Enterotoxigenic profiles and polymerase chain reaction detection of Bacillus cereus group cells and B. cereus strains from foods and food-borne outbreaks

Bacillus cereus is one of the important food pathogens. Since B. cereus group cells, such as B. cereus, B. thuringiensis, B. anthracis and B. mycoides, share many phenotypical properties and a high level of chromosomal sequence similarity, it is interesting to investigate the virulence profiles for B. cereus group cells, including B. cereus strains isolated from foods and samples associated with food-poisoning outbreaks. For this investigation, the presence of enterotoxin genes, such as those of haemolysin BL, B. cereus enterotoxin T and enterotoxin FM, were assayed by polymerase chain reaction (PCR) methods. Meanwhile, their enterotoxin activities were assayed using the BCET-RPLA kit, haemolytic patterns on sheep blood agar and their cytotoxicity to Chinese hamster ovary (CHO) cells. Results showed that there were 12 enterotoxigenic profiles for the 98 B. cereus group strains collected. In addition, if any of the three types of enterotoxins was present in the B. cereus group cells, these cells were shown to be cytotoxic to the CHO cells. Similar enterotoxigenic profiles could be found among strains of B. cereus, B. mycoides and B. thuringiensis. Thus, all B. cereus group strains may be potentially toxigenic and the detection of these cells in foods is important. We thus designed PCR primers, termed Ph1/Ph2, from the sphingomyelinase gene of B. cereus cells. These primers were specific for all B. cereus group strains and could be used for the detection of B. cereus cells contaminated in food samples.     

Germination and proliferation of emetic Bacillus cereus sensu lato strains in milk

The Bacillus cereus sensu lato group includes potentially pathogenic bacteria that are ubiquitous in the environment and, importantly, could also be present in food products. This study focuses on emetic isolates which presumably could cause acute food poisoning and emetic syndrome. Here, we evaluate the ability of psychrotolerant Bacillus weihenstephanensis MC118 (isolated from soil) and mesophilic B. cereus BOD3/9 isolated from milk to germinate and multiply at 7 and 30 °C. Whereas the rates of germination at 30 °C in milk and nutrient broth of MC118 and BOD3/9 were similar, MC118, but not BOD3/9, proliferated to achieve relatively high numbers (～10⁶?colony-forming units/g) within 7 days of incubation at 7 °C. Mesophilic BOD3/9 showed a slight decrease of cell concentration in similar studies at 7 °C. Genotyping with repetitive extragenic palindromic sequence-based PCR and pulsed field gel electrophoresis revealed significant similarities between BOD3/9 and emetic reference B. cereus F4810/72 strain, while the B. weihenstephanensis MC118 isolate was more similar to the B. weihenstephanensis non-emetic reference DSMZ11821 strain. Our data suggest that emetic isolates that are also psychrotolerant, such as MC118, could constitute a hazard in the dairy industry, where milk could be a suitable medium for germination and growth.     

Application of sonication to release DNA from Bacillus cereus for quantitative detection by real-time PCR

A rapid sonication method for lysis of Gram-positive bacteria was evaluated for use in combination with quantitative real-time polymerase chain reaction (PCR) analyses for detection. Other criteria used for evaluation of lysis were microscopic cell count, colony forming units (cfu), optical density at 600 nm and total yield of DNA measured by PicoGreen fluorescence. The aim of this study was complete disruption of cellular structures and release of DNA without the need for lysing reagents and time-consuming sample preparation. The Gram-positive bacterium Bacillus cereus was used as a model organism for Gram-positive bacteria. It was demonstrated by real-time PCR that maximum yield of DNA was obtained after 3 to 5 min of sonication. The yield of DNA was affected by culture age and the cells from a 4-h-old culture in the exponential phase of growth gave a higher yield of DNA after 5 min of sonication than a 24-h-old culture in the stationary phase of growth. The 4-h-old culture was also more sensitive for lysis caused by heating. The maximum yield of DNA, evaluated by real-time PCR, from a culture of the Gram-negative bacterium Escherichia coli, was obtained after 20 s of sonication. However, the yield of target DNA from E. coli rapidly decreased after 50 s of sonication due to degradation of DNA. Plate counting (cfu), microscopic counting and absorbance at 600 nm showed that the number of viable and structurally intact B. cereus cells decreased rapidly with sonication time, whereas the yield of DNA increased as shown by PicoGreen fluorescence and real-time PCR. The present results indicate that 3-5 min of sonication is sufficient for lysis and release of DNA from samples of Gram-positive bacteria.     

Rapid genotypic detection of Bacillus anthracis and the Bacillus cereus group by multiplex real-time PCR melting curve analysis

Bacillus anthracis has four plasmid possible virulence genotypes: pXO1+/pXO2+, pXO1+/pXO2-, pXO1-/pXO2+ or pXO1-/pXO2-. Due to the lack of a specific chromosomal marker for B. anthracis, differentiation of the pXO1-/pXO2- form of B. anthracis from closely related Bacillus cereus group species is difficult. In this study, we evaluate the ability of sspE, pXO1 and pXO2 primers to discriminate individual B. anthracis and the B. cereus group genotypes using multiplex real-time PCR and melting curve analysis. Optimal conditions for successful multiplex assays have been established. Purified DNAs from 38 bacterial strains including 11 strains of B. anthracis and 18 B. cereus group strains were analyzed. Nine of the B. cereus group near-neighbor strains were shown by multilocus sequence typing to be phylogenetically proximate to the B. anthracis clade. We have demonstrated that the four plasmid genotypes of B. anthracis and B. cereus group near-neighbors were differentially and simultaneously discriminated by this assay.     

A new rapid and sensitive detection method for cereulide-producing Bacillus cereus using a cycleave real-time PCR

Aims:  To develop a rapid and sensitive detection method for cereulide-producing Bacillus cereus using a real-time PCR based on the sequence of the cereulide synthesis gene.
Methods and Results:  A total of 56 cereulide-producing B. cereus and 15 cereulide-negative strains were tested. We designed specific primers and probes for the detection of cereulide-producing B. cereus . The new cycleave real-time PCR assay gave positive detections for all of 56 cereulide-producing B. cereus strains, whereas all other strains including 10 systemic infectious disease strains were negative. No cross-reaction was observed and the internal control showed positive for all samples.
Conclusions:  The performance of the assay was highly reproducible and specific for cereulide-producing B. cereus . The positive detection was obtained within only 2 h for cereulide-producing strains. The detection limit of this assay was evaluated as 10⁴ CFU g⁻¹ food sample. The assay also confirmed that strains from systemic infectious cases were cereulide-negative.
Significance and Impact of the Study:  This assay is applicable for contaminated foods as well as specimens from infectious disease cases. We recommend this assay for routine examination of suspected B. cereus food poisonings.     

Identification of emetic toxin producing Bacillus cereus strains by a novel molecular assay

Bacillus cereus causes two types of gastrointestinal diseases: emesis and diarrhea. The emetic type of the disease is attributed to the heat-stable depsipeptide cereulide and symptoms resemble Staphylococcus aureus intoxication, but there is no rapid method available to detect B. cereus strains causing this type of disease. In this study, a polymerase chain reaction (PCR) fragment of unknown function was identified, which was shown to be specific for emetic toxin producing strains of B. cereus. The sequence of this amplicon was determined and a PCR assay was developed on this basis. One hundred B. cereus isolates obtained from different food poisoning outbreaks and diverse food sources from various geographical locations and 29 strains from other species belonging to the B. cereus group were tested by this assay. In addition, 49 non-B. cereus group strains, with special emphasis on food pathogens, were used to show that the assay is specific for emetic toxin producing B. cereus strains. The presented PCR assay is the first molecular tool for the rapid detection of emetic toxin producing B. cereus strains.     

Detection of enterotoxic Bacillus cereus and Bacillus thuringiensis strains by PCR analysis

Many strains of Bacillus cereus cause gastrointestinal diseases, and the closely related insect pathogen B. thuringiensis has also been involved in outbreaks of diarrhea. The diarrheal types of diseases are attributed to enterotoxins. Two different enterotoxic protein complexes, hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE), and an enterotoxic protein, enterotoxin T, have been characterized, and the genes have been sequenced. PCR primers for the detection of these genes were deduced and used to detect the genes in 22 B. cereus and 41 B. thuringiensis strains. At least one gene of each of the two protein complexes HBL and NHE was detected in all of the B. thuringiensis strains, while six B. cereus strains were devoid of all three HBL genes, three lacked at least two of the three NHE genes, and one lacked all three. Five different sets of primers were used for detection of the gene (bceT) encoding enterotoxin T. The results obtained with these primer sets indicate that bceT is widely distributed among B. cereus and B. thuringiensis strains and that the gene varies in sequence among different strains. PCR with the two primer sets BCET1-BCET3 and BCET1-BCET4 unambiguously detected the bceT gene, as confirmed by Southern analysis. The occurrence of the genes within the two complexes is significantly associated, while neither the occurrence of the two complexes nor the occurrence of the bceT gene is significantly associated in the 63 strains. We suggest an approach for detection of enterotoxin-encoding genes in B. cereus and B. thuringiensis based on PCR analysis with the six primer sets for the detection of genes in the HBL and NHE operons and with the BCET1, BCET3, and BCET4 primers for the detection of bceT. PCR analysis of the 16S-23S rRNA gene internal transcribed spacer region revealed identical patterns for all strains studied.     

Diagnostic real-time PCR for detection of Salmonella in food

A robust 5' nuclease (TaqMan) real-time PCR was developed and validated in-house for the specific detection of Salmonella in food. The assay used specifically designed primers and a probe target within the ttrRSBCA locus, which is located near the Salmonella pathogenicity island 2 at centisome 30.5. It is required for tetrathionate respiration in Salmonella. The assay correctly identified all 110 Salmonella strains and 87 non-Salmonella strains tested. An internal amplification control, which is coamplified with the same primers as the Salmonella DNA, was also included in the assay. The detection probabilities were 70% when a Salmonella cell suspension containing 10(3) CFU/ml was used as a template in the PCR (5 CFU per reaction) and 100% when a suspension of 10(4) CFU/ml was used. A pre-PCR sample preparation protocol including a preenrichment step in buffered peptone water followed by DNA extraction-purification was applied when 110 various food samples (chicken rinses, minced meat, fish, and raw milk) were investigated for Salmonella. The diagnostic accuracy was shown to be 100% compared to the traditional culture method. The overall analysis time of the PCR method was approximately 24 h, in contrast to 4 to 5 days of analysis time for the traditional culture method. This methodology can contribute to meeting the increasing demand of quality assurance laboratories for standard diagnostic methods. Studies are planned to assess the interlaboratory performance of this diagnostic PCR method.     

Phenotypic homogeneity of emetic Bacillus cereus isolates in China

Emetic Bacillus cereus strains produce a potent cereulide cytotoxin, which can cause acute and fatal cases of food poisoning. We isolated 18 emetic B. cereus strains from a food poisoning event, and from clinical and non-random food surveillance in China and phenotypic characteristics of haemolysis, starch hydrolysis, salicin fermentation, gelatin liquefaction, cytotoxicity, and susceptibility to antibiotics were assessed. All isolates were positive for haemolysis and gelatin liquefaction, and negative for starch hydrolysis and salicin fermentation. Their haemolytic potentials were intermediate to Bacillus anthracis and B. cereus ATCC 14579 (a non-emetic strain). All isolates were cytotoxic to CHO, Hep-2, and Vero cells, and were sensitive to ampicillin. The homogeneous phenotypes of emetic isolates from China are similar to the corresponding traits of European and Japanese isolates that have been characterized, suggesting highly similar phenotypes of emetic B. cereus worldwide.     

Toxin gene profiling of enterotoxic and emetic Bacillus cereus

Very different toxins are responsible for the two types of gastrointestinal diseases caused by Bacillus cereus: the diarrhoeal syndrome is linked to nonhemolytic enterotoxin NHE, hemolytic enterotoxin HBL, and cytotoxin K, whereas emesis is caused by the action of the depsipeptide toxin cereulide. The recently identified cereulide synthetase genes permitted development of a molecular assay that targets all toxins known to be involved in food poisoning in a single reaction, using only four different sets of primers. The enterotoxin genes of 49 strains, belonging to different phylogenetic branches of the B. cereus group, were partially sequenced to encompass the molecular diversity of these genes. The sequence alignments illustrated the high molecular polymorphism of B. cereus enterotoxin genes, which is necessary to consider when establishing PCR systems. Primers directed towards the enterotoxin complex genes were located in different CDSs of the corresponding operons to target two toxin genes with one single set of primers. The specificity of the assay was assessed using a panel of B. cereus strains with known toxin profiles and was successfully applied to characterize strains from food and clinical diagnostic labs as well as for the toxin gene profiling of B. cereus isolated from silo tank populations.     

PCR法快速鉴定眼源性蜡样芽胞杆菌

目的建立一种快速、灵敏、特异的眼源性蜡样芽胞杆菌PCR检测方法,为蜡样芽胞杆菌性眼内炎患者的快速诊断提供依据。方法选择编码蜡样芽胞杆菌细胞毒素的cytK为靶基因设计引物,建立检测眼源性蜡样芽胞杆菌PCR;PCR产物用琼脂糖凝胶电泳鉴定,基因序列与GenBank比对验证扩增产物;将计数过的5株蜡样芽胞杆菌菌悬液,梯度稀释后分别提取DNA进行PCR扩增,确定检测方法的灵敏度;分别用眼部常见感染菌金黄色葡萄球菌、表皮葡萄球菌、甲型溶血性链球菌、化脓性链球菌、藤黄微球菌、铜绿假单胞菌、大肠埃希菌、普通变形杆菌和白假丝酵母菌以及枯草芽胞杆菌DNA进行特异性试验;进一步将该方法应用到人工污染致病蜡样芽胞杆菌的房水标本中,并分析其灵敏度。结果5株分离自眼内炎患者标本中的蜡样芽胞杆菌均扩增出360bp左右的DNA片段,测序结果与GenBank比对一致;该法检测在5h内完成,方法灵敏度达7．5～15．0CFU／mL;其他菌株检测未出现非特异性扩增;对模拟感染房水标本的PCR鉴定结果与分离培养对比,二者符合率为100％,模拟标本的检测灵敏度与纯菌结果一致。结论cytK基因为靶基因的PCR用于眼源性蜡样芽胞杆菌的快速检测,具有简便、快速、敏感、特异等特点,为眼内炎患者的快速诊断提供依据,在实际检验工作中有良好的应用前景。     

Adaptation of the food-borne pathogen Bacillus cereus to carvacrol

Carvacrol, a natural antimicrobial compound present in the essential oil fraction of oregano and thyme, is bactericidal towards Bacillus cereus. A decrease of the sensitivity of B. cereus towards carvacrol was observed after growth in the presence of non-lethal carvacrol concentrations. A decrease of the melting temperature (Tm) of membranes from 20.5 degrees C to 12.6 degrees C was the immediate effect of the addition of carvacrol. Cells adapted to 0.4 mM carvacrol showed a lower membrane fluidity than nonadapted cells. Adaptation of 0.4 mM carvacrol increased the Tm from 20.5 degrees C to 28.3 degrees C. The addition of carvacrol to cell suspensions of adapted B. cereus cells decreased Tm again to 19.5 degrees C, approximately the same value as for the non-adapted cells in the absence of carvacrol. During adaptation, changes in the fatty acid composition were observed. The relative amount of iso-C13:0, C14:0, and iso-C15:0 increased and cis-C16:1 and C18:0 decreased. The head-group composition also changed, two additional phospholipids were formed and one phospholipid was lacking in the adapted cells. It could be concluded that B. cereus adapts to carvacrol when present at non-lethal concentrations in the growth medium by lowering its membrane fluidity by changing the fatty acid and headgroup composition.     

Development of real-time PCR assays for detection and quantification of Bacillus cereus group species: differentiation of B. weihenstephanensis and rhizoid B. pseudomycoides isolates from milk

Quantitative real-time PCR (qRT-PCR) offers an alternative method for the detection of bacterial contamination in food. This method provides the quantitation and determination of the number of gene copies. In our study, we established an RT-PCR assay using the LightCycler system to detect and quantify the Bacillus cereus group species, which includes B. cereus, B. anthracis, B. thuringiensis, B. weihenstephanensis, B. mycoides, and B. pseudomycoides. A TaqMan assay was designed to detect a 285-bp fragment of the motB gene encoding the flagellar motor protein, which was specific for the detection of the B. cereus group species, excluding B. pseudomycoides, and the detection of a 217-bp gene fragment of a hypothetical protein specific only for B. pseudomycoides strains. Based on three hydrolysis probes (MotB-FAM-1, MotB-FAM-2, and Bpm-FAM-1), it was possible to differentiate B. weihenstephanensis from the B. cereus group species with nonrhizoid growth and B. pseudomycoides from the whole B. cereus group. The specificity of the assay was confirmed with 119 strains belonging to the Bacillus cereus group species and was performed against 27 other Bacillus and non-Bacillus bacteria. A detection limit was determined for each assay. The assays performed well not only with purified DNA but also with DNA extracted from milk samples artificially contaminated with bacteria that belong to the B. cereus group species. This technique represents an alternative approach to traditional culture methods for the differentiation of B. cereus group species and differentiates B. weihenstephanensis and B. pseudomycoides in one reaction.     

Novel cost-efficient real-time PCR assays for detection and quantitation of Listeria monocytogenes

Listeriosis is a serious food-borne infection with mortality rates approaching 30%. Therefore, the rapid, cost-effective, and automated detection of Listeria monocytogenes throughout the food chain continues to be a major concern. Here we describe three novel quantitative real-time PCR assays for L. monocytogenes based on amplification of a target hlyA gene with SYBR Green I chemistry and hydrolysis probe (TaqMan MGB probe). In order to offer sensitive, rapid and robust tool of additional economical value the real-time PCR assays were designed and optimized to only 5 μl-reactions. All assays were evaluated by using different non-reference Listeria strains isolated from various food matrices. Results demonstrated specificity to L. monocytogenes with accurate quantification over a dynamic range of 5-6 log units with R² higher than 0.98 and amplification efficiencies reaching above 92%. The detection and quantification limits were as low as 165 genome equivalents. Comparison of novel assays to commercially available TaqMan® Listeria monocytogenes Detection Kit and previously published studies revealed similar specificity, sensitivity and efficiency, but greater robustness and especially cost-efficiency in the view of smaller reaction volumes and continuous increase in sample throughput.     

TaqMan real-time PCR versus four conventional PCR assays for detection of apple proliferation phytoplasma

A recently developed TaqMan real-time PCR assay for detection of apple proliferation phytoplasma was evaluated in comparison to four conventional PCR-based methods with the aim to assess its potential for research and routine applications. All five protocols were tested in parallel on the same DNA isolates obtained from orchard trees. The performance of the methods was evaluated by means of sensitivity, specificity, susceptibility to inhibition, handling effort, testing time, assay expenses, and potential risk for operator and environment. Compared to the conventional PCR methods, the TaqMan real-time PCR procedure combined the highest test sensitivity with the highest test specificity and was, above all, not susceptible to PCR inhibition. Furthermore, TaqMan real-time PCR had the simplest and fastest testing process, involving a minimum of handling steps. Its disadvantage is the high cost of consumables and reagents, exceeding that of a standard PCR procedure up to four-fold. However, the higher material costs could be compensated by considerably lower personnel costs and by saving expenses for hazardous waste disposal. Due to the simple testing procedure and the output of results as numeric data the TaqMan real-time PCR assay has a high potential for automation, and seems to represent the currently most suitable method for large-scale testing procedures.     

Bactericidal activity of carvacrol towards the food-borne pathogen Bacillus cereus

Carvacrol, a natural plant constituent occurring in oregano and thyme, was investigated for its bactericidal effect towards the food-borne pathogen Bacillus cereus . Carvacrol showed a dose-related growth inhibition of B. cereus . At concentrations of 0·75 mmol l⁻¹ and above, total inhibition of the growth was observed. Below this concentration, carvacrol extended the lag-phase, reduced the specific growth rate and reduced the maximum population density. Incubation for 40 min in the presence of 0·75–3 mmol l⁻¹ carvacrol decreased the number of viable cells of B. cereus exponentially. Spores were found to be approximately 2·3-fold less sensitive to carvacrol than vegetative cells. Bacillus cereus cells showed reduced susceptibility towards carvacrol at pH 7·0 compared with different values between pH 4·5 and 8·5. The culture and exposure temperatures had a significant influence on the survival of vegetative cells. The highest death rate of cells was observed at an exposure temperature of 30 °C. Membrane fluidity was found to be an important factor influencing the bactericidal activity of carvacrol.     

PCR assay of the groEL gene for detection and differentiation of Bacillus cereus group cells

Strains of species in the Bacillus cereus group are potentially enterotoxic. Thus, the detection of all B. cereus group strains is important. As 16S ribosomal DNA sequence analysis cannot adequately differentiate species of the B. cereus group, we explored the potential of the groEL gene as a phylogenetic marker. A phylogenetic analysis of the groEL sequences of 78 B. cereus group strains revealed that the B. cereus group strains were split into two major clusters, one including six B. mycoides and one B. pseudomycoides (cluster II) and the other including two B. mycoides and the rest of the B. cereus group strains (cluster I). Cluster I was further differentiated into two subclusters, Ia and Ib. The sodA gene sequences of representative strains from different clusters were also compared. The phylogenetic tree constructed from the sodA sequences showed substantial similarity to the tree constructed from the groEL sequences. Based on the groEL sequences, a PCR assay for detection and identification of B. cereus group strains was developed. Subsequent restriction fragment length polymorphism (RFLP) analysis verified the PCR amplicons and the differentiation of the B. cereus group strains. RFLP with MboI was identical for all the B. cereus group strains analyzed, while RFLP with MfeI or PstI classified all B. cereus and B. thuringiensis strains into two groups. All cluster II B. mycoides and B. pseudomycoides strains could be discriminated from other B. cereus group bacteria by restriction analysis with TspRI.     

Immunoquantitative real-time PCR for detection and quantification of Staphylococcus aureus enterotoxin B in foods

A real-time immunoquantitative PCR (iqPCR) method for detection of Staphylococcus aureus enterotoxin B (SEB) was developed and evaluated using both pure cultures and foods. The assay consisted of immunocapture of SEB and real-time PCR amplification of the DNA probe linked to the detection antibody. iqPCR was compared to an in-house enzyme-linked immunosorbent assay (ELISA) using the same couple of capture-detection antibodies and to commercial kits for detection of S. aureus enterotoxins (SE). The iqPCR was approximately 1,000 times more sensitive (<10 pg ml(-1)) than the in-house ELISA and had a dynamic range of approximately 10 pg ml(-1) to approximately 30,000 pg ml(-1). iqPCR was not inhibited by any of the foods tested and was able to detect SEB present in these foods. No cross-reactivity with SE other than SEB was observed. Application of iqPCR for detection of SEB in cultures of S. aureus revealed the onset of SEB production after 4 h of incubation at 22, 37, and 42 degrees C, which was in the first half of the exponential growth phase. The total amounts of SEB produced by the two strains tested were larger at 42 degrees C than at 37 degrees C and were strain dependent.     

Prevalence, phenotypic traits and molecular characterization of emetic toxin-producing Bacillus cereus strains isolated from human stools in Korea

Aims: To investigate the prevalence and genotypic/phenotypic characters of emetic toxin‐producing Bacillus cereus strains isolated from sporadic food poisoning cases in Korea. Methods and Results: The prevalence of emetic B. cereus was determined in 56 899 stool samples from sporadic food poisoning cases in Korea between 2004 and 2006. We assessed toxin profiles, phenotypic traits and antibiotic resistance. The molecular subtyping was ascertained using an automated repetitive sequence‐based PCR (rep‐PCR) system, DiversiLab?, with these emetic strains isolated from sporadic food poisoning cases and other emetic strains isolated from an outbreak and food samples. Emetic B. cereus was present in 0·012% of sporadic food poisoning cases. The prevalence of nheABC, hblCDA, cytK and entFM enterotoxin genes among emetic strains was 100, 14·3, 14·3 and 100%, respectively. Most emetic strains were negative for salicin hydrolysis (100%), starch fermentation (85·7%) and haemolysis (85·7%). One emetic isolate, VK7, exhibited several unique traits, such as harbouring the hbl gene and ability to hydrolyse starch. All isolated strains were highly resistant to β‐lactam antibiotics. All emetic strains except VK7 exhibited an identical rep‐PCR banding pattern, while nonemetic strains were classified into various pulsotypes. Conclusions: Most emetic strains except one isolate exhibited similar genotypic/phenotypic traits and subtyping pattern. Automatic rep‐PCR (DiversiLab?) may be used to discriminate emetic strains from nonemetic strains, although we could not distinguish between most emetic strains using that. Significance and Impact of the Study: Result of this study may contribute an extended database on the prevalence and toxigenic traits of emetic B. cereus strains isolated from Korea.