Aeromonas hydrophila is a gram-negative opportunistic pathogen of animals and humans. The pathogenesis of A. hydrophila is multifactorial. Genomic subtraction and markers of genomic islands (GIs) were used to identify putative virulence genes in A. hydrophila PPD134/91. Two rounds of genomic subtraction led to the identification of 22 unique DNA fragments encoding 19 putative virulence factors and seven new open reading frames, which are commonly present in the eight virulence strains examined. In addition, four GIs were found, including O-antigen, capsule, phage-associated, and type III secretion system (TTSS) gene clusters. These putative virulence genes and gene clusters were positioned on a physical map of A. hydrophila PPD134/91 to determine their genetic organization in this bacterium. Further in vivo study of insertion and deletion mutants showed that the TTSS may be one of the important virulence factors in A. hydrophila pathogenesis. Furthermore, deletions of multiple virulence factors such as S-layer, serine protease, and metalloprotease also increased the 50% lethal dose to the same level as the TTSS mutation (about 1 log) in a blue gourami infection model. This observation sheds light on the multifactorial and concerted nature of pathogenicity in A. hydrophila. The large number of putative virulence genes identified in this study will form the basis for further investigation of this emerging pathogen and help to develop effective vaccines, diagnostics, and novel therapeutics.Aeromonas hydrophila is a ubiquitous gram-negative bacterium of aquatic environments, which has been implicated as a causative agent of motile aeromonad septicemia in a variety of aquatic animals (especially freshwater fish species) (2, 37). It causes gastrointestinal and extraintestinal infections in humans, including septicemia, wound infections, and gastroenteritis (16). A number of virulence factors have been identified in A. hydrophila, namely, pili and adhesins (29, 30), O-antigens and capsules (23,48,49), S-layers (10), exotoxins such as hemolysins and enterotoxin (7,14), and a repertoire of exoenzymes which digest cellular components such as proteases, amylases, and lipases (20, 28).The pathogenesis of A. hydrophila is multifactorial. Most studies to date have concentrated on the characterization of a few virulence factors in different animal models by using different strains (10, 34, 42), making it very difficult to evaluate the significance of each gene in the virulence of A. hydrophila. This study aims to identify more putative virulence determinants and characterize them in an integrated manner.Suppressive subtraction hybridization or genomic subtraction offers a genome-level approach to identifying the genetic differences between virulent and avirulent strains of bacteria (22,47). In this study, we used genomic subtraction to identify 22 unique DNA fragments encoding 19 putative virulence factors and seven new open reading frames (ORFs), which are frequently present in a group of virulent strains of A. hydrophila. In...
The sequences of the O-antigen and capsule gene clusters of the virulent Aeromonas hydrophila strain PPD134/91 were determined. The O-antigen gene cluster is 17,296 bp long and comprises 17 genes. Seven pathway genes for the synthesis of rhamnose and mannose, six transferase genes, one O unit flippase gene, and one O-antigen chain length determinant gene were identified by amino acid sequence similarity. PCR and Southern blot analysis were performed to survey the distribution of these 17 genes among 11 A. hydrophila strains of different serotypes. A. hydrophila PPD134/91 might belong to serotype O:18, as represented by JCM3980; it contained all the same O-antigen genes as JCM3980 (97 to 100% similarity at the DNA and amino acid levels). The capsule gene cluster of A. hydrophila PPD134/91 is 17,562 bp long and includes 13 genes, which were assembled into three distinct regions similar to those of the group II capsule gene cluster of Escherichia coli and other bacteria. Regions I and III contained four and two capsule transport genes, respectively. Region II had five genes which were highly similar to capsule synthesis pathway genes found in other bacteria. Both the purified O-antigen and capsular polysaccharides increased the ability of the avirulent A. hydrophila strain PPD35/85 to survive in naïve tilapia serum. However, the purified surface polysaccharides had no inhibitory effect on the adhesion of A. hydrophila PPD134/91 to carp epithelial cells.Surface polysaccharides, such as O-antigen and capsule, are important bacterial cell surface components. The O-antigen polysaccharide is covalently ligated to the lipid A-core complex and extends outward from the cell surface. The capsule is an extracellular polysaccharide enclosing the bacterium while remaining attached to the cell. Both the O-antigen polysaccharide and the capsule are composed of repeating oligosaccharide units (44). They act as prominent antigens and play important roles in the pathogenicity of many bacterial pathogens, such as protecting bacterial cells from complement-mediated serum killing (20, 30), acting as adhesion factors (31), protecting the bacteria from the effects of desiccation (38), and aiding survival in phagocytes (56). The serogrouping of bacterial strains within a genus is determined by the structural variability of surface polysaccharides. For example, Escherichia coli strains are divided into more than 160 serogroups based on the different surface polysaccharides (67). Klebsiella species have been classified into 72 serogroups based on the structural variability of their capsular polysaccharides (39).Aeromonas hydrophila is an important pathogen of a wide variety of aquatic and terrestrial animals, especially fish (4). In fish, it causes hemorrhagic septicemia, which often results in high mortalities in commercial aquaculture. Some strains of A. hydrophila are also reported to cause infections in humans. The clinical symptoms include septicemia (17), meningitis (25), peritonitis (35), pneumonia (32), myonecrosis (34), and diarrh...
We present egglog, a fixpoint reasoning system that unifies Datalog and equality saturation (EqSat). Like Datalog, egglog supports efficient incremental execution, cooperating analyses, and lattice-based reasoning. Like EqSat, egglog supports term rewriting, efficient congruence closure, and extraction of optimized terms. We identify two recent applications -- a unification-based pointer analysis in Datalog and an EqSat-based floating-point term rewriter -- that have been hampered by features missing from Datalog but found in EqSat or vice-versa. We evaluate our system by reimplementing those projects in egglog. The resulting systems in egglog are faster, simpler, and fix bugs found in the original systems.
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