Bacterial Polysaccharides Research Articles

Preclinical development of the quadrivalent meningococcal (ACYW) tetanus toxoid conjugate vaccine, MenQuadfi®

Bacterial capsular polysaccharide vaccines are generally poorly immunogenic in infants and older adults. The immunogenicity of capsular polysaccharide vaccines can be improved by conjugating them to immunogenic carrier proteins. One of the most recently licensed conjugate vaccines is the quadrivalent meningococcal vaccine with serogroups A, C, Y, and W conjugated to a tetanus toxoid protein carrier (MenACYW-TT; MenQuadfi, Sanofi Pasteur, Swiftwater, PA, USA). MenACYW-TT was developed to induce optimal immune responses against each of the meningococcal serogroups A, C, W, and Y, and across all age groups, especially infants and older adults (those aged ≥ 50 years). Here, we detail the early iterative vaccine development approach taken, whereby many different ‘small-scale’ conjugate vaccine candidates were prepared and examined for immunogenicity in a mouse model to identify the most immunogenic vaccine. Additional insights from phase I clinical studies informed further optimization of the vaccine candidates by tailoring their conjugation parameter attributes for the optimal immune response in humans. The parameters studied included: different carrier proteins [PR]; polysaccharide [PS] sizes; conjugation chemistries [linker vs. no-linker; lattice vs. neoglycoprotein; activation/derivatization levels]; conjugate size; PS:PR loading ratio; percent free PS; percent free PR; and O-acetylation content. The lead quadrivalent conjugate vaccine (polysaccharides of > 50 kDa size conjugated to TT at a high PS:PR ratio via reductive amination for serogroups C, W and Y, and carbonyldiimidazole/adipic acid dihydrazide linker chemistry for serogroup A) empirically identified from the extensive preclinical studies, was ultimately confirmed by the robust antibody responses observed in all age groups in the various clinical studies, including in the most challenging infant and older adult age groups, and subsequently led to the licensed formulation.Graphical abstract

Strong seasonal differences of bacterial polysaccharide utilization in the North Sea over an annual cycle

Marine heterotrophic bacteria contribute considerably to global carbon cycling, in part by utilizing phytoplankton-derived polysaccharides. The patterns and rates of two different polysaccharide utilization modes - extracellular hydrolysis and selfish uptake - have previously been found to change during spring phytoplankton bloom events. Here we investigated seasonal changes in bacterial utilization of three polysaccharides, laminarin, xylan and chondroitin sulfate. Strong seasonal differences were apparent in mode and speed of polysaccharide utilization, as well as in bacterial community compositions. Compared to the winter month of February, during the spring bloom in May, polysaccharide utilization was detected earlier in the incubations and a higher portion of all bacteria took up laminarin selfishly. Highest polysaccharide utilization was measured in June and September, mediated by bacterial communities that were significantly different from spring assemblages. Extensive selfish laminarin uptake, for example, was detectible within a few hours in June, while extracellular hydrolysis of chondroitin was dominant in September. In addition to the well-known Bacteroidota and Gammaproteobacteria clades, the numerically minor verrucomicrobial clade Pedosphaeraceae could be identified as a rapid laminarin utilizer. In summary, polysaccharide utilization proved highly variable over the seasons, both in mode and speed, and also by the bacterial clades involved.

The biosynthetic origin of ribofuranose in bacterial polysaccharides.

Bacterial surface polysaccharides are assembled by glycosyltransferase enzymes that typically use sugar nucleotide or polyprenyl-monophosphosugar activated donors. Characterized representatives exist for many monosaccharides but neither the donor nor the corresponding glycosyltransferases have been definitively identified for ribofuranose residues found in some polysaccharides. Klebsiella pneumoniae O-antigen polysaccharides provided prototypes to identify dual-domain ribofuranosyltransferase proteins catalyzing a two-step reaction sequence. Phosphoribosyl-5-phospho-D-ribosyl-α-1-diphosphate serves as the donor for a glycan acceptor-specific phosphoribosyl transferase (gPRT), and a more promiscuous phosphoribosyl-phosphatase (PRP) then removes the residual 5'-phosphate. The 2.5-Å resolution crystal structure of a dual-domain ribofuranosyltransferase ortholog from Thermobacillus composti revealed a PRP domain that conserves many features of the phosphatase members of the haloacid dehalogenase family, and a gPRT domain that diverges substantially from all previously characterized phosphoribosyl transferases. The gPRT represents a new glycosyltransferase fold conserved in the most abundant ribofuranosyltransferase family.

Rheological Response of Polylactic Acid Dispersions in Water with Xanthan Gum.

In this work, the rheological behavior of stable poly(lactic acid) (PLA) dispersions in water, intended for coating applications, was investigated. The newly prepared dispersion consists of PLA particles with an average diameter of 222 ± 2 nm based on dynamic light scattering (DLS) and scanning electron microscopy (SEM) analyses, at concentrations varying in the 5–22 wt % range. Xanthan gum (XG), a bacterial polysaccharide, was used as a thickening agent to modulate the viscosity of the formulations. The rheological properties of the PLA dispersions with different XG and PLA contents were studied in steady shear, amplitude sweep, and frequency sweep experiments. Under steady shear conditions, the viscosity of all the formulations showed a shear-thinning behavior similar to XG solutions in the whole investigated 1–1000 s–1 range, with values dependent on both PLA particles and XG concentrations. Amplitude and frequency sweep data revealed a weak-gel behavior except in the case of the most diluted sample, with moduli dependent on both PLA and XG contents. A unified scaling parameter was identified in the volume fraction (ϕ) of the PLA particles, calculated by considering the dependence of the continuous phase density on the XG concentration. Accordingly, a master curve at different volume fractions was built using the time–concentration–superposition approach. The master curve describes the rheological response of the system over a wider frequency window than the experimentally accessible one and reveals the presence of a superimposed β relaxation process in the high-frequency region.

Conjugate of structurally reassigned pneumococcal serotype 31 polysaccharide with CRM197 elicited potent immune response

Around 100 Streptococcus pneumonia (Spn) serotypes have been discovered, 90% of the severe diseases in children are caused by 13 serotypes. With the success of pneumococcal bacterial polysaccharide conjugate vaccines (PCVs), the burden of pneumococcal disease has been significantly reduced. Serotype 31 is a non-vaccine serotype and has increased in prevalence. By using Nuclear Magnetic Resonance (NMR) as the primary tool, we report the revised serotype 31 polysaccharide (s-31-ps) structure as [→3)-β-D-Galf-(5/6-OAc)-(1 → 3)-β-D-Galp-(1 → 3)-β-L-Rhap-(2-OAc)-(1 → 2)-α-L-Rhap-(1 → 4)-β-D-GlcpA-(1→]n. Furthermore, the reductive amination-conjugate of serotype 31 polysaccharide and cross reacting material (CRM197) protein was prepared in organic solvent (N,N-dimethylformamide, DMF) instead of water. The reaction is faster, and the DMF conjugate elicited comparable immune responses with the aqueous conjugate. S-31-ps conjugate vaccine has the potential of being included in the next-generation PCV vaccines.

Interdependence of Shigella flexneri O Antigen and Enterobacterial Common Antigen Biosynthetic Pathways.

Outer membrane (OM) polysaccharides allow bacteria to resist harsh environmental conditions and antimicrobial agents, traffic to and persist in pathogenic niches, and evade immune responses. Shigella flexneri has two OM polysaccharide populations, being enterobacterial common antigen (ECA) and lipopolysaccharide (LPS) O antigen (Oag); both are polymerized into chains by separate homologs of the Wzy-dependent pathway. The two polysaccharide pathways, along with peptidoglycan (PG) biosynthesis, compete for the universal biosynthetic membrane anchor, undecaprenyl phosphate (Und-P), as the finite pool of available Und-P is critical in all three cell wall biosynthetic pathways. Interactions between the two OM polysaccharide pathways have been proposed in the past where, through the use of mutants in both pathways, various perturbations have been observed. Here, we show for the first time that mutations in one of the two OM polysaccharide pathways can affect each other, dependent on where the mutation lies along the pathway, while the second pathway remains genetically intact. We then expand on this and show that the mutations also affect PG biosynthesis pathways and provide data which supports that the classical mutant phenotypes of cell wall mutants are due to a lack of available Und-P. Our work here provides another layer in understanding the complex intricacies of the cell wall biosynthetic pathways and demonstrates their interdependence on Und-P, the universal biosynthetic membrane anchor. IMPORTANCE Bacterial outer membrane polysaccharides play key roles in a range of bacterial activities from homeostasis to virulence. Two such OM polysaccharide populations are ECA and LPS Oag, which are synthesized by separate homologs of the Wzy-dependent pathway. Both ECA and LPS Oag biosynthesis join with PG biosynthesis to form the cell wall biosynthetic pathways, which all are interdependent on the availability of Und-P for proper function. Our data show the direct effects of cell wall pathway mutations affecting all related pathways when they themselves remain genetically unchanged. This work furthers our understanding of the complexities and interdependence of the three cell wall pathways.

Four temporin-derived peptides exhibit antimicrobial and antibiofilm activities against methicillin-resistant.

Temporin-GHa (GHa) was cloned from , showing a weak antimicrobial activity. In order to improve its bactericidal efficacy, GHaR6R, GHaR7R, GHaR8R and GHaR9W were designed and synthesized. Compared to the parent peptide, the GHa-derived peptides show potent antimicrobial activities against methicillin-resistant (MRSA), which is the main pathogen with high morbidity and mortality that causes various infections in humans. These peptides exert bactericidal actions on MRSA by permeabilizing the cytoplasmic membranes and damaging membrane integrity. All of the four peptides exhibit excellent stability under harsh conditions, including extreme temperature and salts. Furthermore, they inhibit the formation of biofilm and eradicate mature biofilm of MRSA. The GHa-derived peptides decrease bacterial surface hydrophobicity, autoaggregation and polysaccharide intercellular adhesion synthesis in concentration-dependent manner. Real-time quantitative reverse transcription PCR analysis revealed that the peptides downregulate the expression of adhesion genes involved in biofilm formation. Except for GHaR7R, the other three peptides have low hemolytic toxicity against human erythrocytes. In the presence of human erythrocytes, GHaR7R, GHaR8R and GHaR9W interact with MRSA preferentially. GHaR6R, GHaR8R and GHaR9W show less toxicity toward normal cells HL-7702 and hFOB1.19. These results suggest that the GHa-derived peptides may be promising antimicrobial candidates against MRSA infections.

NoteIdentification of 5,7-diacetamido-3,5,7,9-tetradeoxy-d-glycero-l-manno-non-2-ulosonic acid (di-N-acetyl-8-epipseudaminic acid) in the capsular polysaccharide of Acinetobacter baumannii Res546

A structurally diverse capsular polysaccharide that surrounds the bacterial cell plays an important role in virulence of Acinetobacter baumannii, a cause of nosocomial infections worldwide. Various isomers of 5,7-diacylamido-3,5,7,9-tetradeoxynon-2-ulosonic acid have been identified as components of bacterial polysaccharides. In this work, we report on the identification of a new isomer having the d-glycero-l-manno configuration (8-epipseudaminic acid) in the capsular polysaccharide of A. baumannii Res546. The higher sugar was isolated by Smith degradation of the polysaccharide followed by mild acid hydrolysis and identified by a comparison with all isomers using NMR spectroscopy and optical rotation.

Effects of bacterial exopolysaccharide on rheological properties of acid milk curd and its correlates

Exopolysaccharide (EPS) is a general term for the forms of bacterial polysaccharides found outside the cell wall of bacteria. The industrial microbiologist has become interested in exploiting microbial exopolymer production due to an increasing interest and need for novel polysaccharides. The use of bacterial EPS in acid milk curd (AMC) production accounts for a quality improvement of the milk curd in terms of stability during the normal operations of storage and transportation. On the other hand, EPS have been used as prebiotic because of its health-promoting effects in human beings. The objective of this study was to evaluate the rheological properties (RP) and their statistical correlations in acid milk curd (AMC) samples added with bacterial EPS. Rheometric properties are suitable parameters to evaluate food quality such as AMC. Some of the RP normally used in this evaluation include elastic modulus (EM), hardness (HR), breaking energy (BE), viscosity (VIS) and stress. Correlation between pairs of these parameters are important to understand in order to predict statistically other parameters in case they cannot be easily determined. Acid milk curd samples were formulated using 10, 15, and 20 % of reconstituted skim milk (RSM), to which 0, 0.2, 0.4, 0.6, 0.8, and 1 % level of EPS were added, respectively. Samples were pasteurized at 60 o C for 30 min, and at 85 oC for 30s. Afterwards, AMC samples were tempered to 40 °C inoculated with Lactobacillus delbrueckii subsp. bulgaricus B-5b, and incubated at 37 °C for 16 h. Results indicated that viscosity and hardness showed the strongest and highest correlation among all the parameters evaluated (r=0.9272) and it was comparable to that between viscosity and breaking energy (r=0.8349) and breaking energy and hardness (r=0.7694). However, elastic modulus and viscosity showed a rather low correlation (r=0.5394) and very low correlation (r=0.1830), respectively, suggesting that estimation of elastic modulus from viscosity values would be inaccurate. In general, rheometric properties of acid milk curd increased with higher EPS concentrations until 1 % level of EPS addition. Therefore, it is recommended to use BE and HR values, and BE and VIS values to predict accurate measurements of other RP values. Key words: Exopolysaccharide (EPS), acid milk curd (AMC), rheometric properties (RP), statistical correlation (SC)

Virus-like Particle Display of Vibrio choleraeO-Specific Polysaccharide as a Potential Vaccine against Cholera.

Vibrio cholerae, a noninvasive mucosal pathogen, is endemic in more than 50 countries. Oral cholera vaccines, based on killed whole-cell strains of Vibrio cholerae, can provide significant protection in adults and children for 2-5 years. However, they have relatively limited direct protection in young children. To overcome current challenges, in this study, a potential conjugate vaccine was developed by linking O-specific polysaccharide (OSP) antigen purified from V.cholerae O1 El Tor Inaba strain PIC018 with Qβ virus-like particles efficiently via squarate chemistry. The Qβ-OSP conjugate was characterized with mass photometry (MP) on the whole particle level. Pertinent immunologic display of OSP was confirmed by immunoreactivity of the conjugate with convalescent phase samples from humans with cholera. Mouse immunization with the Qβ-OSP conjugate showed that the construct generated prominent and long-lasting IgG antibody responses against OSP, and the resulting antibodies could recognize the native lipopolysaccharide from Vibrio cholerae O1 Inaba. This was the first time that Qβ was conjugated with a bacterial polysaccharide for vaccine development, broadening the scope of this powerful carrier.

Diverse Mechanisms of Protective Anti-Pneumococcal Antibodies.

The gram-positive bacterium Streptococcus pneumoniae is a leading cause of pneumonia, otitis media, septicemia, and meningitis in children and adults. Current prevention and treatment efforts are primarily pneumococcal conjugate vaccines that target the bacterial capsule polysaccharide, as well as antibiotics for pathogen clearance. While these methods have been enormously effective at disease prevention and treatment, there has been an emergence of non-vaccine serotypes, termed serotype replacement, and increasing antibiotic resistance among these serotypes. To combat S. pneumoniae, the immune system must deploy an arsenal of antimicrobial functions. However, S. pneumoniae has evolved a repertoire of evasion techniques and is able to modulate the host immune system. Antibodies are a key component of pneumococcal immunity, targeting both the capsule polysaccharide and protein antigens on the surface of the bacterium. These antibodies have been shown to play a variety of roles including increasing opsonophagocytic activity, enzymatic and toxin neutralization, reducing bacterial adherence, and altering bacterial gene expression. In this review, we describe targets of anti-pneumococcal antibodies and describe antibody functions and effectiveness against S. pneumoniae.

Partial depolymerization of capsular polysaccharides isolated from Streptococcus pneumoniae serotype 2 by various methods

Partial depolymerization of bacterial capsular polysaccharides (CPS) is an essential process carried out before its use as an antigenic preparation in a vaccine industry. Choice of CPS depolymerization methods depends on the process robustness, reproducibility, yield, retention of CPS bioactivity, etc. Partial depolymerization methods based on chemicals, enzymes, mechanical, thermal, etc. have been subject of many investigations before. Partial depolymerization of Streptococcus pneumoniae serotype 2 purified CPS was conducted by methods such as acid hydrolysis, microfluidization, ultrasonication, thermal and microwave. Partial depolymerization of the CPS was evaluated by size exclusion high performance liquid chromatography, whereas structural identity and conformity of CPS was ensured by 1H NMR spectroscopy. The antigenicity of CPS was assessed by bead based competitive inhibition assay. Microwave and thermal methods effectively depolymerized CPS and reduced the concentration of cell wall polysaccharide (CWPS) impurity, but both methods have a negative impact on the antigenicity of CPS. Whereas the trifluoroacetic acid treatment not only depolymerized the CPS but completely removed the CWPS while retaining the antigenicity of 92 ± 4% and this method is advantageous over other methods.

Extraction of the Bacterial Extracellular Polysaccharide FucoPol by Membrane-Based Methods: Efficiency and Impact on Biopolymer Properties.

In this study, membrane-based methods were evaluated for the recovery of FucoPol, the fucose-rich exopolysaccharide (EPS) secreted by the bacterium Enterobacter A47, aiming at reducing the total water consumption and extraction time, while keeping a high product recovery, thus making the downstream procedure more sustainable and cost-effective. The optimized method involved ultrafiltration of the cell-free supernatant using a 30 kDa molecular weight cut-off (MWCO) membrane that allowed for a 37% reduction of the total water consumption and a 55% reduction of the extraction time, compared to the previously used method (diafiltration-ultrafiltration with a 100 kDa MWCO membrane). This change in the downstream procedure improved the product’s recovery (around 10% increase) and its purity, evidenced by the lower protein (8.2 wt%) and inorganic salts (4.0 wt%) contents of the samples (compared to 9.3 and 8.6 wt%, respectively, for the previously used method), without impacting FucoPol’s sugar and acyl groups composition, molecular mass distribution or thermal degradation profile. The biopolymer’s emulsion-forming and stabilizing capacity was also not affected (emulsification activity (EA) with olive oil, at a 2:3 ratio, of 98 ± 0% for all samples), while the rheological properties were improved (the zero-shear viscosity increased from 8.89 ± 0.62 Pa·s to 17.40 ± 0.04 Pa·s), which can be assigned to the higher purity degree of the extracted samples. These findings demonstrate a significant improvement in the downstream procedure raising FucoPol’s recovery, while reducing water consumption and operation time, key criteria in terms of process economic and environmental sustainability. Moreover, those changes improved the biopolymer’s rheological properties, known to significantly impact FucoPol’s utilization in cosmetic, pharmaceutical or food products.

Nano-SiO2 inhibits the marine aquatic pathogen Vibrio parahaemolyticus

In this study, we investigated the effects of Nano-SiO2, ultraviolet (UV) irradiation, and hydrogen peroxide on the growth of four marine aquatic pathogens as well as on their polysaccharide, biofilm formation, protein, and DNA. The results indicated that among all concentrations, 100 μg/L Nano-SiO2 demonstrated the most inhibition of Edwardsiella tarda, Vibrio anguillarum, and Vibrio parahaemolyticus. Ultraviolet irradiation for 3 min had the most obvious inhibitory effect on E. tarda, but neither inhibitory nor promoting effects on the other three strains. High hydrogen peroxide concentrations had obvious inhibitory effects on all bacterial growth. Moreover, Nano-SiO2 had some influence on bacterial polysaccharide properties and some amount of DNA splicing effect, but no effect was noted on proteins. At present, the recognized pathogenic genes of V. parahaemolyticus include tdh, trh, and tlh. We thus studied the effect of Nano-SiO2 on these genes and then conducted infection experiments. The results showed that Nano-SiO2 further reduced infection with V. parahaemolyticus in shrimp.

T-independent antigen induces humoral memory through germinal centers.

T-dependent humoral responses generate long-lived memory B cells and plasma cells (PCs) predominantly through germinal center (GC) reaction. In human and mouse, memory B cells and long-lived PCs are also generated during immune responses to T-independent antigen, including bacterial polysaccharides, although the underlying mechanism for such T-independent humoral memory is not clear. While T-independent antigen can induce GCs, they are transient and thought to be nonproductive. Unexpectedly, by genetic fate-mapping, we find that these GCs actually output memory B cells and PCs. Using a conditional BCL6 deletion approach, we show memory B cells and PCs fail to last when T-independent GCs are precluded, suggesting that the GC experience per se is important for programming longevity of T-independent memory B cells and PCs. Consistent with the fact that infants cannot mount long-lived humoral memory to T-independent antigen, B cells from young animals intrinsically fail to form T-independent GCs. Our results suggest that T-independent GCs support humoral memory, and GC induction may be key to effective vaccines with T-independent antigen.

Bacterial Succinoglycans: Structure, Physical Properties, and Applications.

Succinoglycan is a type of bacterial anionic exopolysaccharide produced from Rhizobium, Agrobacterium, and other soil bacteria. The exact structure of succinoglycan depends in part on the type of bacterial strain, and the final production yield also depends on the medium composition, culture conditions, and genotype of each strain. Various bacterial polysaccharides, such as cellulose, xanthan, gellan, and pullulan, that can be mass-produced for biotechnology are being actively studied. However, in the case of succinoglycan, a bacterial polysaccharide, relatively few reports on production strains or chemical and structural characteristics have been published. Physical properties of succinoglycan, a non-Newtonian and shear thinning fluid, have been reported according to the ratio of substituents (pyruvyl, succinyl, acetyl group), molecular weight (Mw), and measurement conditions (concentration, temperature, pH, metal ion, etc.). Due to its unique rheological properties, succinoglycan has been mainly used as a thickener and emulsifier in the cosmetic and food industries. However, in recent reports, succinoglycan and its derivatives have been used as functional biomaterials, e.g., in stimuli-responsive drug delivery systems, therapeutics, and cell culture scaffolds. This suggests a new and expanded application of succinoglycan as promising biomaterials in biomedical fields, such as tissue engineering, regenerative medicine, and pharmaceuticals using drug delivery.

Small-Molecule Compound SYG-180-2-2 to Effectively Prevent the Biofilm Formation of Methicillin-Resistant Staphylococcus aureus.

The resistance of methicillin-resistant Staphylococcus aureus (MRSA) has augmented due to the abuse of antibiotics, bringing about difficulties in the treatment of infection especially with the formation of biofilm. Thus, it is essential to develop antimicrobials. Here we synthesized a novel small-molecule compound, which we termed SYG-180-2-2 (C21H16N2OSe), that had antibiofilm activity. The aim of this study was to demonstrate the antibiofilm effect of SYG-180-2-2 against clinical MRSA isolates at a subinhibitory concentration (4 μg/ml). In this study, it was showed that significant suppression in biofilm formation occurred with SYG-180-2-2 treatment, the inhibition ranged between 65.0 and 85.2%. Subsequently, confocal laser scanning microscopy and a bacterial biofilm metabolism activity assay further demonstrated that SYG-180-2-2 could suppress biofilm. Additionally, SYG-180-2-2 reduced bacterial adhesion and polysaccharide intercellular adhesin (PIA) production. It was found that the expression of icaA and other biofilm-related genes were downregulated as evaluated by RT-qPCR. At the same time, icaR and codY were upregulated when biofilms were treated with SYG-180-2-2. Based on the above results, we speculate that SYG-180-2-2 inhibits the formation of biofilm by affecting cell adhesion and the expression of genes related to PIA production. Above all, SYG-180-2-2 had no toxic effects on human normal alveolar epithelial cells BEAS-2B. Collectively, the small-molecule compound SYG-180-2-2 is a safe and effective antibacterial agent for inhibiting MRSA biofilm.

Injectable Biological Bulking Agent to Treat Stress Urinary Incontinence in Women: A Feasibility Pilot Study

Background: Urinary incontinence mainly affects women regardless of age and, as it affects their quality of life, influences work, sex life and independence for activities of daily living. The treatment of stress urinary incontinence including urethral volume injection therapy can provide an intermediate option over non-surgical and surgical therapies. One of the mechanisms for stress continence depends on the effective coaptation of the urethra during the increase in intra-abdominal pressure. The bulking agents can be injected transurethral or periurethral retrogradely, using direct vision from a cystoscope. Purpose: To evaluate the feasibility and preliminary outcome performance of the bacterial polysaccharide gel used as biological bulking agent applied in female patients with stress urinary incontinence. Methods: A prospective clinical pilot study was performed, in a single institution, including female patients who were admitted to the urologic outpatient clinic with Stress Urinary Incontinence (SUI) without previous treatments and they were selected and underwent bulking agent procedure. The evaluation was performed at the time of enrollment and 6 months after treatment. The primary outcome was Quality of Life (QOL) using the ICIQ-SF Questionnaire. The amount of urine leakage measured by the 1-HOUR PAD-TEST was the second outcome. Results: Fifteen women (with an average age of 53 years) were submitted to the application of bacterial cellulose gel and she was analyzed. Only two patients presented unchanged incontinence. The study considered as primary outcome the improvement or disappearance of symptoms after six months of intervention. Post-intervention Quality of Life (QOL) questionnaire indicated that all of these patients related a better quality of life (62.5%). Through the PAD-test it was possible to observe a decrease in urinary leak of 85% comparing the results pre and post-intervention (BCA—Bacterial Cellulose Application) with p-value equal to 0.000009. Conclusions: The results of this pilot study suggest that the use of biological bulking agent is a promising approach to treat stress urinary incontinence in female patients. Trial registration: Registration number and date of registration should be instated in this section.

Prevalence of Streptococcus agalactiae capsular types among pregnant women in Rio de Janeiro and the impact of a capsular based vaccine

Streptococcus agalactiae (group B Streptococcus, GBS) remains one major neonatal pathogen, being maternal colonization a risk factor for infection development. Despite effective, the usage of antibiotics to prevent neonatal infections has limitations. The bacterial polysaccharide capsule is a virulence determinant, a target for vaccine directed to pregnant women, and also the most useful epidemiological marker of GBS infections. Capsular polysaccharides are diverse and disease severity varies according to the expressed type. Here, capsular typing of 124 GBS isolates recovered from pregnant women was determined by a multiplex PCR-based method. The most frequent types were Ia (33.0%), II (25.8%) and V (21.8%). Other types found were Ib (8.9%), III (8.9%) and IV (1.6%). While type Ia was prevalent during the whole period (2002-2018), fluctuations in distribution of other types, specially V, were observed over time. Capsular type III, traditionally associated with severe neonatal infections, was poorly detected. Distribution of maternal GBS capsular types in the metropolitan area of Rio de Janeiro, with prevalence of Ia and II, is quite different from other parts of the world. The knowledge about GBS capsular type distribution is essential to predict the theoretical impact of developing capsule-based vaccines in the local population.

Simplified Enzymatic Synthesis of 2-Keto-3-Deoxy-D-Gluconate from D-Gluconate Using the Gluconate Dehydratase from Thermoproteus tenax.

Many research areas, e.g., basic research but also applied fields of biotechnology, biomedicine, and diagnostics often suffer from the unavailability of metabolic compounds. This is mostly due to missing easy and efficient synthesis procedures. We herein describe the biocatalytic/enzymatic production of 2-keto-3-deoxy-D-gluconate, an intermediate of central metabolic pathways in all three domains of life and also of bacterial polysaccharides, lipopolysaccharides, and cell wall components. The method is based on the gluconate dehydratase from the hyperthermophilic crenarchaeon Thermoproteus tenax, which can be easily recombinantly overproduced in Escherichia coli and-due to its intrinsic thermostability-rapidly be purified by two precipitation steps. The enzyme completely converts D-gluconate to solely stereochemically pure KDG, taking benefits from the enol-keto-tautomerism of the primary reaction product. The final product can then easily be separated from the protein by ultrafiltration. The simple one-step procedure, which is suitable at least for the lab-scale/gram-scale production of KDG, replaces lengthy multi-step reactions and is easily scalable. This approach also illustrates the great application potential of Archaea with their unusual metabolic pathways and enzymes for the synthesis of added value products.