Molecular analysis of the rfb O antigen gene cluster of Salmonella enterica serogroup O:6,14 and development of a serogroup-specific PCR assay

The Kauffmann-White scheme for serotyping Salmonella recognizes 46 somatic (O) antigen groups, which together with detection of the flagellar (H) antigens form the basis for serotype identification. Although serotyping has become an invaluable typing method for epidemiological investigations of Salmonella, it does have some practical limitations. We have been characterizing the genes required for O and H antigen biosynthesis with the goal of developing a DNA-based system for the determination of serotype in Salmonella. The majority of the enzymes involved in O antigen biosynthesis are encoded by the rfb gene cluster. We report the sequencing of the rfb region from S. enterica serotype Sundsvall (serogroup O:6,14). The S. enterica serotype Sundsvall rfb region is 8.4 kb in length and comprises six open reading frames. When compared with other previously characterized rfb regions, the serogroup O:6,14 sequence is most related to serogroup C(1). On the basis of DNA sequence similarity, we identified two genes from the mannose biosynthetic pathway, two mannosyl transferase genes, the O unit flippase gene and, possibly, the O antigen polymerase. The whole cluster is derived from a low-G+C-content organism. Comparative sequencing of an additional serogroup O:6,14 isolate (S. enterica serotype Carrau) revealed a highly homologous sequence, suggesting that O antigen factors O:24 and O:25 (additional O factors associated with serogroup O:6,14) are encoded outside the rfb gene cluster. We developed a serogroup O:6,14-specific PCR assay based on a region of the putative wzx (O antigen flippase) gene. This provides the basis for a sensitive and specific test for the rapid identification of Salmonella serogroup O:6,14.     

Molecular Analysis of the rfb O Antigen Gene Cluster of Salmonella enterica Serogroup O:6,14 and Development of a Serogroup-Specific PCR Assay

The Kauffmann-White scheme for serotyping Salmonella recognizes 46 somatic (O) antigen groups, which together with detection of the flagellar (H) antigens form the basis for serotype identification. Although serotyping has become an invaluable typing method for epidemiological investigations of Salmonella, it does have some practical limitations. We have been characterizing the genes required for O and H antigen biosynthesis with the goal of developing a DNA-based system for the determination of serotype in Salmonella. The majority of the enzymes involved in O antigen biosynthesis are encoded by the rfb gene cluster. We report the sequencing of the rfb region from S. enterica serotype Sundsvall (serogroup O:6,14). The S. enterica serotype Sundsvall rfb region is 8.4 kb in length and comprises six open reading frames. When compared with other previously characterized rfb regions, the serogroup O:6,14 sequence is most related to serogroup C₁. On the basis of DNA sequence similarity, we identified two genes from the mannose biosynthetic pathway, two mannosyl transferase genes, the O unit flippase gene and, possibly, the O antigen polymerase. The whole cluster is derived from a low-G+C-content organism. Comparative sequencing of an additional serogroup O:6,14 isolate (S. enterica serotype Carrau) revealed a highly homologous sequence, suggesting that O antigen factors O:24 and O:25 (additional O factors associated with serogroup O:6,14) are encoded outside the rfb gene cluster. We developed a serogroup O:6,14-specific PCR assay based on a region of the putative wzx (O antigen flippase) gene. This provides the basis for a sensitive and specific test for the rapid identification of Salmonella serogroup O:6,14.     

Rapid microarray‐based DNA genoserotyping of Escherichia coli

In this study, an improvement in the oligonucleotide‐based DNA microarray for the genoserotyping of Escherichia coli is presented. Primer and probes for additional 70 O antigen groups were developed. The microarray was transferred to a new platform, the ArrayStrip format, which allows high through‐put tests in 96‐well formats and fully automated microarray analysis. Thus, starting from a single colony, it is possible to determine within a few hours and a single experiment, 94 of the over 180 known O antigen groups as well as 47 of the 53 different H antigens. The microarray was initially validated with a set of defined reference strains that had previously been serotyped by conventional agglutination in various reference centers. For further validation of the microarray, 180 clinical E. coli isolates of human origin (from urine samples, blood cultures, bronchial secretions, and wound swabs) and 53 E. coli isolates from cattle, pigs, and poultry were used. A high degree of concordance between the results of classical antibody‐based serotyping and DNA‐based genoserotyping was demonstrated during validation of the new 70 O antigen groups as well as for the field strains of human and animal origin. Therefore, this oligonucleotide array is a diagnostic tool that is user‐friendly and more efficient than classical serotyping by agglutination. Furthermore, the tests can be performed in almost every routine lab and are easily expanded and standardized.     

Characterization and application of a murine monoclonal antibody that reacts specifically with the serogroup D1Salmonella

A murine hybridoma cell line that produces monoclonal antibody (mAb) against the serogroup D1 Salmonella lipopolysaccharide (LPS) antigen was established. The trisaccharide tyvelose alpha 1----3 mannose alpha 1----4 rhamnose was shown to be involved in the reactive epitope of the mAb since this mAb reacted strongly with strains of serogroup D1 Salmonella but not with Salmonella strains from the O serogroups of A, B, and D2, and sodium meta-periodate was found to destroy the reactivity of the serogroup D1 O-antigen with the mAb. As such this mAb was found to be a useful serotyping reagent for the identification of serogroup D1 Salmonella, and for the differentiation of strains of serogroups D1 and D2 Salmonella which have identical flagellar H antigens.     

Serotyping of Vibrio anguillarum.

A serotyping scheme based on the detection of O antigens by slide agglutination in fish-pathogenic strains of Vibrio anguillarum is presented. Over a period of 5 years 270 Vibrio strains from feral and cultured fish, 189 strains from the environment, and 36 strains from invertebrates were collected. The strains were divided into 10 distinct serotypes (O1 through O10). More than 90% of the fish-pathogenic strains, but only 40% of the environmental strains, were typable; 71% of the strains isolated from cultured rainbow trout were serotype O1, whereas 78% of the strains isolated from feral fish were serotype O2. No dominating environmental serotype was found. A serotyping system for V. anguillarum is proposed. A total of 90 strains received from culture collections and laboratories in different countries were typed according to the present system.     

Serotyping of Vibrio anguillarum

A serotyping scheme based on the detection of O antigens by slide agglutination in fish-pathogenic strains of Vibrio anguillarum is presented. Over a period of 5 years 270 Vibrio strains from feral and cultured fish, 189 strains from the environment, and 36 strains from invertebrates were collected. The strains were divided into 10 distinct serotypes (O1 through O10). More than 90% of the fish-pathogenic strains, but only 40% of the environmental strains, were typable; 71% of the strains isolated from cultured rainbow trout were serotype O1, whereas 78% of the strains isolated from feral fish were serotype O2. No dominating environmental serotype was found. A serotyping system for V. anguillarum is proposed. A total of 90 strains received from culture collections and laboratories in different countries were typed according to the present system.     

Development of an O-antigen serotyping scheme for Cronobacter sakazakii

Cronobacter sakazakii is an opportunistic pathogen that can cause severe infections. Serotyping provides a basis for the categorization of bacterial strains and is an important tool for epidemiological and surveillance purposes. In this study, of the 135 Cronobacter strains tested initially, 119 were identified as C. sakazakii and used. A serotyping scheme for C. sakazakii that classifies strains based on their different O antigens was developed. Seven antisera that exhibited high agglutinin titers (>640) were produced. O2 and O6 antisera were specific for their homologous strains, O4 and O7 antisera gave heterologous titers with O1 and O6 antigens, respectively, and O1, O3, and O5 antisera cross-reacted with each other and require preabsorption with the other two antigens. All of these 119 C. sakazakii strains were clearly assigned to these seven serotypes. O1 and O2 are the dominant serotypes, comprising 69.7% of the isolates. We also characterized the O-antigen gene clusters using restriction fragment length polymorphism (RFLP). The grouping of C. sakazakii strains based on their RFLP banding patterns correlated well with the grouping of strains based on our serotyping scheme. The serotype scheme presented here could prove to be a useful tool for serotyping C. sakazakii isolates.     

Distribution of Vibrio cholerae O1 antigen biosynthesis genes among O139 and other non-O1 serogroups of Vibrio cholerae

The organization and distribution of the genes responsible for O antigen biosynthesis in various serogroups of Vibrio cholerae were investigated using several DNA probes derived from various regions of the genes responsible for O1 antigen biosynthesis. Based on the reactivity pattern of the probes against the various serogroups, the cluster of genes responsible for the O1 antigen biosynthesis could be broadly divided into six groups, designated as class 1-6. The class 3 cluster of genes corresponding to gmd to wbeO, wbeT and a part of wbeU was specific for only the O1 serogroup. The other cluster of genes (class 1, 2, 4-6) reacted with other serogroups of V. cholerae. These data indicate that serotype conversion in V. cholerae does not depend on a simple mutational event but may involve horizontal gene transfer not only between V. cholerae strains but also between V. cholerae and species other than V. cholerae.     

基因芯片技术检测3种食源性致病微生物方法的建立

建立一种运用多重PCR和基因芯片技术检测和鉴定志贺氏菌、沙门氏菌、大肠杆菌O157的方法, 为3种食源性致病菌的快速检测和鉴定提供了准确、快速、灵敏的方法。分别选取编码志贺氏菌侵袭性质粒抗原H基因(ipaH)、沙门氏菌肠毒素(stn)基因和致泻性大肠杆菌O157志贺样毒素(slt)基因设计引物和探针, 进行三重PCR扩增, 产物与含特异性探针的芯片杂交。对7种细菌共26株菌进行芯片检测, 仅3种菌得到阳性扩增结果, 证明此方法具有很高的特异性。3种致病菌基因组DNA和细菌纯培养物的检测灵敏度约为8 pg。对模拟食品样品进行直接检测, 结果与常规细菌学培养结果一致, 检测限为50 CFU/mL。结果表明：所建立的基因芯片检测方法特异性好, 灵敏度高, 为食源性致病菌的检测提供了理想手段, 有良好的应用前景。     

Examination of retail chickens and sausages in Britain for vero cytotoxin-producing Escherichia coli.

Samples from chickens and pork sausages were examined for the presence of Vero cytotoxin-producing Escherichia coli by using DNA probes for the Vero cytotoxin genes. Hybridization was detected in 25% of the 184 sausage samples, but none of the chickens was positive. No E. coli O157:H7 strains were isolated, and serotyping showed that the Vero cytotoxin-producing E. coli strains belonged to eight different O serogroups and that six strains had an unidentifiable O antigen.     

Examination of retail chickens and sausages in Britain for vero cytotoxin-producing Escherichia coli

Samples from chickens and pork sausages were examined for the presence of Vero cytotoxin-producing Escherichia coli by using DNA probes for the Vero cytotoxin genes. Hybridization was detected in 25% of the 184 sausage samples, but none of the chickens was positive. No E. coli O157:H7 strains were isolated, and serotyping showed that the Vero cytotoxin-producing E. coli strains belonged to eight different O serogroups and that six strains had an unidentifiable O antigen.     

Molecular serotyping of Escherichia coli O111:H8

Accurate Escherichia coli serotyping is critical for pathogen diagnosis and surveillance of non-O157 Shiga-toxigenic strains, however few laboratories have this capacity. The molecular serotyping protocol described in this paper targets the somatic and flagellar antigens of E. coli O111:H8 used in traditional serotyping, and can be performed routinely in the laboratory.     

Molecular serotyping of Escherichia coli O26:H11

Serotyping is the foundation of pathogenic Escherichia coli diagnostics; however, few laboratories have this capacity. We developed a molecular serotyping protocol that targets, genetically, the same somatic and flagellar antigens of E. coli O26:H11 used in traditional serotyping. It correctly serotypes strains untypeable by traditional methods, affording primary laboratories serotyping capabilities.     

Presence of rfe genes in Escherichia coli: their participation in biosynthesis of O antigen and enterobacterial common antigen.

In Salmonella, ilv-linked rfe genes participate in the biosynthesis of the enterobacterial common antigen (CA) as well as of certain types of O antigen (serogroups C1 and L). rff genes, probably in the same cluster with rfe, are required for CA synthesis (P.H. M?kel? et al., in preparation). Several Escherichia coli strains were studied to determine whether they also have rfe-rff genes that are involved in the synthesis of O antigen and CA, or of CA only. In a first approach, E, coli K-12 F-prime factors carrying the genes ilv and argH or argE and presumably rfe-rff genes were introduced into CA-negative Salmonella mutants that are blocked in CA synthesis because of mutated rfe or rff genes. All resulting ilv+ hybrids were CA positive. In recipients with group C1-derived rfb genes, the synthesis of O6,7-specific antigen was also restored. This result shows that E. coli K-12 has rfe and rff genes providing the functions required in the synthesis of CA and Salmonella 6,7-specific polysaccharide. By introduction of defective rfe regions from suitable Salmonella donors into E. coli O8, 09, and O100 strains, the synthesis of CA as well as of the O-specific polysaccharides was blocked. This indicates that in the E. coli strains tested the rfe genes are involved in the synthesis of both O antigen and CA. This suggestion was confirmed by the finding of E. coli rough mutants that had simultaneously become CA negative. In transduction experiments it could be shown that the appearance of the rough and CA- phenotype was due to a defect in the ilv-linked rfe region.     

Molecular Serotyping of Escherichia coli O26:H11

Serotyping is the foundation of pathogenic Escherichia coli diagnostics; however, few laboratories have this capacity. We developed a molecular serotyping protocol that targets, genetically, the same somatic and flagellar antigens of E. coli O26:H11 used in traditional serotyping. It correctly serotypes strains untypeable by traditional methods, affording primary laboratories serotyping capabilities.     

Genetic analyses of the putative O and K antigen gene clusters of pandemic Vibrio parahaemolyticus

Pandemic V. parahaemolyticus strains have rapidly changed their serotypes, but its determinants, especially K antigen, and the genes involved in serotype have been an open question. The purpose of this study was to gain insights into these points. Although V. parahaemolyticus is known to be lacking O-side chain on its lipopolysaccharide, and O antigens are thought to be represented by core OS, the genome sequence of V. parahaemolyticus O3:K6 strain RIMD2210633 suggests that this bacterium potentially synthesizes O-side chain. To explore possible relatedness between this O-side chain biosynthesis gene cluster, which is similar in the serotypes of Vibrio cholerae, and of V. parahaemolyticus, we amplified both core OS and O-side chain gene clusters of the strains belonging to various serotypes of V. parahaemolyticus by long PCR and performed PCR RFLP analyses. The results of our RFLP analyses suggest that the core OS biosynthesis gene cluster is related to the O antigens of pandemic V. parahaemolyticus and that the putative O-side chain gene cluster is related to K antigens of pandemic V. parahaemolyticus. We then determined the sequence of these regions of a pandemic O4:K68 strain, and compared it with the corresponding sequence of RIMD2210633. In addition, PCR analysis showed the putative O4 and K68 antigen gene clusters are unique to the strains belonging to the O4 and K68 serotype respectively. The data implies that the pandemic O4:K68 V. parahaemolyticus strain emerged from the pandemic O3:K6 strain by replacement of the putative O and K antigen gene clusters.     

Properties of wild-type strains of enterotoxigenic Escherichia coli which produce colonization factor antigen II, and belong to serogroups other than O6

Enterotoxigenic strains of Escherichia coli, which belonged to serogroups other than O6 and produced colonization factor antigen II, usually produced only coli surface antigen 3 (CS3) and gave weak mannose-resistant haemagglutination of bovine erythrocytes. A non-autotransferring plasmid, NTP165, from a strain of E. coli O168. H16 coded for heat-stable enterotoxin, heat-labile enterotoxin and CS antigens. The CS antigens expressed after acquisition of plasmid NTP165 depended on the recipient strain: a biotype A strain of serotype O6. H16 expressed CS1 and CS3; a biotype C strain of serotype O6. H16 expressed CS2 and CS3; strain K12 and strain E19446 of serotype O139. H28 expressed only CS3. An exceptional wild-type strain, E24377, of serotype O139. H28 produced CS1 and CS3 when isolated; a variant of E24377 which had lost the plasmid coding for CS antigens produced both CS1 and CS3 after the introduction of NTP165.     

Somatic and flagellar antigens ofSerratia ficaria from the United States and the Mediterranean region

Somatic (O) and flagellar (H) antigens of 37Serratia ficaria strains were studied. All strains shared a common H antigen (H1). Four O antigens were identified that defined four serovars (O1:H1, O2:H1, O3:H1, and O4:H1). All American strains studied (isolated from the fig-fig wasp biological cycle or from a human patient) belonged to serotype O1:H1. Strains from the Mediterranean region (Sicily, Tunisia, France) were not so antigenically uniform and all four serotypes were found in figs from Sicily.     

Molecular Analysis of a Salmonella Enterica Group E1 Rfb Gene Cluster: O Antigen and the Genetic Basis of the Major Polymorphism

Salmonella enterica is highly polymorphic for the O antigen, a surface polysaccharide that is subject to intense selection by the host immune system. This polymorphism is used for serotyping Salmonella isolates. The genes encoding O antigen biosynthesis are located in the rfb gene cluster. We report here the cloning and sequence of the 19-kb rfb region from strain M32 (serovar anatum, group E1) and compare it with that of strain LT2 (serovar typhimurium, group B). Genes for biosynthetic pathways common to both strains are conserved and have very similar sequences. In contrast, the five genes for CDP-abequose synthesis, present in strain LT2, are absent in strain M32; three open reading frames (ORFs) of strain LT2, thought to include genes for transferases, are not present in strain M32 but are replaced by three different ORFs with little or low level of similarity. Both rfb gene clusters are low in G + C content, indicating that they were transferred from a common ancestral species with low G + C content to S. enterica relatively recently (in the evolutionary sense). We discuss the recombination and lateral transfer events which may have been involved in the evolution of the polymorphism.     

Development of primers to O-antigen biosynthesis genes for specific detection of Escherichia coli O157 by PCR.

The chemical composition of each O-antigen subunit in gram-negative bacteria is a reflection of the unique DNA sequences within each rfb operon. By characterizing DNA sequences contained with each rfb operon, a diagnostic serotype-specific probe to Escherichia coli O serotypes that are commonly associated with bacterial infections can be generated. Recently, from an E. coli O157:H7 cosmid library, O-antigen-positive cosmids were identified with O157-specific antisera. By using the cosmid DNAs as probes, several DNA fragments which were unique to E. coli O157 serotypes were identified by Southern analysis. Several of these DNA fragments were subcloned from O157-antigen-positive cosmids and served as DNA probes in Southern analysis. One DNA fragment within plasmid pDS306 which was specific for E. coli O157 serotypes was identified by Southern analysis. The DNA sequence for this plasmid revealed homology to two rfb genes, the first of which encodes a GDP-mannose dehydratase. These rfb genes were similar to O-antigen biosynthesis genes in Vibrio cholerae and Yersinia enterocolitica serotype O:8. An oligonucleotide primer pair was designed to amplify a 420-bp DNA fragment from E. coli O157 serotypes. The PCR test was specific for E. coli O157 serotypes. PCR detected as few as 10 cells with the O157-specific rfb oligonucleotide primers. Coupled with current enrichment protocols, O157 serotyping by PCR will provide a rapid, specific, and sensitive method for identifying E. coli O157.