Production of the Kanagawa hemolysin by Vibrio parahaemolyticus in a synthetic medium.

A synthetic medium capable of supporting growth of Vibrio parahaemolyticus is described. Growth yields and generation times were comparable to growth in a complex medium, although Kanagawa hemolysin was undetectable in the synthetic medium. Upon the addition of single amino acids to this synthetic medium, only D-tryptophan induced production of the hemolysin. L-Tryptophan was found to inhibit the action of the D-stereoisomer. The response to D-tryptophan was pH dependent; the greatest hemolysin expression occurred at pH values below 6.5. The addition of 100 micrometers D-tryptophan to early-logarithmic-phase cultures caused an inhibition of growth and of substrate utilization, both of which lasted 7 h. During this time, hemolysin was produced only intracellularly. The hemolysin appeared in the supernatant only when growth recommenced. The hemolysins within the cell and in the supernatant were both inactivated by antiserum raised against the standard Kanagawa hemolysin.     

Zebrafish as a useful model for zoonotic Vibrio parahaemolyticus pathogenicity in fish and human

Vibrio parahaemolyticus is an important aquatic zoonotic pathogen worldwide that causes vibriosis in many marine fish, and sepsis, gastroenteritis and wound infection in humans. However, the pathogenesis of different sources of V. parahaemolyticus is not fully understood. Here, we examined the pathogenicity and histopathology of fish (V. parahaemolyticus 1.2164) and human (V. parahaemolyticus 17) strains in a zebrafish (Danio rerio). We found that different infection routes resulted in different mortality in zebrafish. Moreover, death due to V. parahaemolyticus 1.2164 infection occurred quicker than that caused by V. parahaemolyticus 17 infection. Hematoxylin-eosin staining of liver, kidney and intestine sections showed histological lesions in all three organs after infection with either strain. V. parahaemolyticus 1.2164 caused more severe damage than V. parahaemolyticus 17. In particular, V. parahaemolyticus 1.2164 treatment induced more serious hydropic degeneration and venous sinus necrosis in the liver than V. parahaemolyticus 17 treatment. The expression levels of three proinflammatory cytokines, interleukin 1β (il1β), interferon phi 1 (ifnϕ1) and tumor necrosis factor α (tnfα), as determined by quantitative real-time PCR, were upregulated in all examined tissues of infected fish. Notably, the peak levels of tnfα were significantly higher than those of il1β and ifnϕ1, suggesting, together with pathological results, that tnfα and il1β play an important role in acute sepsis. High amounts of tnfα may be related to acute liver necrosis, while ifnϕ1 may respond to V. parahaemolyticus and play an antibacterial role for chronically infected adult zebrafish. Taken together, our results suggest that the zebrafish model of V. parahaemolyticus infection is useful for studying strain differences in V. parahaemolyticus pathogenesis.     

Comparative genomic analysis of European and Middle Eastern community-associated methicillin-resistant Staphylococcus aureus (CC80:ST80-IV) isolates by high-density microarray

Infections as a result of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) are an issue of increasing global healthcare concern. In Europe, this principally involves strains of multi-locus sequence type clonal complex 80 sequence type 80 with methicillin resistance in a staphylococcal chromosomal cassette (SCC mec ) type IV arrangement (CC80:ST80-IV). As with other CA-MRSA strains, CC80:ST80-IV isolates tend to appear uniform when analysed by common molecular typing methods (e.g. pulsed field gel electrophoresis, multi-locus sequence type, SCC mec ). To explore whether DNA sequence-based differences exist, we compared the genetic composition of six CC80:ST80-IV isolates of diverse chronological and geographic origin (i.e. Denmark and the Middle East) using an Affymetrix high-density microarray that was previously used to analyse CA-MRSA USA300 isolates. The results revealed a high degree of homology despite the diversity in isolation date and origin, with isolate differences primarily in conserved hypothetical open reading frames and intergenic sequences, but also including regions of known function. This included the confirmed loss of SCC mec recombinase genes in two Danish isolates representing potentially new SCC mec types. Microarray analysis grouped the six isolates into three relatedness pairs, also identified by pulsed field gel electrophoresis, which were consistent with both the clinical and molecular data.     

Comparison of the modified Elek test and Wagatsuma agar for determination of the Kanagawa phenomenon of Vibrio parahaemolyticus.

The modified Elek test and Wagatsuma agar were compared for their ability to detect the Kanagawa activity of 142 strains of Vibrio parahaemolyticus. The performance of the modified Elek test was on a par with that of the Wagatsuma agar as far as positivity was concerned, and the test was far superior to Wagatsuma agar in eliminating doubtful results. The results of the modified Elek test were not unduly influenced by the different types of agar used.     

Purification and characterization of a hemolysin produced by a clinical isolate of Kanagawa phenomenon-negative Vibrio parahaemolyticus and related to the thermostable direct hemolysin.

A clinical isolate (strain 4037) of Kanagawa phenomenon-negative Vibrio parahaemolyticus was studied. Although the strain was judged to be Kanagawa phenomenon-negative by various conventional tests, it produced a new hemolysin (named Vp-TRH, for thermostable direct hemolysin [Vp-TDH]-related hemolysin) that was related to the Vp-TDH produced by ordinary Kanagawa phenomenon-positive V. parahaemolyticus. Vp-TRH was purified by ammonium sulfate fractionation and successive column chromatographies on DEAE-cellulose, hydroxyapatite, and Mono Q. The molecular weight of Vp-TRH was estimated as 48,000 by Sephadex G-100 gel filtration, and the molecular weight of the subunit was estimated to be 23,000 by sodium dodecyl sulfate-slab gel electrophoresis. Thus, like Vp-TDH, Vp-TRH seems to be composed of two subunits. The isoelectric point of Vp-TRH was determined to be 4.6. Vp-TRH showed lytic activities different from those of Vp-TDH on erythrocytes from various animals, especially those from calves, chickens, and sheep. The hemolytic activity of Vp-TRH was labile on heat treatment at 60 degrees C for 10 min, unlike that of Vp-TDH. The immunological similarities, but not the identities of Vp-TRH and Vp-TDH, were demonstrated by Ouchterlony, neutralization, and latex agglutination tests. Thus, we conclude that this clinical isolate of Kanagawa phenomenon-negative V. parahaemolyticus produces a new type of hemolysin that is similar, but not identical, to Vp-TDH, which is usually produced by Kanagawa phenomenon-positive V. parahaemolyticus.     

Restriction fragment length polymorphism of the tdh and trh genes in clinical Vibrio parahaemolyticus strains.

The restriction fragment length polymorphism of the genes encoding thermostable direct hemolysin (tdh) and thermostable direct hemolysin-related hemolysin (trh) was analyzed for 137 strains of Vibrio parahaemolyticus isolated from specimens from diarrheal patients in Thailand. The HindIII restriction fragment patterns of tdh and trh were grouped into five and four types, respectively. A strong association between the restriction fragment patterns of tdh and trh was observed with V. parahaemolyticus strains.     

Correlation between the presence of the VcB variable tandem repeat region and the VPI-1 pathogenicity island in <Emphasis Type="Italic">Vibrio cholerae</Emphasis>

Primers specially designed for the purpose were used to establish the correlation between the presence of the VcB variable tandem repeat region located on chromosome II of Vibrio cholerae and the VPI-1 pathogenicity island located on chromosome I. It was demonstrated that the studied region of deletion is flanked by direct repeats and contains a gene encoding uropathogenic protein belonging to the type VI secretion system, as well as the transposase and integrase genes. The obtained results allow it to be suggested that the studied region may be a previously unknown pathogenicity islet.     

Infectious CTXPhi and the vibrio pathogenicity island prophage in Vibrio mimicus: evidence for recent horizontal transfer between V. mimicus and V. cholerae

Vibrio mimicus differs from Vibrio cholerae in a number of genotypic and phenotypic traits but like V. cholerae can give rise to diarrheal disease. We examined clinical isolates of V. mimicus for the presence of CTXPhi, the lysogenic filamentous bacteriophage that carries the cholera toxin genes in epidemic V. cholerae strains. Four V. mimicus isolates were found to contain complete copies of CTXPhi. Southern blot analyses revealed that V. mimicus strain PT5 contains two CTX prophages integrated at different sites within the V. mimicus genome whereas V. mimicus strains PT48, 523-80, and 9583 each contain tandemly arranged copies of CTXPhi. We detected the replicative form of CTXPhi, pCTX, in all four of these V. mimicus isolates. The CTX prophage in strain PT5 was found to produce infectious CTXPhi particles. The nucleotide sequences of CTXPhi genes orfU and zot from V. mimicus strain PT5 and V. cholerae strain N16961 were identical, indicating contemporary horizontal transfer of CTXPhi between these two species. The receptor for CTXPhi, the toxin-coregulated pilus, which is encoded by another lysogenic filamentous bacteriophage, VPIPhi, was also present in the CTXPhi-positive V. mimicus isolates. The nucleotide sequences of VPIPhi genes aldA and toxT from V. mimicus strain PT5 and V. cholerae N16961 were identical, suggesting recent horizontal transfer of this phage between V. mimicus and V. cholerae. In V. mimicus, the vibrio pathogenicity island prophage was integrated in the same chromosomal attachment site as in V. cholerae. These results suggest that V. mimicus may be a significant reservoir for both CTXPhi and VPIPhi and may play an important role in the emergence of new toxigenic V. cholerae isolates.     

Comparative and functional genomic analyses of the pathogenicity of phytopathogen Xanthomonas campestris pv. campestris

Xanthomonas campestris pathovar campestris (Xcc) is the causative agent of crucifer black rot disease, which causes severe losses in agricultural yield world-wide. This bacterium is a model organism for studying plant-bacteria interactions. We sequenced the complete genome of Xcc 8004 (5,148,708 bp), which is highly conserved relative to that of Xcc ATCC 33913. Comparative genomics analysis indicated that, in addition to a significant genomic-scale rearrangement cross the replication axis between two IS1478 elements, loss and acquisition of blocks of genes, rather than point mutations, constitute the main genetic variation between the two Xcc strains. Screening of a high-density transposon insertional mutant library (16,512 clones) of Xcc 8004 against a host plant (Brassica oleraceae) identified 75 nonredundant, single-copy insertions in protein-coding sequences (CDSs) and intergenic regions. In addition to known virulence factors, full virulence was found to require several additional metabolic pathways and regulatory systems, such as fatty acid degradation, type IV secretion system, cell signaling, and amino acids and nucleotide metabolism. Among the identified pathogenicity-related genes, three of unknown function were found in Xcc 8004-specific chromosomal segments, revealing a direct correlation between genomic dynamics and Xcc virulence. The present combination of comparative and functional genomic analyses provides valuable information about the genetic basis of Xcc pathogenicity, which may offer novel insight toward the development of efficient methods for prevention of this important plant disease.     

Genotyping of Salmonella enterica serovar Typhi strains isolated from 1959 to 2006 in China and analysis of genetic diversity by genomic microarray

Aim

To determine the genotype of Salmonella enterica serovar Typhi (S. Typhi) strains in China and analyze their genetic diversity.

Methods

We collected S. Typhi strains from 1959 to 2006 in five highly endemic Chinese provinces and chose 40 representative strains. Multilocus sequence typing was used to determine the genotypes or sequence types (ST) and microarray-based comparative genomic hybridization (M-CGH) to investigate the differences in gene content among these strains.

Results

Forty representative S. Typhi strains belonged to 4 sequence types (ST1, ST2, ST890, and ST892). The predominant S. Typhi genotype (31/40) was ST2 and it had a diverse geographic distribution. We discovered two novel STs – ST890 and ST892. M-CGH showed that 69 genes in these two novel STs were divergent from S. Typhi Ty2, which belongs to ST1. In addition, 5 representative Typhi strains of ST2 isolated from Guizhou province showed differences in divergent genes.

Conclusion

We determined two novel sequence types, ST890 and ST892, and found that ST2 was the most prevalent genotype of S. Typhi in China. Genetic diversity was present even within a highly clonal bacterial population.Salmonella enterica serovar Typhi (S. Typhi) is a Gram-negative, human-restricted enteroinvasive pathogen that causes typhoid fever (1,2). The harm caused by S. Typhi has been greatly reduced by the application of antibiotics, but typhoid fever is still a common disease in tropical and subtropical regions, and many drug-resistant strains of S. Typhi have been discovered. In the recent years, there have been more than 16 million cases reported annually worldwide. Even in the United States and other developed countries, there are still outbreaks of typhoid fever caused by S. Typhi (3). This is in part due to the ability of S. Typhi to rapidly evolve through either horizontal gene transfer mechanisms or produce a cloud of related strains by using highly mutable genes (4). Thus, there is an urgent need for improved molecular diagnostics to discriminate among the large numbers of related strains. There are many methods for genotyping of Salmonella, and polymerase chain reaction (PCR)-based typing methods are very prevalent. A multiplex PCR-based reverse line blot hybridization system can enhance outbreak investigations and surveillance of Salmonella infections (5). Recently, real-time PCR-based single nucleotide polymorphism typing method has been used for global epidemiological analysis of S. Typhi (6).Multilocus sequence typing (MLST), which is based on the analysis of DNA sequence polymorphisms in a group of housekeeping genes, is the most widely used method for bacterial strain genotyping (7). Each unique sequence of a housekeeping gene is assigned an allele number, and an allele profile of a strain is defined as the set of allele numbers for that strain. Each unique allele profile is assigned a sequence type (ST) number. Strains that have the same ST number are identical at all of the sequenced loci and are considered to be members of the same clone. MLST, unlike earlier molecular typing methods, such as pulsed-field gel electrophoresis (PFGE), has high discriminative power, allows easy standardization of data acquisition and analysis across laboratories, and has a high degree of portability of the resulting sequence data (8). MLST was used to investigate the genotype of S. Typhi as early as in 2002 (9). Up to now, 51 S. Typhi strains have been recorded in the Salmonella enterica MLST Database (10) and classified into 8 STs (ST1, ST2, ST3, ST8, ST890, ST892, ST911, and ST980), but most of them belong to ST1 (15/51) and ST2 (29/51).One well accepted definition of procaryotic species, based on whole-genome DNA-DNA hybridization, is that it is an entity comprised of strains sharing a reassociation value of approximately 70% or greater (11). Genomic diversity and relatedness of closely related organisms has since recently been determined with microarrays, which have higher resolution than traditional DNA-DNA hybridization methods (12). Microarray-based comparative genomic hybridization (M-CGH) is in widespread use in relatedness determination of procaryotic species (13-15). CGH usually uses the whole genome open reading frame (ORF) array-based hybridization approach (16).Typhoid fever is endemic in developing countries, such as China. However, there are few reports of genotyping of S. Typhi in China (17). These reports mostly used PFGE, which is currently the method of choice for genotyping of sporadic or epidemic Salmonella isolates. S. typhi strains isolated from Shenzhen in China showed 22 distinct PFGE patterns with variable genetic diversity (17). We speculated that genetic diversity of S. Typhi strains may be largely present throughout the last several decades in China. Therefore, we collected S. Typhi strains from 1959 to 2006 and chose 40 strains representative in terms of genetic diversity isolated from 5 highly endemic Chinese provinces. The aim of this study was to identify the genotype of 40 representative S. Typhi strains by MLST and evaluate their genetic diversity by M-CGH.     

Comparative genomic sequence analysis between a standard challenge strain and a vaccine strain of duck enteritis virus in China

Here, we present the complete genomic sequence of the Chinese standard challenge strain (CSC) of duck enteritis virus (DEV), which was isolated in China in 1962. The DEV CSC genome is 162,131 bp long and contains 78 predicted open reading frames (ORFs). Comparison of the genomic sequences of DEV CSC and DEV live vaccine strain K at passage 63 (DEV K p63) revealed that the DEV CSC genome is 4,040 bp longer than the DEV K p63 genome, mainly because of 3,513-bp and 528-bp insertions at the 5′ and 3′ ends of the unique long segment, respectively. At the nucleotide level, 63 of the 76 ORFs in the DEV CSC genome were 100 % identical to the ORFs in the DEV K p63 genome. Two ORFs (UL56 and US10) had frameshift mutations in the C-terminal regions, while LORF5 was unique to the DEV K p63 genome. It is difficult to assign attenuated virulence to changes in specific genes. However, the complete DEV CSC genome will further advance our understanding of the genes involved in virulence and evolution. The DEV CSC genome sequence has been deposited in GenBank under accession number JQ673560.     

Comparative genomic analysis for the presence of potential enterococcal virulence factors in the probiotic Enterococcus faecalis strain Symbioflor 1

Enterococci are members of the natural microbiota of animal and human intestinal tracts and are capable of causing opportunistic infections. They are also used as starter cultures in the food industry as well as in health supplements and probiotics by the pharmaceutical industry. This Janus-faced status requires a careful evaluation on the basis of pathogenic traits to ensure the safety of the strain used to produce food and pharmaceuticals. We performed gapped-genome sequencing of a probiotic strain Enterococcus faecalis Symbioflor 1 and present initial results deriving from comparative genome analysis with that of the previously sequenced pathogenic clinical isolate E. faecalis V583. There was strong overall conservation of synteny between both strains and a detailed analysis revealed the absence of large genomic regions from the chromosome of the probiotic strain, indicating gene loss. Genes absent from the Symbioflor 1 strain included those encoding the enterococcal cytolysin, enterococcal surface protein, and gelatinase (coccolysin) as well as hyaluronidase and the peptide antibiotic AS-48. This data was confirmed using PCR primers specific for the respective genes. However, other enterococcal determinants such as aggregation substance, collagen adhesion protein, the ability to resist oxygen anions as well as capsule formation were detected. The presence of these traits may be advantageous for the strain Symbioflor 1 since they potentially enable colonization and proliferation of the bacterium on mucosal surfaces thereby conferring on it probiotic traits.     

Comparative genomic hybridization in childhood acute lymphoblastic leukemia: correlation with interphase cytogenetics and loss of heterozygosity analysis

We used comparative genomic hybridization (CGH) to study DNA copy number changes in 71 children with acute lymphoblastic leukemia (ALL) including 50 B-lineage and 21 T-ALLs. Forty-two patients (59%) showed genomic imbalances whereby gains were more frequently observed than losses (127 vs. 29). Gains most commonly affected the entire chromosomes 21 and 10 (19.7% each), 6, 14, 18, X (15.5% each), 17 (14.1%) and 4 (11.3%). Highly hyperdiploid karyotypes (chromosome number >50) occurred more frequently in B-lineage than in T-lineage ALL (24% vs. 4.8%). In both cell lineages deletions were mainly detected on 9p (14.1%) and 12p (8.4%), and on 6q in T-lineage ALL (4.2%). These findings were compared with loss of heterozygosity (LOH) of 6q, 9p, 11q, and 12p previously performed in 56 of the 71 patients. Among 54 sites of LOH, CGH revealed losses of the respective chromosome arms in 17 LOH-positive regions (31.5%). G-banding analysis and interphase cytogenetics with subregional probes for 14 loci confirmed the presence of genomic imbalances as detected by CGH. We, therefore, conclude that, in the absence of cytogenetic data, CGH represents a suitable method for identifying hyperdiploid karyotypes as well as prognostically relevant deletions in ALL patients.     

Comparative genomic analysis for the presence of potential enterococcal virulence factors in the probiotic Enterococcus faecalis strain Symbioflor 1

Enterococci are members of the natural microbiota of animal and human intestinal tracts and are capable of causing opportunistic infections. They are also used as starter cultures in the food industry as well as in health supplements and probiotics by the pharmaceutical industry. This Janus-faced status requires a careful evaluation on the basis of pathogenic traits to ensure the safety of the strain used to produce food and pharmaceuticals. We performed gapped-genome sequencing of a probiotic strain Enterococcus faecalis Symbioflor 1 and present initial results deriving from comparative genome analysis with that of the previously sequenced pathogenic clinical isolate E. faecalis V583. There was strong overall conservation of synteny between both strains and a detailed analysis revealed the absence of large genomic regions from the chromosome of the probiotic strain, indicating gene loss. Genes absent from the Symbioflor 1 strain included those encoding the enterococcal cytolysin, enterococcal surface protein, and gelatinase (coccolysin) as well as hyaluronidase and the peptide antibiotic AS-48. This data was confirmed using PCR primers specific for the respective genes. However, other enterococcal determinants such as aggregation substance, collagen adhesion protein, the ability to resist oxygen anions as well as capsule formation were detected. The presence of these traits may be advantageous for the strain Symbioflor 1 since they potentially enable colonization and proliferation of the bacterium on mucosal surfaces thereby conferring on it probiotic traits.     

EAggEC中小肠结肠炎耶尔森菌强毒力岛结构和功能研究

目的了解小肠结肠炎耶尔森菌（Yersinia enterocolitica,Yen）强毒力岛在肠聚集性大肠杆菌（EAggEC）中的结构和摄铁功能，为进一步研究细菌毒力的进化奠定理论基础。方法PCR扩增、原位杂交及SDS-PAGE。结果在检测的6株EAggEC有5株检出了毒力岛，而且这些阳性菌株中的毒力岛都位于asntRNA（天门冬氨酸tRNA）位点；在缺铁条件下，EAggRC能够表达和耶尔森菌相同的摄铁蛋白HMWP1和HMWP2。结论小肠结肠炎耶尔森菌强毒力岛在肠聚集性大肠杆菌中有阳性率较高的分布，并具有相同的摄铁功能，很可能EAggRC和耶尔森菌中的强毒力岛有相同的转移机制。     

Composition and gene expression of the cag pathogenicity island in Helicobacter pylori strains isolated from gastric carcinoma and gastritis patients in Costa Rica

The composition and in vitro expression of the cag pathogenicity island genes in a group of Helicobacter pylori strains obtained from patients suffering from chronic gastritis-associated dyspepsia (n = 26) or gastric carcinoma (n = 17) were analyzed. No significant difference in the distribution of the 10 studied regions was found between the cases and the controls. Nine strains did not harbor any of the selected regions: eight (30.8%) isolated from patients with gastritis only and one (5.9%) from a patient with gastric carcinoma. No association was found between the number of repeated sequences at the 3' end of the cagA gene or the presence of tyrosine phosphorylation motifs and the clinical origin of the strains. The virB10 homolog gene was the sole gene studied to be significantly expressed more often in cancer strains than in gastritis strains (P = 0.03).     

Comparative genomic analysis of Campylobacter jejuni strains reveals diversity due to genomic elements similar to those present in C. jejuni strain RM1221

Analysis of the complete genomic sequence of Campylobacter jejuni strain RM1221 identified four large genomic elements, Campylobacter jejuni-integrated elements (CJIEs), that were absent from C. jejuni strain NCTC 11168. To further investigate the genomic diversity of Campylobacter, we conducted a comparative genomic analysis from a collection of 67 C. jejuni and 12 Campylobacter coli strains isolated from various geographical locations and clinical and veterinary sources. Utilizing PCR, we demonstrated that 55% of the C. jejuni strains examined were positive for at least one RM1221-like genomic element and 27% were positive for two or more of these CJIEs. Furthermore, many C. coli strains were positive for either genomic element CJIE1 or CJIE3. To simultaneously assess for the presence or absence of several genes that comprise the various CJIEs, we developed a multistrain C. jejuni DNA microarray that contained most of the putative coding sequences for strains NCTC 11168 and RM1221. A comparative genomic hybridization (CGH) analysis of 35 of the 67 C. jejuni strains confirmed the presence of genomic elements similar to those in strain RM1221. Interestingly, the DNA microarray analysis demonstrated that these genomic elements in the other C. jejuni strains often exhibited modular patterns with some regions of the CJIEs present and other regions either absent or highly divergent compared to strain RM1221. Our CGH method also identified 18 other intraspecies hypervariable regions, such as the capsule and lipooligosaccharide biosynthesis regions. Thus, the inclusion of genes from these integrated genomic elements and the genes from the other intraspecies hypervariable regions contributes to a better assessment of the diversity in C. jejuni and may increase the usefulness of DNA microarrays as an epidemiological genotyping tool. Finally, we also showed that in CJIE1, a Campylobacter Mu-like phage, is located differentially in other strains of C. jejuni, suggesting that it may integrate essentially randomly.     

Tissue microarray analysis reveals strong clinical evidence for a close association between loss of annexin A1 expression and nodal metastasis in gastric cancer

Aims Annexin A1 (ANXA1) is a calcium- and phospholipid-binding protein that has been implicated in the regulation of inflammation, cell proliferation, and apoptosis. Its role in tumor development and progression is controversial, whereas its role in gastric cancer is unknown. We investigated ANXA1 expression and determined its clinical significance in gastric cancer. Methods and results Tissue microarray blocks containing primary gastric cancer, lymph node metastasis, and adjacent normal mucosa specimens obtained from 1,072 Chinese patients were constructed. Expression of ANXA1 in these specimens was analyzed using immunohistochemistry. Complete loss of ANXA1 expression was observed in 691 (64%) of the 1,072 primary tumors and 146 (86%) of 169 nodal metastases. Loss of ANXA1 expression was significantly associated with advanced T stage, lymph node metastasis, advanced disease stage, and poor histological differentiation. Loss of ANXA1 expression correlated significantly with poor survival rates in both univariate and multivariate analyses. Conclusions ANXA1 expression decreased significantly as gastric cancer progressed and metastasized, suggesting the importance of ANXA1 as a negative biomarker for gastric cancer development and progression.