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In children, the incidence of pneumococcal meningitis has decreased since the introduction of pneumococcal conjugate vaccine (PCV7 and PCV13). However, since the introduction of the vaccine, developed countries have seen the emergence of non-PCV13 serotypes. However, invasive pneumococcal disease (IPD) caused by PCV13-targeted serotypes still represents an important public health problem in resource-limited countries. To develop a rapid, simple, and cost-effective assay to detect serotypes of Streptococcus pneumoniae, we developed a novel loop-mediated isothermal amplification (LAMP) assay based on the sequences available for the 13 capsular types that are included in PCV13: 1, 3, 4, 5, 6 A, 6B, 7 F, 9 V, 14, 18 C, 19 A, 19 F, and 23 F. We evaluated test reactivity, specificity, sensitivity and performance, and compared the results between established LAMP and conventional PCR assays. To support its clinical use, the detection limits of the LAMP assay were evaluated using bacterial genomic DNA-spiked cerebrospinal fluid (CSF) and blood specimens. We confirmed the specificity of the LAMP assay using 41 serotypes of pneumococcal strains. The sensitivity of the LAMP assay was 10 to 100 copies per reaction, compared to 10 to 104 copies per reaction for PCR assays. The detection limits of the LAMP assay were comparable when using DNA-spiked CSF and blood specimens, as compared to using purified DNA as the template. In conclusion, a rapid and simple LAMP-based pneumococcal serotyping method has been developed. This is the first report of a LAMP method for a PCV13 serotype-specific identification assay, which could be a promising step to facilitate epidemiological studies of pneumococcal serotyping.

Streptococcus pyogenes is a genetically diverse pathogen, with over 200 different genotypes defined by emm typing, but only a minority of these genotypes are responsible for the majority of human infection in high-income countries. Two prevalent genotypes associated with disease rose to international dominance following recombination of a toxin locus that conferred increased expression. Here, we found that recombination of this locus and promoter has occurred in other diverse genotypes, events that may allow these genotypes to expand in the population. We identified an association between the loss of hyaluronic acid capsule synthesis and high toxin expression, which we propose may be associated with an adaptive advantage. As S. pyogenes pathogenesis depends both on capsule and toxin production, new variants with altered expression may result in abrupt changes in the molecular epidemiology of this pathogen in the human population over time., Gene transfer and homologous recombination in Streptococcus pyogenes has the potential to trigger the emergence of pandemic lineages, as exemplified by lineages of emm1 and emm89 that emerged in the 1980s and 2000s, respectively. Although near-identical replacement gene transfer events in the nga (NADase) and slo (streptolysin O) loci conferring high expression of these toxins underpinned the success of these lineages, extension to other emm genotype lineages is unreported. The emergent emm89 lineage was characterized by five regions of homologous recombination additional to nga-slo, including complete loss of the hyaluronic acid capsule synthesis locus hasABC, a genetic trait replicated in two other leading emm types and recapitulated by other emm types by inactivating mutations. We hypothesized that other leading genotypes may have undergone similar recombination events. We analyzed a longitudinal data set of genomes from 344 clinical invasive disease isolates representative of locations across England, dating from 2001 to 2011, and an international collection of S. pyogenes genomes representing 54 different genotypes and found frequent evidence of recombination events at the nga-slo locus predicted to confer higher toxin genotype. We identified multiple associations between recombination at this locus and inactivating mutations within hasAB, suggesting convergent evolutionary pathways in successful genotypes. This included common genotypes emm28 and emm87. The combination of no or low capsule and high expression of nga and slo may underpin the success of many emergent S. pyogenes lineages of different genotypes, triggering new pandemics, and could change the way S. pyogenes causes disease.

Background The aim of this study was to analyze the microbiological characteristics of nasopharyngeal carriage Haemophilus influenzae isolates collected from children with respiratory infections in Beijing hospital and Youyang Hospital of China. Methods The serotypes of all isolates were determined using latex agglutinated antisera (a‐f). The minimum inhibitory concentrations (MICs) of 11 antibiotics were determined using E‐test strips. For the beta‐lactamase‐negative ampicillin‐resistant (BLNAR) isolates, ftsI gene was sequenced based on fragments amplified by PCR. STs of H influenzae isolates were determined by multi‐locus sequence typing. Results The overall carriage rate of H influenzae in the study population was 9.1% (362/3984). One hundred and ninety H influenzae isolates which were selected in our study were non‐typeable (NTHi) and 44 (23.2%) of them were positive for β‐lactamase. All isolates were susceptible to ceftriaxone and levofloxacin. Susceptibility rates to erythromycin and sulfamethoxazole‐trimethoprim in Beijing were significantly higher than Youyang (P

Nine Klebsiella pneumoniae isolates coproducing NDM-1 and OXA-232 carbapenemases were successively isolated from a single patient. Although they were isolated simultaneously and were isogenic, they presented different colony phenotypes (matt and mucoid). All nine isolates were resistant to most antibiotics except colistin and fosfomycin. In addition, matt-type isolates were resistant to tigecycline. No differences were detected in the cps cluster sequences, except for the insertion of IS5 in the wzb gene of two matt-type isolates. In vitro virulence assays based on production of capsular polysaccharide, biofilm formation, and resistance to human serum indicated that the mucoid-type isolates were significantly more virulent than the matt-type. In addition, mucoid-type isolates showed higher survival rates than the matt-type ones in infection experiments in the fruit fly, suggesting a higher virulence of K. pneumoniae isolates with a mucoid phenotype. To our knowledge, this is the first report of K. pneumoniae colonies with different phenotypes being isolated from the same sample. In addition, we show that virulence varies with colony phenotype. Dissemination of K. pneumoniae isolates expressing both antibiotic resistance and high virulence would constitute a great threat.

In this national comprehensive seroprevalence study, indirect ELISA test was used to evaluate Haemophilus influenzae type b (Hib) anti-polyribosyl-ribitol phosphate (PRP) total antibodies (IgM, IgG, IgA) in 1,003 sera samples from routine medical check-up of healthy individuals attending the local medical facility in Jeddah, Saudi Arabia in the period from February 2014 to January 2016. Serum anti-CPS antibodies confer immunity against invasive Hib disease. An anti-CPS concentration of ⩾ 0.15 μg/mL is believed to be a serological indication for short-term immunity protection against invasive Hib disease, while a concentration of ⩾ 1.0 μg/mL is believed to be long-term protective. Results showed higher level of anti-Hib IgG (2.41 μg/ml average geometric mean concentration (GMC) regardless of age and gender, followed by levels of IgM (0.91 μg/ml) and IgA (0.34 μg/ml), reflecting the community immunity against Hib. Low anti-Hib level (< 0.15 μg/ml of anti-PRP IgG) in elderly people (males aged 57-91 years and females aged 35-64 years) may indicate a need for a booster dose of Hib vaccine to elderly people in the community. The IgG prevalence over IgM, and IgM prevalence over IgA indicate the major role of IgG over IgM and IgA in keeping immunity in the track against Hib. Low level of IgM and IgA comparing to IgG may indicate the absence of Hib acute infections in the population.

Whole genome sequencing analysis of 100 Neisseria meningitidis serogroup A isolates has revealed that the csaABCD-ctrABCD-ctrEF capsule polysaccharide synthesis locus represents a spontaneous point mutation hotspot. Structural and functional properties of the capsule of 11 carriage and two disease isolates with non-synonymous point mutations or stop codons in capsule synthesis genes were analyzed for their capsular polysaccharide expression, recognition by antibodies and sensitivity to bactericidal killing. Eight of eleven carriage isolates presenting capsule locus mutations expressed no or reduced amounts of capsule. One isolate with a stop codon in the O-acetyltransferase gene expressed non-O-acetylated polysaccharide, and was not recognized by anti-capsule antibodies. Capsule and O-acetylation deficient mutants were resistant to complement deposition and killing mediated by anti-capsular antibodies, but not by anti-lipopolysaccharide antibodies. Two capsule polymerase mutants, one carriage and one case isolate, showed capsule over-expression and increased resistance against bactericidal activity of both capsule- and lipopolysaccharide-specific antibodies. Meningococci have developed multiple strategies for changing capsule expression and structure, which is relevant both for colonization and virulence. Here we show that point mutations in the capsule synthesis genes substantially contribute to the repertoire of genetic mechanisms in natural populations leading to variability in capsule expression.

Capsular type K54 ofKlebsiella pneumoniaeis associated with hypervirulence and we sought to discover the basis for this among isolates submitted to the UK reference laboratory between 2012 and 2017. Isolates were typed by variable number tandem repeat analysis, and capsular type and virulence elements sought by PCR. The most prevalent type found (15/31 isolates) corresponded to clonal group (CG) 29 and included five representatives carryingrmpA,rmpA2(regulators of mucoid phenotype),iutAandiroD(from the aerobactin and salmochelin siderophore clusters) associated with virulence plasmids. These included isolate KpvK54, recovered from pus. The remaining isolates did not carry a virulence plasmid. We also noted 11 further related isolates, including NCTC 9159, not of capsular type K54, but nevertheless sometimes associated with sepsis and abscesses. Whole-genome sequencing showed that KpvK54 carried a large virulence plasmid and an ICEKp3-like structure carrying the yersiniabactin cluster, absent in NCTC 9159. Comparative chromosomal analysis with an additional four genomes showed that KpvK54 shared further genes with K1-ST23 hypervirulent isolates, and with LS358, a K54-ST29 isolate from liver abscess puncture fluid. While CG29 isolates displayed varying degrees of virulence, some, especially those with the virulence plasmid (all K54), were clearly associated with hypervirulence.

This study characterizes KPC-2 producing Klebsiella pneumoniae belonging to ST101. Whole genome sequencing using the Ion Torrent PGM platform with 400 bp chemistry was performed. bla KPC-2 was found on an IncFIIK plasmid associated with ISKpn6 and ISKpn7 without Tn4401. This is the first report of KPC-2 K. pneumoniae from bacteremia in India. The isolate also coded for other resistance genes such as aadA1, aadA2, armA, aac(3)-Ild, aac(6′)-Ild for aminoglycoside; bla SHV-11 , bla TEM-1B , bla OXA-9, for β-lactams and aac(6′)-Ild, oqxA, oqxB, qnrB1 for fluoroquinolones. It belonged to the K17 capsular type. India is endemic to New Delhi metallo-β-lactamase and OXA48-like carbapenemases and K. pneumoniae carbapenemase (KPC) is seldom reported. With high rates of carbapenem resistance, emergence of KPC in India will challenge patient management. The isolate was susceptible to colistin. The patient had a fatal outcome.

Haemophilus influenzae is a major pathogenic bacteria causing invasive disease, which is classified into six capsular serotypes (a-f) and non-typeable (NT) strains. Capsular serotyping of H. influenzae is traditionally determined by serological methods and more recently by PCR methods. However, these methods are time-consuming and expensive. In the present study, matrix-associated laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was evaluated as an alternative method for capsular serotyping of H. influenzae clinical strains. We created an in-house database of all six serotypes and NT H. influenzae strains using the main spectrum creation standard method set to the default parameters in MADI-TOF MS. We evaluated the performance of the in-house database using 79 clinical strains already identified by PCR and 58 prospectively collected clinical strains. Measurements were performed using the Bruker MALDI BioTyper system. The peak list was matched against the reference library using the integrated pattern algorithm of the software. The best-matched spectrum was considered the serotyping result. All 137 test strains were correctly identified as H. influenzae using MALDI-TOF MS. The sensitivity and specificity for identification for type b, type e, and type f capsular serotypes and NT H. influenzae using MALDI-TOF MS were 100%/94.3%, 94.7%/97.9%, 97.4%/97.9%, and 85.5%/99.2%, respectively. Our findings indicate that MALDI-TOF MS is a useful alternative method for capsular serotyping of H. influenzae strains. This method is faster and more cost-effective than traditional methods and will therefore be useful for routine applications in clinical laboratories.

Shiga toxin-producing Escherichia coli (STEC) has been associated with illnesses and outbreaks linked to fresh vegetables, prompting a growing public health concern. Most studies regarding interactions of STEC on fresh produce focused on E. coli O157:H7. Limited information is available about survival or fitness of E. coli O104:H4, non-O157 pathogen that was linked to one of the largest outbreaks of hemolytic uremic syndrome in 2011. In this study, survival of E. coli O104:H4 was evaluated on Arabidopsis thaliana plant and lettuce for 5 days compared with E. coli O157:H7, and expression of pathogenesis-realted gene (PR1; induction of plant defense response) was examined by reverse transcription quantitative PCR, and potential influence of capsular polysaccharide (CPS) on the bacterial fitness on plant was investigated. Populations of E. coli O104:H4 strains (RG1, C3493, and LpfA) on Arabidopsis and lettuce were significantly (P 0.05) greater than those of E. coli O157:H7 strains (7386 and sakai) at day 5 post-inoculation, indicating E. coli O104:H4 may have better survival ability on the plants. In addition, the E. coli O104:H4 strains produced significantly (P 0.05) higher amounts of CPS compared with the E. coli O157:H7 strains. RG1 strain (1.5-fold) initiated significantly (P 0.05) lower expression of PR1 gene indicating induction of plant defense response compared with E. coli O157:H7 strains 7386 (2.9-fold) and sakai (2.7-fold). Collectively, the results in this study suggests that different level of CPS production and plant defense response initiated by each STEC strain might influence the bacterial survival or persistence on plants. The present study provides better understanding of survival behavior of STEC, particularly E. coli O104:H4, using a model plant and vegetable under pre-harvest conditions with plant defense response.

The constitutively expressed hyaluronic acid capsule is an important virulence factor of Streptococcus equi, the cause of equine strangles. Study of the genomic sequence of CF22caps-, a non-encapsulated mutant of S. equi CF22 generated by gamma (Co60) irradiation revealed a non-sense mutation in fasC (SEQ_0302), a sensor kinase gene in FasBCAX an operon with an important regulatory role in expression of streptococcal secreted virulence and matrix binding proteins. The mutation was associated with a significant (p < .05) decrease in transcription of hasA, the synthase gene essential for hyaluronic acid synthesis and, conversely, with small increases in transcription of skc, covR and seM. The early growth phase of CF22caps- was also delayed compared to the CF22caps+ parent. In contrast to the human pathogen, S. pyogenes, capsule synthesis in S. equi therefore appears to be controlled by FasBCAX and not by CovRS.

Staphylococcus aureus produces capsular polysaccharides (CPs) both in vivo and under defined culture conditions being serotypes 5 and 8 the most prevalent. S. aureus isolates that fail to produce CP5 or CP8 are defined as non-typeable (NT). Loss of capsule expression, however, may lead to S. aureus persistence in a chronically infected host. The prevalence of NT strains of S. aureus isolated from bovine mastitis varies according to the geographic origin of the strain. The aims of this work were to detect phenotypically and genotypically the capsular profile of 144 S. aureus isolated from bovine mastitis in Argentina, Chile, and Uruguay and explore the factors that are considered to be associated with capsule expression as presence of IS257, IScap, and agr typing of non-related collection. The detection of the IS257, IScap, cap genes, and agr typing was performed using PCR. The detection and quantification of capsular polysaccharide production were performed by ELISA assays. We found that 96% of the S. aureus isolates investigated carried cap5(8) genes but over 75% of strains do not express capsule in the three countries studied. However, only 6 isolates from Argentina carried the IScap element that totally suppressed the expression of the capsule, suggesting that other factors could influence on CP expression. Moreover, the agrI/NT association was statistically significant suggesting that this profile is a phenomenon observed not only in other parts of the world but also in our region. Fil: Ambroggio, Maria Belen. Universidad Nacional del Litoral; Argentina Fil: Perrig, Melina Soledad. Universidad Nacional del Litoral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina Fil: Camussone, Cecilia María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; Argentina Fil: Pujato, Nazarena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral; Argentina Fil: Bertón, Alicia. Lactodiagnóstico Sur Uruguay; Uruguay Fil: Gianneechini, Edgardo. Laboratory Veterinary Direction “Miguel C. Rubino”; Uruguay Fil: Alvarez, Silvia. Udder Health; Chile Fil: Marcipar, Iván Sergio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral; Argentina Fil: Calvinho, Luis Fernando. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; Argentina. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias; Argentina Fil: Barbagelata, María Sol. Universidad Nacional del Litoral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina

Background/Purpose: Klebsiella pneumoniae is one of the leading causes of device-related infections (DRIs), which are associated with attachment of bacteria to these devices to form a biofilm. The latter is composed of not only bacteria but also extracellular polymeric substances (EPSes) consisting of extracellular DNAs, polysaccharides, and other macromolecules. The phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) regulates diverse processes of bacterial physiology. In the genome of K. pneumoniae MGH 78578, we found an uncharacterized enzyme II complex homolog of PTS: KPN00353 (EIIA homolog), KPN00352 (EIIB homolog), and KPN00351 (EIIC homolog). The aim of this study was to characterize the potential physiological role of KPN00353, KPN00352, and KPN00351 in biofilm formation by K. pneumoniae. Methods/Results: We constructed the PTS mutants and recombinant strains carrying the gene(s) of PTS. The recombinant K. pneumoniae strain overexpressing KPN00353–KPN00352–KPN00351 produced more extracellular matrix than did the vector control according to transmission and scanning electron microscopy. Judging by quantification of biofilm formation, of extracellular DNA (eDNA), and of capsular polysaccharide, the recombinant strain overexpressing KPN00353-KPN00352-KPN00351 produced more biofilm and capsular polysaccharide after overnight culture and more eDNA in the log phase as compared to the vector control. Conclusion: The genes, KPN00353–KPN00352–KPN00351, encode a putative enzyme II complex in PTS and positively regulate biofilm formation by enhancing production of eDNA and capsular polysaccharide in K. pneumoniae. Five proteins related to chaperones, to the citric acid cycle, and to quorum sensing are upregulated by the KPN00353–KPN00352–KPN00351 system. Keywords: Klebsiella, PTS, Biofilm, eDNA, Polysaccharide

Purpose Asplenic patients are at increased risk for post-splenectomy infection caused by Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae type B (Hib), and vaccination rates against these organisms remain low. The purpose of this study was to evaluate vaccination rates before and after implementation of a pharmacist-driven electronic vaccination tracking system. Methods This retrospective cohort analysis compared adult splenectomy patients before and after implementation of a pharmacist-driven tracking system with a primary outcome of complete initial vaccination. The system included use of an i-Vent to track and communicate vaccination status and a bundled vaccination order set. Complete initial vaccination was defined as documented administration of the following vaccines: pneumococcal, meningococcal, and Hib. Secondary outcomes included complete follow-up vaccination and factors associated with incomplete vaccination. Results A total of 261 patients were included for analysis (142 pre-intervention, 119 post-intervention). The most common indication for splenectomy was malignancy (52.1% pre-intervention, 47.9% post-intervention). Complete initial vaccination rates increased by almost 10% post-intervention from 68.3% to 77.3% (p = 0.11). There was a statistically significant increase with guideline recommended pneumococcal (13-valent) as part of the initial vaccination series (p Conclusion Implementation of a pharmacist-driven electronic vaccination tracking system and bundled order set may increase rates of vaccination among asplenic patients. Although this improvement was not statistically significant, it is still clinically impactful. One limitation of the study was many outpatient oncology pharmacists were not utilizing the tracking tool at the time of data collection. Projected vaccination rates are likely higher now that more pharmacists are aware of this tool.

Haemophilus influenzae type b (Hib) conjugate vaccines have drastically reduced disease incidence worldwide. Protection against Hib infection has relied on the serum bactericidal activity (SBA) of antibodies to the Hib capsular polysaccharide (polyribosylribitol phosphate). However, licensure usually relies on measuring induction of antibodies to PRP as a surrogate for SBA. In a phase III clinical trial we compared a PRP-conjugate vaccine using the nontoxic diphtheria toxin mutant, CRM197, as carrier protein with the licensed tetanus toxoid conjugate when administered subcutaneously as a three dose primary series in Japanese infants. As an addition to the phase III study, we have now evaluated SBA and show PRP-CRM197 induces higher levels of SBA than PRP-T four weeks after the primary series, with a statistically significant correlation with anti-PRP titers. This data confirms the superior immunogenicity of PRP-CRM197 compared with PRP-T assessed as SBA following a three-dose primary series by subcutaneous administration. Clinical trial registry: Registered on ClinicalTrials.gov (NCT01379846).

In tumorigenesis, CXCL12 level increases sharply because of tumor tissue hypoxia. As CXCR4 cells, endothelial progenitor cells (EPCs) are mobilized to tumor bed through the CXCL12/CXCR4 axis and are involved in tumor angiogenesis. In this process, either glycosaminoglycan (GAG) or heparan sulfate (HS) carried by membrane proteoglycans is implicated. Exogenous soluble HS mimetics can act as a competitive inhibitor of membranous HS, thereby preventing the formation of a normal signal axis. In this work, the effect of HS mimetics on the CXCL12-induced EPCs in vitro was investigated. HS mimetics, named as K5PSs, were obtained from sulfated Escherichia coli K5 polysaccharide/heparosan, and EPCs were collected from rat bone marrow. Results showed that CXCL12 could promote EPCs viability. This promotion might be related to its regulation of cell cycle and anti-apoptosis activity; it also could promote EPCs migration and secretion of pro-angiogenesis factors. All its functions were obtained by activation of MAPK/ERK pathway, FAK pathway, and PI3k/AKT pathway. However, its effect on EPCs was attenuated by K5PSs, and the existence of sulfate groups both at 2-O-position and N-position in K5PSs is essential to inhibit its effect on EPCs. This work suggested that K5PSs could be applied in anti-tumor treatment through inhibiting tumor angiogenesis.

Hypermucoviscous (HV) strains of capsular types K1, K2 and K57 are the most virulent representatives of the Klebsiella pneumoniae species. Eight novel bacteriophages lytic for HV K. pneumoniae were isolated and characterized. Three bacteriophages, KpV41, KpV475, and KpV71 were found to have a lytic activity against mainly K. pneumoniae of capsular type K1. Two phages, KpV74, and KpV763 were lytic for K2 capsular type K. pneumoniae , and the phage KpV767 was specific to K57-type K. pneumoniae only. Two more phages, KpV766, and KpV48 had no capsular specificity. The phage genomes consist of a linear double-stranded DNA of 40,395–44,623 bp including direct terminal repeats of 180–246 bp. The G + C contents are 52.3–54.2 % that is slightly lower than that of genomes of K. pneumoniae strains being used for phage propagation. According to the genome structures, sequence similarity and phylogenetic data, the phages are classified within the genus Kp32virus and Kp34virus of subfamily Autographivirinae, family Podoviridae . In the phage genomes, genes encoding proteins with putative motifs of polysaccharide depolymerase were identified. Depolymerase genes of phages KpV71 and KpV74 lytic for hypermucoviscous K. pneumoniae of K1 and K2 capsular type, respectively, were cloned and expressed in Escherichia coli , and the recombinant gene products were purified. The specificity and polysaccharide-degrading activity of the recombinant depolymerases were demonstrated.

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.

Staphylococcus aureus produces an antiphagocytic polysaccharide capsule to evade neutrophil-mediated killing. Many vaccines against encapsulated bacterial pathogens require generation of functional anti-capsular antibodies to mediate protection against infection and disease. Here it is shown that the generation of such antibody responses to S. aureus in vivo and in vitro requires the presence of O-acetyl modifications on the capsular polysaccharides. O-acetylation of S. aureus capsular polysaccharide therefore should be monitored carefully during vaccine development and production. This finding may provide additional insight into the previous failure of a S. aureus capsular polysaccharide conjugate vaccine.

Introduction: North American indigenous populations experience a high burden of invasive bacterial infections. Because Streptococcus pneumoniae and Haemophilus influenzae have multiple antigenic variants, the existing vaccines cannot prevent all cases. This study addresses the current epidemiology of invasive H. influenzae and pneumococcal disease (IPD) in a region of Northwestern Ontario, Canada with a relatively high (82%) indigenous population. Methods: Data were retrieved from a retrospective chart review at a hospital servicing a population of 29Â 000 (82% indigenous), during January 2010âJuly 2015. Results: Ten cases of invasive H. influenzae disease and 37 cases of IPD were identified. The incidence of both in the study population (6.3 and 23.1/100Â 000/year, respectively) exceeded national rates (1.6 and 9.0/100Â 000/year). H. influenzae serotype a (Hia) was the most common (50%), followed by non-typeable H. influenzae (20%). In adults, 77% of IPD cases were caused by serotypes included in the 23-valent pneumococcal polysaccharide vaccine. All paediatric IPD cases were caused by serotypes not included in the 13-valent pneumococcal conjugate vaccine. The case-fatality rate was 10% for invasive H. influenzae and 2.7% for IPD. Most cases exhibited substantial co-morbidity. Conclusions: In Northwestern Ontario, the incidence of invasive Hia disease exceeds that of H. influenzae type b (Hib) in the pre-Hib vaccine era. This provides strong support for the development of a new Hia vaccine. Improved pneumococcal vaccination of high-risk adults in the region is warranted. Keywords: Pneumococcal, Streptococcus pneumoniae, Haemophilus influenzae, Invasive disease, Canada, Indigenous

Several Cronobacter species are opportunistic pathogens that cause infections in humans. This study evaluated the phenotypic characteristics of 57 Cronobacter strains (C. sakazakii n = 41, C. malonaticus n = 10, C. dublinensis n = 4, and C. muytjensii n = 2) isolated from food (n = 54) and clinical specimens (n = 3) in Brazil. These strains included sequence types (ST): ST395-ST398, ST402, ST413 and ST433-ST439, isolated from food samples, and three C. malonaticus clinical strains previous isolated from an outbreak which were ST394 (n = 1) and ST440 (n = 2). Strains were tested for capsule production, biofilm formation, protease activity, hemolytic activity, cell-cell aggregation, and desiccation resistance. Capsule formation was observed with all Cronobacter strains. Forty-four (77.2%) strains showed proteolytic activity on milk agar. All strains showed β-hemolysis against erythrocytes from guinea pig, horse and rabbit. Using erythrocytes from sheep, the majority of strains (53/57; 92.9%) showed α-hemolysis and the remaining, β-hemolysis. All Cronobacter strains produced weak biofilms in microtiters polystyrene plates, which were independent of temperature (4, 25 and 37 °C) and/or growth conditions. In glass tubes, formation of either a moderate or strong biofilm was observed in 15/57 (26.3%), 19/57 (33.3%) and 27/57 (47.4%), at 4, 25 and 37 °C, respectively. Desiccation treatment decreased Cronobacter viability by 1.55 to > 3.87 Log10 CFU/mL. Cell-cell aggregation was observed in 17 (29.8%) strains. This study showed that the Cronobacter species evaluated showed differing phenotypes, independent of their origin (clinical or not) and ST. Further studies are necessary to elucidate the factors affecting phenotype expression. This may identify novel bacterial targets that could be useful in the development of strategies to control Cronobacter in food chain and to prevent cases of infections.

Cryptococcus neoformans is an encapsulated fungal pathogen that causes cryptococcosis, which is a major opportunistic infection in immunosuppressed individuals. Mammalian β-galactoside-binding protein Galectin-3 (Gal-3) modulates the host innate and adaptive immunity, and plays significant roles during microbial infections including some fungal diseases. Here we show that this protein plays a role also in C. neoformans infection. We find augmented Gal-3 serum levels in human and experimental infections, as well as in spleen, lung, and brain tissues of infected mice. Gal-3-deficient mice are more susceptible to cryptococcosis than WT animals, as demonstrated by the higher fungal burden and lower animal survival. In vitro experiments show that Gal-3 inhibits fungal growth and exerts a direct lytic effect on C. neoformans extracellular vesicles (EVs). Our results indicate a direct role for Gal-3 in antifungal immunity whereby this molecule affects the outcome of C. neoformans infection by inhibiting fungal growth and reducing EV stability, which in turn could benefit the host., The protein Galectin-3 modulates host immunity and plays roles during infections. Here, Almeida et al. show that this protein contributes to host defence against infection with the fungal pathogen Cryptococcus neoformans by inhibiting fungal growth and inducing lysis of fungal extracellular vesicles.