Construction of Otherwise Isogenic Serotype 6B, 7F, 14, and 19F Capsular Variants of Streptococcus pneumoniae Strain TIGR4

The polysaccharide capsule is the primary virulence factor in Streptococcus pneumoniae. There are at least 90 serotypes of S. pneumoniae, identified based on the immunogenicity of different capsular sugars. The aim of this study was to construct pneumococcal strains that are isogenic except for capsular type. Serotype 4 strain TIGR4 was rendered unencapsulated by recombinational replacement of the capsular polysaccharide synthesis (cps) locus with the bicistronic Janus cassette (C. K. Sung, J. P. Claverys, and D. A. Morrison, Appl. Environ. Microbiol. 67:5190-5196, 2001). In subsequent transformation with chromosomal DNA, the cassette was replaced by the cps locus derived from a strain of a different serotype, either 6B, 7F, 14, or 19F. To minimize the risk of uncontrolled recombinational replacements in loci other than cps, the TIGRcps::Janus strain was “backcross” transformed three times with chromosomal DNA of subsequently constructed capsular type transformants. Capsular serotypes were confirmed in all new capsule variants by the Quellung reaction. Restriction fragment length polymorphism (RFLP) analysis of the cps locus confirmed the integrity of the cps region transformed into the TIGR strain, and RFLP of the flanking regions confirmed their identities with the corresponding regions of the recipient. Transformants had in vitro growth rates greater than or equal to that of TIGR4. All four strains were able to colonize C57BL/6 mice (female, 6 weeks old) for at least 7 days when mice were intranasally inoculated with 6 × 10⁶ to 8 × 10⁶ CFU. The constructed capsular variants of TIGR4 are suitable for use in studies on the role of S. pneumoniae capsular polysaccharide in immunity, colonization, and pathogenesis.     

Discovery of Streptococcus pneumoniae Serotype 6 Variants with Glycosyltransferases Synthesizing Two Differing Repeating Units

Streptococcus pneumoniae is a persistent, opportunistic commensal of the human nasopharynx and is the leading cause of community-acquired pneumonia. It expresses an anti-phagocytic capsular polysaccharide (PS). Genetic variation of the capsular PS synthesis (cps) locus is the molecular basis for structural and antigenic heterogeneity of capsule types (serotypes). Serogroup 6 has four known members (6A–6D) with distinct serologic properties, homologous cps loci, and structurally similar PSs. cps of serotypes 6A/6B have wciNα, encoding α-1,3-galactosyltransferase, whereas serotypes 6C/6D have wciNβ encoding α-1,3-glucosyltransferase. Two atypical serogroup 6 isolates (named 6X11 and 6X12) have been discovered recently in Germany. Flow cytometric studies using monoclonal antibodies show that 6X11 has serologic properties of 6B/6D, whereas 6X12 has 6A/6C. NMR studies of their capsular PSs revealed that 6X11 and 6X12 have two different repeating units with a distribution of ∼40:60 6B:6D and 75:25 6A:6C PS, respectively. Sequencing of the wciNα gene in 6X12 and 6X11 revealed single and double nucleotide substitutions, respectively, resulting in the amino acid changes A150T and D38N. Substitution of alanine with threonine at position 150 in a 6A strain was associated with hybrid serologic and chemical profiles like 6X12. The hybrid serotypes represented by 6X12 and 6X11 strains are now named serotypes 6F and 6G. Single amino acid changes in cps genes encoding glycosyltransferases can alter substrate specificities, permit biosynthesis of heterogeneous capsule repeating units, and result in new hybrid capsule types that may differ in their interaction with the immune system of the host.     

Population-Based Analysis of Invasive Nontypeable Pneumococci Reveals That Most Have Defective Capsule Synthesis Genes

Since nasopharyngeal carriage of pneumococcus precedes invasive pneumococcal disease, characteristics of carriage isolates could be incorrectly assumed to reflect those of invasive isolates. While most pneumococci express a capsular polysaccharide, nontypeable pneumococci are sometimes isolated. Carriage nontypeables tend to encode novel surface proteins in place of a capsular polysaccharide synthetic locus, the cps locus. In contrast, capsular polysaccharide is believed to be indispensable for invasive pneumococcal disease, and nontypeables from population-based invasive pneumococcal disease surveillance have not been extensively characterized. We received 14,328 invasive pneumococcal isolates through the Active Bacterial Core surveillance program during 2006–2009. Isolates that were nontypeable by Quellung serotyping were characterized by PCR serotyping, sequence analyses of the cps locus, and multilocus sequence typing. Eighty-eight isolates were Quellung-nontypeable (0.61%). Of these, 79 (89.8%) contained cps loci. Twenty-two nontypeables exhibited serotype 8 cps loci with defects, primarily within wchA. Six of the remaining nine isolates contained previously-described aliB homologs in place of cps loci. Multilocus sequence typing revealed that most nontypeables that lacked capsular biosynthetic genes were related to established non-encapsulated lineages. Thus, invasive pneumococcal disease caused by nontypeable pneumococcus remains rare in the United States, and while carriage nontypeables lacking cps loci are frequently isolated, such nontypeable are extremely rare in invasive pneumococcal disease. Most invasive nontypeable pneumococci possess defective cps locus genes, with an over-representation of defective serotype 8 cps variants.     

Streptococcus pneumoniae Serotype 11D Has a Bispecific Glycosyltransferase and Expresses Two Different Capsular Polysaccharide Repeating Units

Streptococcus pneumoniae (pneumococcus) expresses a capsular polysaccharide (CPS) that protects against host immunity and is synthesized by enzymes in the capsular polysaccharide synthesis (cps) locus. Serogroup 11 has six members (11A to -E) and the CPS structure of all members has been solved, except for serotype 11D. The cps loci of 11A and 11D differ by one codon (N112S) in wcrL, which putatively encodes a glycosyltransferase that adds the fourth sugar of the CPS repeating unit (RU). Gas chromatography and nuclear magnetic resonance analysis revealed that 11A and 11D PSs contain identical CPS RUs that contain αGlc as the fourth sugar. However, ∼25% of 11D CPS RUs contain instead αGlcNAc as the fourth sugar, suggesting that 11D wcrL encodes a bispecific glycosyltransferase. To test the hypothesis that codon 112 of WcrL determines enzyme specificity, and therefore the fourth sugar in the RU, we generated three isogenic pneumococcal strains with 11A cps loci containing wcrL encoding Ser-112 (MBO128) or Ala-112 (MBO130). MBO128 was serologically and biochemically identical to serotype 11D. MBO130 has a unique serologic profile; has as much αGlcNAc as 11F, 11B, and 11C CPS do; and may represent a new serotype. These findings demonstrate how pneumococci alter their CPS structure and their immunologic properties with a minimal genetic change.     

The Polysaccharide Capsule of Streptococcus pneumonia Partially Impedes MyD88-Mediated Immunity during Pneumonia in Mice

Toll-like receptors (TLR) and the downstream adaptor protein MyD88 are considered crucial for protective immunity during bacterial infections. Streptococcus (S.) pneumoniae is a human respiratory pathogen and a large majority of clinical pneumococcal isolates expresses an external polysaccharide capsule. We here sought to determine the role of pneumococcal capsule in MyD88-mediated antibacterial defense during S. pneumonia pneumonia. Wild type (WT) and Myd88^-/- mice were inoculated intranasally with serotype 2 S. pneumoniae D39 or with an isogenic capsule locus deletion mutant (D39∆cps), and analysed for bacterial outgrowth and inflammatory responses in the lung. As compared to WT mice, Myd88^-/- mice infected with D39 demonstrated a modestly impaired bacterial clearance accompanied by decreased inflammatory responses in the lung. Strikingly, while WT mice rapidly cleared D39∆cps, Myd88^-/- mice showed 10⁵-fold higher bacterial burdens in their lungs and dissemination to blood 24 hours after infection. These data suggest that the pneumococcal capsule impairs recognition of TLR ligands expressed by S. pneumoniae and thereby partially impedes MyD88-mediated antibacterial defense.     

Genomic Analysis Reveals the Molecular Basis for Capsule Loss in the Group B Streptococcus Population

The human and bovine bacterial pathogen Streptococcus agalactiae (Group B Streptococcus, GBS) expresses a thick polysaccharide capsule that constitutes a major virulence factor and vaccine target. GBS can be classified into ten distinct serotypes differing in the chemical composition of their capsular polysaccharide. However, non-typeable strains that do not react with anti-capsular sera are frequently isolated from colonized and infected humans and cattle. To gain a comprehensive insight into the molecular basis for the loss of capsule expression in GBS, a collection of well-characterized non-typeable strains was investigated by genome sequencing. Genome based phylogenetic analysis extended to a wide population of sequenced strains confirmed the recently observed high clonality among GBS lineages mainly containing human strains, and revealed a much higher degree of diversity in the bovine population. Remarkably, non-typeable strains were equally distributed in all lineages. A number of distinct mutations in the cps operon were identified that were apparently responsible for inactivation of capsule synthesis. The most frequent genetic alterations were point mutations leading to stop codons in the cps genes, and the main target was found to be cpsE encoding the portal glycosyl trasferase of capsule biosynthesis. Complementation of strains carrying missense mutations in cpsE with a wild-type gene restored capsule expression allowing the identification of amino acid residues essential for enzyme activity.     

Restriction Fragment Length Polymorphisms Detected with Novel DNA Probes Differentiate among Diverse Lineages of Serogroup 4 Listeria monocytogenes and Identify Four Distinct Lineages in Serotype 4b

Listeria monocytogenes of serotype 4b has been implicated in numerous outbreaks of food-borne listeriosis and in ca. 40% of sporadic cases. Strains of this serotype appear to be relatively homogeneous genetically, and molecular markers specific for distinct serotype 4b lineages have not been frequently identified. Here we show that DNA fragments derived from the putative mannitol permease locus of Listeria monocytogenes had an unexpectedly high potential to differentiate among different strains of serotype 4b when used as probes in Southern blotting of EcoRI-digested genomic DNA, yielding four distinct restriction fragment length polymorphism (RFLP) patterns. Strains of two epidemic-associated lineages, including the major epidemic clone implicated in several outbreaks in Europe and North America, had distinct RFLPs which differed from those of all other serotype 4b strains that we screened but which were encountered among strains of serotypes 1/2b and 3b. In addition, three serogroup 4 lineages were found to have unique RFLPs that were not encountered among any other L. monocytogenes strains. One was an unusual lineage of serotype 4b, and the other two were members of the serotype 4a and 4c group. The observed polymorphisms may reflect evolutionary relationships among lineages of L. monocytogenes and may facilitate detection and population genetic analysis of specific lineages.     

Development of Multiplex PCR Assays for the Identification of the 33 Serotypes of Streptococcus suis

Streptococcus suis is an important zoonotic agent causing severe diseases in pigs and humans. To date, 33 serotypes of S . suis have been identified based on antigenic differences in the capsular polysaccharide. The capsular polysaccharide synthesis (cps) locus encodes proteins/enzymes that are responsible for capsular production and variation in the capsule structures are the basis of S . suis serotyping. Multiplex and/or simplex PCR assays have been developed for 15 serotypes based on serotype-specific genes in the cps gene cluster. In this study, we developed a set of multiplex PCR (mPCR) assays to identify the 33 currently known S . suis serotypes. To identify serotype-specific genes for mPCR, the entire genomes of reference strains for the 33 serotypes were sequenced using whole genome high-throughput sequencing, and the cps gene clusters from these strains were identified and compared. We developed a set of 4 mPCR assays based on the polysaccharide polymerase gene wzy, one of the serotype-specific genes. The assays can identify all serotypes except for two pairs of serotypes: 1 and 14, and 2 and 1/2, which have no serotype-specific genes between them. The first assay identifies 12 serotypes (serotypes 1 to 10, 1/2, and 14) that are the most frequently isolated from diseased pigs and patients; the second identifies 10 serotypes (serotypes 11 to 21 except 14); the third identifies the remaining 11 serotypes (serotypes 22 to 31, and 33); and the fourth identifies a new cps cluster of S . suis discovered in this study in 16 isolates that agglutinated with antisera for serotypes 29 and 21. The multiplex PCR assays developed in this study provide a rapid and specific method for molecular serotyping of S . suis .     

Capsular Polysaccharide Production in Enterococcus faecalis and Contribution of CpsF to Capsule Serospecificity

Many bacterial species produce capsular polysaccharides that contribute to pathogenesis through evasion of the host innate immune system. The gram-positive pathogen Enterococcus faecalis was previously reported to produce one of four capsule serotypes (A, B, C, or D). Previous studies describing the four capsule serotypes of E. faecalis were based on immunodetection methods; however, the underlying genetics of capsule production did not fully support these findings. Previously, it was shown that capsule production for serotype C (Maekawa type 2) was dependent on the presence of nine open reading frames (cpsC to cpsK). Using a novel genetic system, we demonstrated that seven of the nine genes in the cps operon are essential for capsule production, indicating that serotypes A and B do not make a capsular polysaccharide. In support of this observation, we showed that serotype C and D capsule polysaccharides mask lipoteichoic acid from detection by agglutinating antibodies. Furthermore, we determined that the genetic basis for the difference in antigenicity between serotypes C and D is the presence of cpsF in serotype C strains. High-pH anion-exchange chromatography with pulsed amperometric detection analysis of serotype C and D capsules indicated that cpsF is responsible for glucosylation of serotype C capsular polysaccharide in E. faecalis.Enterococcus faecalis is a gram-positive bacterium commonly found as a commensal organism in the gastrointestinal tracts of most mammals. E. faecalis is one of the leading causes of hospital-acquired urinary tract infections, bacteremia, and surgical-site infections (29). The development of multiple antibiotic resistances, including resistance to vancomycin, makes treatment of enterococcal infections difficult (11). The 2004 National Nosocomial Infections Surveillance report indicated that nearly 30% of enterococci isolated from clinical settings were resistant to vancomycin, constituting a 12% rise from the previous 5 years (26). The development of alternative therapies to treat enterococcal infections has frequently been suggested due to rising percentages of antibiotic-resistant enterococcal strains (13-15, 19).Capsular polysaccharides are major contributors to the virulence of many microorganisms. The presence of capsule allows these microbes to escape detection and clearance by the host immune system (9, 27, 30, 41). There have been several publications regarding the role of cell wall polysaccharides in the pathogenesis of enterococcal infections (10, 13, 17, 37, 43). Several attempts have been made to establish a serotyping system for E. faecalis capsular polysaccharides (16, 23, 35, 36). These serotyping schemes include differences in capsular polysaccharide antigens but are also based on differences in surface antigens, including lipoteichoic acid (16, 38). To date, only one study has linked genetic evidence with capsule production (12). Two loci that have been reported to contain putative genes for capsule production are the epa and cps operons (10, 42). The polysaccharide produced by the epa locus is thought to be the cell wall rhamnopolymer (10), but it cannot be detected on the surface of the bacterium (43). Although rhamnopolymer production is reported to be abrogated by mutation (43), the full nature of rhamnopolymer production is yet to be determined for many E. faecalis strains. Probing the genomes of serotype A and B strains with a probe specific to the cps locus, including the genes cpsA and cpsB, identified a single ClaI restriction fragment for serotypes A and B (16). However, multiple ClaI restriction fragments were identified in serotypes C and D (16), suggesting that the genes responsible for capsule production in serotypes C and D were absent in serotypes A and B. Furthermore, the hybridization pattern between serotype C and D strains indicated a single restriction fragment polymorphism, but the basis on which genes were different between the two serotypes was not fully characterized (16). Studies based on the serotyping scheme proposed by Hufnagel et al. (17) have shown that serotype C and D strains are much more resistant to opsonophagoctyosis by neutrophils in the presence of normal human serum. More recently, a study by McBride et al. indicated that serotype C clinical isolates harbored a greater repertoire of antibiotic resistance cassettes and were more likely to possess multiple virulence factors than the other serotypes, suggesting that the presence of the capsule is associated with pathogenic lineages of E. faecalis (17, 24).It is essential to understand the underlying mechanisms of capsule production in E. faecalis because of ongoing efforts to develop alternative therapies targeting capsule. Here, we used a novel vector system for creating isogenic, in-frame deletion mutants to analyze the genetic basis for capsule production and serotype specificity. Our results show that only serotype C and D strains of E. faecalis produce capsular polysaccharides, based on the observation that deletions of cpsC, cpsD, cpsE cpsG, and cpsI abolish the production of capsule. In conjunction with these observations, we also demonstrated that the presence of capsule prevents detection of lipoteichoic acid on the surface of serotype C and D strains but not on unencapsulated strains. Our data also show that CpsF is responsible for the difference in serospecificity between serotype C and D strains.     

A new variant of the capsule 3 cluster occurs in Streptococcus pneumoniae from deceased wild chimpanzees

The presence of new Streptococcus pneumoniae clones in dead wild chimpanzees from the Taï National Park, Côte d''Ivoire, with previous respiratory problems has been demonstrated recently by DNA sequence analysis from samples obtained from the deceased apes. In order to broadenour understanding on the relatedness of these pneumococcal clones to those from humans, the gene locus responsible for biosynthesis of the capsule polysaccharide (CPS) has now been characterized. DNA sequence analysis of PCR fragments identified a cluster named cps3_Taï containing the four genes typical for serotype 3 CPS, but lacking a 5′-region of ≥2 kb which is degenerated in other cps3 loci and not required for type 3 biosynthesis. CPS3 is composed of a simple disaccharide repeat unit comprising glucose and glucuronic acid (GlcUA). The two genes ugd responsible for GlcUA synthesis and wchE encoding the type 3 synthase are essential for CPS3 biosynthesis, whereas both, galU and the 3′-truncated gene pgm are not required due to the presence of homologues elsewhere in the genome. The DNA sequence of cps3_Taï diverged considerably from those of other cps3 loci. Also, the gene pgm _Taï represents a full length version with a nonsense mutation at codon 179. The two genes ugd _Taï and wchE _Taï including the promoter region were transformed into a nonencapsulated laboratory strain S. pneumoniae R6. Transformants which expressed type 3 capsule polysaccharide were readily obtained, documenting that the gene products are functional. In summary, the data indicate that cps3_Taï evolved independent from other cps3 loci, suggesting the presence of specialized serotype 3 S. pneumoniae clones endemic to the Taï National Park area.     

Chemical Structures of Streptococcus pneumoniae Capsular Polysaccharide Type 39 (CPS39), CPS47F,and CPS34 Characterized by Nuclear Magnetic Resonance Spectroscopy and Their Relation to CPS10A

Structural characterization of Streptococcus pneumoniae capsular polysaccharides (CPS) is a prerequisite for unraveling both antigenic and genetic relationships that exist between different serotypes. In the current study, comparative structural studies of S. pneumoniae CPS serogroup 10 (CPS10) were extended to include genetically related S. pneumoniae CPS34, CPS39, and CPS47F. High-resolution heteronuclear nuclear magnetic resonance (NMR) spectroscopy confirmed the published structure of CPS34 and, in conjunction with glycosyl composition analyses, revealed the following repeat unit structures of the other serotypes, which have not been previously characterized: Open in a separate window Common and unique structural features of these polysaccharides, including different positions of O-acetylation, were unambiguously associated with specific genes in each corresponding cps locus. The only exception involved the gene designated wcrC, which is associated with the α1-2 transfer of Gal pyranoside (Galp) to ribitol-5-phosphate in the synthesis of CPS10A, CPS47F, and CPS34 but with α1-1 transfer of Gal to ribitol-5-phosphate in the synthesis of CPS39. The corresponding gene in the cps39 locus, although related to wcrC, more closely resembled a previously identified gene (i.e., wefM) of Streptococcus oralis that is associated with α1-1 transfer of Galp to ribitol-5-phosphate. These and other recent findings identify linkages from α-Galp to ribitol-5-phosphate and from this residue to adjacent Gal furanoside (Galf) as important sites of CPS structural and genetic diversity.     

KIAA1462, A Coronary Artery Disease Associated Gene,Is a Candidate Gene for Late Onset Alzheimer Disease in APOE Carriers

Alzheimer disease (AD) is a devastating neurodegenerative disease affecting more than five million Americans. In this study, we have used updated genetic linkage data from chromosome 10 in combination with expression data from serial analysis of gene expression to choose a new set of thirteen candidate genes for genetic analysis in late onset Alzheimer disease (LOAD). Results in this study identify the KIAA1462 locus as a candidate locus for LOAD in APOE4 carriers. Two genes exist at this locus, KIAA1462, a gene associated with coronary artery disease, and “rokimi”, encoding an untranslated spliced RNA The genetic architecture at this locus suggests that the gene product important in this association is either “rokimi”, or a different isoform of KIAA1462 than the isoform that is important in cardiovascular disease. Expression data suggests that isoform f of KIAA1462 is a more attractive candidate for association with LOAD in APOE4 carriers than “rokimi” which had no detectable expression in brain.     

Bacterial Load of Pneumococcal Serotypes Correlates with Their Prevalence and Multiple Serotypes Is Associated with Acute Respiratory Infections among Children Less Than 5 Years of Age

Background

Among pneumococcal serotypes, some serotypes are more prevalent in the nasopharynx than others; determining factors for higher prevalence remain to be fully explored. As non-vaccine serotypes have emerged after the introduction of 7-valent conjugate vaccines, study of serotype specific epidemiology is in need. When two or more serotypes co-colonize, they evolve rapidly to defend host''s immune responses; however, a clear association of co-colonization with a clinical outcome is lacking.

Methods

Children less than 5 years old who were admitted to hospital due to acute respiratory infections (ARI) (n = 595) and healthy children (n = 350) were recruited. Carriage of pneumococcus was determined by culture and lytA PCR in the nasopharyngeal samples. Serotype/serogroup detection and its quantification were done by the nanofluidic real time PCR system. Spearman''s correlation and logistic regression were used to examine a correlation of serotype/serogroup specific bacterial load with its prevalence and an association of co-colonization with ARI respectively.

Results

Serotype/serogroup specific bacterial load was correlated with its prevalence, both in ARI cases (Spearman''s rho = 0.44, n = 186; P<0.0001) and healthy children (Spearman''s rho = 0.41, n = 115; P<0.0001). The prevalence of multiple serotypes was more common in ARI cases than in healthy children (18.5% vs 7.1%; aOR 2.92, 95% CI: 1.27–6.71; P = 0.01). The dominant serotype in the co-colonization had a 2 log10 higher bacterial load than the subdominant serotype, both in ARI cases (P<0.001) and healthy children (P<0.05).

Conclusions

High bacterial load in the nasopharynx may help transmit pneumococci among hosts, and increase the chance of successful acquisition and colonization. Co-colonization of multiple serotypes of pneumococci is linked with ARI, which infers the interactions of multiple serotypes may increase their pathogenicity; however, they may compete for growth in number.     

Requirement of NMB0065 for connecting assembly and export of sialic acid capsular polysaccharides in Neisseria meningitidis

Capsule expression in Neisseria meningitidis is encoded by the cps locus comprised of genes required for biosynthesis and surface translocation. Located adjacent to the gene encoding the polysialyltransferase in serogroups expressing sialic acid-containing capsule, NMB0065 is likely a member of the cps locus, but it is not found in serogroups A or X that express non-sialic acid capsules. To further understand its role in CPS expression, NMB0065 mutants were created in the serogroups B, C and Y strains. The mutants were as sensitive as unencapsulated strains to killing by normal human serum, despite producing near wild-type levels of CPS. Absence of surface expression of capsule was suggested by increased surface hydrophobicity and confirmed by immunogold electron microscopy, which revealed the presence of large vacuoles containing CPS within the cell. GC–MS and NMR analyses of purified capsule from the mutant revealed no apparent changes in polymer structures and lipid anchors. Mutants of NMB0065 homologues in other sialic acid CPS expressing meningococcal serogroups had similar phenotypes. Thus, NMB0065 (CtrG) is not involved in biosynthesis or lipidation of sialic acid-containing capsule but encodes a protein required for proper coupling of the assembly complex to the membrane transport complex allowing surface expression of CPS.     

Genetic analysis of the capsular polysaccharide synthesis locus in 15 Streptococcus suis serotypes

The capsular polysaccharide (CPS) synthesis locus of 13 Streptococcus suis serotypes (serotype 1, 3, 4, 5, 7, 8, 9, 10, 14, 19, 23, 25 and 1/2) was sequenced and compared with that of serotype 2 and 16. The CPS synthesis locus of these 15 serotypes falls into two genetic groups. The locus is located on the chromosome between orfZ and aroA. All the translated proteins in the CPS synthesis locus were clustered into 127 homology groups using the tribemcl algorithm. The general organization of the locus suggested that the CPS of S.?suis could be synthesized by the Wzy-dependent pathway. The capsule of serotypes 3, 4, 5, 7, 9, 10, 19 and 23 was predicted to be amino-polysaccharide. Sialic acid was predicted to be present in the capsule of serotypes 1, 2, 14, 16 and 1/2. The characteristics of the CPS synthesis locus suggest that some genes may have been imported into S.?suis (or their ancestors) on multiple occasions from different and unknown sources.     

Elucidation of structural and antigenic properties of pneumococcal serotype 11A, 11B, 11C, and 11F polysaccharide capsules

Despite the emerging impact of serogroup 11 serotypes in Streptococcus pneumoniae epidemiology, the structures of serogroup 11 capsule types have not been fully elucidated, particularly the locations of O-acetyl substitutions. Here, we report the complete structures of the serotype 11B, 11C, and 11F polysaccharides and a revision to the serotype 11A capsular polysaccharide using nuclear magnetic resonance (NMR). All structures shared a linear, tetrasaccharide backbone with a pendant phosphopolyalcohol. Three of four saccharides are conserved in all serotypes. The individual serotype capsules differed in the identity of one saccharide, the pendant phosphopolyalcohol, and the O-acetylation pattern. Though the assigned locations of O-acetate substitutions in this study differed from those of previous reports, our findings were corroborated with strong correlations to serology and genetics. We examined the binding of serotyping sera to serogroup 11 polysaccharides by using flow cytometry and an inhibition-type enzyme-linked immunosorbent assay (ELISA) and found that de-O-acetylation of capsular polysaccharides by mild hydrolysis decreases its immunoreactivity, supporting the crucial role of O-acetylation in the antigenicity of these polysaccharides. Due to strong correlations between polysaccharide structures and capsule biosynthesis genes, we were able to assign target substrates for the O-acetyltransferases encoded by wcwC, wcwR, wcwT, and wcjE. We identified antigenic determinants for serogroup 11 serotyping sera and highlight the idea that conventional serotyping methods are not capable of recognizing all putative variants of S. pneumoniae serogroup 11.     

Invasive Salmonella dublin Infections Associated With Drinking Raw Milk

Salmonella dublin is a serotype of Salmonella that is host-adapted to cattle and rarely infects people. In one year (1980-1981) we diagnosed five cases of salmonellosis due to S dublin at the Veterans Administration Medical Center, San Diego. Four patients had positive blood cultures and one died. A sixth patient, diagnosed in 1978, had a mycotic aortic aneurysm but survived. Compared with nine patients who had Salmonella infections due to other serotypes, the S dublin patients were older, had a greater number of underlying chronic illnesses and were more seriously ill with their infections. Four of the six S dublin cases occurred in association with drinking “certified” raw milk from a commercial dairy.Two microbiologic features of S dublin strains circulating in San Diego were distinctive. They failed to ferment arabinose and could not be grown in a minimal medium using citrate as the sole carbon source. Chronically ill elderly patients should be cautioned against drinking raw milk, an increasingly popular “health food.”     

Sequence diversity within the capsular genes of Streptococcus pneumoniae serogroup 6 and 19

The main virulence factor of Streptococcus pneumoniae is the capsule. The polysaccharides comprising this capsule are encoded by approximately 15 genes and differences in these genes result in different serotypes. The aim of this study was to investigate the sequence diversity of the capsular genes of serotypes 6A, 6B, 6C, 19A and 19F and to explore a possible effect of vaccination on variation and distribution of these serotypes in the Netherlands. The complete capsular gene locus was sequenced for 25 serogroup 6 and for 20 serogroup 19 isolates. If one or more genes varied in 10 or more base pairs from the reference sequence, it was designated as a capsular subtype. Allele-specific PCRs and specific gene sequencing of highly variable capsular genes were performed on 184 serogroup 6 and 195 serogroup 19 isolates to identify capsular subtypes. This revealed the presence of 6, 3 and a single capsular subtype within serotypes 6A, 6B and 6C, respectively. The serotype 19A and 19F isolates comprised 3 and 4 capsular subtypes, respectively. For serogroup 6, the genetic background, as determined by multi locus sequence typing (MLST) and multiple-locus variable number of tandem repeat analysis (MLVA), seemed to be closely related to the capsular subtypes, but this was less pronounced for serogroup 19 isolates. The data also suggest shifts in the occurrence of capsular subtypes within serotype 6A and 19A after introduction of the 7-valent pneumococcal vaccine. The shifts within these non-vaccine serotypes might indicate that these capsular subtypes are filling the niche of the vaccine serotypes. In conclusion, there is considerable DNA sequence variation of the capsular genes within pneumococcal serogroup 6 and 19. Such changes may result in altered polysaccharides or in strains that produce more capsular polysaccharides. Consequently, these altered capsules may be less sensitive for vaccine induced immunity.     

Unusual features in organisation of capsular polysaccharide-related genes of C. jejuni strain X

PCR probing of the genome of Campylobacter jejuni strain X using conserved capsular polysaccharide (CPS)-related genes allowed elucidation of a complete sequence of the respective gene cluster (cps). This is the largest known Campylobacter cps cluster (38 kb excluding flanking kps regions), which includes a number of genes not detected in other Campylobacter strains. Sequence analysis suggests genetic rearrangements both within and outside the cps gene cluster, a mechanism which may be responsible for mosaic organisation of sugar transferase-related genes leading to structural variability of the capsular polysaccharide (CPS).     

Long-Term Epidemiology of Streptococcus pneumoniae Serogroup 6 in a Region of Southern Europe with Special Reference to Serotype 6E

Streptococcus pneumoniae serotype 6E has recently been described, but its long-term epidemiology is not well known. From 1981–2013, 704 serogroup 6 clinical isolates were obtained in Gipuzkoa, Basque Country, Spain. All invasive and one in four non-invasive isolates were included. Overall, 75, 97, 51 and 45 serotypes 6A, 6B, 6C and 6E isolates, respectively, were detected. No serotype 6D isolates were identified. The prevalence of serotypes 6E and 6B, but not that of serotypes 6A and 6C, declined after the introduction of pneumococcal conjugate vaccines. Serotype 6E isolates showed the highest resistance rate. Most serotype 6E isolates were ST90.