YciR, a Specific 3'-Phosphodiesterase, Plays a Role in the Pathogenesis of Uropathogenic Escherichia coli CFT073.

Urinary tract infections (UTIs), with the characteristics of recurrence and resistance to antibiotics due to misuse, remain a common health and economic issue for patients. Uropathogenic Escherichia coli (UPEC), which is capable of evading the immune response by forming intracellular bacterial communities (IBCs) in the cytoplasm of bladder epithelial cells (BECs) after invasion, has been shown to be the prevailing cause of UTIs. Cyclic dimeric guanosine monophosphate (c-di-GMP) is a small molecule responsible for eliciting the innate immune response of the host only if it has not been degraded by some phosphodiesterases (PDEs), such as YciR. The relationship between YciR and c-di-GMP levels in UPEC is inconclusive. In this study, we investigated the gene expression profile of UPEC in BECs and identified yciR as an upregulated gene. Western blot revealed that YciR enhanced the virulence of UPEC by inhibiting the phosphorylation of NF-κB. The expression of yciR could be repressed by HupB in a directly binding manner. We identified YciR, a novel PDE, and defined its possible function in innate immune evasion. We also demonstrated that YciR is an HupB-dependent PDE that degrades c-di-GMP and that a low concentration of c-di-GMP might make NF-κB less phosphorylated, thereby reducing the host's pro-inflammatory response. This is the first time that YciR has been identified as a virulence factor in the pathogenesis of UPEC. These findings further increase our understanding of the pathogenesis of UPEC and provide a theoretical basis for further studies.

Pan-Genome Analysis of Delftia tsuruhatensis Reveals Important Traits Concerning the Genetic Diversity, Pathogenicity, and Biotechnological Properties of the Species.

Delftia tsuruhatensis strains have long been known to promote plant growth and biological control. Recently, it has become an emerging opportunistic pathogen in humans. However, the genomic characteristics of the genetic diversity, pathogenicity, and biotechnological properties have not yet been comprehensively investigated. Here, a comparative pan-genome analysis was constructed. The open pan-genome with a large and flexible gene repertoire exhibited a high degree of genetic diversity. The purifying selection was the main force to drive pan-genome evolution. Significant differences were observed in the evolutionary relationship, functional enrichment, and degree of selective pressure between the different components of the pan-genome. A high degree of genetic plasticity was characterized by the determinations of diverse mobile genetic elements (MGEs), massive genomic rearrangement, and horizontal genes. Horizontal gene transfer (HGT) plays an important role in the genetic diversity of this bacterium and the formation of genomic traits. Our results revealed the occurrence of diverse virulence-related elements associated with macromolecular secretion systems, virulence factors associated with multiple nosocomial infections, and antimicrobial resistance, indicating the pathogenic potential. Lateral flagellum, T1SS, T2SS, T6SS, Tad pilus, type IV pilus, and a part of virulence-related genes exhibited general properties, whereas polar flagellum, T4SS, a part of virulence-related genes, and resistance genes presented heterogeneous properties. The pan-genome also harbors abundant genetic traits related to secondary metabolism, carbohydrate active enzymes (CAZymes), and phosphate transporter, indicating rhizosphere adaptation, plant growth promotion, and great potential uses in agriculture and biological control. This study provides comprehensive insights into this uncommon species from the genomic perspective. IMPORTANCE D. tsuruhatensis is considered a plant growth-promoting rhizobacterium (PGPR), an organic pollutant degradation strain, and an emerging opportunistic pathogen to the human. However, the genetic diversity, the evolutionary dynamics, and the genetic basis of these remarkable traits are still little known. We constructed a pan-genome analysis for D. tsuruhatensis and revealed extensive genetic diversity and genetic plasticity exhibited by open pan-genome, diverse mobile genetic elements (MGEs), genomic rearrangement, and horizontal genes. Our results highlight that horizontal gene transfer (HGT) and purifying selection are important forces in D. tsuruhatensis genetic evolution. The abundant virulence-related elements associated with macromolecular secretion systems, virulence factors, and antimicrobial resistance could contribute to the pathogenicity of this bacterium. Therefore, clinical microbiologists need to be aware of D. tsuruhatensis as an opportunistic pathogen. The genetic profiles of secondary metabolism, carbohydrate active enzymes (CAZymes), and phosphate transporter could provide insight into the genetic armory of potential applications for agriculture and biological control of D. tsuruhatensis in general.

Attachment of Enterohemorrhagic Escherichia coli to Host Cells Reduces O Antigen Chain Length at the Infection Site That Promotes Infection.

Many enteropathogenic bacteria express a needle-like type III secretion system (T3SS) that translocates effectors into host cells promoting infection. O antigen (OAg) constitutes the outer layer of Gram-negative bacteria protecting bacteria from host immune responses. Shigella constitutively shortens the OAg molecule in its three-dimensional conformation by glucosylation, leading to enhanced T3SS function. However, whether and how other enteropathogenic bacteria shorten the OAg molecule that probably facilitates infection remain unknown. For the first time, we report a smart mechanism by which enterohemorrhagic Escherichia coli specifically reduces the size of the OAg molecule at the infection site upon sensing mechanical signals of intestinal epithelial cell attachment via the membrane protein YgjI. YgjI represses expression of the OAg chain length regulator gene fepE via the global regulator H-NS, leading to shortened OAg chains and injection of more T3SS effectors into host cells. However, bacteria express long-chain OAg in the intestinal lumen benefiting their survival. Animal experiments show that blocking this regulatory pathway significantly attenuates bacterial virulence. This finding enhances our understanding of interactions between the surfaces of bacterial and host cells and the way this interaction enhances bacterial pathogenesis. IMPORTANCE Little is known about the regulation of cell wall structure of enteropathogenic bacteria within the host. Here, we report that enterohemorrhagic Escherichia coli regulates its cell wall structure during the infection process, which balances its survival in the intestinal lumen and infection of intestinal epithelial cells. In the intestinal lumen, bacteria express long-chain OAg, which is located in the outer part of the cell wall, leading to enhanced resistance to antimicrobial peptides. However, upon epithelial cell attachment, bacteria sense this mechanical signal via a membrane protein and reduce the OAg chain length, resulting in enhanced injection into epithelial cells of T3SS effectors that mediate host cell infection. Similar regulation mechanisms of cell wall structure in response to host cell attachment may be widespread in pathogenic bacteria and closely related with bacterial pathogenesis.

A Novel Small RNA Promotes Motility and Virulence of Enterohemorrhagic Escherichia coli O157:H7 in Response to Ammonium.

Enterohemorrhagic Escherichia coli serotype O157:H7 (O157) is a critical, foodborne, human intestinal pathogen that causes severe acute hemorrhagic diarrhea, abdominal cramping, and even death. Small RNAs (sRNAs) are noncoding regulatory molecules that sense environmental changes and trigger various virulence-related signaling pathways; however, few such sRNAs have been identified in O157. Here, we report a novel sRNA, EsrF that senses high ammonium concentrations in the colon and enhances O157 pathogenicity by promoting bacterial motility and adhesion to host cells. Specifically, EsrF was found to directly interact with the 5' untranslated regions of the flagellar biosynthetic gene, flhB, mRNA and increase its abundance, thereby upregulating expression of essential flagellar genes, including flhD, flhC, fliA, and fliC, leading to elevated O157 motility and virulence. Meanwhile, an infant rabbit model of O157 infection showed that deletion of esrF and flhB significantly attenuates O157 pathogenicity. Furthermore, NtrC-the response regulator of the NtrC/B two-component system-was found to exert direct, negative regulation of esrF expression. Meanwhile, high ammonium concentrations in the colon release the inhibitory effect of NtrC on esrF, thereby enhancing its expression and subsequently promoting bacterial colonization in the host colon. Our work reveals a novel, sRNA-centered, virulence-related signaling pathway in O157 that senses high ammonium concentrations. These findings provide novel insights for future research on O157 pathogenesis and targeted treatment strategies.IMPORTANCE The process by which bacteria sense environmental cues to regulate their virulence is complex. Several studies have focused on regulating the expression of the locus of enterocyte effacement pathogenicity island in the typical gut pathogenic bacterium, O157. However, few investigations have addressed the regulation of other virulence factors in response to intestinal signals. In this study, we report our discovery of a novel O157 sRNA, EsrF, and demonstrate that it contributed to bacterial motility and virulence in vitro and in vivo through the regulation of bacterial flagellar synthesis. Furthermore, we show that high ammonium concentrations in the colon induced esrF expression to promote bacterial virulence by releasing the repression of esrF by NtrC. This study highlights the importance of sRNA in regulating the motility and pathogenicity of O157.

Genomic insights into evolution of pathogenicity and resistance of multidrug-resistant Raoultella ornithinolytica WM1.

Raoultella ornithinolytica is a poorly understood opportunistic pathogen, and the underlying mechanisms of its multidrug resistance and pathogenicity have not yet been comprehensively investigated. The multidrug-resistant (MDR) strain WM1 was isolated from the blood of a male patient in Tianjin, China, in 2018. Here, we describe the complete genome and provide a genomic analysis of R. ornithinolytica WM1. The isolate was resistant to all tested antimicrobials except amikacin, tobramycin, and tigecycline. Two plasmids, pWM1-1 (IncHI5) and pWM1-2 (IncR), carried multidrug-resistance regions. A large antimicrobial resistance island region resided on pWM1-1 and exhibited mosaic structures resulting from the acquisition of complex integrations of variable regions, including genes conferring resistance to multiple classes of antimicrobials. Moreover, WM1 possessed virulence-related elements that encode several virulence factors, including type I fimbriae, Escherichia coli common pilus, type II and VI secretion systems, yersiniabactin, enterobactin, and surface polysaccharide, indicating pathogenic potential. Furthermore, the core genome phylogeny and pan-genome analyses revealed extensive genetic diversity. Our analysis indicates the need for stringent infection control, antimicrobial stewardship, periodic resistance monitoring, and rational medication to address potential threats posed by MDR R. ornithinolytica strains.

Chemotaxis and Shorter O-Antigen Chain Length Contribute to the Strong Desiccation Tolerance of a Food-Isolated Cronobacter sakazakii Strain.

Cronobacter sakazakii is an opportunistic pathogen causing a lethality rate as high as 80% in infants. Desiccation tolerance ensures its survival in powdered infant formula (PIF) and contributes to the increased exposure to neonates, resulting in neonatal meningitis, septicemia, and necrotizing enterocolitis. This study showed that a food-isolated C. sakazakii G4023 strain exhibited a stronger desiccation tolerance than C. sakazakii ATCC 29544 strain. Considering the proven pathogenicity of G4023, it could be a big threat to infants. Transcriptome and proteome were performed to provide new insights into the desiccation adaptation mechanisms of G4023. Integrated analyses of these omics suggested that 331 genes were found regulated at both transcriptional and protein levels (≥2.0- and ≥1.5-fold, respectively). Deletion of chemotaxis system encoded genes cheA and cheW resulted in decreased tolerance in both short- and long-term desiccation. Reduced O-antigen chain length contributed to the biofilm formation and desiccation tolerance in the short term rather than the long term. In addition, biosynthesis of flagella, arginine and its transport system, and Fe/S cluster were also observed regulated in desiccated G4023. A better understanding of desiccation adaptation mechanisms of G4023 could in turn guide the operations during production and preservation of PIF or other food to reduce survival odds of G4023 and lower its exposure to get to infants.

Comparative Genomic Analysis Reveals Genetic Mechanisms of the Variety of Pathogenicity, Antibiotic Resistance, and Environmental Adaptation of Providencia Genus.

The bacterial genus Providencia is Gram-negative opportunistic pathogens, which have been isolated from a variety of environments and organisms, ranging from humans to animals. Providencia alcalifaciens, Providencia rettgeri, and Providencia stuartii are the most common clinical isolates, however, these three species differ in their pathogenicity, antibiotic resistance and environmental adaptation. Genomes of 91 isolates of the genus Providencia were investigated to clarify their genetic diversity, focusing on virulence factors, antibiotic resistance genes, and environmental adaptation genes. Our study revealed an open pan-genome for the genus Providencia containing 14,720 gene families. Species of the genus Providencia exhibited different functional constraints, with the core genes, accessory genes, and unique genes. A maximum-likelihood phylogeny reconstructed with concatenated single-copy core genes classified all Providencia isolates into 11 distant groups. Comprehensive and systematic comparative genomic analyses revealed that specific distributions of virulence genes, which were highly homologous to virulence genes of the genus Proteus, contributed to diversity in pathogenicity of Providencia alcalifaciens, Providencia rettgeri, and Providencia stuartii. Furthermore, multidrug resistance (MDR) phenotypes of isolates of Providencia rettgeri and Providencia stuartii were predominantly due to resistance genes from class 1 and 2 integrons. In addition, Providencia rettgeri and Providencia stuartii harbored more genes related to material transport and energy metabolism, which conferred a stronger ability to adapt to diverse environments. Overall, our study provided valuable insights into the genetic diversity and functional features of the genus Providencia, and revealed genetic mechanisms underlying diversity in pathogenicity, antibiotic resistance and environmental adaptation of members of this genus.

Comparative Genomic Analysis of Citrobacter and Key Genes Essential for the Pathogenicity of Citrobacter koseri.

Citrobacter species are opportunistic bacterial pathogens that have been implicated in both nosocomial and community-acquired infections. Among the genus Citrobacter, Citrobacter koseri is often isolated from clinical material, and has been known to cause meningitis and brain abscess in neonates and immunocompromised individuals. The virulence determinants of Citrobacter, however, remain largely unknown. Based on traditional methods, the genus Citrobacter has been divided into 11 species, but this has been problematic. Here, we determined an improved, detailed, and more accurate phylogeny of the genus Citrobacter based on whole genome sequence (WGS) data from 129 Citrobacter genomes, 31 of which were sequenced in this study. A maximum likelihood (ML) phylogeny constructed with core genome single-nucleotide polymorphisms (SNPs) classified all Citrobacter isolates into 11 distinct groups, with all C. koseri strains clustering into a single group. For comprehensive and systematic comparative genomic analyses, we investigated the distribution of virulence factors, resistance genes, and macromolecular secretion systems among the Citrobacter genus. Moreover, combined with group-specific genes analysis, we identified a key gene cluster for iron transport, which is present in the C. koseri group, but absent in other the groups, suggesting that the high-pathogenicity island (HPI) cluster may be important for the pathogenicity of C. koseri. Animal experiments showed that loss of the HPI cluster significantly decreased C. koseri virulence in mice and rat. Further, we provide evidence to explain why Citrobacter freundii is less susceptible than C. koseri to several antibiotics in silico. Overall, our data reveal novel virulence clusters specific to the predominantly pathogenic C. koseri strains, which form the basis for elucidating the virulence mechanisms underlying these important pathogens.

Changing Molecular Epidemiology of Vibrio cholerae Outbreaks in Shanghai, China.

The 7th cholera pandemic began in 1961 in Sulawesi, Indonesia, and then spread around the world in at least three waves. However, the lack of genome sequences for Vibrio cholerae strains under long-term surveillance in East Asia, especially in China, has restricted our understanding of the dynamics of the intracountry and intercountry evolution and transmission of the 7th-pandemic clones. In this study, we obtained the genome sequences of 60 V. cholerae strains isolated in Shanghai, the largest port in the world and the largest city in China, from 1961 to 2011. Our whole-genome-based phylogeny of 7th-pandemic strains revealed that all but one fell into five "stages," most of which are single clades and share independent ancestors. Each stage dominated in succession for a period, with little overlap between them. In addition, two near-identical Shanghai strains belonging to a pre-7th-pandemic precursor and 4 nontoxigenic O1/O139 strains attributed to independent recombination events at the O-antigen loci were present. The major lineages of the 7th pandemic in Shanghai appeared to be closely related to V. cholerae strains isolated from South or Southeast Asia. Stage succession was consistently related to changes in society and human activity, implying that human-caused niche change may play a vital role in the cholera dynamics in Shanghai.IMPORTANCE V. cholerae is the causative agent of cholera, a life-threatening disease characterized by severe, watery diarrhea. The 7th pandemic started in Indonesia in 1961 and spread globally, currently infecting 1.3 million to 4 million people annually. Here, we applied whole-genome sequencing to analyze a long-term collection of V. cholerae clinical strains to reveal the phylogenetic background and evolutionary dynamics of the 7th pandemic in Shanghai, which had undergone breathtakingly rapid development in the last half-century. All but one of the Shanghai 7th-pandemic strains fell into five "stages" that were dominant in Shanghai and appeared to be closely related to 7th-pandemic strains of South or Southeast Asia. Our findings extended the understanding of the dynamics of the evolution and transmission of the 7th-pandemic clones in East Asia and the relationship between social changes and cholera epidemiology.

Comparative genomic analysis of the Hafnia genus reveals an explicit evolutionary relationship between the species alvei and paralvei and provides insights into pathogenicity.

BACKGROUND: The Hafnia genus is an opportunistic pathogen that has been implicated in both nosocomial and community-acquired infections. Although Hafnia is fairly often isolated from clinical material, its taxonomy has remained an unsolved riddle, and the involvement and importance of Hafnia in human disease is also uncertain. Here, we used comparative genomic analysis to define the taxonomy of Hafnia, identify species-specific genes that may be the result of ecological and pathogenic specialization, and reveal virulence-related genetic profiles that may contribute to pathogenesis. RESULTS: One complete genome sequence and 19 draft genome sequences for Hafnia strains were generated and combined with 27 publicly available genomes. We provided high-resolution typing methods by constructing phylogeny and population structure based on single-copy core genes in combination with whole genome average nucleotide identity to identify two distant Hafnia species (alvei and paralvei) and one mislabeled strain. The open pan-genome and the presence of numerous mobile genetic elements reveal that Hafnia has undergone massive gene rearrangements. Presence of species-specific core genomes associated with metabolism and transport suggests the putative niche differentiation between alvei and paralvei. We also identified possession of diverse virulence-related profiles in both Hafnia species., including the macromolecular secretion system, virulence, and antimicrobial resistance. In the macromolecular system, T1SS, Flagellum 1, Tad pilus and T6SS-1 were conserved in Hafnia, whereas T4SS, T5SS, and other T6SSs exhibited the evolution of diversity. The virulence factors in Hafnia are related to adherence, toxin, iron uptake, stress adaptation, and efflux pump. The identified resistance genes are associated with aminoglycoside, beta-lactam, bacitracin, cationic antimicrobial peptide, fluoroquinolone, and rifampin. These virulence-related profiles identified at the genomic level provide insights into Hafnia pathogenesis and the differentiation between alvei and paralvei. CONCLUSIONS: Our research using core genome phylogeny and comparative genomics analysis of a larger collection of strains provides a comprehensive view of the taxonomy and species-specific traits between Hafnia species. Deciphering the genome of Hafnia strains possessing a reservoir of macromolecular secretion systems, virulence factors, and resistance genes related to pathogenicity may provide insights into addressing its numerous infections and devising strategies to combat the pathogen.

Genomics and Experimental Analysis Reveal a Novel Factor Contributing to the Virulence of Cronobacter sakazakii Strains Associated With Neonate Infection.

BACKGROUND: Cronobacter sakazakii causes meningitis and necrotizing enterocolitis in premature infants. However, its virulence determinants, especially those specific for strains associated with neonate infections, remain largely unknown. METHODS: In this study, we performed a comparative genomic analysis of 209 C. sakazakii genomes, and 8 clonal groups (CGs) were revealed. RESULTS: CG1 and CG2 were found to be significantly associated with neonate infections, and significantly prevalent genes in these 2 CGs were identified. Of these, a gene encoding the LysR-type regulator, CklR, was shown to contribute to bacterial pathogenicity based on animal experiments. We found that CklR directly binds and activates the suf Fe-S cluster biosynthesis operon, and high expression of the suf operon increases bacterial resistance to oxidative stress, which increases survival within the host. This leads to a high degree of bacteremia, which contributes to the development of meningitis. CONCLUSIONS: Our work revealed a novel virulence factor specific to predominant pathogenic C. sakazakii strains.

Erratum to: "Structural Relationships Between Genetically Closely Related O-Antigens of Escherichia coli and Shigella spp." [Biochemistry (Moscow), 81, 600 (2016)].

This corrects the article DOI: 10.1134/S0006297916060067.

Establishment of a suspension array for Pseudomonas aeruginosa O-antigen serotyping.

Pseudomonas aeruginosa (P. aeruginosa) is widespread in the environment. It readily infects hospital and immunocompromised patients. Traditional biochemical and immunological diagnoses of P. aeruginosa infection cannot meet clinical demands. The variability of O-antigens is the primary basis for the serotyping schemes of many gram-negative bacteria, which is the most widely used method for pathogenic epidemiological purposes. In this study, we developed a suspension array that can accurately diagnose and identify 19 different P. aeruginosa O-antigen serotypes except O15, whose O-antigen gene cluster has not been characterized. In this assay, wzx/wzy genes were selected as the sero-specific gene for 18 serotypes other than O6, for which the glycosyltransferase gene wbpP was utilized. Meanwhile, the wzyß gene was added to separate O2/16 from O5/18/20, and the insertion sequence (IS) in wzx was used to separate O17 from O11. Eighty-two clinical isolates were screened to test our assay. A total of 65 isolates (79.3%) could be serotyped, and the result were confirmed to be correct by sequencing. Sensitivity analysis indicated that at least 5 ng DNA or 103 CFU cells could be detected using our suspension array. To our knowledge, this is the first report on serotyping P. aeruginosa by suspension array and may be of great value in the clinical diagnostics of P. aeruginosa infection.

Identifying genetic diversity of O antigens in Aeromonas hydrophila for molecular serotype detection.

Aeromonas hydrophila is a globally occurring, potentially virulent, gram-negative opportunistic pathogen that is known to cause water and food-borne diseases around the world. In this study, we use whole genome sequencing and in silico analyses to identify 14 putative O antigen gene clusters (OGCs) located downstream of the housekeeping genes acrB and/or oprM. We have also identified 7 novel OGCs by analyzing 15 publicly available genomes of different A. hydrophila strains. From the 14 OGCs identified initially, we have deduced that O antigen processing genes involved in the wzx/wzy pathway and the ABC transporter (wzm/wzt) pathway exhibit high molecular diversity among different A. hydrophila strains. Using these genes, we have developed a multiplexed Luminex-based array system that can identify up to 14 A. hydrophila strains. By combining our other results and including the sequences of processing genes from 13 other OGCs (7 OGCs identified from publicly available genome sequences and 6 OGCs that were previously published), we also have the data to create an array system that can identify 25 different A. hydrophila serotypes. Although clinical detection, epidemiological surveillance, and tracing of pathogenic bacteria are typically done using serotyping methods that rely on identifying bacterial surface O antigens through agglutination reactions with antisera, molecular methods such as the one we have developed may be quicker and more cost effective. Our assay shows high specificity, reproducibility, and sensitivity, being able to classify A. hydrophila strains using just 0.1 ng of genomic DNA. In conclusion, our findings indicate that a molecular serotyping system for A. hydrophila could be developed based on specific genes, providing an important molecular tool for the identification of A. hydrophila serotypes.

Development of rapid and simple experimental and in silico serotyping systems for Citrobacter.

AIM: Members of the genus Citrobacter are important opportunistic pathogens responsible for high mortality rate. Therefore, in this study, we aimed to develop efficient and accurate Citrobacter typing schemes for clinical detection and epidemiological surveillance. MATERIALS & METHODS: Using genomic and experimental analyses, we located the O-antigen biosynthesis gene clusters in Citrobacter genome for the first time, and used comparative genomic analyses to reveal the specific genes in different Citrobacter serotypes. RESULTS: Based on the specific genes in O-antigen biosynthesis gene clusters of Citrobacter, we established experimental and in silico serotyping systems for this bacterium. CONCLUSION: Both serotyping tools are reliable, and our observations are biologically and clinically relevant for understanding and managing Citrobacter infection.

Development of a molecular serotyping scheme and a multiplexed luminex-based array for Providencia.

Providencia is an opportunistic human pathogen that belongs to the Enterobacteriaceae family. The bacterial cell surface O-antigen is one of the most structurally variable cell constituents and serves as a basis for serotyping gram-negative bacteria. In this work, the genomes of 12 Providencia strains were sequenced, and genes driving O-antigen biosynthesis were analyzed. The O-antigen-synthesizing genes of Providencia are located in the O-antigen gene cluster (OGC) between the cpxA and yibK genes. The gene functions predicted in silico agreed with the known O-antigen structures. All clusters were found to contain both wzx and wzy and exhibit a high degree of heterogeneity. Based on the sero-specific genes, we developed a molecular serotyping system to detect 23 serotypes (from the present and previous studies) for the first time. Five Proteus strains, five Morganella strains, five uropathogenic Escherichia coli (UPEC) strains and 32 Providencia strains with other serotypes were used to assess the specificity of our multiplexed Luminex-based array. Five serogroups (O3, O8, O19, O38 and O52 strains) were used to determine the sensitivity of the suspension array. The detection sensitivity was 0.1 ng genomic DNA, 103 CFU/ml in pure culture, or 104 CFU/ml in mock urine specimens. Furthermore, 29 publicly available Providencia genomes (which have not been serotyped) were analyzed, and 23 novel putative OGC types were identified. In total, we identified 35 new OGCs and developed a molecular serotyping system based on the sero-specific genes. The established classification system can support promising applications in basic research, clinical diagnosis, and epidemiological surveillance.

Structural Relationships Between Genetically Closely Related O-Antigens of Escherichia coli and Shigella spp.

Gene clusters for biosynthesis of 24 of 34 basic O-antigen forms of Shigella spp. are identical or similar to those of the genetically closely related bacterium Escherichia coli. For 18 of these relatedness was confirmed chemically by elucidation of the O-antigen (O-polysaccharide) structures. In this work, structures of the six remaining O-antigens of E. coli O32, O53, O79, O105, O183 (all related to S. boydii serotypes), and O38 (related to S. dysenteriae type 8) were established using (1)H and (13)C NMR spectroscopy. They were found to be identical to the Shigella counterparts, except for the O32- and O38-polysaccharides, which differ in the presence of O-acetyl groups. The structure of the E. coli O105-related O-polysaccharide of S. boydii type 11 proposed earlier is revised. The contents of the O-antigen gene clusters of the related strains of E. coli and Shigella spp. and different mechanisms of O-antigen diversification in these bacteria are discussed in view of the O-polysaccharide structures established. These data illustrate the value of the O-antigen chemistry and genetics for elucidation of evolutionary relationships of bacteria.

Structure of the ß-l-fucopyranosyl phosphate-containing O-specific polysaccharide of Escherichia coli O84.

Fine structure of the O-polysaccharide chain of the lipopolysaccharide (O-antigen) defines the serospecificity of bacterial cells, which is the basis for O-serotyping of medically and agriculturally important gram-negative bacteria including Escherichia coli. In order to obtain the O-polysaccharide for structural analysis, the lipopolysaccharide was isolated from cells of E. coli O84a by phenol/water extraction and degraded with mild acid. However, the O-polysaccharide was cleaved at a highly acid-labile ß-l-fucopyranosyl phosphate (ß-l-Fucp-1-P) linkage to give mainly a pentasaccharide that corresponded to the O-polysaccharide repeat. Therefore, the lipopolysaccharide and the pentasaccharide as well as their O-deacylated derivatives were studied using sugar analysis, NMR spectroscopy, and (for oligosaccharides) ESI HR MS, and the O84-polysaccharide structure was established. The O-polysaccharide is distinguished by the presence of ß-l-Fucp-1-P and randomly di-O-acetylated 6-deoxy-d-talose, which are found for the first time in natural carbohydrates. The gene cluster for the O84-antigen biosynthesis was analysed and its content was found to be consistent with the O-polysaccharide structure.