O-Antigen Gene Clusters of Plesiomonas shigelloides Serogroups and Its Application in Development of a Molecular Serotyping Scheme.

Plesiomonas shigelloides is a Gram-negative, flagellated, rod-shaped, ubiquitous, and facultative anaerobic bacterium. It has been isolated from various sources, such as freshwater, surface water, and many wild and domestic animals. P. shigelloides is associated with diarrheal diseases of acute secretory gastroenteritis, an invasive shigellosis-like disease, and a cholera-like illness in humans. At present, 102 somatic antigens and 51 flagellar antigens of P. shigelloides have been recognized; however, very little is known about variations of O-antigens among P. shigelloides species. In this study, 12 O-antigen gene clusters of P. shigelloides, O2H1a1c (G5877), O10H41 (G5892), O12H35 (G5890), O23H1a1c (G5263), O25H3 (G5879), O26H1a1c (G5889), O32H37 (G5880), O33H38 (G5881), O34H34 (G5882), O66H3 (G5270), O75H34 (G5885), and O76H39 (G5886), were sequenced and analyzed. The genes that control O-antigen synthesis are present as chromosomal gene clusters that maps between rep and aqpZ, and most of the synthesis and translocation of OPS (O-specific polysaccharide) belongs to Wzx/Wzy pathway with the exception of O12, O25, and O66, which use the ATP-binding cassette (ABC) transporter pathway. Phylogenetic analysis of wzx and wzy show that the wzx and wzy genes are specific to individual O-antigens and can be used as targets in molecular typing. Based on the sequence data, an O-antigen specific suspension array that detects 12 distinct OPS' has been developed. This is the first report to catalog the genetic features of P. shigelloides O-antigen variations and develop a suspension array for the molecular typing. The method has several advantages over traditional bacteriophage and serum agglutination methods and lays the foundation for easier identification and detection of additional O-antigen in the future.

Plesiomonas shigelloides sipD mutant, generated by an efficient gene transfer system, is less invasive.

Plesiomonas shigelloides is widely associated with human diarrheal disease. Research on this pathogen has been hampered by the absence of an effective genetic manipulation system. In the present study, an efficient and precise conjugation transfer procedure, mediated by suicide vector pRE112 was used to overcome this limitation. The efficiency of generating double recombinants was average 74.3%, and the conjugation protocol may be applied to other P. shigelloides strains. We also identified that the SipD protein of P. shigelloides G5884 (serotype O45) is 65% similar to the SipD in Salmonella pathogenicity island 1 (SPI-1), which is a key element of the type III secretion system related to Salmonella invasion. A P. shigelloides sipD null mutant was generated via the conjugation system, using the suicide vector pRE112. The isogenic mutant strain lacking sipD showed a 50% reduction in its capacity to invade Caco-2 cells.

Genetic diversity of K-antigen gene clusters of Escherichia coli and their molecular typing using a suspension array.

Capsular polysaccharides (CPSs), or K-antigens, are the major surface antigens of Escherichia coli. More than 80 serologically unique K-antigens are classified into 4 groups (Groups 1-4) of capsules. Groups 1 and 4 contain the Wzy-dependent polymerization pathway and the gene clusters are in the order galF to gnd; Groups 2 and 3 contain the ABC-transporter-dependent pathway and the gene clusters consist of 3 regions, regions 1, 2 and 3. Little is known about the variations among the gene clusters. In this study, 9 serotypes of K-antigen gene clusters (K2ab, K11, K20, K24, K38, K84, K92, K96, and K102) were sequenced and correlated with their CPS chemical structures. On the basis of sequence data, a K-antigen-specific suspension array that detects 10 distinct CPSs, including the above 9 CPSs plus K30, was developed. This is the first report to catalog the genetic features of E. coli K-antigen variations and to develop a suspension array for their molecular typing. The method has a number of advantages over traditional bacteriophage and serum agglutination methods and lays the foundation for straightforward identification and detection of additional K-antigens in the future.