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Isolates of Enterobacteriaceae collected from the same patient can lose carbapenem susceptibility during antimicrobial therapy, but little attention has been given to how this conversion takes place. In the current study, we retrospectively analyzed microbiological and clinical data from patients with enterobacterial infections at a tertiary hospital in Shanghai, China. After screening 4795 patients and 7120 Enterobacteriaceae isolates over the 3-year study period, we found the change from carbapenem susceptible to carbapenem resistant in 41 pairs of isolates, of which 35 pairs (85.4%) were K. pneumoniae and 25 (61.0%) were from the same anatomic sites. Thirty-six isolate pairs showed different pulsed-field gel electrophoresis patterns between the carbapenem-susceptible and the corresponding resistant strain, and 5 pairs displayed identical pulsed-field gel electrophoresis patterns. Thirty-three (91.7%) of the 36 pairs of Enterobacteriaceae isolates were carbapenem-resistant K. pneumoniae with blaKPC-2, and 28 pairs (90.3%) of K. pneumoniae isolates had different sequence types (STs), with ST11 the most common ST found in carbapenem-resistant K. pneumoniae isolates. Forty of the 41 patients had received antimicrobial therapy such as carbapenems, cephalosporins, and fluoroquinolones, before the isolation of carbapenem-resistant Enterobacteriaceae. These results demonstrated that strain replacement is the main cause of emerging carbapenem resistance in Enterobacteriaceae during hospitalization. The loss of carbapenem susceptibility was not mainly due to in vivo development of carbapenem resistance.

Background Streptococcus suis is an encapsulated zoonotic pathogen. Increasing antimicrobial resistance invokes the need for effective vaccines. Despite many attempts to develop an effective vaccine, none is currently available. Methods A capsular polysaccharide (CPS)-expressing attenuated mutant 2015033 was constructed by deleting 5 virulence-associated factors (sly, scpA, ssnA, fhb, and ssads) in an infective S. suis strain SC19. The safety and immune effect of 2015033 were determined both in vitro and in vivo. Results Deletion of 5 genes did not impact the growth ability and CPS generation of 2015033, and the mutant exhibited no cytotoxicity in different cell models. 2015033 was more easily eliminated by innate immunity both in vitro and in vivo. In addition, 2015033 showed a diminished invasive ability in different mouse organs (brain, lung, and liver) and avirulent properties in mice associated with weak inflammation-inducing ability. Immunization with 2015033 triggered T cell-dependent immunity, suppressed streptococcal toxic shock-like syndrome, and conferred sequence type-independent protection to mice during infection. Conclusions This study presents the feasibility of the strategy of multigene deletion for the development of promising live vaccines against invasive encapsulated pathogens.

Group B streptococci (GBS) are encapsulated, β-hemolytic bacteria that are a common cause of infections in human newborns and certain adults. Two factors important for GBS virulence are the sialic acid capsular polysaccharide that promotes immune evasion and the hemolytic pigment that induces host cell cytotoxcity. These virulence factors are often oppositely regulated by the CovR/CovS two-component system. Clinical GBS strains exhibiting hyperhemolysis and low capsule due to pathoadaptive covR/S mutations have been isolated from patients. Given the importance of capsule to GBS virulence, we predicted that a decrease or loss of capsule would attenuate the virulence of covR/S mutants. Surprisingly, hyperhemolytic GBS with low or no capsule exhibit increased virulence, intracellular persistence, and blood-brain barrier penetration, which was independent of a Trojan horse mechanism of barrier penetration. Additionally, intracellular persistence enabled both hemolytic and hyperhemolytic GBS to evade antibiotics routinely used to treat these infections. The finding that diminished capsule expression promotes GBS virulence, intracellular persistence, and antibiotic evasion has important implications for sustained antibiotic therapy and efficacy of capsule-based vaccines.

BACKGROUND: Klebsiella pneumoniae causing community-acquired pyogenic liver abscess complicated with metastatic meningitis and endophthalmitis has emerged recently, most frequently associated with the K1 capsular type. METHODS: A bacteriophage (NTUH-K2044-K1-1) that infects K. pneumoniae NTUH-K2044 (capsular type K1) was isolated and characterized. RESULTS: The phage infected all K1 strains, and none of the strains with other capsular types. Capsule deletion mutants were not lysed by this phage, suggesting that the capsule was essential for phage infection. Complete genome sequencing revealed the phage was a novel phiKMV-like virus. The gene-encoding capsule depolymerase was identified. The recombinant enzyme demonstrated specific lysis of the K1 capsule. Treatment with the phage or the recombinant enzyme provided significantly increased survival in mice infected with NTUH-K2044 strain, including one treated after the detection of a neck abscess by imaging. No obvious disease was observed after administration of this phage in mice. Phage was retained at detectable levels in liver, spleen, brain, and blood 24 hours after administration in mice. CONCLUSIONS: These results demonstrate this phage and its capsule depolymerase exhibit specificity for capsular type K1 and can be used for the diagnosis and treatment of K1 K. pneumoniae infections.

Staphylococcus aureus produces numerous virulence factors but little is known about their in vivo regulation during an infection.The production of capsule and α-toxin, and the expression of their respective genes, cap5 and hla, were analyzed by comparing CYL11481 (derivative of Newman) and its isogenic regulatory mutants in vitro. The temporal expression of cap5 and hla and the regulatory genes in vivo was carried out using a rat infective endocarditis model.In vitro analyses showed that capsule was positively regulated by MgrA, Agr, Sae, ArlR, and ClpC, and negatively by CodY and SbcDC. The α-toxin was positively regulated by MgrA, Agr, Sae, ArlR, and SbcDC but negatively by ClpC and CodY. In vivo analyses showed that cap5 expression correlated best with mgrA expression, whereas hla expression correlated best with sae expression. Mutation in mgrA drastically reduced cap5 expression in vivo.Our results suggest that, in vitro, Agr is the most important regulator for capsule and α-toxin production, as well as for cap5 transcription, but SaeR is the most critical for hla transcription. However, in vivo, MgrA is the major transcriptional regulator of capsule, but not α-toxin, whereas saeR expression correlates best with hla expression.

The phenotype of B cells that respond to vaccination with the purified pneumococcal polysaccharide (PPS) has been a topic of debate. We have recently identified the phenotype of cells from healthy young volunteers as CD27(+)IgM(+) B cells. However, the PPS-responding B-cell population has not yet been identified in high-risk populations, such as elderly individuals. Previous studies have shown that elderly individuals have a lower percentage of immunoglobulin M memory B cells than healthy young adults. In this study, we directly characterized the phenotype of PPS-specific B cells before and after vaccination with PPS vaccine (PPV) in elderly adults, using fluorescently labeled PPS14 and PPS23F. In contrast to our observations in healthy young volunteers, the PPS-responding B-cell population consisted primarily of switched memory (CD27(+)IgM(-)) B cells. In concurrence with these findings, postvaccination immunoglobulin M concentrations were not significantly increased in this population, and the opsonophagocytic response was decreased, compared with that in young adults. These findings identify a significant shift in the phenotype of the B-cell population in response to PPV among elderly individuals.

Background . Changes in serotype prevalence among pneumococcal populations result from both serotype replacement and serotype (capsular) switching. Temporal changes in serotype distributions are well documented, but the contribution of capsular switching to such changes is unknown. Furthermore, it is unclear to what extent vaccine-induced selective pressures drive capsular switching. Methods . Serotype and multilocus sequence typing data for 426 pneumococci dated from 1937 through 2007 were analyzed. Whole-genome sequence data for a subset of isola tes were used to investigate capsular switching events. Results . We identi fi ed 36 independent capsular switch events, 18 of which were explored in detail with whole- genome sequence data. Recombination fragment lengths were estimated for 11 events and ranged from approximately 19.0 kb to ≥ 58.2 kb. Two events took place no later than 1960, and the imported DNA included the capsular locus and the nearby penicillin-binding protein genes pbp2x and pbp1a . Conclusions . Capsular switching has been a regular occurrence among pneumococcal populations throughout the past 7 decades. Recombination of large DNA fragments (>30 kb), sometimes including the capsular locus and penicil- lin-binding protein genes, predated both vaccine introducti on and widespread antibiotic use. This type of recombina- tion has likely been an intrinsic feature throughout the history of pneumococcal evolution.

Streptococcus pneumoniae, the pneumococcus, is both a common human nasopharyngeal commensal and a highly significant opportunistic pathogen. Most pathogenic pneumococci express a protective polysaccharide capsule, which shields pneumococci from innate immunity [1] and greatly increases its transmissibility and virulence. However, anti-polysaccharide capsule antibodies can mediate type-specific bacterial clearance and provide protection against colonization and infection. This selective pressure has likely contributed to the evolution of at least 93 antigenically distinct capsule types, which are commonly referred to as serotypes [2–4]. Each serotype has a unique chemical structure, and for nearly all of the 93 serotypes, capsular synthesis is mediated by the genes within the capsule synthesis (cps) locus. [5]. Studies on serogroup 15 and the newly discovered serotype 11E demonstrate that bacteria expressing certain capsule types can arise independently and recurrently. Antigenic distinctions between serotypes 15B and 15C is attributed to phase-variable O-acetylation of capsule polysaccharide mediated by wciZ, which encodes a putative transmembane O-acetyltransferase of the PF01757 protein family and is truncated in 15C cps loci as a result of a reversible slipped-strand mutation [6, 7]. The rate of in vitro conversion between serotypes 15B and 15C is proposed to be as high as 1 in 300 [8]. Similarly, we recently determined the genetic distinction between serotype 11A and serotype 11E to be the integrity of wcjE, which is located at the 3′ end of the cps locus and also encodes a PF01757 O-acetyltransferase [3]. Unlike serotype 15C isolates that contain reversible mutations in wciZ, however, each serotype 11E clinical isolate so far examined harbors a unique irreversible null mutation to wcjE, indicating that the serotype has originated independently in separate hosts. To determine whether this mechanism of recurrent serotype emergence applies to other serotypes, we genetically and serologically characterized clinical isolates expressing the vaccine-target serotype 9V and the closely related serotype 9A. Historically, serotypes 9V and 9N have accounted for the majority of pneumococcal serogroup 9 infections, and 9A has accounted for only a small fraction. The 3′ end of the serotype 9V cps locus contains a wcjE gene that is highly homologous (92.2% sequence identity over 1,029 base-pair overlap) to the 11A wcjE described in our previous study [3, 9]. The only dissimilarity between the published cps loci of serotypes 9V and 9A (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF402095","term_id":"21552720","term_text":"AF402095"}}AF402095 and {"type":"entrez-nucleotide","attrs":{"text":"CR931645","term_id":"68642573","term_text":"CR931645"}}CR931645) is a single slipped-strand nucleotide deletion in 9A wcjE [10]. Because the genetic analysis was limited to single serotype 9V and 9A isolates, it is unknown whether this particular wcjE mutant allele is typically found within serotype 9A strains. Moreover, wcjE inactivation has not been confirmed as the molecular basis for serological differences between serotypes 9V and 9A. Whether wcjE is active in 9V capsule synthesis remains unclear because none of the multiple O-acetate substitutions on serotype 9V polysaccharide repeat units [11] (Figure 1) are identical to the wcjE-associated structure in serotype 11A, that is, O-acetate substitution of the 6 carbon of 11A β-galactose–a modification absent on 11E polysaccharide [12]. And even though the published serotype 9A polysaccharide structure has no O-acetate substitutions (Figure 1C) [11], a recent study suggested the presence of some O-acetyl groups on 9A polysaccharide [10], consistent with the fact that both 9V and 9A cps loci contain another functionally intact O-acetyltransferase, wcjD (Figure 1A). Figure 1. Diagram depicting the serotype 9V capsule synthesis (cps) locus and the published structures of serotype 9V and 9A capsule polysaccharide of Streptococcus pneumoniae. A, Depiction of the 9V cps locus according to Van Selm et al in 2002 [9] . The 2 putative ... Here, we show that serotype 9A is derived from serotype 9V through inactivation of wcjE. Furthermore, we show that each 9A isolate contains a unique mutation to wcjE. With the use of a novel flow-cytometric serotyping assay, we show that serotype 9A strains display diverse serological profiles that are dependent on the type of mutation to wcjE. We concluded that multiple mutations present in clinical 9A wcjE alleles can mediate variable expression levels of epitope previously associated only with serotype 9V.

Recently, 2 serologically and biochemically distinct subtypes-11Aalpha and 11Abeta-were discovered among serotype 11A isolates of Streptococcus pneumoniae. Sequence comparison of the capsular polysaccharide synthesis (cps) loci of the 2 subtypes identified disruption of the wcjE gene, a putative O-acetyltransferase, as the genetic hallmark of the 11Abeta phenotype. Directed disruption of wcjE in vitro in an 11Aalpha strain switched the strain to the 11Abeta phenotype, confirming the role played by the gene in the divergence between the subtypes. Furthermore, sequences from 7 11Abeta clinical strains each contained unrelated disruptive mutations in the wcjE gene, displaying an unprecedented degree of genetic heterogeneity in a pneumococcal serotype. We propose to name the 11Aalpha subtype as serotype 11A and the 11Abeta subtype as 11E, a new serotype. Our findings also suggest that the diversity of pneumococcal capsules is much greater than was previously recognized.

Background Mucoviscosity-associated gene A (magA) is proposed to play a decisive role in the pathogenesis of liver abscesses due to Klebsiella pneumoniae. Although some investigators consider MagA to be a putative O-antigen ligase, it is also reportedly associated with the K1 antigen. Methods Using magA-positive serotype K1 K. pneumoniae STL43 isolated from a patient with liver abscess, we constructed 3 bacterial mutants by targeting genes within the same transcription unit, including magA, wcaG, and rfbP. The virulence of these mutants was determined by neutrophil phagocytosis and inoculation of mice. Transmission electron microscopy and Western blot analysis were used to define their surface polysaccharides. Results STL43 was resistant, and all 3 mutants were highly susceptible, to phagocytosis. None of the mutant strains caused death in mice at the lethal dose of STL43. In contrast to previous reports, transmission electron microscopy revealed that all 3 mutants were nonencapsulated. Analysis of surface polysaccharides revealed that all 3 mutants retained their O antigen but lost their K antigen/capsule. Furthermore, amino acid analysis showed that MagA shared a conserved domain of Wzy, the serotype-specific capsular polysaccharide polymerase. Conclusions In accordance with the bacterial polysaccharide gene nomenclature (BPGN) scheme, MagA should be renamed Wzy(KpK1), the capsular polymerase specific to K. pneumoniae serotype K1.

Background. A quadrivalent meningococcal conjugate vaccine (MCV4) was licensed in the United States in 2005; no serogroup B vaccine is available. Neisseria meningitidis changes its capsular phenotype through capsular switching, which has implications for vaccines that do not protect against all serogroups. Methods. Meningococcal isolates from 10 Active Bacterial Core surveillance sites from 2000 through 2005 were analyzed to identify changes occurring after MCV4 licensure. Isolates were characterized by multilocus sequence typing (MLST) and outer membrane protein gene sequencing. Isolates expressing capsular polysaccharide different from that associated with the MLST lineage were considered to demonstrate capsular switching. Results. Among 1160 isolates, the most common genetic lineages were the sequence type (ST)-23, ST-32, ST-11, and ST-41/44 clonal complexes. Of serogroup B and Y isolates, 8 (1.5%) and 3 (0.9%), respectively, demonstrated capsular switching, compared with 36 (12.9%) for serogroup C (P

Background. Clumping factor A (ClfA) is a Staphylococcus aureus cell wall-associated adhesin that mediates staphylococcal binding to fibrinogen and platelets. Our goals were to determine whether expression of capsular polysaccharide (CP) affected ClfA-mediated adherence of S. aureus and to assess whether the length of the ClfA repeat region influenced this interaction. Methods. ClfA constructs with repeat regions of different lengths were introduced into isogenic S. aureus strains that expressed CP5, CP8, or no CP. S. aureus binding to fibrinogen was assessed in rabbit plasma and on fibrinogen-coated microtiter plates. Adherence of S. aureus strains to platelets was evaluated by flow cytometry and confocal microscopy. Results. As the length of the ClfA repeat region increased, binding of acapsular S. aureus to fibrinogen-coated microtiter plates was enhanced. By contrast, encapsulated S. aureus expressing the full-length ClfA were poorly adherent. The acapsular S. aureus mutant strain showed a 2-fold increase in platelet binding, compared with the isogenic encapsulated strains. By contrast, platelet aggregation was unaffected by CP production. Conclusion. CP expression inhibits S. aureus ClfA-mediated binding to fibrinogen and platelets, and a full-length repeat region cannot overcome this inhibition. These findings have important implications for vaccine development, given that CP may mask surface adhesins.

Antibodies to pneumococcus are thought to represent the primary mechanism of naturally acquired resistance to colonization. Here, however, we show that, in patients with chronic obstructive pulmonary disease (COPD), resistance to pneumococcal colonization is not associated with higher concentrations of serum anti-capsular or -noncapsular antibodies. We compared preacquisition serum antibody concentrations to capsular antigens 6B, 7F, 14, 19F, and 23F from patients with COPD who did and did not acquire pneumococcal respiratory tree colonization over the course of 2 years. Colonized patients did not have lower anti-capsular antibody concentrations than control subjects who did not acquire pneumococcus. We found no difference in functional antibody concentrations between colonized patients and control subjects. Furthermore, colonized patients had significantly higher preacquisition concentration of antibody directed against the whole cell and pneumococcal surface protein A than control subjects. We thus conclude that, in adult patients with COPD, resistance to pneumococcal colonization is unlikely to be determined by higher serum antibody concentrations to pneumococcal antigens.

During reinforced surveillance of acute bacterial meningitis in Burkina Faso, meningococcal strains of phenotype NG:NT:NST were isolated from cerebrospinal fluid samples from 3 patients. The strains were negative for the ctrA gene but were positive for the crgA gene. Molecular typing revealed that the strains harbored the capsule null locus (cnl) and belonged to the multilocus sequence type (ST)-192. PorA sequencing showed that all strains were either P1.18-11,42; P1.18,42-1; P1.18-11,42-1; P1.18-11,42-3; or P1.18-12,42-1. Sequencing also showed that all strains were negative for the FetA receptor gene. Serum killing assays showed these strains to be resistant, with the resistance comparable with that of a fully capsular serogroup B strain, MC58. The same strains were found in 14 healthy carriers in the general population of Bobo-Dioulasso (100% of ST-192 isolates tested for cnl). The presence of cnl meningococci that can escape serum killing and cause invasive disease is of concern for future vaccination strategies and should promote rigorous surveillance of cnl meningococcal disease.

Background. Primary pyogenic liver abscess (PLA) caused by Klebsiella pneumoniae is an emerging infectious disease. Capsular serotype K1 and the magA gene have been reported to be associated with this disease. Methods. The prevalence of magA was determined by polymerase chain reaction (PCR). The sequences of the magA flanking region were completed by inverse PCR and direct sequencing. Serotyping was performed by double immunodiffusion. Insertion mutations and trans-complementation were used to define the K1 genetic determination region. Results. Thirty-five of 42 strains from patients with PLA were magA positive, whereas only 1 of 32 non-PLA strains was magA positive. All 36 magA-positive strains were serotype K1, and the 38 magA-negative strains were not (36/36 vs. 0/38; ). Sequencing of the magA flanking region revealed a putative capsular polysaccharide P ! .0001 synthesis (cps) region; this region was 25 kb in length and contained 20 open reading frames (ORFs); of these ORFs, 9 were cotranscribed as part of an operon and differed from both MGH78578 and the Chedid strain. Mutation of 4 genes in this region turned the mutant strains anti-K1 negative. Trans-complementation restored the K1 phenotype. Conclusions. The operon containing magA is responsible for capsular serotype K1 of K. pneumoniae. Several loci in the operon are unique determinants of K1 strains.

Colonization of the colon and vagina is thought to be important in the pathogenesis of group B Streptococcus (GBS) infection. However, little is known about the strategies used by GBS to translocate through the epithelial barrier during the onset of disease. We used differentiated epithelial cells grown on transwell inserts as a model of the epithelial barrier. Bacterial translocation occurred without a detectable decrease in transepithelial resistance. Whereas acapsular GBS was better able to adhere to and invade epithelial cells, the percentage of bacteria translocating across the epithelial monolayer was independent of the presence of the capsule. Transmission electron microscopy showed the intimate association of GBS with intercellular junctions and the capacity to cross the monolayer by a paracellular mechanism. This process consisted of an active and transient opening of cell junctions. Indeed, GBS was preferentially found along the cell perimeter, where it colocalized with junctional protein complexes.

We obtained bronchoalveolar lavage (BAL) fluid from 45 Malawian adults, to measure the concentrations of innate pulmonary immune factors that are important in lung defense against infection. Increased concentrations of the beta -chemokine RANTES were found in BAL fluid from the human immunodeficiency virus (HIV)-1-infected subjects, compared with those in BAL fluid from the HIV-1-uninfected control subjects (mean, 86 pg/mL vs. 0 pg/mL; P.001). Lysozyme concentrations were also elevated in the HIV-1-infected subjects, compared with those in the HIV-1-uninfected control subjects (1.9 mu g/mL vs. 1.1 mu g/mL; P=.03), but were not elevated in the HIV-1-infected subjects who had recently recovered from invasive pneumococcal disease. Concentrations of lactoferrin and secretory leukocyte protease inhibitor (SLPI) were not different when the subjects were compared by HIV-1 serostatus. Concentrations of RANTES (R2=0.68 and P.0001) and SLPI (R2=0.29 and P=.001) correlated with BAL fluid HIV-1 load but not with plasma HIV-1 load.

Most invasive Haemophilus influenzae type b strains possess a duplication of the capsulation locus. Further amplification resulting in as many as 5 copies has been described. To verify whether amplification is involved in vaccine failure, the number of copies of the locus was determined by Southern blotting in 90 strains from children with true vaccine failure (TVF) between 1993 and 1999 and in 139 strains from unvaccinated children (50 collected between 1993 and 1999 and 89 collected between 1991 and 1992, before routine immunization was introduced). A significantly greater proportion of strains from TVFs contained multiple copies, compared with strains from control children (24% vs. 10%; P = .0379), which suggests that amplification of the capb locus may be a contributory factor in vaccine failure. The presence of multiple-copy strains was associated with disease other than meningitis.

Background. Increases in antimicrobial resistance in Escherichia coli have been paralleled by an increasing incidence of E. coli sepsis, suggesting a possible link between resistance and virulence.Methods. All 76 multidrug-resistant (MDR) E. coli isolates (i.e., those resistant to ⩾3 antimicrobial agents, including ampicillin, ceftazidime, trimethoprim-sulfamethoxazole, gentamicin, and ciprofloxacin) reported to the Tracking Resistance in the United States Today studies during 2000–2001 and 76 closely matched pansusceptible control isolates were studied. Extended virulence profiles and E. coli phylogenetic group (A, B1, B2, or D) were compared between groups.Results. The MDR isolates, which represented predominantly non-B2 phylogenetic groups (91%), exhibited significantly reduced molecular virulence, compared with the predominantly group B2-derived control isolates (58%). Only 30% of MDR isolates, compared with 61% of control isolates (P < .001 ), qualified as extraintestinal pathogenic E. coli (ExPEC), and even these isolates exhibited significantly lower virulence scores than did susceptible ExPEC (7.25 vs. 9.0; P = .001 ). Phylogenetic differences accounted for the apparent virulence differences between MDR and control isolates.Conclusions. These findings argue against a direct link between virulence traits and antimicrobial resistance in E. coli. Instead, they call into question why non-B2 strains are more commonly MDR, with differential exposure to selection pressure (including in agriculture) as one possible explanation.

We examined the antibody responses elicited in rats after transcutaneous immunization (TCI) with the Haemophilus influenzae type b (Hib)-cross-reacting material (CRM(197)) glycoconjugate vaccine coadministered with cholera toxin or mutants of heat-labile enterotoxin of Escherichia coli (LTK63 and LTR72) as adjuvants. The glycoconjugate vaccine was immunogenic, eliciting high antibody responses to the capsular polysaccharide of Hib and to diphtheria toxin. Passively transferred immune serum protected infant rats against challenge with the Hib Eagan strain and exhibited strong neutralizing activity against diphtheria toxin both in vitro and in vivo. The finding that TCI of rats can elicit antibody responses surpassing the minimum levels required for protective immunity against Hib and diphtheria suggests that this immunization strategy holds a lot of promise for future pediatric use. However, further studies are required to confirm the potential of TCI with glycoconjugate vaccines in humans.

Despite the characterization of some Burkholderia cepacia complex exopolysaccharides (EPSs), little is known about the role of EPSs in the pathogenicity of B. cepacia complex organisms. We describe 2 Burkholderia cenocepacia (genomovar III) isolates obtained from a patient with cystic fibrosis (CF): the nonmucoid isolate C8963 and the mucoid isolate C9343. Both isolates had identical random amplified polymorphic DNA patterns. C9343 produced a capsule composed of the EPSs PS-I and PS-II, as well as alpha -1,6-glucan. These isolates exhibited several phenotypic differences: C8963 synthesized octanoyl-homoserine lactone and produced biofilms, but C9343 did not; in a mouse model of pulmonary infection, C8963 was cleared more rapidly than was C9343; and C9343 interacted poorly with macrophages and neutrophils, compared with C8963, suggesting that the C9343 capsule interfered with cell-surface interactions. Overproduction of EPS by C9343 resulted in a mucoid appearance and interfered with cell-surface interactions and clearance in an animal model. This mucoid colonial appearance could enhance the persistence and virulence of this important CF-related pathogen.

Four genes, cpsA-cpsD, at the 5' end of the capsular polysaccharide (CPS) biosynthesis locus are conserved in nearly all of the 90 known serotypes of Streptococcus pneumoniae. In the present study, the impact that mutations in cpsA, cpsB, and cpsD have on CPS production and on virulence in mice infected via systemic and intranasal routes was investigated. Strains exhibiting rough colony morphologies (in which either the cpsB or cpsD gene had been deleted) were avirulent, but a smooth, partially encapsulated strain (in which the cpsA gene had been deleted) was as virulent as the wild-type strain. Interestingly, mucoid strains containing mutations affecting the [YGX](3)-repeat domain of CpsD were unable to cause bacteremia after intranasal challenge of CD1 mice, even though such strains were capable of killing BALB/c mice after intraperitoneal challenge. In our model, the ability of S. pneumoniae to regulate, via CpsD phosphorylation, CPS production was required for its transition from the lung to the bloodstream.

Penicillin and vancomycin induce a lytic response in Streptococcus pneumoniae that requires the N-acetylmuramyl-l-alanine amidase LytA. We show that clinical isolates of pneumococci of capsular serotypes 1, 4, 6B, and 23F were generally less lytic to penicillin than pneumococci of serotypes 14 and 3. In addition, most 9V isolates were less lytic to vancomycin, compared with isolates of other serotypes. Parent-mutant pairs expressing and not expressing capsular serotypes 2, 4, and 9V were compared for antibiotic-induced lysis. The nonencapsulated variants were considerably more lytic after beta-lactam and/or vancomycin treatment, and antibiotic tolerance was seen only in the context of capsule expression. Conversion from a nonlytic to a lytic phenotype, after loss of capsule expression, required an intact lytA autolysin gene. Exogenous addition of purified LytA gave a lower lytic response in capsulated strains, compared with that in nonencapsulated mutants. Spontaneous autolysis in stationary phase also was negatively affected by capsule expression in an autolysin-dependent manner. Long-term starvation in the stationary phase of the vancomycin- and penicillin-tolerant isolate I95 yielded nonencapsulated mutants that had lost antibiotic tolerance and were lytic to penicillin and vancomycin. The 9V capsular locus of I95 and one of these stationary phase-selected mutants were completely sequenced. The only difference found was a 1-bp frameshift deletion in the cps9vE gene of the lytic mutant, encoding a uridine diphosphate-glucosyl-1-phosphate transferase. Two additional independently isolated lytic mutants of I95 from the stationary phase also contained mutations in the same region of cps9vE, which identified it as a mutational hot spot. This report demonstrates that capsular polysaccharides negatively influence the lytic process and contribute to antibiotic tolerance in clinical isolates of pneumococci.

We tested the hypothesis that human immunodeficiency virus (HIV)-infected adults have a specific defect in anti-pneumococcal capsular polysaccharide (Pn-specific) immunoglobulin (Ig) in fluid obtained from the lower respiratory tract. Higher levels of total IgG and IgM were present in bronchoalveolar lavage samples from HIV-infected subjects than in those from HIV-uninfected subjects. Pn-specific IgG and IgM in bronchoalveolar lavage samples were not significantly different between HIV-infected and -uninfected subjects. After pneumococcal infection, HIV-infected patients had higher bronchoalveolar lavage levels of Pn-specific IgG than HIV-infected patients without recent infection (geometric means, 387 vs. 30 ng/mL, P=.001).

After the introduction and extensive dissemination of the multidrug-resistant Streptococcus pneumoniae clone Spain(6B)-2 between 1989 and the early to mid-1990s, the prevalence of pneumococcal isolates expressing intermediate resistance to penicillin, mainly of capsular types 6, 19, and 23, also began to increase in Iceland. The purpose of this study was to investigate whether these isolates originated in Iceland or represented strains imported to the country. Isolates were characterized by pulsed-field gel electrophoresis; multilocus sequence typing; determination of pbp1a, pbp2b, and pbp2x gene restriction patterns; and partial sequencing of these pbp genes. The results indicate that, although singular events suggesting horizontal transfer of pbp genes (and capsular genes) were detected, the majority of clones circulating in the country had genetic backgrounds also detected abroad. The major mechanism of dissemination of penicillin resistance in Iceland appears to be the repeated introduction of multiple lineages, followed by clonal spread.

Streptococcus pneumoniae (ie, pneumococcus) is a ubiquitous colonizer of the nasopharynx whose incidental invasion of the blood stream results in invasive pneumococcal disease (IPD). A prerequisite for invasion is expression of a polysaccharide (PS) capsule, of which >90 capsule serotypes have been identified [1]. The propensity of a colonizing pneumococcal strain to cause IPD (ie, its invasiveness) can vary greatly according to which serotype is expressed [2–4]. Accordingly, some prevalent serotypes, such as serotype 11A (ST11A), appear to be less invasive than others [5, 6]. The causal link between pneumococcal serotype and invasiveness in humans remains elusive. Recent discovery and characterization of ST11E, which was previously mistyped as ST11A [7], suggest that minor changes in capsule structure affect pneumococcal survival in different host tissues. ST11A and ST11E PS structures are identical except for the presence of O-acetylation (OAc) of carbon-6 of β-galactose (βGal6-OAc) on ST11A PS [8]. This OAc is dependent on the O-acetyltransferase gene wcjE encoded in the ST11A capsule synthesis (cps) locus [7]. Peculiarly, each ST11E strain contains a different inactivating mutation to wcjE [7, 9]. Furthermore, ST11E strains are rarely isolated from the nasopharynx but can account for up to 50% of IPD isolates typed as ST11A by the Quellung reaction [9]. This unique genetic heterogeneity and epidemiological behavior suggests that ST11E strains are not transmitted between hosts but instead independently evolve from colonizing ST11A strains during host invasion. Thus, loss of wcjE-dependent capsule OAc may benefit survival in blood. We hypothesize that a protective serum factor specifically recognizes ST11A but not ST11E, resulting in a decreased ability of ST11A pneumococci to cause IPD. Ficolin-1, -2, and -3 are serum-associated pattern-recognition molecules in humans [10, 11]. Functionally and structurally similar to mannose-binding lectin (MBL), ficolins form complexes with MBL-associated serine proteases and, upon binding, initiate the lectin complement pathway and direct opsonophagocytosis [12, 13]. Reportedly, binding of human ficolin-2 (L-ficolin) to pneumococci initiates the complement cascade in vitro [14, 15], and mice deficient in expression of ficolin-A, a putative ficolin-2 ortholog, display increased susceptibility to experimental IPD [15, 16]. However, genetic polymorphisms putatively decreasing ficolin-2 function [17, 18] are not associated with increased susceptibility to IPD [19], and reports of ficolin-2 ligand specificity and complement deposition on pneumococci are inconsistent [14, 15, 20]. Here, we evaluated the relationship between ficolin-2 and the clinical behavior of pneumococcal serotypes.

Haemophilus influenzae type b (Hib) capsular polysaccharide (PS) induces protective antibodies but is T independent and poorly immunogenic in infants. Conjugate vaccines of Hib PS linked to proteins, such as CRM(197), increase the PS antibody titer and elicit immunologic memory. To define the conjugate-induced memory T cell response, 19 adults were immunized with Hib-CRM(197), and antibody titers, carrier protein-specific CD4(+) T cell proliferation, and cytokine production were measured. Hib-CRM(197) induced PS and CRM(197) antibodies, vigorous T cell recall responses, and production of cytokines, including interleukin (IL)-2, IL-5, IL-10, and interferon-gamma. There was marked variability in PS antibody titer, despite consistent CRM(197)-specific recall responsiveness, which correlated with peak IgM and IgA PS antibody titers. Correlations were also found between IL-2 and IL-5 and IgA PS antibody levels. Hib-CRM(197) induced a rapid increase in CRM(197)-specific memory T cells and mixed Th1/Th2 cytokines, which may regulate the isotype and quantity of PS antibody.

CsrS/CsrR is a 2-component system in Streptococcus pyogenes that negatively regulates hyaluronic capsule and several exotoxins. To detect spontaneous mutations in csrRS, mucoid and large colony variants of M1 strain MGAS166 were isolated from experimental murine skin infections. By use of complementation with a csrRS(+) plasmid, relevant mutations were also detected in 7 of 12 human clinical isolates. The presence of spontaneous mutants in mouse infection was associated with larger, more necrotic lesions. Most spontaneous changes in CsrR resulted from single amino acid substitutions, whereas most csrS mutations were frameshift or nonsense mutations. In 2 instances, IS1548 insertions were found in csrS. Experimental inoculation of mixtures of wild-type (wt) and csrRS(-) bacteria yielded larger, more necrotic lesions than did either strain at twice the inoculum, which suggests that these variants may exhibit pathogenic synergy. Spontaneous emergence of csrRS(-) mutants in vivo enhances the virulence of wt bacteria and increases severity of murine skin infection.

JRS4(HE), a highly encapsulated, mouse-passaged variant of group A streptococcal strain JRS4, was characterized. The mucoid phenotype of JRS4(HE) was preserved after extensive passage in vitro. The level and size of csrRS transcript in JRS4(HE) was similar to that of JRS4, yet JRS4(HE) expressed high levels of has and sagA and exhibited an increased activity of streptolysin S. These findings indicate that the CsrRS repressor system was inactive in JRS4(HE). JRS4(HE) adhered to HEp-2 cells at the stationary phase but did not internalize these cells. At midlogarithmic phase, JRS4(HE) neither adhered to nor internalized cells, because of an increased amount of hyaluronic acid. Mice injected subcutaneously with JRS4(HE) developed large, deep necrotic lesions. In contrast, mice challenged with JRS4 developed small, superficial lesions. Despite the use of a high inoculum, mice challenged with JRS4(HE) did not develop a lethal bacteremic infection. It is concluded that inactivation of CsrRS in vivo is insufficient to cause a spreading necrotic disease.

Concentrations of serum anti-Haemophilus influenzae type b (anti-Hib) capsular polysaccharide (CPS) >/=0.15 and >/=1.0 microgram/mL are widely used as surrogates for protection against invasive Hib disease. However, the relationship between serum anti-Hib CPS following immunization and protection against colonization is not known, making it difficult to evaluate new Hib vaccines or combination vaccines. In the Dominican Republic, nasopharyngeal swabs were collected from 546 9-month-old infants who had received Hib conjugate vaccine at ages 2, 4, and 6 months and from 600 unvaccinated infants of the same age. The prevalence of Hib colonization was lower among vaccinated infants than among unvaccinated infants (0.9% vs. 2.3%). Among vaccinated infants, protection against colonization was significantly correlated with anti-Hib CPS concentrations >/=5 microgram/mL 1 month following the third dose of vaccine. These results suggest that the concentration of serum anti-Hib CPS needed for protection against colonization is greater than that needed for protection for invasive disease.

IgG to capsular polysaccharide (CPS) of Streptococcus pneumoniae is thought to provide the greatest degree of protection against pneumococcal disease. Serum obtained at hospital admission from 14 (27%) of 51 patients with bacteremic pneumococcal pneumonia and 11 (37%) of 30 with nonbacteremic pneumococcal pneumonia contained IgG to CPS of the infecting serotype; these percentages are similar to the prevalence of IgG to CPS in a control population. However, when compared with antibody from healthy adults, this IgG had far less capacity to opsonize the infecting pneumococcal serotype for phagocytosis in vitro by normal human polymorphonuclear leukocytes or to protect mice against experimental challenge. Failure to opsonize correlated closely with failure to protect mice, and each of these parameters correlated well with poor avidity for CPS. Future vaccine studies may need to examine the functional capacity of antibodies as a surrogate for infection, in addition to measuring their concentration in serum.

Two animal models were used to study maternal transfer of antibody to a group B Streptococcus (GBS) type III polysaccharide‐tetanus toxoid (III-TT) conjugate. The III-TT vaccine protected all 27 mouse pups born to vaccinated dams against a GBS challenge. In a separate study of vaccinated mouse dams and pups, maternal sera contained all 4 subclasses of polysaccharide-specific IgG, with IgG1 accounting for 83% of total IgG. Specific IgG subclass distribution ( ) in newborn pups closely resembled that in their IgG1 11 IgG2a = IgG2b = IgG3 mothers. Seven of 9 female baboons given the III-TT vaccine had 5- to 36-fold increases in specific antibody from baseline levels; they transferred 26%‐185% of specific antibody to their offspring. Matched maternal and neonatal sera obtained at delivery were functionally equivalent in an in vitro opsonophagocytosis assay. These preclinical studies provide further evidence for effective immunogenicity of GBS conjugate vaccine and efficient transport of functionally active maternal antibody. Group B Streptococcus (GBS) is the leading cause of bacteremia and meningitis among neonates in the United States. Early-onset GBS disease occurs within 7 days of birth, with most cases manifesting in the first 24 h [1]. Late-onset GBS disease develops after age 1 week, with a declining frequency of cases by age 3 months. Although the factors increasing the risk for late-onset GBS disease are not well understood, earlyonset GBS disease is acquired during the birthing process from mothers who are genitally or rectally colonized with GBS. The unique susceptibility of the neonate to GBS disease provided the rationale for the development of a maternal vaccine. We hoped that the administration of an effective vaccine to pregnant women at the beginning of the third trimester would elicit high levels of functional, type-specific IgG that would cross the placenta and protect neonates from disease. Indeed, a GBS polysaccharide vaccine administered to pregnant women at 31 weeks of gestation, although not optimally immunogenic, resulted in transfer of polysaccharide-specific functional IgG to their newborns [2].

Several multidrug-resistant (MDR) isolates of Streptococcus pneumoniae recovered from a variety of clinical sources and expressing serotypes 9N, 14, 19F, and 3 could not be distinguished from the capsular type 23 Spanish/USA epidemic clone in antibiotype, pulsed-field gel electrophoretic pattern, and restriction fragment length polymorphism types of their penicillin binding protein genes 1A, 2X, and 2B. When tested in a mouse model of virulence, isolates expressing capsular type 3 were lethal for 100% of infected animals within 2 days after intraperitoneal injection of 10(2) cfu/mouse. In contrast, several capsular type 23F isolates belonging to the MDR Spanish/USA clone and recovered from the same site were not lethal, even when injected at the dose of 10(7) cfu/mouse. These data suggest that the pneumococcal isolates expressing serotypes 9N, 14, 19F, and 3 represent products of in vivo capsular transformation events in which the MDR epidemic capsular type 23F Spanish/USA clone was the recipient.

The pneumococcus undergoes spontaneous phase variation between an opaque and a transparent colony form. In an animal model of systemic infection following intraperitoneal inoculation of mice, the opaque phenotype was significantly more virulent than the transparent for each of 3 strains examined. The opaque phenotype was associated with 1.2- to 5.6-fold greater amounts of capsular polysaccharide compared with the transparent using a sandwich ELISA. A similar technique comparing the amount of total teichoic acid showed that the transparent phenotype had 2.1- to 3.8-fold more immunodetectable teichoic acid. This difference was confirmed by comparing the incorporation of [3H]choline into teichoic acid. Cell fractionation revealed that variation in quantity of incorporated choline was due to differences in cell wall-associated teichoic acid. Results suggest that the pneumococcus phase varies between a virulent form with more capsular polysaccharide and less teichoic acid and an avirulent form with less capsular polysaccharide and more teichoic acid.

Opsonophagocytic capacity and concentrations of antibodies to the capsular polysaccharide (PS) of Streptococcus pneumoniae serotypes 6A, 6B, 19F, and 19A were determined for sera from adults immunized with 14- and 23-valent S. pneumoniae capsular PS vaccines. The concentration of anticapsular PS antibodies strongly correlated (r = .72 to r = .91) with the opsonophagocytic activities. However, 10%-20% of serum samples display strikingly less opsonophagocytic activity for the cross-reactive serotypes than expected on the basis of their antibody concentrations to the cross-reactive serotypes. From 1 poorly opsonic serum sample, kappa and lambda fractions of anti-6B antibodies were purified by affinity chromatography. Kappa (but not lambda) type antibody was found to have less opsonophagocytic capacity than expected. Thus, ELISA measurements of antibody responses to pneumococcal capsular PSs may not be reflective of opsonophagocytic function of the antibody.

Staphylococcus aureus capsular polysaccharide type 5 (CP5) expression was investigated in lung tissue and nasal polyps of two cystic fibrosis (CF) patients, in rats, and in vitro using ELISA and IFA. In CF tissues, S. aureus expressed protein A and teichoic acid but only 1%-5% of cells expressed CP5. When rats were challenged with CP5-positive S. aureus in the granuloma pouch model, only 1%-5% of CP5-positive cells were detectable in pouch exudates. CF and pouch isolates, however, reexpressed CP5 (70%-90% of cells) when grown in vitro with air. Addition of > or = 1% CO2 to air or to O2/N2 gas mixtures reduced CP5 expression significantly (P < .001) in a dose-dependent manner (6%-1% CP5-positive cells). The results show that S. aureus does not produce CP5 in CF airways and in rat granuloma pouches and that CO2 is an environmental signal that regulates CP5 expression.

Cross-protection among pneumococcal serotypes within serogroups was measured in the chinchilla otitis media (OM) model because several serotypes that cause OM in children are closely related biochemically. Chinchillas were given tetravalent vaccine composed of pneumococcal capsular polysaccharides (PS; types 6B, 14, 19F, 23F) conjugated to an outer membrane protein complex, and 89% to 96% developed a > or = 2-fold serum IgG rise against vaccine PS. Vaccine efficacy was tested by inoculating middle ear hypotympanic bullae with Streptococcus pneumoniae types 6B, 6A, 19F, or 19A. OM severity in the vaccinated groups challenged with types 6B, 6A, and 19F but not 19A was significantly better than in the respective placebo groups. Culture-positive pneumococcal OM occurred in 38%, 62%, 0, and 78% of vaccinated chinchillas challenged with types 6B, 6A, 19F, and 19A, respectively, but in 88% of type 6B- and 100% of type 6A-, 19A-, and 19F-challenged placebo chinchillas.

The occurrence of mutations in methicillin-resistant Staphylococcus aureus (MRSA) during persistent infection leads to antimicrobial resistance but may also impact host-pathogen interactions. Here, we investigate the host-pathogen consequences of 2 mutations arising in clinical MRSA during persistent infection: RpoB H481Y, which is linked to rifampicin resistance, and RelA F128Y, which is associated with an active stringent response. Allelic exchange experiments showed that both mutations cause global transcriptional changes, leading to upregulation of capsule production, with attenuated virulence in a murine bacteremia model and reduced susceptibility to both antimicrobial peptides and whole-blood killing. Disruption of capsule biosynthesis reversed these impacts on innate immune function. These data clearly link MRSA persistence and reduced virulence to the same mechanisms that alter antimicrobial susceptibility. Our study highlights the wider consequences of suboptimal antimicrobial use, where drug resistance and immune escape mechanisms coevolve, thus increasing the likelihood of treatment failure.

Background. Herd immunity is important in the effectiveness of conjugate polysaccharide vaccines against encapsulated bacteria. A large multicenter study investigated the effect of meningococcal serogroup C conjugate vaccine introduction on the meningococcal population. Methods. Carried meningococci in individuals aged 15-19 years attending education establishments were investigated before and for 2 years after vaccine introduction. Isolates were characterized by multilocus sequence typing, serogroup, and capsular region genotype and changes in phenotypes and genotypes assessed. Results. A total of 8462 meningococci were isolated from 47 765 participants (17.7%). Serogroup prevalence was similar over the 3 years, except for decreases of 80% for serogroup C and 40% for serogroup 29E. Clonal complexes were associated with particular serogroups and their relative proportions fluctuated, with 12 statistically significant changes (6 up, 6 down). The reduction of ST-11 complex serogroup C meningococci was probably due to vaccine introduction. Reasons for a decrease in serogroup 29E ST-254 meningococci (from 1.8% to 0.7%) and an increase in serogroup B ST-213 complex meningococci (from 6.7% to 10.6%) were less clear. Conclusions. Natural fluctuations in carried meningococcal genotypes and phenotypes a can be affected by the use of conjugate vaccines, and not all of these changes are anticipatable in advance of vaccine introduction. © The Author 2011.

Streptococcus suis is an emerging zoonotic agent of septicemia and meningitis. Knowledge on host immune responses toward S. suis and strategies used by this pathogen for subversion of these responses is scarce. Here, S. suis modulation of dendritic cell (DC) functions were assessed for the first time. Using S. suis knockout mutants in capsular polysaccharide (CPS) expression, it was shown that CPS blocks DC phagocytosis and impairs cytokine release by hindering cell wall components. Mutants impaired in D-alanylation of lipoteichoic acid (LTA) or N-deacetylation of peptidoglycan (PG) further demonstrated the importance of cell wall in modulation of DC activation. Notably, LTA/PG modifications were identified as major players in resistance to complement-dependent killing by DCs. Finally, S. suis hemolysin was partially involved in cytokine release and also contributed to bacterial escape of opsonophagocytosis. Overall, S. suis uses its arsenal of virulence factors to modulate DC functions and escape immune surveillance.

Background The RTS,S/AS01(E) malaria candidate vaccine is being developed for immunization of African infants through the Expanded Program of Immunization (EPI). Methods This phase 2, randomized, open, controlled trial conducted in Ghana, Tanzania, and Gabon evaluated the safety and immunogenicity of RTS,S/AS01(E) when coadministered with EPI vaccines. Five hundred eleven infants were randomized to receive RTS,S/AS01(E) at 0, 1, and 2 months (in 3 doses with diphtheria, tetanus, and whole-cell pertussis conjugate [DTPw]; hepatitis B [HepB]; Haemophilus influenzae type b [Hib]; and oral polio vaccine [OPV]), RTS,S/AS01(E) at 0, 1, and 7 months (2 doses with DTPwHepB/Hib+OPV and 1 dose with measles and yellow fever), or EPI vaccines only. Results The occurrences of serious adverse events were balanced across groups; none were vaccine-related. One child from the control group died. Mild to moderate fever and diaper dermatitis occurred more frequently in the RTS,S/AS01(E) coadministration groups. RTS,S/AS01(E) generated high anti-circumsporozoite protein and anti-hepatitis B surface antigen antibody levels. Regarding EPI vaccine responses upon coadministration when considering both immunization schedules, despite a tendency toward lower geometric mean titers to some EPI antigens, predefined noninferiority criteria were met for all EPI antigens except for polio 3 when EPI vaccines were given with RTS,S/AS01(E) at 0, 1, and 2 months. However, when antibody levels at screening were taken into account, the rates of response to polio 3 antigens were comparable between groups. Conclusion RTS,S/AS01(E) integrated in the EPI showed a favorable safety and immunogenicity evaluation. Trial registration. ClinicalTrials.gov identifier: NCT00436007 . GlaxoSmithKline study ID number: 106369 (Malaria-050).

The major pneumococcal virulence determinant is its capsule, and pneumococcal epidemiology is based on 91 capsular serotypes, each corresponding to the structure of the capsular polysaccharide determined by the type-specific capsular genome. Here, we provide the beginnings of an approach to intertwine serotype epidemiology, capsular regulatory gene characteristics on the basis of existing sequence information, and the reanalysis of published epidemiological data. We present an approach to explain epidemiological characteristics of serotypes on the basis of genetic differences in their capsular regulatory genes. The part of the capsular genome that regulates capsular expression falls into 2 highly divergent sequence clans: the ancestral pneumococcal capsular regulatory gene sequences (present in 49 serotypes) and laterally transferred sequences (present in 32 serotypes). Our survey of epidemiological data showed a tendency of the ancestral type of the capsular regulatory genome to be associated with carriage and the laterally transferred sequences to be associated with invasive disease isolates. The regulatory gene region shows mosaic structures that have signatures of recent recombination events, reminiscent of structures known from antibiotic resistance genes.

Nasopharyngeal carriage of Streptococcus pneumoniae is required for transmission of the bacteria and for invasive disease. There have been conflicting reports as to whether protection against carriage is serotype specific and which immune mechanisms drive carriage. Analyzing longitudinal carriage data from Israeli toddlers in day care, we found a lower risk of colonization with types 6A, 14, and 23F after previous exposure to the homologous type. Nonsignificant trends suggesting possible protection derived from prior exposure were found for types 19A and 23A. Furthermore, we found that, for types 14 and 23F, this specific protection correlated with increased serotype-specific antibody concentration. We found no evidence of specific protection for type 6B, group 15, or type 19F. Our findings imply that at least some serotypes generate anti-capsular antibodies that can reduce the risk of carriage in unimmunized toddlers.

The distribution of serotypes of group B streptococci (GBS) isolated from 67 infants with early-onset sepsis are described. Case-infants were assembled from 13 hospitals across the United States from 15 July 1995 to 5 February 1997 through prospective active surveillance. The distribution of GBS serotypes was Ia, 40%; Ib, 9%; II, 6%; III, 27%; V, 15%; and nontypeable, 3%. Type V occurred more frequently in the northeast region (New York and New Jersey) than in other regions (29% vs. 9%, P = .06). Conversely, type III occurred significantly less frequently in the northeast region than other regions (10% vs. 35%, P = .04). GBS types Ia, III, and V accounted for 82% of the isolates. This report supports previous observations about the emergence of GBS type V, but our data caution that conclusions about serotype distributions based on one geographic location or on a small number of patients may not be generally applicable. Continued monitoring seems necessary for the design of a GBS vaccine.

We investigated the immunogenicity of a 7-valent conjugate pneumococcal vaccine (CPV) in human immunodeficiency virus - infected Ugandan adults and measured the effect of past pneumococcal polysaccharide vaccine (PPV) receipt given as part of a controlled trial. Two doses of CPV, 4 weeks apart, were given to 54 past PPV recipients and 55 past placebo recipients (84% female; median CD4 cell count, 251 cells/mu L [range, 1 - 936 cells/mu L]). Postvaccination anticapsular immunoglobulin G (IgG) concentrations were directly correlated with CD4 cell count (P < .01 for all serotypes). There were significant increases in anticapsular IgG concentrations for all serotypes after the first dose (P < .01) and for all serotypes except 14 and 9V after the second dose. Past receipt of PPV did not affect vaccine response.

Streptococcus pneumoniae isolates of serotypes 1, 4, 6B, 7F, 14, and 19F belonging to clonal types with known invasive disease potential in humans were used to infect C57BL/6 and BALB/c mice. Most isolates were able to colonize the nasopharynx for 7 days. One serotype 19F isolate of the clonal type ST162 had higher bacterial numbers than other isolates and clonal types of the same serotype. Serotype 4 clones caused the most-severe invasive disease, whereas serotype 1 clones caused low-level bacteremia without disease symptoms. BALB/c mice were more likely than C57BL/6 mice to develop meningitis. Disease kinetics varied significantly between clonal types. Although most induced a robust tumor necrosis factor response, some isolates of serotype 1 and 7F did not, suggesting that invasive disease caused by different clonal types may result in different degrees of host response. Capsular serotype, other clonal properties, and host factors are important for the development of pneumococcal disease.

Thirty-nine previously treated Hodgkin's disease (HD) patients were immunized with 7-valent pneumococcal conjugate vaccine (7-OMPC) followed by one dose of 23-valent polysaccharide pneumococcal vaccine (23-PS). To determine the priming effect of 7-OMPC vaccine, their antibody responses to six serotypes contained in both vaccines were compared to those of 57 HD patients who received 23-PS vaccine only. The geometric mean antibody concentrations after immunization with 23-PS vaccine were significantly higher for five of the six measured serotypes in HD patients primed with 7-OMPC vaccine compared with responses in HD patients who received 23-PS vaccine only. The mean of the six antibody concentrations was significantly higher for the primed group at 12.5 micrograms/mL and 7.76 micrograms/mL, respectively (P = .015). Priming with a conjugate vaccine should be considered as a strategy to protect high-risk adults.

BACKGROUND: Neisseria meningitidis is a diverse commensal bacterium that occasionally causes severe invasive disease. The relationship between meningococcal genotype and capsular polysaccharide, the principal virulence factor and vaccine component, was investigated in carried meningococci isolated from 8000 children and young adults in Bavaria, Germany. METHODS: Of the 830 meningococci isolated (carriage rate, 10.4%) by microbiological techniques, 822 were characterized by serogrouping, multilocus sequence typing, and genetic analysis of the capsule region. Statistical and population genetic analyses were applied to these data. RESULTS: The rapid increase in carriage rates with age of carrier, the low prevalence of hyperinvasive meningococci, and the relative prevalence of the 4 disease-associated serogroups were consistent with earlier observations. There was no genetic structuring of the meningococcal population by age of carrier or sampling location; however, there was significant geographic structuring of the meningococci isolated in civil, but not military, institutions. The rate of capsule gene expression did not vary with age of carrier or meningococcal genotype, except for serogroup C, for which increased expression was associated with ST-11 (formerly ET-37) complex meningococci. CONCLUSIONS: Serogroup C capsule expression during carriage may contribute to the invasive character of ST-11 complex meningococci and to the high efficacy of meningococcal serogroup C conjugate polysaccharide vaccine.

We assessed a rat model to evaluate the immunogenicity of Haemophilus influenzae type b (Hib) conjugate vaccines and the effect on Hib immunogenicity of combining 2 Hib vaccines (Hib-tetanus toxoid [TT]-A and Hib-TT-B) with diphtheria-TT-acellular pertussis (DTaP)(3) or DTaP(5)/inactivated poliovirus (IPV) vaccines. Rats were immunized subcutaneously with Hib alone or with Hib and DTaP-based vaccines; anti-Hib capsular polysaccharide IgG, poly-ribosyl-ribitol-phosphate (PRP), IgG subclass, and cellular immune responses were evaluated. Results showed a significant reduction in the antibody response to PRP when Hib-TT-A was administered in combination with DTaP(3) and showed changes in the anti-PRP IgG subclass distribution between the separate and combination groups. However, combining Hib-TT-B with DTaP(5)/IPV did not reduce the anti-PRP antibody response. These results suggest that the model can predict the effect of combined administration of Hib and DTaP vaccines on Hib immunogenicity and would be suitable for preclinical studies of mechanisms of interference in Hib/DTaP vaccines.

Background Infections caused by group B streptococcal (GBS) type V are increasingly common. Capsular polysaccharide (CPS)-protein conjugate GBS vaccines are immunogenic in healthy adults, but type V vaccines have not previously been tested. Methods Thirty-five healthy, nonpregnant women were randomized to receive an intramuscular dose of GBS type V CPS-tetanus toxoid (TT) vaccine (n=15), GBS type V CPS-cross-reactive material (CRM(197)) conjugate vaccine (n=15), or placebo (n=5) (double-masked design). Levels of serum antibodies to type V CPS were measured by ELISA, and functional activity was measured by opsonophagocytosis. Results The vaccines were well tolerated. Significant increases in type V CPS-specific immunoglobulin G (IgG) were elicited by both vaccines, peaking at 4-8 weeks and persisting for 26 weeks. Four-fold or greater increases in type V CPS-specific IgG concentrations were noted in postimmunization serum samples obtained from 93% of subjects in each vaccine group. These concentrations persisted in > or =85% of conjugate-vaccine recipients 104 weeks later. Type V CPS-specific immunoglobulin M was a dominant isotype of immune response to each conjugate. Postimmunization serum samples promoted opsonophagocytic killing of GBS type V in vitro, whereas those from placebo recipients did not. Conclusion GBS type V conjugate vaccines are safe and immunogenic and would be appropriate for inclusion in a candidate multivalent GBS vaccine.