Serotype distribution of invasive Streptococcus pneumoniae in Canada during the introduction of the 13-valent pneumococcal conjugate vaccine, 2010

A baseline serotype distribution was established by age and region for 2058 invasive Streptococcus pneumoniae isolates collected during the implementation period of the 13-valent pneumococcal conjugate vaccine (PCV13) program in many parts of Canada in 2010. Serotypes 19A, 7F, and 3 were the most prevalent in all age groups, accounting for 57% in <2 year olds, 62% in 2-4 year olds, 45% in 5-14?year olds, 44% in 15-49?year olds, 41% in 50-64?year olds, and 36% in ≥65?year olds. Serotype 19A was most predominant in Western and Central Canada representing 15% and 22%, respectively, of the isolates from those regions, whereas 7F was most common in Eastern Canada with 20% of the isolates. Other prevalent serotypes include 15A, 23B, 12F, 22F, and 6C. PCV13 serotypes represented 65% of the pneumococci isolated from?<2 year olds, 71% of 2-4 year olds, 61% of 5-14 year olds, 60% of 15-49 year olds, 53% of 50-64 year olds, and 49% of the?≥65?year olds. Continued monitoring of invasive pneumococcal serotypes in Canada is important to identify epidemiological trends and assess the impact of the newly introduced PCV13 vaccine on public health.     

Molecular characterization of Streptococcus pneumoniae,particularly serotype19A/ST320, which emerged in Krasnoyarsk,Russia

Streptococcus pneumoniae , a common human pathogen, colonizes the nasopharynx and causes diseases including acute otitis media (AOM). Herein, pneumococcal serotype distributions in children before and after PCV7 vaccination and in patients with pneumococcal disease in Siberian Russia (Krasnoyarsk) are reported. Analyses included antimicrobial susceptibility testing, sequence typing (ST), pulsed field gel electrophoresis, virulence‐related surface protein gene (VSG) typing with novel primers and structural analysis by scanning electron microscopy. In healthy children (HC) prior to administration of PCV7, drug‐susceptible serotype23F/ST1500 was a major pneumococcal genotype. In the PCV7 trial, multidrug‐resistant serotype19A/ST320 emerged in vaccinees after PCV7, exhibiting a PCV7‐induced serotype replacement. Multidrug‐resistant serotype19A/ST320 was evident in patients with AOM. Community‐acquired pneumonia (CAP) isolates showed genetic similarities to the AOM (ST320) genotype, constituting a common non‐invasive AOM–CAP group. In contrast, meningitis isolates were more divergent. Overall, 25 ST types were identified; five (20%) of which were Krasnoyarsk‐native. Regarding VSGs, PI‐1 (rlrA /rrgB ), PI‐2 (pitA /B ), psrP and cbpA were present at 54.3%, 38.6%, 48.6%, and 95.7%, respectively, with two major VSG content types, PI‐1^?/PI‐2^?/psrP ⁺/cbpA ⁺ and PI‐1⁺/PI‐2⁺/psrP ^‐/cbpA ⁺, being found for HC and non‐invasive diseases, respectively. A major clone of serotype19A/ST320 (PI‐1⁺/PI‐2⁺) produced the longest pneumococcal wire (pilus) structures in colonies. ST1016 (PI‐1^?/PI‐2^?) in HC had HEp‐2 cell‐adherent pili. These results suggest that serotype19A/ST320 and related genotypes, with the VSG content type PI‐1⁺/PI‐2⁺/psrP ^?/cbpA ⁺, emerged in vaccinees after PCV7 in Siberia, accompanying diseases in non‐vaccinated children, and that some genotypes (serotypes19A/ST320 and 18/ST1016) produced novel pneumococcal structures, predicting their roles in colony formation and adherence.

     

Purification of capsular polysaccharide produced by Streptococcus pneumoniae serotype 19A

Streptococcus pneumoniae is a major cause of invasive infection in young infants and older adults. There are currently 90 capsular serotypes identified and 23 serotypes (1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F, and 33F) are responsible for about 90% of invasive disease. Among the more than 90 different S. pneumoniae serotypes, serotype 19A is globally very prevalent. A simplified purification procedure including adjustment of cell lysate pH to 4.5, fractionation with 50- 80% ethanol, and dialysis rendered capsular polysaccharide (CPS) in a yield of 31.32 +/- 3.11 mg from 1 l culture (75% recovery after lyses). The product contained only 69.6 microng of protein (99.78% purity) and 0.8 mg (sum of the precipitants from 50~60%, 60~70%, and 70~80%) of nucleic acid (97.45% purity). The purified CPS was conjugated with bovine serum albumin; the product size ranged from 100 to 180 kDa.     

Comparative genomics for identification of clone-specific sequence blocks in Streptococcus pneumoniae

The partial genome sequences of a serotype 3 and a serotype 2 pneumococcal strain were compared to the complete type 4 pneumococcal genome. Over 500000 and 150000 base pairs of the partial genome data, obtained from published patents, were analysed respectively. Global alignment showed that nearly the whole genome is highly conserved in accordance with data of multilocus sequence typing of housekeeping genes. The search for clone-specific genes revealed 17 new open reading frames in the type 3 strain, while no new open reading frame was detected in the type 2 strain. Allelic variation of genes was restricted by the use of crude sequence data, but still permitted identification of some new alleles and the observation that all surface proteins present in the partial genome data were highly conserved. In both strains we observed also a variety of chromosomal rearrangements and variations due to mobile genetic elements. All together, this comparative genomic approach gives a genome-based overview of strain relatedness and a prospective on what could be expected when sequencing other pneumococcal strains.     

Comparative genomic analyses of seventeen Streptococcus pneumoniae strains: insights into the pneumococcal supragenome

The distributed-genome hypothesis (DGH) states that pathogenic bacteria possess a supragenome that is much larger than the genome of any single bacterium and that these pathogens utilize genetic recombination and a large, noncore set of genes as a means of diversity generation. We sequenced the genomes of eight nasopharyngeal strains of Streptococcus pneumoniae isolated from pediatric patients with upper respiratory symptoms and performed quantitative genomic analyses among these and nine publicly available pneumococcal strains. Coding sequences from all strains were grouped into 3,170 orthologous gene clusters, of which 1,454 (46%) were conserved among all 17 strains. The majority of the gene clusters, 1,716 (54%), were not found in all strains. Genic differences per strain pair ranged from 35 to 629 orthologous clusters, with each strain's genome containing between 21 and 32% noncore genes. The distribution of the orthologous clusters per genome for the 17 strains was entered into the finite-supragenome model, which predicted that (i) the S. pneumoniae supragenome contains more than 5,000 orthologous clusters and (ii) 99% of the orthologous clusters (~3,000) that are represented in the S. pneumoniae population at frequencies of ≥0.1 can be identified if 33 representative genomes are sequenced. These extensive genic diversity data support the DGH and provide a basis for understanding the great differences in clinical phenotype associated with various pneumococcal strains. When these findings are taken together with previous studies that demonstrated the presence of a supragenome for Streptococcus agalactiae and Haemophilus influenzae, it appears that the possession of a distributed genome is a common host interaction strategy.     

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 x 10(6) to 8 x 10(6) 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.     

A functional genomics approach to establish the complement of carbohydrate transporters in Streptococcus pneumoniae

The aerotolerant anaerobe Streptococcus pneumoniae is part of the normal nasopharyngeal microbiota of humans and one of the most important invasive pathogens. A genomic survey allowed establishing the occurrence of twenty-one phosphotransferase systems, seven carbohydrate uptake ABC transporters, one sodium:solute symporter and a permease, underlining an exceptionally high capacity for uptake of carbohydrate substrates. Despite high genomic variability, combined phenotypic and genomic analysis of twenty sequenced strains did assign the substrate specificity only to two uptake systems. Systematic analysis of mutants for most carbohydrate transporters enabled us to assign a phenotype and substrate specificity to twenty-three transport systems. For five putative transporters for galactose, pentoses, ribonucleosides and sulphated glycans activity was inferred, but not experimentally confirmed and only one transport system remains with an unknown substrate and lack of any functional annotation. Using a metabolic approach, 80% of the thirty-two fermentable carbon substrates were assigned to the corresponding transporter. The complexity and robustness of sugar uptake is underlined by the finding that many transporters have multiple substrates, and many sugars are transported by more than one system. The present work permits to draw a functional map of the complete arsenal of carbohydrate utilisation proteins of pneumococci, allows re-annotation of genomic data and might serve as a reference for related species. These data provide tools for specific investigation of the roles of the different carbon substrates on pneumococcal physiology in the host during carriage and invasive infection.     

Nasal carriage of Streptococcus pneumoniae among Hungarian children before the wide use of the conjugate vaccine

Streptococcus pneumoniae is responsible for a significant amount of morbidity and mortality worldwide, especially among children <5 years. Healthy carriers are the most important sources of infections and the carriage also peaks in the first years of life, especially among children attending communities. In this study, for the first time in Hungary, we surveyed the nasal carriage of healthy children, just before the use of the conjugate vaccine started increasing.Nasal specimens of 358 children were cultured and pneumococci isolated. The strains were serotyped with antisera and PCR, genotyped by PFGE and their antibiotic sensitivity determined by agar dilution method.The carriage rate was 37.71%. The isolates were sensitive to most tested antibiotics, except for macrolides. In this cohort of specimens still the widespread, so-called "pediatric serotypes" dominated (14, 19F, 23F, 6A, 6B in ranking order), but three of the previously rare types: 15B, 11A and 13 were represented already by 21.5% of all strains and also a few other rare non-vaccine types (e.g. 10A or 37) were detected.The calculated vaccine coverage was 55.6% for PCV-7, 69.6% for PCV-13 and 86.7% for Pneumovax. In this cohort, only 15.9% of the children (n = 57) were vaccinated. The carriage rate of PCV-7 vaccinated children was significantly lower (30.4%) than that of the non-vaccinated group (39.2%). The clonality of the isolates was significant within each group, revealing the extensive bacterium exchange among children.     

Synthesis of a trisaccharide component of the capsular polysaccharide of Streptococcus pneumoniae type 19F

2-O-[4-O-(2-Acetamido-2-deoxy-beta-D-mannopyranosyl)-alpha-D- glucopyranosyl]-alpha,beta-L-rhamnopyranose, a structural component of the capsular polysaccharide of Streptococcus pneumoniae type 19F, has been synthesized by sequential glycosylation reactions using the glycosyl acceptor 2,2,2-trichloroethyl 3,4-di-O-benzyl-alpha-L-rhamnopyranoside (prepared from the known 2-O-acetyl-3,4-di-O-benzyl-alpha-L-rhamnopyranosyl chloride), and the glycosyl donors 4-O-acetyl-2,3,6-tri-O-benzyl-alpha-D-glucopyranosyl chloride and 4,6-di-O-acetyl-2-azido-3-O-benzyl-2-deoxy-alpha-D-mannopyranosyl bromide (prepared in seven steps from the known methyl 2-azido-4,6-O-benzylidene-2-deoxy-alpha-D-altropyranoside). The corresponding 8-(methoxycarbonyl)octyl glycoside has also been synthesized, by coupling of 8-(methoxycarbonyl)octyl trifluoromethanesulfonate and the sodium salt of 2-O-[4-O-(2-acetamido-4,6-di-O-acetyl-3-O-benzyl-2-deoxy-beta-D- mannopyranosyl)-2,3,6-tri-O-benzyl-alpha-D-glucopyranosyl]-3,4-di-O- benzyl-alpha,beta-L-rhamnopyranose.     

Comparative Genetics of Capsular Polysaccharide Biosynthesis in Streptococcus pneumoniae Types Belonging to Serogroup 19

The genetic basis for the structural diversity of capsule polysaccharide (CPS) in Streptococcus pneumoniae serogroup 19 (consisting of types 19F, 19A, 19B, and 19C) has been determined for the first time. In this study, the genetic basis for the 19A and 19C serotypes is described, and the structures of all four serogroup 19 cps loci and their flanking sequences are compared. Transformation studies show that the structural difference between the 19A and 19F CPSs is likely to be a consequence of differences between their respective polysaccharide polymerase genes (cps19aI and cps19fI). The CPS of type 19C differs from that of type 19B by the addition of glucose. We have identified a single gene difference between the two cps loci (cps19cS), which is likely to encode a glucosyl transferase. The arrangement of the genes within the cps19 loci is highly conserved, with 13 genes (cps19A to -H and cps19K to -O) common to all four serogroup 19 members. These cps genes encode functions required for the synthesis of the shared trisaccharide component of the group 19 CPS repeat unit structures. Furthermore, the genetic differences between the group 19 cps loci identified are consistent with the CPS structures of the individual serotypes. Functions have been assigned to nearly all of the cps19 gene products, based on either gene complementation or similarity to other proteins with known functions, and putative biosynthetic pathways for production of all four group 19 CPSs have been proposed.     

Invasive pneumococcal disease in Portugal prior to and after the introduction of pneumococcal heptavalent conjugate vaccine

The rates of invasive pneumococcal disease (IPD), serotype distribution and antimicrobial susceptibility prior to and after the introduction of the heptavalent pneumococcal conjugate vaccine in Portuguese children were evaluated. The changes in incidence of IPD in children under 1 year old between the two periods of the study was not significant (P=0.53), despite the 21% decline. In children under 18 years old there was a 27.7% decrease in vaccine serotypes. All nonvaccine serotypes increased 71.4%. The decrease in vaccine serotypes was more impressive during the first year of life (-54.8%) than for children between 1 and 5 years of age (-19.1%). Among children under 1 year old, penicillin nonsusceptible isolates declined between the two periods of the study (47.2% vs. 25.0%) (P=0.03), as did those of cefotaxime and ceftriaxone nonsusceptible isolates. No changes were observed for isolates nonsusceptible to tetracycline and macrolides. The serotypes of these nonsusceptible isolates differed after the introduction of vaccine (P=0.01). Multiresistance increased 57.1% after the introduction of vaccine. Multiresistant isolates with vaccine serotype declined 42.9% (P<0.001), and nonvaccine serotypes appeared during the vaccination period (P<0.001). These findings suggest a replacement of vaccine serotypes by nonvaccine serotypes, mainly among nonsusceptible isolates.     

Characterization of the dihydrolipoamide dehydrogenase from Streptococcus pneumoniae and its role in pneumococcal infection

In the present study, we have characterized the dihydrolipoamide dehydrogenase (DLDH) of Strepto-coccus pneumoniae and its role during pneumococcal infection. We have also demonstrated that a lack of DLDH results in a deficiency in alpha-galactoside metabolism and galactose transport. DLDH is an enzyme that is classically involved in the three-step conversion of 2-oxo acids to their respective acyl-CoA derivatives, but DLDH has also been shown to have other functions. The dldh gene was virtually identical in three pneumococcal strains examined. Besides the functional domains and motifs associated with this enzyme, analysis of the pneumococcal dldh gene sequence revealed the presence of an N-terminal lipoyl domain. DLDH-negative bacteria totally lacked DLDH activity, indicating that this gene encodes the only DLDH in S. pneumoniae. These DLDH-negative bacteria grew normally in vitro but were avirulent in sepsis and lung infection models in mice, indicating that DLDH activity is necessary for the survival of pneumococci within the host. The lack of virulence was not associated with a loss of 2-oxo acid dehydrogenase activity, as the wild-type pneumococcal strains did not contain activity of any of the known 2-oxo acid enzyme complexes. Instead, studies of carbohydrate utilization demonstrated that the DLDH-negative bacteria were impaired for alpha-galactoside and galactose metabolism. The DLDH mutants lost their ability to oxidize or grow with galactose or melibiose as sole carbon source and showed reduced oxidation and growth on raffinose or stachyose. The bacteria had an 85% reduction in alpha-galactosidase activity and showed virtually no transport of galactose into the cells, which can explain these phenotypic changes. The DLDH-negative bacteria produced only 50% of normal capsular polysaccharide, a phenotype that may be associated with impaired carbohydrate metabolism.     

Comparative genomics inferred two distinct populations of piscine pathogenic Streptococcus agalactiae,serotype Ia ST7 and serotype III ST283, in Thailand and Vietnam

The genomes of Streptococcus agalactiae (group B streptococcus; GBS) collected from diseased fish in Thailand and Vietnam over a nine-year period (2008–2016) were sequenced and compared (n = 21). Based on capsular serotype and multilocus sequence typing (MLST), GBS isolates are divided into 2 groups comprised of i) serotype Ia; sequence type (ST)7 and ii) serotype III; ST283. Population structure inferred by core genome (cg)MLST and Bayesian clustering analysis also strongly indicated distribution of two GBS populations in both Thailand and Vietnam. Deep phylogenetic analysis implied by CRISPR array's spacer diversity was able to cluster GBS isolates according to their temporal and geographic origins, though ST7 has varying CRISPR1-spacer profiles when compared to ST283 strains. Based on overall genotypic features, Thai ST283 strains were closely related to the Singaporean ST283 strain causing foodborne illness in humans in 2015, thus, signifying zoonotic potential of this GBS population in the country.     

Development of a DNA microarray to identify the Streptococcus pneumoniae serotypes contained in the 23-valent pneumococcal polysaccharide vaccine and closely related serotypes

Streptococcus pneumoniae is a major worldwide human pathogen. This investigation has developed a reliable and accurate DNA microarray method for inter-species differentiation of S. pneumoniae and intra-species differentiation of the 23 groups of S. pneumoniae including serotypes represented in the 23-valent pneumococcal vaccine and the other 20 closely related serotypes. In addition to 16S rDNA probes, serotype- or serogroup-specific probes targeting the capsular polysaccharide synthesis (cps) genes, wzy or capA were generated. We adopted a two-step multiplex PCR to improve the sensitivity of detection to a level of 10(5) cfu/ml in pure culture or 50 ng DNA. A total of 169 isolates (from China, Australia, Canada and New Zealand) including 147 belonging to 23-valent vaccine and closely related serotypes of S. pneumoniae, 11 belonging to other serotypes and 11 of different species commonly isolated from respiratory tract were tested to verify the method. The DNA microarray method developed provides a sensitive means to rapidly identify the members of the most common S. pneumoniae serotypes in patients and to monitor their distribution in different patient groups and geographic locations. Such information is needed for disease surveillance and to monitor vaccine efficacy.     

Biological role of the pneumococcal amidase. Cloning of the lytA gene in Streptococcus pneumoniae

A pneumococcal recombinant plasmid, pRG2, containing the lytA gene that codes for the pneumococcal N-acetylmuramoyl-L-alanine amidase has been constructed using the pneumococcal plasmid pLS1 as a vector. pRG2 was introduced by genetic transformation into a mutant of Streptococcus pneumoniae (M31) that has a complete deletion of the lytA gene. The transformed strain (M51) grew at a normal growth rate as 'diplo' cells and underwent autolysis at the end of the exponential phase of growth, two properties that had been lost in the deleted mutant M31. M51 lysed very rapidly at the end of the exponential phase when the cells were grown in choline-containing medium probably because of the higher level of amidase activity present in this strain as compared to the lysis-prone strain M11. These findings show that the expression of the plasmid-linked gene was placed under the mechanism(s) of control of the cell during the exponential phase. Our results demonstrate that the physiological role of the pneumococcal amidase was to catalyze the separation of the daughter cells at the end of the cell division to produce diplo cells; in addition we have also confirmed the basic role of this autolysin in the bacteriolytic nature of beta-lactam antibiotics.     

Differential idiotype utilization for the in vivo type 14 capsular polysaccharide-specific Ig responses to intact Streptococcus pneumoniae versus a pneumococcal conjugate vaccine

Murine IgG responses specific for the capsular polysaccharide (pneumococcal capsular polysaccharide serotype 14; PPS14) of Streptococcus pneumoniae type 14 (Pn14), induced in response to intact Pn14 or a PPS14-protein conjugate, are both dependent on CD4(+) T cell help but appear to use marginal zone versus follicular B cells, respectively. In this study, we identify an idiotype (44.1-Id) that dominates the PPS14-specific IgG, but not IgM, responses to intact Pn14, isolated PPS14, and Group B Streptococcus (strain COH1-11) expressing capsular polysaccharide structurally identical to PPS14. The 44.1-Id, however, is not expressed in the repertoire of natural PPS14-specific Abs. In distinct contrast, PPS14-specific IgG responses to a soluble PPS14-protein conjugate exhibit minimal usage of the 44.1-Id, although significant 44.1-Id expression is elicited in response to conjugate attached to particles. The 44.1-Id elicited in response to intact Pn14 was expressed in similar proportions among all four IgG subclasses during both the primary and secondary responses. The 44.1-Id usage was linked to the Igh(a), but not Igh(b), allotype and was associated with induction of relatively high total PPS14-specific IgG responses. In contrast to PPS14-protein conjugate, avidity maturation of the 44.1-Id-dominant PPS14-specific IgG responses was limited, even during the highly boosted T cell-dependent PPS14-specific secondary responses to COH1-11. These results indicate that different antigenic forms of the same capsular polysaccharide can recruit distinct B cell clones expressing characteristic idiotypes under genetic control and suggest that the 44.1-Id is derived from marginal zone B cells.     

Maintaining vaccine delivery following the introduction of the rotavirus and pneumococcal vaccines in Thailand

Although the substantial burdens of rotavirus and pneumococcal disease have motivated many countries to consider introducing the rotavirus vaccine (RV) and heptavalent pneumococcal conjugate vaccine (PCV-7) to their National Immunization Programs (EPIs), these new vaccines could affect the countries' vaccine supply chains (i.e., the series of steps required to get a vaccine from their manufacturers to patients). We developed detailed computational models of the Trang Province, Thailand, vaccine supply chain to simulate introducing various RV and PCV-7 vaccine presentations and their combinations. Our results showed that the volumes of these new vaccines in addition to current routine vaccines could meet and even exceed (1) the refrigerator space at the provincial district and sub-district levels and (2) the transport cold space at district and sub-district levels preventing other vaccines from being available to patients who arrive to be immunized. Besides the smallest RV presentation (17.1 cm3/dose), all other vaccine introduction scenarios required added storage capacity at the provincial level (range: 20 L-1151 L per month) for the three largest formulations, and district level (range: 1 L-124 L per month) across all introduction scenarios. Similarly, with the exception of the two smallest RV presentation (17.1 cm3/dose), added transport capacity was required at both district and sub-district levels. Added transport capacity required across introduction scenarios from the provincial to district levels ranged from 1 L-187 L, and district to sub-district levels ranged from 1 L-13 L per shipment. Finally, only the smallest RV vaccine presentation (17.1 cm3/dose) had no appreciable effect on vaccine availability at sub-districts. All other RV and PCV-7 vaccines were too large for the current supply chain to handle without modifications such as increasing storage or transport capacity. Introducing these new vaccines to Thailand could have dynamic effects on the availability of all vaccines that may not be initially apparent to decision-makers.     

Biochemical characterization of the CDP-D-arabinitol biosynthetic pathway in Streptococcus pneumoniae 17F

Streptococcus pneumoniae is a major human pathogen associated with many diseases worldwide. Capsular polysaccharides (CPSs) are the major virulence factor. The biosynthetic pathway of D-arabinitol, which is present in the CPSs of several S. pneumoniae serotypes, has never been identified. In this study, the genes abpA (previously known as abp1) and abpB (previously known as abp2), which have previously been reported to be responsible for nucleoside diphosphate (NDP)-D-arabinitol (the nucleotide-activated form of D-arabinitol) synthesis, were cloned. The enzyme products were overexpressed, purified, and analyzed for their respective activities. Novel products produced by AbpA- and AbpB-catalyzing reactions were detected by capillary electrophoresis, and the structures of the products were elucidated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. As a result, abpA was identified to be a D-xylulose-5-phosphate cytidylyltransferase-encoding gene, responsible for the transfer of CTP to D-xylulose-5-phosphate (D-Xlu-5-P) to form CDP-D-xylulose, and abpB was characterized to be a CDP-D-xylulose reductase-encoding gene, responsible for the conversion of CDP-D-xylulose to CDP-D-arabinitol as the final product. The kinetic parameters of AbpA for the substrates D-Xlu-5-P and CTP and those of AbpB for the substrate CDP-D-xylulose and the cofactors NADH or NADPH were measured, and the effects of temperature, pH, and cations on the two enzymes were analyzed. This study confirmed the involvement of the genes abpA and abpB and their products in the biosynthetic pathway of CDP-D-arabinitol.