O-antigen Gene Research Articles

Structure and gene cluster of the O-antigen of Escherichia coli O165 containing 5-N-acetyl-7-N-[(R)-3-hydroxybutanoyl]pseudaminic acid.

Upon mild acid degradation of the lipopolysaccharide of Escherichia coli O165, the O-polysaccharide chain was cleaved at the glycosidic linkage of 5-N-acetyl-7-N-[(R)-3-hydroxybutanoyl]pseudaminic acid (Pse5Hb7Ac). Analysis of the resulting linear tetrasaccharide and alkali-treated lipopolysaccharide by (1)H/(13)C 1D and 2D nuclear magnetic resonance spectroscopy enabled elucidation of the following structure of the O-polysaccharide: →8)-α-Psep5Hb7Ac-(2 → 6)-β-d-Galp-(1 → 4)-β-d-Glсp-(1 → 3)-α-d-GlсpNAc-(1→. The β-d-Galp-(1 → 4)-β-d-Glсp-(1 → 3)-d-GlсpNAc structural element is also present in the O-polysaccharide of E. coli O82. The content of the O-antigen gene cluster of E. coli O165 was found to be consistent with the O-polysaccharide structure established. Functions of proteins encoded in the gene cluster, including enzymes involved in the Pse5Hb7Ac biosynthesis and glycosyltransferases, were putatively assigned by comparison with sequences in available databases.

Structural comparison of O-antigen gene clusters of Legionella pneumophila and its application of a serogroup-specific multiplex PCR assay.

The Legionella pneumophila serogroups O1, O4, O6, O7, O10 and O13 are pathogenic strains associated with pneumonia. The surface O-antigen gene clusters of L. pneumophila serogroups O4, O6, O7, O10 and O13 were sequenced and analyzed, with the function annotated on the basis of homology to that of the genes of L. pneumophila serogroup O1 (L. pneumophila subsp. pneumophila str. Philadelphia 1). The gene locus of the six L. pneumophila serogroups contains genes of yvfE, neuABCD, pseA-like for nucleotide sugar biosynthesis, wecA for sugar transfer, and wzm as well as wzt for O-antigen processing. The detection of O-antigen genes allows the fine differentiation at species and serogroup level without the neccessity of nucleotide sequencing. The O-antigen-processing genes wzm and wzt, which were found to be distinctive for different for different serogroups, have been used as the target genes for the detection and identification of L. pneumophila strains of different O serogroups. In this report, a multiplex PCR assay based on wzm or wzt that diferentiates all the six serogroups by amplicon size was developed with the newly designed specific primer pairs for O1 and O7, and the specific primer pairs for O4, O6, O10, and O13 reported previously. The array was validated by analysis of 34 strains including 15 L. pneumophila O-standard reference strains, eight reference strains of other Legionella non-pneumophila species, six other bacterial species, and five L. pneumophila environmental isolates. The detection sensitivity was one ng genomic DNA. The accurate and sensitive assay is suitable for the identification and detection of strains of these serogroups in environmental and clinical samples.

Structure and genetics of biosynthesis of the glycosyl phosphate-containing O-polysaccharide of Escherichia coli O160

On mild acid degradation of the lipopolysaccharide of Escherichia coli O160, the O-polysaccharide was cleaved by acid-labile glycosyl phosphate linkages in the main chain. The resultant oligosaccharide and the alkali-treated lipopolysaccharide were studied by sugar analysis along with 1H and 13C NMR spectroscopies, and the following structure of the branched pentasaccharide repeating unit of the O-polysaccharide was established: [Display omitted] The O-antigen gene cluster of E. coli O160 was found to be consistent with the O-polysaccharide structure established.

Structure and gene cluster of the O-antigen of Escherichia coli O43

The O-polysaccharide (O-antigen) of Escherichia coli O43 was isolated from the lipopolysaccharide and studied by chemical methods, including sugar analyses, Smith degradation, and solvolysis with anhydrous trifluoroacetic acid, along with 1H and 13C NMR spectroscopy. The following structure of the pentasaccharide repeating unit of the O-polysaccharide was established: [Display omitted] Functions of genes in the O-antigen gene cluster of E. coli O43 were assigned by a comparison with sequences in the available databases and found to be in agreement with the O-polysaccharide structure.

Identification serologically, chemically and genetically of two Escherichia coli strains as candidates for new O serogroups.

Escherichia coli strains are normally identified by the combination of their O and H (and sometimes K) antigens, and serotyping based on the antigens is believed to be crucial for clinical detection and epidemiological investigation. Two E. coli strains, G5413 and G5287, were isolated from faecal samples of female patients with diarrhoea and were not agglutinated with any antisera that cover the well-known O serogroups of E. coli. We elucidated the O-polysaccharide (OPS) structures and analysed the O-antigen gene clusters of these bacteria. The OPS structure of G5413 established by monosaccharide analysis and NMR spectroscopy was found to be unique amongst known bacterial polysaccharide structures. The O-antigen gene cluster of this strain was sequenced and did not match sequence data with any of the 184 O serogroups that have been recognized internationally. Gene functions were tentatively assigned and were appropriate to the OPS structure. Based on these data, we suggest G5413 as a candidate for a new E. coli O serogroup. Both the OPS structure and O-antigen gene cluster of G5287 were identical to those of E. coli L-19, a candidate for another new O serogroup characterized by us recently. Recognition of these two provisional O serogroups increases the number of known O-antigen forms of E. coli to 186.

Diversity of O Antigens within the Genus Cronobacter: from Disorder to Order.

Cronobacter species are Gram-negative opportunistic pathogens that can cause serious infections in neonates. The lipopolysaccharides (LPSs) that form part of the outer membrane of such bacteria are possibly related to the virulence of particular bacterial strains. However, currently there is no clear overview of O-antigen diversity within the various Cronobacter strains and links with virulence. In this study, we tested a total of 82 strains, covering each of the Cronobacter species. The nucleotide variability of the O-antigen gene cluster was determined by restriction fragment length polymorphism (RFLP) analysis. As a result, the 82 strains were distributed into 11 previously published serotypes and 6 new serotypes, each defined by its characteristic restriction profile. These new serotypes were confirmed using genomic analysis of strains available in public databases: GenBank and PubMLST Cronobacter. Laboratory strains were then tested using the current serotype-specific PCR probes. The results show that the current PCR probes did not always correspond to genomic O-antigen gene cluster variation. In addition, we analyzed the LPS phenotype of the reference strains of all distinguishable serotypes. The identified serotypes were compared with data from the literature and the MLST database (www.pubmlst.org/cronobacter/). Based on the findings, we systematically classified a total of 24 serotypes for the Cronobacter genus. Moreover, we evaluated the clinical history of these strains and show that Cronobacter sakazakii O2, O1, and O4, C. turicensis O1, and C. malonaticus O2 serotypes are particularly predominant in clinical cases.

Expression of the Yersinia enterocolitica O:3 LPS O-antigen and outer core gene clusters is RfaH-dependent.

The antiterminator RfaH is required for the expression of LPS, capsule, haemolysin, exotoxin, haemin uptake receptor and F pilus. As these structures are critical for bacterial virulence, loss of RfaH usually leads to attenuation. Here, we inactivated the rfaH gene of Yersinia enterocolitica O:3 to study its role in this enteropathogen. RNA sequencing of the WT and ΔrfaH strain transcriptomes revealed that RfaH acted as a highly specific regulator that enhanced the transcription of the operons involved in biosynthesis of LPS O-antigen and outer core (OC), but did not affect the expression of enterobacterial common antigen. Interestingly, the transcriptome of the ΔrfaH strain was very similar to that of an O-antigen-negative mutant. This indicated that some of the changes seen in the ΔrfaH strain, such as the genes involved in outer membrane homeostasis or in the stress-response-associated Cpx pathway, were actually due to indirect responses via the loss of O-antigen. The decreased amount of LPS on the ΔrfaH strain cell surface resulted in an attenuated stress response, and lower resistance to compounds such as SDS and polymyxin B. However, the ΔrfaH strain was significantly more resistant to complement-mediated killing by normal human serum. Taken together, our results revealed a novel role of RfaH acting as a highly specific regulator of O-antigen and OC of LPS in Y. enterocolitica O:3. It may be speculated that RfaH might have an in vivo role in controlling tissue-specific expression of bacterial surface oligo/polysaccharides.

Structure of the O-polysaccharide of Escherichia coli O87

The following structure of the O-polysaccharide of Escherichia coli HS1/2 serving as a primary receptor for bacteriophage DT57-12 was elucidated by sugar analysis along with 1D and 2D 1H and 13C NMR spectroscopy: [Display omitted] This structure is shared by E. coli O87 type strain. Putatively assigned functions of genes in the O-antigen gene cluster of E. coli O87 are consistent with the O-polysaccharide structure established.

Structure of fructans from three strains of Cronobacter dublinensis

Partially O-acetylated fructan was isolated by phenol—water extraction of cells of Cronobacter dublinensis G2732 followed by anion-exchange chromatography on DEAE-Toyopearl 650M. Structure of the isolated fructan was established by 2D 1H and 13C NMR spectroscopy. The same fructan but lacking O-acetyl groups was isolated from two other C. dublinensis strains, G3947 and G4061. Genes for the fructan biosynthesis were not found at the typical Cronobacter O-antigen gene cluster located between the conserved genes galF and gnd and are evidently located elsewhere on the chromosome.

Whole genome analysis to detect potential vaccine-induced changes on Shigella sonnei genome

Shigellosis or bacillary dysentery is endemic worldwide and is a significant cause of death in children less than five years of age in developing countries. There are no licensed Shigella vaccines and glycoconjugates are among the leading candidate vaccines against shigellosis today.We used whole genome sequence analysis (WGA) to find out whether immunization, with an investigational Shigella sonnei glycoconjugate, could induce selective pressure leading to changes in the genome of S. sonnei. An outbreak of culture-proven S. sonnei shigellosis which occurred immediately after vaccination in one of the cohorts of volunteers participating in a phase III trial of the vaccine in Israel created a unique condition in which the epidemic agent “co-existed” with the developing immune responses induced by the vaccine and natural infection among vaccinees who developed S. sonnei shigellosis. By comparing the whole genomes of S. sonnei isolated from vaccinees and from volunteers in the control group, we show at a very high sensitivity that a potent S. sonnei glycoconjugate that conferred 74% protective efficacy against the homologous disease did not induce changes in the genome of S. sonnei and in particular on the O-antigen gene cluster.

Structure and gene cluster of the O-antigen of Escherichia coli O140

An acidic O-polysaccharide (O-antigen) was isolated from the lipopolysaccharide of Escherichia coli O140 and studied by sugar analysis along with 1D and 2D 1H and 13C NMR spectroscopy. The following structure of the branched hexasaccharide repeating unit was established: ▪The O-antigen gene cluster of E. coli O140 was sequenced. The gene functions were tentatively assigned by a comparison with sequences in the available databases and found to be in full agreement with the E. coli O140 polysaccharide structure.

Structures and gene clusters of the closely related O-antigens of Escherichia coli O46 and O134, both containing d-glucuronoyl-d-allothreonine

The O-polysaccharides (O-antigens) were isolated by mild acid degradation of the lipopolysaccharide (LPS) of Escherichia coli O46 and O134. The structures of their linear tetrasaccharide repeating units were established by sugar analysis along with 1D and 2D 1H and 13C NMR spectroscopy: ▪.where d-aThr indicates d-allothreonine and R indicates O-acetyl substitution (∼70% on aThr and∼15% on GalNAc) in E. coli O46 whereas the O-acetylation is absent in E. coli O134. Functions of genes in the essentially identical O-antigen gene clusters of E. coli O46 and O134 were tentatively assigned by a comparison with sequences in available databases and found to be in agreement with the O-polysaccharide structures established.

Escherichia coli O-Antigen Gene Clusters of Serogroups O62, O68, O131, O140, O142, and O163: DNA Sequences and Similarity between O62 and O68, and PCR-Based Serogrouping.

The DNA sequence of the O-antigen gene clusters of Escherichia coli serogroups O62, O68, O131, O140, O142, and O163 was determined, and primers based on the wzx (O-antigen flippase) and/or wzy (O-antigen polymerase) genes within the O-antigen gene clusters were designed and used in PCR assays to identify each serogroup. Specificity was tested with E. coli reference strains, field isolates belonging to the target serogroups, and non-E. coli bacteria. The PCR assays were highly specific for the respective serogroups; however, the PCR assay targeting the O62 wzx gene reacted positively with strains belonging to E. coli O68, which was determined by serotyping. Analysis of the O-antigen gene cluster sequences of serogroups O62 and O68 reference strains showed that they were 94% identical at the nucleotide level, although O62 contained an insertion sequence (IS) element located between the rmlA and rmlC genes within the O-antigen gene cluster. A PCR assay targeting the rmlA and rmlC genes flanking the IS element was used to differentiate O62 and O68 serogroups. The PCR assays developed in this study can be used for the detection and identification of E. coli O62/O68, O131, O140, O142, and O163 strains isolated from different sources.

Structure and genetics of the O-antigen of Enterobacter cloacae G3054 containing di-N-acetylpseudaminic acid

Mild acid degradation of the lipopolysaccharide of Enterobacter cloacae G3054 resulted in the cleavage of the O-polysaccharide at the linkage of residues of 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic acid (di-N-acetylpseudaminic acid, Pse5Ac7Ac) in the main chain. The resultant oligosaccharide and an alkali-treated lipopolysaccharide were studied by sugar analysis along with 1H and 13C NMR spectroscopy, and the following structure of the branched pentasaccharide O-unit of the O-polysaccharide was established: [Display omitted] The O-antigen gene cluster of E. cloacae G3054 between conserved genes galF and gnd was sequenced. Most genes necessary for the O-antigen synthesis were found in the cluster and their functions were tentatively assigned by comparison with sequences in the available databases.

Development of three multiplex PCR assays targeting the 21 most clinically relevant serogroups associated with Shiga toxin-producing E. coli infection in humans.

Escherichia coli serogroups O5, O15, O26, O45, O55, O76, O91, O103, O104, O111, O113, O118, O121, O123, O128, O145, O146, O157, O165, O172, and O177 are the O-antigen forms of the most clinically relevant Shiga toxin-producing E. coli (STEC) serotypes. In this study, three multiplex PCR assays able to specifically detect these 21 serogroups were developed and validated. For this purpose, the O-antigen gene clusters of E. coli O5 and O76 were fully sequenced, their associated genes were identified on the basis of homology, and serogroup-specific primers were designed. After preliminary evaluation, these two primer pairs were proven to be highly specific and suitable for the development of PCR assays for O5 and O76 serogroup identification. Specific primers were also designed for serogroups O15, O45, O55, O91, O104, O113, O118, O123, O128, O146, O157, O165, O172, and O177 based on previously published sequences, and previously published specific primers for serogroups O26, O103, O111, O121, and O145 were also included. These 21 primer pairs were shown to be specific for their target serogroup when tested against E. coli type strains representing 169 known O-antigen forms of E. coli and Shigella and therefore suitable for being used in PCR assays for serogroup identification. In order to validate the three multiplex PCR assays, 22 E. coli strains belonging to the 21 covered serogroups and 18 E. coli strains belonging to other serogroups were screened in a double-blind test and their sensitivity was determined as 1 ng chromosomal DNA. The PCR assays developed in this study could be a faster, simpler, and less expensive strategy for serotyping of the most clinically relevant STEC strains in both clinical microbiology and public health laboratories, and so their development could benefit for clinical diagnosis, epidemiological investigations, surveillance, and control of STEC infections.

A new pyrosequencing assay for rapid detection and genotyping of Shiga toxin, intimin and O157-specific rfbE genes of Escherichia coli

Shiga toxin (stx)-producing Escherichia coli (STEC) contamination in food and water is one of the most recognized concerns and a major financial burden in human hygiene control worldwide. Rapid and highly reliable methods of detecting and identifying STEC causing gastroenteric illnesses are crucial to prevent foodborne outbreaks. A number of tests have been developed and commercialized to detect STEC using molecular microbiology techniques. Most of these are designed to identify virulence factors such as Shiga toxin and intimin as well as E. coli O and H antigen serotype specific genes. In order to screen pathogenic STEC without relying on O:H serotyping, we developed a rapid detection and genotyping assay for STEC virulence genes using a PCR-pyrosequencing application. We adapted the PyroMark Q24 Pyrosequencing platform for subtyping 4 major virulence genes, Shiga toxin 1 and 2 (stx1 and stx2), intimin (eae) and O157-antigen gene cluster target rfbE, using Single Nucleotide Polymorphism (SNP) analysis. A total of 224 E. coli strains including isolates from Canadian environment, food and clinical cases were examined. Based on the multiple alignment analysis of 30–80 base nucleotide pyrogram reads, three alleles of the Shiga toxin 1a gene (stx1a) (stx1a-I, stx1a-II, stx1a-III) were identified. Results of the stx1, stx2, eae and rfbE genotyping revealed that each group of O:H serotype shares distinctive characteristics that could be associated with the virulence of each genotype. O157:H7/NM carries stx1a-II (94%), stx2a (82%), λ/γ1-eae (100%) and rfbE type-H7/NM (100%). Whereas isolates of the “Top-6” serotypes (O26, O45, O103, O111, O121, O145) had a high incidence of stx1a-I (90%) and stx2a (100%). stx1a-III (60%) was only observed in non Top-7 (Top-6 plus O157) STEC and Shigella spp. The entire assay, from extracting DNA from colonies on a plate to the generation of sequence information, can be completed in 5h. The method of profiling these 4 STEC pathogenic genotypes as demonstrated in this paper is rapid, easily performed, informative and cost-effective, and thus has a potential to be deployed in the food industry for the routine screening of potentially pathogenic STEC isolates.

Structural and genetic relationships of closely related O-antigens of Cronobacter spp. and Escherichia coli: C. sakazakii G2594 (serotype O4)/E. coli O103 and C. malonaticus G3864 (serotype O1)/E. coli O29

O-Antigen (O-polysaccharide) variation is the basis for bacterial serotyping and is important in bacterial virulence and niche adaptation. In this work, we present structural and genetic evidences for close relationships between the O-antigens of the Cronobacter spp. and Escherichia coli. Cronobacter sakazakii G2594 (serotype O4) and Cronobacter malonaticus G3864 (serotype O1) are structurally related to those of E. coli O103 and O29, respectively, and some other members of the Enterobacteriaceae family differing in the patterns of lateral glucosylation (C. sakazakii G2594) or O-acetylation (C. malonaticus G3864). The O-antigen gene clusters of the corresponding Cronobacter and E. coli strains contain the same genes with high-level similarity, and the structural differences within both O-antigen pairs were suggested to be due to modification genes carried by prophages.

Related structures of the O-polysaccharides of Cronobacter dublinensis G3983 and G3977 containing 3-(N-acetyl-l-alanyl)amino-3,6-dideoxy-d-galactose

Cronobacter spp. are emerging opportunistic human pathogens linked with life-threatening infections predominantly in neonates. O-Antigen (O-polysaccharide) is highly variable and plays an important role in virulence and niche adaptation. In this work, short-chain O-polysaccharides consisting on the average of 2–3 repeating units were obtained by mild acid or mild alkaline degradation of the lipopolysaccharides of C. dublinensis G3983 and G3977 and studied by composition analysis, Smith degradation, and 1H and 13C NMR spectroscopy. The following structures of the O-polysaccharides were established: [Display omitted] where R indicates H in strain G3983 or α-d-Glcp in strain G3977, d-Fuc3NAlaAc indicates 3-(N-acetyl-l-alanyl)amino-3,6-dideoxy-d-galactose. Both strains share the O-antigen gene cluster, which is identical to that of C. dublinensis O1 (Foodborne Pathog. Dis.2013, 10, 343–352). The assigned gene functions are in agreement with the O-antigen structure of C. dublinensis G3983, and the side-chain glucosylation of the O-antigen of C. dublinensis G3977 is evidently encoded elsewhere in the genome.

Structural and genetic studies of the O-antigen of Escherichia coli O163

An acidic O-polysaccharide (O-antigen) of Escherichia coli O163 was obtained by mild acid hydrolysis of the lipopolysaccharide and studied by sugar analysis and Smith degradation along with 1D and 2D 1H and 13C NMR spectroscopy. The following structure of the linear tetrasaccharide repeating unit was established, which is unique among known structures of bacterial polysaccharides: [Display omitted] Functions of genes in the O-antigen gene cluster of E. coli O163 were tentatively assigned by comparison with sequences in the available databases and found to be in agreement with the O-polysaccharide structure. Relationships between O-antigen structures and gene clusters of E. coli O163 and Salmonella enterica O41 are discussed.

Prevalence of molecular markers for Salmonella and Shiga toxigenic Escherichia coli (STEC) in whole-muscle beef cuts sold at retail markets in Costa Rica

The present study sought to determine the prevalence of molecular markers for Salmonella and Shiga toxigenic Escherichia coli (STEC) serogroups O26, O45, O103, O111, O121, O145, and O157 in whole-muscle beef cuts sold at retail in urban and semi-rural areas of Costa Rica. A total of 279 (171 urban, 108 semi-rural) samples were purchased from 93 butcher shops between August of 2012 and August of 2013 and tested for the presence of molecular markers characteristic of Salmonella and STEC using the DuPont Qualicon BAX® System. The overall prevalence of Salmonella and STEC markers was 3.6% (10/279) and 4.7% (13/279), respectively. Salmonella markers were more frequently found in semi-rural (6.5%; 7/108) than in urban (1.8%; 3/171) areas. Similarly, STEC markers were more commonly detected in semi-rural (7.4%; 8/108) than in urban (2.9%; 5/171) areas. A marginal association was found between Salmonella markers and sampling location (P = 0.0496), whereas the presence of STEC markers and sampling location were considered independent (P = 0.1416). Among the 13 positive samples for STEC, 38 O-antigen gene fragments were amplified, with serogroups O45 and O121 being the predominant (28.9 and 21.1%, respectively). Markers for somatic antigens O111 and O157 were not detected in any of the samples. The present investigation is the first of its kind in Central America and shows that both Salmonella and STEC may be commonly present in whole-muscle raw beef cuts sold at retail markets in Costa Rica. Consequently, consumers may be directly at risk of foodborne illness due if meat products are not adequately cooked and handled in food service or domestic settings. Future work should emphasize continuous monitoring of the presence of these and other zoonotic pathogens in meat and meat products and on the implementation and validation of adequate food safety controls along the farm-to-table continuum.