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AIMS The goal of this study was to characterize biopolymers from two modes of the Herbaspirillum lusitanum P6-12 growth: planktonic, in which cells are free swimming, and biofilm life style, in which the cells are sessile. METHODS AND RESULTS Differences in biopolymers composition from planktonic and biofilm cells of H. lusitanum strain P6-12 were analysed using Fourier transform infrared spectroscopy (FTIR), sodium dodecyl sulphate-polyacrylamide gel electrophoresis, gas-liquid chromatography and spectrophotometry. A high degree of polymer separation and purification was achieved by ultracentrifugation, and column chromatography allowed us to identify the chemical differences between biopolymers from biofilm and planktonic H. lusitanum. It was shown that planktonic cells of H. lusitanum P6-12 when cultivated in a liquid medium to the end of the exponential phase of growth, produced two high-molecular-weight glycoconjugates (were arbitrarily called CPS-I and CPS-II) of a lipopolysaccharide (LPS) nature and a lipid-polysacharide complex (were arbitrarily called EPS). The EPS, CPS-I, CPS-II had different monosaccharide and lipid compositions. The extracellular polymeric matrix (EPM) produced by the biofilm cells was mostly proteinaceous, with a small amount of carbohydrates (up to 3%). From the biofilm culture medium, a free extracellular polymeric substance (was arbitrarily called fEPS) was obtained that contained proteins and carbohydrates (up to 7%). The cells outside the biofilm had capsules containing high-molecular-weight glycoconjugate (was arbitrarily called CPSFBC ) that consisted of carbohydrates (up to 10%), proteins (up to 16%) and lipids (up to 70%). CONCLUSIONS During biofilm formation, the bacteria secreted surface biopolymers that differed from those of the planktonic cells. The heterogeneity of the polysaccharide containing biopolymers of the H. lusitanum P6-12 surface is probably conditioned by their different functions in plant colonization and formation of an efficient symbiosis, as well as in cell adaptation to existence in plant tissues. SIGNIFICANCE AND IMPACT OF THE STUDY The results of the study permit a better understanding of the physiological properties of the biopolymers, for example, in plant-microbe interactions.

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.

The capsule (cap) of Streptococcus suis is an anti-phagocytic element and is believed to be one of the major virulence factors of S. suis. However, we have found both cap-positive and cap-negative isolates in porcine endocarditis. The cap-negative isolates show strong adherence to platelets. Here, we compared the genome sequences of multiple cap-negative isolates with those of a cap-positive isolate from a single endocarditis case. The cap-positive and cap-negative isolates from the same pig were phylogenetically closest compared with those from other pigs. Some of the cap-negative isolates from the same pig showed different mutations in capsular polysaccharide synthesis (cps) genes, suggesting that these isolates arisen in the pig after infection. Different mutations in whole genomes were also found among isolates with identical mutations in cps genes, indicating that mutations in cps genes and the whole genome occurred independently. The calculated mutation frequencies were similar to those reported for other bacteria, suggesting all those mutations occurred spontaneously. Since cap-negative isolates are rarely found in lesions of other diseases, such as meningitis, these results suggest that endocarditis lesions may simply favored the cap-negative mutants to survive the niches, leading to their persistence in the lesions.

The Active Anthrax Detect (AAD) Rapid Test lateral flow immunoassay is a point-of-care assay that was under investigational use for detecting Bacillus anthracis capsular polypeptide (polyglutamic acid) in human blood, serum and plasma. Small sample volumes, rapid results and no refrigeration required allow for easy use in either the field or laboratory. Although the test was developed for use in suspect cases of human inhalation anthrax, its features also make it a potentially powerful tool for testing suspect animal cases. We tested animal tissue samples that were confirmed or ruled out for B. anthracis. The AAD Rapid Tests were also deployed in the field, testing animal carcasses during an anthrax outbreak in hippopotami (Hippopotamus amphibius) and Cape buffalo (Syncerus caffer) in Namibia. Evaluation of all samples showed a specificity of 82% and sensitivity of 98%. However, when the assay was used on specimens from only fresh carcasses (dead for Significance and Impact of the Study In countries where anthrax is endemic, many human outbreaks are often caused by epizootics. Earlier detection of infected animals may allow for identification of exposed people, early implementation of prevention and control methods, and ultimately lessen the number of people and animals affected. Detection of Bacillus anthracis in animal tissues using a simple, rapid and field-deployable method would allow for faster outbreak response. We evaluated a simple sample collection and processing method for use with the Active Anthrax Detect Rapid Test lateral flow immunoassay to screen dead animals for anthrax.

Observations of bacteria at the single-cell level have revealed many instances of phenotypic heterogeneity within otherwise clonal populations, but the selective causes, molecular bases, and broader ecological relevance remain poorly understood. In an earlier experiment in which the bacterium Pseudomonas fluorescens SBW25 was propagated under a selective regime that mimicked the host immune response, a genotype evolved that stochastically switched between capsulation states. The genetic cause was a mutation in carB that decreased the pyrimidine pool (and growth rate), lowering the activation threshold of a preexisting but hitherto unrecognized phenotypic switch. Genetic components surrounding bifurcation of UTP flux toward DNA/RNA or UDP-glucose (a precursor of colanic acid forming the capsules) were implicated as key components. Extending these molecular analyses—and based on a combination of genetics, transcriptomics, biochemistry, and mathematical modeling—we show that pyrimidine limitation triggers an increase in ribosome biosynthesis and that switching is caused by competition between ribosomes and CsrA/RsmA proteins for the mRNA transcript of a positively autoregulated activator of colanic acid biosynthesis. We additionally show that in the ancestral bacterium the switch is part of a program that determines stochastic entry into a semiquiescent capsulated state, ensures that such cells are provisioned with excess ribosomes, and enables provisioned cells to exit rapidly from stationary phase under permissive conditions.

Background Group B Streptococcus (GBS) frequently colonizes pregnant women and can cause sepsis and meningitis in young infants. If colonization was prevented through maternal immunization, a reduction in perinatal GBS disease might be possible. A GBS type III capsular polysaccharide (CPS)-tetanus toxoid conjugate (III-TT) vaccine was evaluated for safety and efficacy in preventing acquisition of GBS colonization. Methods Healthy, nonpregnant women aged 18–40 years and screened to be GBS III vaginal and rectal culture negative were randomized to receive III-TT conjugate or tetanus diphtheria toxoid vaccine in a multicenter, observer-blinded trial. GBS vaginal and rectal cultures and blood were obtained bimonthly over 18 months. Serum concentrations of GBS III CPS-specific antibodies were determined using enzyme-linked immunosorbent assay. Results Among 1525 women screened, 650 were eligible for the intent-to-treat analysis. For time to first acquisition of vaginal GBS III, vaccine efficacy was 36% (95% confidence interval [CI], 1%–58%; P = .044), and for first rectal acquisition efficacy was 43% (95% CI, 11% to 63%; P = .014). Two months post-immunization, geometric mean concentrations of serum GBS type III CPS-specific immunoglobulin G were 12.6 µg/mL (95% CI, 9.95 to 15.81) in GBS III-TT recipients, representing a 4-fold increase from baseline in 95% of women, which persisted. Both vaccines were well tolerated. Conclusions GBS CPS III-TT conjugate vaccine significantly delayed acquisition of vaginal and rectal GBS III colonization. In addition to its use for maternal immunization to passively protect infants with maternally derived antibodies, a multivalent vaccine might also serve to reduce fetal and neonatal exposure to GBS. Clinical Trials Registration NCT00128219.

Neisseria meningitidis Group X is an emerging cause of bacterial meningitis in Sub-Saharan Africa. The capsular polysaccharide of Group X is a homopolymer of N-acetylglucosamine α(1-4) phosphate and is a vaccine target for prevention of disease associated with this meningococcal serogroup. We have demonstrated previously that the formation of the polymer is catalyzed by a phosphotransferase which transfers N-acetylglucosamine-1-phosphate from UDP-N-acetylglucosamine to the 4-hydroxyl of the N-acetylglucosamine on the nonreducing end of the growing chain. In this study, we use substrate analogs of UDP-GlcNAc to define the enzyme/donor substrate interactions critical for catalysis. Our kinetic analysis of the phosphotransferase reaction is consistent with a sequential mechanism of substrate addition and product release. The use of novel uracil modified analogs designed by Wagner et al. enabled us to assess whether the CsxA-catalyzed reaction is consistent with a donor dependent conformational change. As expected with this model for glycosyltransferases, UDP-GlcNAc analogs with bulky uracil modifications are not substrates but are inhibitors. An analog with a smaller iodo uracil substitution is a substrate and a less potent inhibitor. Moreover, our survey of analogs with modifications on the N-acetylglucosamine residue of the sugar nucleotide donor highlights the importance of substituents at C2 and C4 of the sugar residue. The hydroxyl group at C4 and the structure of the acyl group at C2 are very important for specificity and substrate interactions during the polymerization reaction. While most analogs modified at C2 were inhibitors, acetamido analogs were also substrates suggesting the importance of the carbonyl group.

The ability to produce polysaccharides with diverse biological functions is widespread in bacteria. In lactic acid bacteria (LAB), production of polysaccharides has long been associated with the technological, functional and health-promoting benefits of these microorganisms. In particular, the capsular polysaccharides and exopolysaccharides have been implicated in modulation of the rheological properties of fermented products. For this reason, screening and selection of exocellular polysaccharide-producing LAB has been extensively carried out by academia and industry. To further exploit the ability of LAB to produce polysaccharides, an in-depth understanding of their biochemistry, genetics, biosynthetic pathways, regulation and structure-function relationships is mandatory. Here, we provide a critical overview of the latest advances in the field of glycosciences in LAB. Surprisingly, the understanding of the molecular processes involved in polysaccharide synthesis is lagging behind, and has not accompanied the increasing commercial value and application potential of these polymers. Seizing the natural diversity of polysaccharides for exciting new applications will require a concerted effort encompassing in-depth physiological characterization of LAB at the systems level. Combining high-throughput experimentation with computational approaches, biochemical and structural characterization of the polysaccharides and understanding of the structure-function-application relationships is essential to achieve this ambitious goal.

The introduction of pentavalent vaccine containing Haemophilus influenzae type b antigen in Indonesia’s National Immunization Program occurred nearly three decades after the vaccine was first available in the United States and 16 years after Indonesia added hepatitis B vaccine into the program. In this study, we analyzed the process that led to the decision to introduce pentavalent vaccine in Indonesia. Using process tracing and case comparison, we used qualitative data gathered through interviews with key informants and data extracted from written sources to identify four distinct but interrelated processes that were involved in the decision making: (a) pentavalent vaccine use policy process, (b) financing process, (c) domestic vaccine development process and (d) political process. We hypothesized that each process is associated with four necessary conditions that are jointly sufficient for the successful introduction of pentavalent vaccine in Indonesia, namely (a) an evidence-based vaccine use recommendation, (b) sufficient domestic financing capacity, (c) sufficient domestic vaccine manufacturing capacity and (d) political support for introduction. This analysis of four processes that led to the decision to introduce a new vaccine in Indonesia may help policy makers and other stakeholders understand and manage activities that can accelerate vaccine introduction in the future.

Background Bacillus Calmette-Guerin (BCG) vaccine is widely used for the prevention of tuberculosis, despite limited efficacy. Most immunological studies of BCG or Mycobacterium tuberculosis strains grow bacteria in the presence of detergent, which also strips the mycobacterial capsule. The impact of the capsule on vaccine efficacy has not been explored. Methods We tested the influence of detergent in cultures of BCG and M. tuberculosis strains on the outcome of vaccination experiments on mice and transcriptional responses on M. tuberculosis Results Vaccination of mice with encapsulated BCG promoted a more potent immune response relative to vaccination with unencapsulated BCG, including higher polysaccharide-specific capsule antibody titers, higher interferon γ and interleukin 17 splenic responses, and more multifunctional CD4(+) T cells. These differences correlated with variability in the bacterial burden in lung and spleen of mice infected with encapsulated or unencapsulated M. tuberculosis The combination of vaccination and challenge with encapsulated strains resulted in the greatest protection efficacy. The transcriptome of encapsulated M. tuberculosis was similar to that of starvation, hypoxia, stationary phase, or nonreplicating persistence. Conclusions The presence of detergent in growth media and a capsule on BCG were associated with differences in the outcome of vaccination, implying that these are important variables in immunological studies.

Background Many studies have documented lower vaccine efficacy among children in low-income countries, compared with their counterparts in high-income countries. This disparity is especially apparent with respect to oral vaccines such as rotavirus and oral polio vaccines. One potential contributing factor is the presence of maternal antenatal helminth infections, which can modulate the infant's developing immune system. Methods Using a multiplex immunoassay, we tested plasma immunoglobulin A (IgA) or immunoglobulin G (IgG) levels specific for antigens in 9 routinely administered childhood vaccines among 1639 children aged approximately 13 months enrolled in the ECUAVIDA (Ecuador Life) birth cohort study in Ecuador. We compared vaccine responses in 712 children of mothers who tested positive for helminth infections in the last trimester of pregnancy to responses in 927 children of mothers without helminth infection. Results Plasma IgA levels specific for antigens in rotavirus vaccine and oral polio vaccine containing poliovirus serotypes 1 and 3 were all significantly higher in children of helminth-infected mothers, compared with children of uninfected mothers. Plasma IgG levels specific for diphtheria, tetanus, pertussis, measles, rubella, and Haemophilus influenzae type b vaccine antigens were comparable between the 2 groups. Conclusions Antenatal maternal helminth infections were not associated with reduced antibody responses to infant vaccines, but rather with modestly increased IgA responses to oral vaccines.

Klebsiella pneumoniae clonal group (CG) 258, comprising sequence types (STs) 258, 11, and closely related variants, is associated with dissemination of the K. pneumoniae carbapenemase (KPC). Hospital outbreaks of KPC CG258 infections have been observed globally and are very difficult to treat. As a consequence, there is renewed interest in alternative infection control measures such as vaccines and phage or depolymerase treatments targeting the K. pneumoniae polysaccharide capsule. To date, 78 immunologically distinct capsule variants have been described in K. pneumoniae. Previous investigations of ST258 and a small number of closely related strains suggested that capsular variation was limited within this clone; only two distinct ST258 capsule polysaccharide synthesis (cps) loci have been identified, both acquired through large-scale recombination events (>50 kb). In contrast to previous studies, we report a comparative genomic analysis of the broader K. pneumoniae CG258 (n = 39). We identified 11 different cps loci within CG258, indicating that capsular switching is actually common within the complex. We observed several insertion sequences (IS) within the cps loci, and show further intraclone diversification of two cps loci through IS activity. Our data also indicate that several large-scale recombination events have shaped the genomes of CG258, and that definition of the complex should be broadened to include ST395 (also reported to harbor KPC). As only the second report of extensive intraclonal cps variation among Gram-negative bacterial species, our findings alter our understanding of the evolution of these organisms and have key implications for the design of control measures targeting K. pneumoniae capsules.

BACKGROUND Human immunodeficiency virus (HIV)-exposed infants are at increased risk of invasive Group B Streptococcus (GBS) disease; however, the reason for this increased susceptibility has not been characterized. METHODS We compared GBS capsular and surface-protein maternal immunoglobin G antibody concentrations and cord-maternal ratios between HIV-infected and HIV-uninfected mother-newborn dyads. RESULTS Median capsular antibody concentrations (µg/mL) were lower in HIV-infected than HIV-uninfected women for serotypes Ib (P = .033) and V (P = .040); and for pilus island (PI)-1 (P = .016), PI-2a (P = .015), PI-2b (P = .015), and fibrinogen-binding protein A (P < .001). For serotypes Ia and III, cord-maternal ratios were 37.4% (P < .001) and 32.5% (P = .027) lower in HIV-infected compared to HIV-uninfected mother-newborn dyads. The adjusted odds of having capsular antibody concentration ≥2 µg/mL when comparing HIV-infected to -uninfected women were 0.33 (95% confidence interval [CI], .15-.75) and 0.34 (95% CI, .12-1.00) for serotypes Ia and III, respectively. Antibody levels and cord-maternal ratios were independent of CD4(+) lymphocyte counts or HIV-1 viral load. CONCLUSIONS The lower GBS antibody concentrations and reduced transplacental antibody transfer in HIV-infected women, which likely contribute to their infants being at heightened susceptibility for invasive GBS disease, could possibly be mitigated by vaccination with a GBS conjugate vaccine currently under clinical development.

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. Vaccines against Streptococcus pneumoniae, Neisseria meningitidis, and Hemophilus influenzae type b induce functional opsonic or bactericidal antibodies to surface capsular polysaccharides (CP). Targeting the comparable Staphylococcus aureus CP seems logical, but to date such efforts have failed in human trials. Studies using immunization-induced animal antibodies have documented interference in opsonic and protective activities of antibodies to CP by antibodies to another S. aureus cell surface polysaccharide, poly-N-acetyl glucosamine (PNAG). Here we evaluated whether natural antibody to PNAG in normal human serum (NHS) had a similar deleterious effect. Methods. Functional and/or protective activities of antibody to S. aureus CP and PNAG antigens in patients with bacteremia, in mice immunized with combinations of CP and PNAG conjugate vaccines, and in serum samples of healthy subjects with natural antibody to PNAG, to which immunization-induced animal antibodies to CP antigens were added, were evaluated. Results. Antibodies to PNAG and CP that mutually interfered with opsonic killing of S. aureus were detected in 9 of 15 bacteremic patients. Active immunization of mice with combinations of PNAG and CP conjugate antigens always induced antibodies that interfered with each other’s functional activity. Non-opsonic natural antibodies to PNAG found in NHS interfered with the functional and protective activities of immunization-induced antibody to CP antigens during experimental infection with S. aureus. Conclusions. Both immunization-induced animal antibodies and natural antibodies to PNAG in NHS interfere with the protective activities of immunization-induced antibody to S. aureus CP5 and CP8 antigens, representing potential barriers to successful use of CP-specific vaccines.

Background: Since immunomodulators and antitumor necrosis factor (TNF) agents are increasingly used to treat inflammatory bowel disease (IBD), it is recommended to administer antipneumococcal vaccination to prevent opportunistic pneumonia. There is some evidence that concomitant immunosuppression may impair the immune response to vaccination. We aimed to evaluate the response rates to pneumococcal vaccination in four different treatment groups (mesalamine, azathioprine, infliximab, infliximab plus azathioprine). Methods: In all, 96 patients with IBD (54 with Crohn's disease; 42 with ulcerative colitis) were administered a 23-valent polysaccharide pneumococcal vaccine (PSV-23). The levels of antipneumococcal antibodies were measured prior to and at least 3 weeks after vaccination. Response rates and risk factors for impaired immunosuppression were investigated. Patients on mesalamine were used as a control group. Results: Patients administered infliximab or the combination immunosuppressive therapy had significantly lower response rates to vaccination (57.6% and 62.5%, respectively) compared with the group on mesalamine (88.6%; P < 0.05 for both comparisons). Azathioprine alone did not influence the response rate to vaccination (78.9%; P = 0.43 vs. mesalamine group). Mean antibody titers after vaccination were significantly lower in patients under infliximab or combined immunosuppression than controls (P < 0.05). Immunosuppression with infliximab or combination therapy significantly decreased the likelihood of responding to vaccination (odds ratio [OR] = 0.17, 95% confidence interval [CI] 0.04–0.64, P = 0.009, and OR = 0.21, 95% CI 0.05–0.91, P = 0.038, respectively). Pneumococcal vaccination was generally safe and well tolerated. Conclusions: Anti-TNF therapy alone or in combination with azathioprine impairs the response to pneumococcal vaccination in patients with IBD. All patients with IBD should therefore be vaccinated before starting anti-TNF therapy. (Inflamm Bowel Dis 2012;)

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

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.

CPS are major virulence factors in infections caused by Neisseria meningitidis and form the basis for meningococcal serogroup designation and protective meningococcal vaccines. CPS polymers are anchored in the meningococcal outer membrane through a 1,2-diacylglycerol moiety, but the innate immunostimulatory activity of CPS is largely unexplored. Well-established human and murine macrophage cell lines and HEK/TLR stably transfected cells were stimulated with CPS, purified from an endotoxin-deficient meningococcal serogroup B NMB-lpxA mutant. CPS induced inflammatory responses via TLR2- and TLR4-MD-2. Meningococcal CPS induced a dose-dependent release of cytokines (TNF-α, IL-6, IL-8, and CXCL10) and NO from human and murine macrophages, respectively. CPS induced IL-8 release from HEK cells stably transfected with TLR2/6, TLR2, TLR2/CD14, and TLR4/MD-2/CD14 but not HEK cells alone. mAb to TLR2 but not an isotype control antibody blocked CPS-induced IL-8 release from HEK-TLR2/6-transfected cells. A significant reduction in TNF-α and IL-8 release was seen when THP-1- and HEK-TLR4/MD-2-CD14- but not HEK-TLR2- or HEK-TLR2/6-transfected cells were stimulated with CPS in the presence of Eritoran (E5564), a lipid A antagonist that binds to MD-2, and a similar reduction in NO and TNF-α release was also seen in RAW 264.7 cells in the presence of Eritoran. CD14 and LBP enhanced CPS bioactivity, and NF-κB was, as anticipated, the major signaling pathway. Thus, these data suggest that innate immune recognition of meningococcal CPS by macrophages can occur via TLR2- and TLR4-MD-2 pathways.

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 The incidence of invasive Haemophilus influenzae infection decreased dramatically since the introduction of the H. influenzae serotype b (Hib) conjugate vaccine. H. influenzae invasive disease continues to occur and cause significant morbidity and mortality in children aged

Aims: To identify enzymes associated with bacteriophages infecting cystic fibrosis (CF) strains of Pseudomonas aeruginosa that are able to degrade extracellular alginic acids elaborated by the host bacterium. Methods and Results: Plaques produced by 21 Ps. aeruginosa-specific phages were screened for the presence of haloes, an indicator of capsule hydrolytic activity. Four phages produced haloed plaques, and one (PT-6) was investigated further. PT-6 was shown by electron microscopy to belong to Podoviridae family C1, to reduce the viscosity of four alginate preparations using a rolling ball viscometer and to release uronic acid-containing fragments from the polymers, as judged by spectrophotometry and thin layer chromatography. The alginase was partially purified by gel filtration chromatography and shown to be a 37 kDa polypeptide. Conclusions: Infection of CF strains of Ps. aeruginosa by phage PT-6 involves hydrolysis of the exopolysaccharide secreted by the host. Significance and Impact of the Study: The alginase produced by PT-6 has the potential to increase the well-being of CF suffers by improving the surface properties of sputum, accelerating phagocytic uptake of bacteria and perturbing bacterial growth in biofilms.

The high cost of the available pneumococcal conjugated vaccines has been an obstacle in implementing vaccination programs for children in developing countries. As an alternative, Malley et al. proposed a vaccine consisting of inactivated whole-cells of unencapsulated S. pneumoniae, which provides serotype-independent protection and involves lower production costs. Although the pneumococcus has been extensively studied, little research has focused on its large-scale culture, thus implying a lack of knowledge of process parameters, which in turn are essential for its successful industrial production. The strain Rx1Al- eryR was originally cultured in Todd-Hewitt medium (THY), which is normally used for pneumococcus isolation, but is unsuitable for human vaccine preparations. The purposes of this study were to compare the strains Rx1Al- eryR and kanR, develop a new medium, and generate new data parameters for scaling-up the process. In static flasks, cell densities were higher for eryR than kanR. In contrast, the optical density (OD) of the former decreased immediately after reaching the stationary phase, and the OD of the latter remained stable. The strain Rx1Al- kanR was cultivated in bioreactors with medium based on either acid-hydrolyzed casein (AHC) or enzymatically hydrolyzed soybean meal (EHS). Biomass production in EHS was 2.5 times higher than in AHC, and about ten times higher than in THY. The process developed for growing the strain Rx1Al- kanR in pH-controlled bioreactors was shown to be satisfactory to this fastidious bacterium. The new culture conditions using this animal-free medium may allow the production of the pneumococcal whole-cell vaccine.

BACKGROUND. Most studies of the dynamics of maternal group B Streptococcus (GBS) colonization have relied on capsular serotyping to define GBS acquisition or loss. Newer molecular methods that distinguish GBS clones may expand our knowledge and influence vaccination strategies. We used multilocus sequence typing (MLST) and GBS capsular gene cluster (cps) genotyping to investigate the dynamics of perinatal GBS colonization. METHODS. A total of 338 GBS isolates obtained from 212 colonized women who were enrolled in a prior prospective cohort study were serotyped and genotyped by MLST and cps typing before (visit 1) and 6 weeks after (visit 2) delivery. RESULTS. Of the 212 women, 126 were colonized at both visits, whereas 66 lost and 20 acquired GBS by visit 2. MLST of the 338 strains identified 29 sequence types marking distinct bacterial clones. A change in sequence type or cps and serotype occurred in 23 (18.3%) of the 126 women who were colonized at both visits. Specific sequence types were associated with GBS loss and persistence. Older maternal age and exclusive intrapartum antibiotic use were associated with persistent colonization. CONCLUSIONS. Although most GBS-positive pregnant women were stably colonized during the peripartum period, we detected changes in capsule expression and recolonization with antigenically distinct GBS clones over time by applying MLST. Combining the epidemiologic and molecular typing data revealed host factors and clones associated with persistent colonization, as well as a clone that was more readily lost. This knowledge is useful for the development of prevention and intervention strategies to reduce the likelihood of maternal GBS colonization.

Background: Despite the excellent immunogenicity of Haemophilus influenzae type b (Hib) conjugate vaccines, breakthrough cases of Hib disease still affect a small proportion of vaccinated children in the United Kingdom. We performed a retrospective study to compare the avidity of antibody directed against the Hib polysaccharide capsule (PRP) in children who experienced Hib vaccine failure in the United Kingdom among 3 historical cohorts and with age-matched healthy control subjects. Methods: Serum samples from vaccinated children with invasive Hib disease were collected beginning in 1992 as part of enhanced surveillance for Hib disease following vaccine introduction. A total of 251 children who experienced Hib vaccine failure were identified from 3 historical cohorts (1992-1995, 1996-1999, and 2000-2003). The anti-PRP antibody concentration and avidity from healthy age-matched control subjects was obtained for the 3 contemporary time points (1995, 1999, and 2002). Serum anti-PRP antibody concentration was measured in each of the samples using a standard Hib ELISA, and antibody avidity was determined using thiocyanate elution. Results. Within the first 60 days after disease onset, there was no change in the anti-PRP antibody avidity, and there was no statistically significant difference in the geometric mean Hib antibody avidity over the 3 study periods. However, the children who experienced Hib vaccine failure had significantly lower Hib antibody avidity than did healthy control subjects, despite a marked antibody response following infection. Conclusions. Children who experience Hib disease despite vaccination appear to have a defect in immunological priming, leading to a qualitative difference in Hib-specific memory B cells. Low anti-PRP antibody avidity decreases the functional activity of anti-PRP antibody in the sera of these children experiencing vaccine failure, leading to disease susceptibility.

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.

Recombinant human betacellulin binds strongly to heparin, requiring of the order of 0.8 M NaCl for its elution from a heparin affinity matrix. This is in complete contrast to the prototypic member of its cytokine superfamily, epidermal growth factor, which fails to bind to the column at physiological pH and strength. We used a well-established heparin binding ELISA to demonstrate that fucoidan and a highly sulfated variant of heparan sulfate compete strongly for heparin binding. Low sulfated heparan sulfates and also chondroitin sulfates are weaker competitors. Moreover, although competitive activity is reduced by selective desulfation, residual binding to extensively desulfated heparin remains. Even carboxyl reduction followed by extensive desulfation does not completely remove activity. We further demonstrate that both hyaluronic acid and the E. coli capsular polysaccharide K5, both of which are unsulfated polysaccharides with unbranched chains of alternating N-acetylglucosamine linked beta(1-4) to glucuronic acid, are also capable of a limited degree of competition with heparin. Heparin protects betacellulin from proteolysis by LysC, but K5 polysaccharide does not. Betacellulin possesses a prominent cluster of basic residues, which is likely to constitute a binding site for sulfated polysaccharides, but the binding of nonsulfated polysaccharides may take place at a different site.

Streptococcus iniae causes disease in fish and humans and the presence of capsule is associated with virulence. Tn917 transposon mutagenesis was performed to identify capsule-associated genes and a mutant was isolated, with an insertion in a genetic locus encoding a two-component signal transduction system (TCS), which we termed sivS/R. sivS and sivR encode a 506-amino-acid (aa) putative histidine kinase and a 223-aa putative response regulator, respectively. In order to investigate the role of sivS/R, a deletion-insertion mutant was constructed using a PCR ligation technique. Real-time PCR showed that transcription of cpsA, the first gene in the S. iniae capsule operon, was reduced in the mutant, indicating that sivS/R regulates expression of this gene at the transcriptional level. Whole human blood killing assays demonstrated that unlike the parent, the mutant was susceptible to phagocytosis. Transmission electron microscopy showed exopolysaccharide on the surface of the parent strain but not the mutant which showed aberrant asymmetric septae that resulted in clumps of abnormal-shaped cells. Exponential growth rates of the mutant and parent strain were similar, although the mutant exhibited a longer lag phase. We conclude that sivS/R regulates capsule expression, thus affecting the ability to evade phagocytosis.

Recently, we showed that the in vitro lag phase correlates with the pneumococcal serotype. This study investigated the role of capsule genes in bacterial growth using strain D39. Deletion of the entire capsule operon induced a significantly prolonged lag phase in Todd Hewitt broth (P=0.0002). However, partial deletions showed a different influence on the lag phase. Supplementation of media with 5% fetal bovine serum restored normal growth, at least partially, in mutants with a prolonged lag phase. Therefore, pneumococcal capsule gene products influence bacterial growth in vitro in strain D39.

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.

Campylobacter jejuni is a common cause of bacterial enteritis. The surface capsular polysaccharides are important for this bacterium to survive in the environment, but little is known about their involvement in bacterium-host interactions. This study showed that the C. jejuni capsular polysaccharides play an important role in adherence to and invasion of human embryonic epithelial cells. However, no significant role of capsular polysaccharides was shown in colonization of the chicken gut.

Bacterially induced carbonate mineralization has been proposed as a new method for the restoration of limestones in historic buildings and monuments. We describe here the formation of calcite crystals by extracellular polymeric substances isolated from Bacillus firmus and Bacillus sphaericus. We isolated bacterial outer structures (glycocalix and parietal polymers), such as exopolysaccharides (EPS) and capsular polysaccharides (CPS) and checked for their influence on calcite precipitation. CPS and EPS extracted from both B. firmus and B. sphaericus were able to mediate CaCO3 precipitation in vitro. X-ray microanalysis showed that in all cases the formed crystals were calcite. Scanning electron microscopy showed that the shape of the crystals depended on the fractions utilized. These results suggest the possibility that biochemical composition of CPS or EPS influences the resulting morphology of CaCO3. There were no precipitates in the blank samples. CPS and EPS comprised of proteins and glycoproteins. Positive alcian blue staining also reveals acidic polysaccharides in CPS and EPS fractions. Proteins with molecular masses of 25-40 kDa and 70 kDa in the CPS fraction were highly expressed in the presence of calcium oxalate. This high level of synthesis could be related to the binding of calcium ions and carbonate deposition.

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.

SummaryA number of studies have shown that the ratio of IgA1 and IgA2 subclasses in secretions can depend upon the nature of the antigen inducing their production. In order to evaluate the effect of the nature of the antigen on the subclass distribution of the naturally occurring salivary IgA antibodies against Streptococcus pneumoniae, we used enzyme immunoassay to measure the levels of natural IgA, IgA1 and IgA2 antibodies to pneumococcal capsular polysaccharide type 14 (PS14) and pneumococcal surface adhesin A (PsaA) in saliva of children during their first 2 years of life. The sum of anti-PS14 and anti-PsaA IgA1 and IgA2 correlated significantly with the antigen-specific total IgA, which showed that IgA1 and IgA2 add up to IgA. IgA1 was the predominant subclass for both antigens. The median of anti-PS14 and anti-PsaA IgA1 was higher than that of IgA2, and the antigen-specific IgA1 was found in a larger proportion of samples than IgA2. The ratio of IgA1 to IgA2 (IgA1/IgA2 ratio) was lower for anti-PS14 than for anti-PsaA, suggesting that the PS antigen induced more IgA2 than the protein antigen. The possible impact of the IgA subclass distribution on protection of mucosal surfaces by natural or vaccine-induced antibodies needs to be determined.

The polysaccharide capsule of Streptococcus pneumoniae is the main virulence factor of this microorganism. Although the study of the genes responsible for the synthesis of the pneumococcal capsule enabled genetic and molecular analysis, the precise structure, organization, and functioning of these genes have only been investigated very recently. The genes implicated in the production of the type 3 capsule have been sequenced, expressed and their corresponding products biochemically characterized. In addition, partial information on the genes responsible for the biosynthesis of the capsules of pneumococcal types 1, 14 or 19 F is currently available.

KpsM is an integral membrane protein involved in the translocation of the polysialic acid capsule of Escherichia coli K1. The kpsMG93E allele is a point mutation in the first cytoplasmic loop (Cl) of KpsM which partially disrupts translocation of the capsule. While producing polymer of wild-type length, strains harboring the G93E allele exhibit a decreased production of capsular polymer and a reduced rate of polymer translocation to the cell surface.

Fully virulent Bacillus anthracis bacilli are encapsulated and toxinogenic. These bacteria carry two plasmids, pXO1, and pXO2, encoding toxins and capsule synthetic-enzymes (capB, C, A, dep), respectively. The PXO1 plasmid strongly enhances capsule formation. This influence was studied by analysing the expression of a capB-lacZ fusion in various backgrounds. The beta-galactosidase activities were similar in a delta atxA strain and a pXO1 cured strain. Moreover, the capB-lacZ expression level could be restored, in a pXO1 cured strain, by addition of atxA in trans. Thus, we conclude that the pX01 influence on capsule synthesis is mediated by AtxA, the pXO1-encoded trans-activator of the toxin gene expression.

Several septicemic Escherichia coli O78 strains, isolated from different sources, were characterized phenotypically and genotypically. Two avian isolates, one of which is known to carry the AC/I fimbriae, hybridized with the sfa determinant in colony dot-blot assay. Southern hybridizations with specific sfa probes, following pulsed-field gel electrophoresis (PFGE), showed positive hybridization to the same fragment in each of these strains. Determination of the N-terminal amino acid sequence of the AC/I major subunit gene revealed high similarity to the sequence of the SfaA-II protein. These data suggest that the adhesin gene cluster, coding for AC/I fimbriae, belongs to the S-fimbrial adhesin family.

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.

Cryptococcus neoformans has been divided into five serotypes (A, B, C, D and AD) by the slide agglutination test against antigens of the polysaccharide capsule component. The isolates of serotype AD show positive reactions with both serotype A and D antigens. In this study, the nucleotide sequences of the capsule-associated genes CAP10, CAP59, CAP60 and CAP64 from five serotypes of C. neoformans were analyzed for their phylogenetic relationships, with special regard to serotype AD. The nucleotide sequence analyses showed that serotype AD had two different sequences in these four genes. Phylogenetic analysis revealed that these genes from serotype AD are included in the two clusters of serotypes A and D, but not in serotypes B or C. Southern blot analysis of genomic DNAs of serotypes A, D and AD digested with BamH I by hybridizing with a CAP64 probe indicated that both bands of fragments detected in serotypes A and D were also detected in serotype AD. These results confirm that serotype AD could be a mixture of serotypes A and D in the four CAP genes, consistent with its putative origin detected by the hybridization of these two serotypes. The present results indicated that serotypes of C. neoformans could be identified by the phylogenetic analyses of CAP genes.

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.

This study investigated whether cell size correlates with phenotype and proliferative capacity in human corneal epithelial cells. Primary cultured human corneal epithelial cells were sorted by flow cytometry based on forward scatter profile in comparison with the profile of beads of known size. Four fractions (A, B, C, and D) of cells ranging in size from 10 to 16, 17 to 23, 24 to 30, and ≥31 μm in diameter, respectively, were collected to evaluate their 5-bromo-2-deoxyuridine (BrdU) label retention properties, cell phenotype, and clonal growth capacity on a 3T3 fibroblast feeder layer. Among these four populations, cell size was shown to positively correlate with the expression of the differentiation markers keratin (K) 3, K12, and involucrin and inversely with the levels of stem cell–associated markers ΔNp63 and ABCG2 and with colony-forming efficiency (CFE) and growth capacity. Population A with the smallest size, accounting for 11.0% ± 4.5% of the entire population, contained the greatest number of BrdU label-retaining slow-cycling cells, displayed the highest percentage of cells immunopositive to p63 and ABCG2 and negative to K3 and involucrin, expressed the highest levels of ΔNp63 and ABCG2 mRNA and the lowest levels of K3, K12, and involucrin, and possessed the highest CFE and growth capacity. These findings suggest that cell size correlates with cell differentiation phenotypes and proliferative capacity in human corneal epithelial cells. The smallest cells in population A seem to be enriched for putative stem cells, and small cell size may represent one of the important properties of adult corneal epithelial stem cells.

SummaryCapsular polysaccharide from Actinobacillus actinomycetemcomitans Y4 (Y4 CP) induces bone resorption in a mouse organ culture system and osteoclast formation in mouse bone marrow cultures, as reported in previous studies. We also found that Y4 CP inhibits the release of interleukin (IL)-6 and IL-8 from human gingival fibroblast (HGF). Thus Y4 CP induces various responses in localized tissue and leads to the secretion of several cytokines. However, the effects of Y4 CP on human monocytes/macrophages are still unclear. In this study, THP-1 cells, which are a human monocytic cell line, were stimulated with Y4 CP, and we measured gene expression in inflammatory cytokine and signal transduction pathways. IL-1β and tumour necrosis factor (TNF)-α mRNA were induced from Y4 CP-treated THP-1 cells. IL-1β mRNA expression was increased according to the dose of Y4 CP, and in a time-dependent manner. IL-1β mRNA expression induced by Y4 CP (100 µg/ml) was approximately 7- to 10-fold greater than that in the control by real-time PCR analysis. Furthermore, neither PD98059, a specific inhibitor of extracellular signal-regulated kinase nor SB203580, a specific inhibitor of p38 kinase prevented the IL-1β expression induced by Y4 CP. However, JNK Inhibitor II, a specific inhibitor of c-Jun N-terminal kinase (JNK) prevented the IL-1β mRNA expression induced by Y4 CP in a concentration-dependent manner. These results indicate that Y4 CP-mediated JNK pathways play an important role in the regulation of IL-1β mRNA. Therefore, Y4 CP-transduced signals for IL-1β induction in the antibacterial action of macrophages may provide a therapeutic strategy for periodontitis.

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.