringens is a causative agent of food-borne gastroenteritis for which C. perfringens enterotoxin (CPE) has been considered an essential factor. Recently, we experienced two outbreaks of food-borne gastroenteritis in which non-CPE producers of C. perfringens were strongly suspected to be the cause. Here, we report a novel enterotoxin produced by C. perfringens isolates, BEC (binary enterotoxin of C. perfringens). Culture supernatants of the C. perfringens strains showed fluid-accumulating activity in rabbit ileal loop and suckling mouse assays. Purification of the enterotoxic substance in the supernatants and high-throughput sequencing of genomic DNA of the strains revealed BEC, composed of BECa and BECb. BECa and BECb displayed limited amino acid sequence similarity to other binary toxin family members, such as the C. perfringens iota toxin. The becAB genes were located on 54.5-kb pCP13-like plasmids. Recombinant BECb (rBECb) alone had fluid-accumulating activity in the suckling mouse assay. Although rBECa alone did not show enterotoxic activity, rBECa enhanced the enterotoxicity of rBECb when simultaneously administered in suckling mice. The entertoxicity of the mutant in which the becB gene was disrupted was dramatically decreased compared to that of the parental strain. rBECa showed an ADP-ribosylating activity on purified actin. Although we have not directly evaluated whether BECb delivers BECa into cells, rounding of Vero cells occurred only when cells were treated with both rBECa and rBECb. These results suggest that BEC is a novel enterotoxin of C. perfringens distinct from CPE, and that BEC-producing C. perfringens strains can be causative agents of acute gastroenteritis in humans. Additionally, the presence of becAB on nearly identical plasmids in distinct lineages of C. perfringens isolates suggests the involvement of horizontal gene transfer in the acquisition of the toxin genes.C lostridium perfringens, a spore-forming anaerobic rod, is a member of the normal intestinal flora in humans and animals and a component of soil and sewage microbiota (1-4). C. perfringens is the causative agent of various human diseases, including gas gangrene and food-borne gastroenteritis (5-10). The pathogenicity of C. perfringens is attributed to various toxins produced by the organism, including alpha, beta, epsilon, and iota toxins that classify C. perfringens isolates into five toxin types (A to E), theta toxin, NetB, and C. perfringens enterotoxin (CPE) (10-13).CPE, which is mainly produced by type A C. perfringens, is associated with human gastrointestinal (GI) illnesses, such as food-borne gastroenteritis, antibiotic-associated diarrhea, and sporadic diarrhea (14-16). CPE is a 35-kDa protein, and the cpe gene is located in the chromosome or on a plasmid (17)(18)(19)(20). After orally ingested CPE-positive C. perfringens reaches the GI tract, sporulating C. perfringens produces CPE, and the toxin causes clinical symptoms, such as diarrhea and abdominal cramping. In the clinical diagnosis of gastroente...

Histamine fish poisoning is caused by histamine-producing bacteria (HPB). Klebsiella pneumoniae and Klebsiella oxytoca are the best-known HPB in fish. However, 22 strains of HPB from fish first identified as K. pneumoniae or K. oxytoca by commercialized systems were later correctly identified as Raoultella planticola (formerly Klebsiella planticola) by additional tests. Similarly, five strains of Raoultella ornithinolytica (formerly Klebsiella ornithinolytica) were isolated from fish as new HPB. R. planticola and R. ornithinolytica strains were equal in their histamine-producing capabilities and were determined to possess the hdc genes, encoding histidine decarboxylase. On the other hand, a collection of 61 strains of K. pneumoniae and 18 strains of K. oxytoca produced no histamine.Histamine fish poisoning (HFP) caused by eating spoiled fish happens throughout the world (2, 3). HFP is usually a rather mild illness; however, serious complications, such as cardiac and respiratory manifestations, occur rarely in individuals with preexisting conditions (12). The implicated fish are mainly of the families Scomberesocidae and Scombridae (the so-called scombroid fish) and contain large amounts of histamine (21). A hazardous level of histamine is produced by the microbial decarboxylation of the free histidine in the muscular tissue of fish. Enteric bacteria have been reported to be the dominant histamine-producing bacteria (HPB) in fish (19). In 1979, Taylor et al. reported that histamine-producing Klebsiella pneumoniae strain T2 was isolated from spoiled tuna sashimi (20). K. pneumoniae has been the best-known HPB ever since that report, and Klebsiella oxytoca is also known as an HPB from fish (13). However, K. pneumoniae strain T2 was later sent to the American Type Culture Collection (now in Manassas, Va.) and identified as Klebsiella planticola (ATCC 43176) in 1987. This strain has been reported to possess the hdc genes, encoding pyridoxal phosphate-dependent histidine decarboxylase (8).In 1981, Bagley et al. proposed the name Klebsiella planticola for "Klebsiella species 2" to distinguish it from both K. pneumoniae and K. oxytoca (1). Moreover, K. planticola, together with Klebsiella ornithinolytica and Klebsiella terrigena, was classified in the new genus Raoultella in 2001 (5). Nevertheless, Raoultella planticola cannot be distinguished from K. pneumoniae or K. oxytoca by using commercialized systems, such as API 20E (Biomérieux, Marcy l'Etoile, France). Additional tests are necessary to differentiate R. planticola from Klebsiella species (14, 15). Similarly, in 1989 the name Klebsiella ornithinolytica was proposed for "NIH group 12" at the National Institute of Health, Tokyo, Japan, and "Klebsiella group 47" at the Centers for Disease Control, Atlanta, Ga., which showed positive reactions in indole production and ornithine decarboxylase tests (6, 18). K. ornithinolytica was also classified in the genus Raoultella.K. pneumoniae and K. oxytoca had been shown to be HPB in several reports, whereas R. planticola had ...

Histamine-producing bacteria (HPB) such as Photobacterium phosphoreum and Raoultella planticola possess histidine decarboxylase (HDC), which converts histidine into histamine. Histamine fish poisoning (HFP) is attributable to the ingestion of fish containing high levels of histamine produced by HPB. Because freezing greatly decreases the histamine-producing ability of HPB, especially of P. phosphoreum, it has been speculated that HFP is caused by HDC itself from HPB cells autolyzing during frozen storage, even when HPB survive frozen storage. Here we constructed recombinant HDCs of P. phosphoreum, Photobacterium damselae, R. planticola, and Morganella morganii and investigated the ability of HDCs to produce sufficient histamine to cause HFP. To elucidate the character of these HDCs, we examined the specific activity of each recombinant HDC at various temperatures, pH levels, and NaCl concentrations. Further, we also investigated the stability of each HDC under different conditions (in reaction buffer, tuna, and dried saury) at various temperatures. P. damselae HDC readily produced sufficient histamine to cause HFP in fish samples. We consider that if HDC is implicated as an independent cause of HFP in frozen-thawed fish, the most likely causative agent is HDC of P. damselae.Histamine fish poisoning (HFP) is a food-borne chemical intoxication caused by the ingestion of histidine-rich scombroid fish such as tuna, bonito, and mackerel in which histamine-producing bacteria (HPB) produce a large amount of histamine (1,22). HPB isolated from fish implicated in actual HFP incidents reported to date include Raoultella planticola, Morganella morganii, Hafnia alvei, and Photobacterium phosphoreum (11,15). Enteric bacteria such as R. planticola and M. morganii are reported to be the dominant HPB in fish, whereas P. phosphoreum and Photobacterium damselae are frequently isolated from fish subjected to appropriate examination for marine bacteria, such as by the addition of NaCl to the culture medium and avoidance of exposure of the bacterium to high temperatures (11,16,17). The former is a psychrotrophic bacterium in the marine environment which has greater histamine-producing activity than enteric bacteria at low temperatures (11). The latter is also a prolific HPB but a mesophilic marine bacterium (13).It has been shown that accumulation of histamine by HPB occurs after the level of bacterial growth exceeds 10 7 CFU/ml in culture medium (19). Viable cell counts of P. phosphoreum and R. planticola have been reported to decrease during frozen storage (2,8). Moreover, the histidine-producing capability of HPB decreases due to injury from freezing, even if the HPB survive frozen storage (6, 21). Tuna, a scombroid fish frequently implicated in HFP, is commonly frozen during fishing and distribution (3). Other scombroid fish are also frequently frozen in current practice. These factors have led to speculation that the accumulation of histamine in thawed fish arises from the release of histidine decarboxylase (HDC) from the autol...

Azo dyes are regarded as pollutants because they are not readily reduced under aerobic conditions. Bacillus sp. OY1-2 transforms azo dyes into colorless compounds, and this reduction is mediated by a reductase activity for the azo group in the presence of NADPH. A 1.2-kbp EcoRI fragment containing the gene that encodes azoreductase was cloned by screening the genomic library of Bacillus sp. OY1-2 with digoxigenin-labeled probe designed from the N-terminal amino acid sequence of the purified enzyme. An open reading frame encoding the azoreductase, consisting of 178 amino acids, was predicted from the nucleotide sequence. In addition, because only a Bacillus subtillis hypothetical protein was discovered in the public databases (with an amino acid identity of 52.8%), the gene encoding the azoreductase cloned in this study was predicted to be a member of a novel family of reductases. Southern blot analysis revealed that the azoreductase gene exists as a single copy gene on a chromosome. Escherichia coli-expressing recombinant azoreductase gave a ten times greater reducing activity toward azo dyes than the original Bacillus sp. OY1-2. In addition, the expressed azoreductase purified from the recombinant E. coli lysate by Red-Sepharose affinity chromatography showed a similar activity and specificity as the native enzyme. This is the first report describing the sequencing and characterization of a gene encoding the azo dye-reducing enzyme, azoreductase, from aerobic bacteria and its expression in E. coli.