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Background Streptococcus pneumoniae (S. pneumoniae) of serogroup 19 are mainly represented by serotypes 19A and 19F, which are associated with antimicrobial resistance and disease. The wzy gene, a component of the pneumococcal capsular locus, is the target to differentiate serotypes 19A and 19F by PCR-based capsular typing. In the last decade, allelic variants of the wzy19F gene have been described, leading to misinterpretation of capsular typing results. Methods A collection of 154 serotype 19F S. pneumoniae strains recovered from carriage and disease in Brazil was evaluated to identify and characterize wzy19F variant isolates. Results Eleven (7%) wzy19F variant isolates were detected and identified as belonging to ST810 (n = 10) or ST13673 (n = 1; single-locus variant of ST810). They were mostly recovered from diseased patients, susceptible to the antimicrobial agents tested (except for one multidrug-resistant strain) and did not harbor pili genes. Sequences of the wzy19F gene of these variants were identical to each other and to those previously described in Brazil, but slightly different from wzy19F variants identified in other countries. Conclusion This study indicated that wzy19F variants present a geographically driven distribution and was the first to uncover phenotypic and genetic features of a wzy19F variant lineage occurring in Brazil since 1989.

KL55, KL74, and KL85 capsular polysaccharide (CPS) biosynthesis loci in Acinetobacter baumannii BAL_204, BAL_309, and LUH5543 genomes, respectively, are related and each contains genes for l -Rhap and d -GlcpA synthesis. The CPSs were isolated and studied by sugar analysis, Smith degradation, and 1H and 13C NMR spectroscopy. The K55 and K74 CPSs are built up of branched octasaccharide repeats (K units) containing one residue each of d -GlcpA and d -GlcpNAc and six residues of l -Rhap. The K55 unit differs from the K74 unit in the linkage between D-GlcpA and an l -Rhap residue in the K unit (1 → 3 versus 1 → 2) and linkage between K units. However, most K units in the isolated K74 CPS were modified by β-elimination of a side-chain α- l -Rhap-(1 → 3)-α- l -Rhap disaccharide from position 4 of GlcA to give 4-deoxy- l -threo-hex-4-enuronic acid (1:~3 ratio of intact and modified units). The K85 CPS has a branched heptasaccharide K unit similar to the K74 unit but with one fewer α- l -Rhap residue in the side chain. In contrast to previous findings on A. baumannii CPSs, each K locus includes fewer glycosyltransferase (Gtr) genes than the number required to form all linkages in the K units. Hence, one Gtr appears to be multifunctional catalysing formation of two 1 → 2 and one 1 → 3 linkages between the l -Rha residues.

Klebsiella pneumoniae produces a thick capsule layer composed of extracellular polysaccharides protecting the bacterial cells from clearance by innate host immunity during infection. Here we characterize the interactions of a structurally diverse set of host defense peptides with K. pneumoniae extracellular polysaccharides. Remarkably, we found that all host defense peptides were active against a diverse set of K. pneumoniae strains, including hypermucoviscous strains with extensive capsule production, and aggregated with extracted capsule. Interestingly, the polyproline peptide bac7 (1-35), was the most potent antimicrobial and induced the most capsule aggregation. In addition to capsule aggregation, we found that bac7 (1-35) could also disrupt pre-formed hypermucoviscous K. pneumoniae biofilm. Further analysis using scanning electron microscopy revealed the biofilm matrix of a hypermucoviscous strain is removed by bac7 (1-35) exposing associated bacterial cells. This is the first description of a host defense peptide interacting with capsular and biofilm extracellular polysaccharides to expose cells from a K. pneumoniae biofilm matrix and suggests that features of polyproline peptides may be uniquely suited for extracellular polysaccharide interactions.

Streptococcus pyogenes is a globally prominent human-specific pathogen responsible for an enormous burden of human illnesses, including >600 million pharyngeal and >100 million skin infections each year. Despite intensive efforts that focus on invasive indications, much remains unknown about this bacterium in its natural state during colonization of the nasopharynx and skin. Using acute experimental infection models in HLA-transgenic mice, we evaluated how the hyaluronic acid (HA) capsule contributes to S. pyogenes MGAS8232 infection within these limited biological niches. Herein, we demonstrate that HA capsule expression promotes bacterial burden in murine nasal turbinates and skin lesions by resisting neutrophil-mediated killing. HA capsule production is encoded by the hasABC operon and compared to wildtype S. pyogenes infections, mice infected with a ΔhasA mutant exhibited over a 1000-fold CFU reduction at 48-hours post-nasal challenge, and a 10,000-fold CFU reduction from skin lesions 72-hours post-skin challenge. HA capsule expression contributed substantially to skin lesion size development following subdermal inoculations. In the absence of capsule expression, S. pyogenes revealed drastically impeded growth in whole human blood and increased susceptibility to killing by isolated neutrophils ex vivo, highlighting its important role in resisting phagocytosis. Furthermore, we establish that neutrophil depletion in mice recovered the reduced burden by the ΔhasA mutant in both the nasopharynx and skin. Together, this work confirms that the HA capsule is a key virulence determinant during acute infections by S. pyogenes and demonstrates that its predominant function is to protect S. pyogenes against neutrophil-mediated killing.

Capsular polysaccharides (CPSs) are important antigenic targets against bacterial infections. As T-independent antigens, however, CPSs elicit short-lived immune responses in adults and are poorly immunogenic in young children. Coupling CPS with protein carriers enhances anti-CPS responses and generates long-lasting immune memory. However, the mechanisms whereby carrier proteins accomplish this are not fully understood. Here, we dissect different mechanisms whereby carrier proteins enhance anti-CPS immunity. We show how coupling CPS with protein carriers modifies the interaction of CPS with antigen-presenting cells, enables a dual-activation mechanism for CPS-specific B cells via interaction with CPS- or carrier-specific T helper cells, and potentiates the recall of anti-CPS responses by engaging memory T helper cells during subsequent vaccination or bacterial exposure. Our findings provide new insights into the immunological basis of carrier-mediated anti-CPS immunity and may help in the design of more effective CPS-based vaccines.

Many encapsulated bacteria use capsules to cause invasive diseases. However, it remains largely unknown how the capsules enhance bacterial virulence under in vivo infection conditions. Here we show that the capsules primarily target the liver to enhance bacterial survival at the onset of blood-borne infections. In a mouse sepsis model, the capsules enabled human pathogens Streptococcus pneumoniae and Escherichia coli to circumvent the recognition of liver-resident macrophage Kupffer cells (KCs) in a capsular serotype-dependent manner. In contrast to effective capture of acapsular bacteria by KCs, the encapsulated bacteria are partially (low-virulence types) or completely (high-virulence types) “untouchable” for KCs. We finally identified the asialoglycoprotein receptor (ASGR) as the first known capsule receptor on KCs to recognize the low-virulence serotype-7F and -14 pneumococcal capsules. Our data identify the molecular interplay between the capsules and KCs as a master controller of the fate and virulence of encapsulated bacteria, and suggest that the interplay is targetable for therapeutic control of septic infections.

In this study, several different depolymerases encoded in the prophage regions of

Acinetobacter baumannii isolate LUH5552 carries the KL89 capsule biosynthesis gene cluster. Capsular polysaccharide (CPS) isolated from LUH5552 was analyzed by sugar analysis, Smith degradation, and one- and two-dimensional

For neonates, group B Streptococcus is life threatening. Current prevention strategies remain insufficient, especially for cases of late-onset sepsis, where intrapartum antibiotic prophylaxis has demonstrated no benefit. One promising approach is the vaccination of pregnant women, which offers protective immunity via transplacental transmission of neutralizing antibodies. Our nationwide, prospective surveillance study aimed to characterize the prevalence of pilus antigen, capsular polysaccharide serotypes, and antibiotic resistance from invasive GBS infections in neonates and compare these results with those from children and adults in Germany. Our study includes 173 neonatal isolates of a total of 450 reported cases during the study period (incidence: 0.34/1000 live births), in addition to 2 pediatric and 803 adult isolates. The comparison between neonatal and adult isolates reveals age-dependent differences in capsular serotype and pilus type distribution and differences in antibiotic resistance patterns.

Background Whole genome sequencing has emerged as a useful tool for identification and molecular characterization of pathogens. MinION (Oxford Nanopore) is a real-time third generation sequencer whose portability, affordability and speed in data production make of it an attractive device for whole genome sequencing. The objective of this study is to evaluate MinION sequencer for pathogen identification and molecular characterization of Streptococcus pneumoniae isolated at a children’s Hospital. Whole genome sequencing of 32 Streptococcus pneumoniae invasive isolates, previously characterized by standard methods (Quellung reaction, Multiplex PCR and Sanger-MLST), were performed. DNA was extracted using ZymoBIOMICS DNA Microprep kit. Quantification and purity of DNA was assessed by Qubit and Nanodrop, respectively. Library preparation was performed using the Rapid Barcoding Kit. Real-time workflow EPI2ME platform “What’s it in my pot” was used for species identification. Fast5 sequences were converted into FASTQ by Albacore software. Reads were assembled using CANU software. PathogenWatch, genomic epidemiology and pubmlst online tools were used for capsular typing and/or whole genome-MLST profile. Results Rapid identification of Streptococcus pneumoniae was achieved by “What’s in my pot”. Capsular typing was correctly assigned with PathogenWatch in all 32 isolates at serogroup level and 24 at serotype level. Whole genome-MLST results obtained by genomic epidemiology and pubmlst were consistent with double locus variant clonal complex obtained by Sanger-MLST in 31 isolates. Conclusion MinION sequencer provides a rapid, cost-effective and promising pathway for performing WGS by a pocked-sized device for epidemiological purposes but improving its sequencing accuracy will make it more appealing to be used in clinical microbiology laboratories.

With the introduction of the pneumococcal conjugate vaccine (PCV), the number of cases of non-vaccine type pneumococci and non-encapsulated Streptococcus pneumoniae (NESp) infection have increased. In order to clarify how pspK-harbouring NESp might have emerged, we characterised NESp and analysed the correlation between transformation and non-encapsulation. A total of 26 NESp strains were used in this study. The genetic backgrounds were compared using multilocus sequence typing (MLST). The ΔpspK::ermB strain, in which pspK was replaced by ermB in NESp, was constructed by homologous recombination. The genomic DNA of the ΔpspK::ermB strain was transformed into two types of encapsulated S. pneumoniae via transformation. The fitness of the parent and non-encapsulated transformants was compared using the growth curve. All NESp had pspK instead of capsular coding regions and were classified into 14 types by MLST, which indicated that NESp had several genetic backgrounds. Transformation of ΔpspK::ermB genomic DNA resulted in 10−4‒10−5 non-encapsulated transformants. Non-encapsulated transformants could grow faster than the encapsulated parent strain. The acquisition of pspK region via transformation contributed to the loss of encapsulation with high frequency. The present results suggest that non-encapsulation through pspK acquisition could be a potential mechanism to evade PCV.

Nature Communications 11(1), 4723 (1-12) (2020). doi:10.1038/s41467-020-18464-y, $O$-Acetylation of the capsular polysaccharide (CPS) of Neisseria meningitidis serogroup A (NmA) is critical for the induction of functional immune responses, making this modification mandatory for CPS-based anti-NmA vaccines. Using comprehensive NMR studies, we demonstrate that $O$-acetylation stabilizes the labile anomeric phosphodiester-linkages of the NmA-CPS and occurs in position C3 and C4 of the $N$-acetylmannosamine units due to enzymatic transfer and non-enzymatic ester migration, respectively. To shed light on the enzymatic transfer mechanism, we solved the crystal structure of the capsule $O$-acetyltransferase CsaC in its apo and acceptor-bound form and of the CsaC-H228A mutant as trapped acetyl-enzyme adduct in complex with CoA. Together with the results of a comprehensive mutagenesis study, the reported structures explain the strict regioselectivity of CsaC and provide insight into the catalytic mechanism, which relies on an unexpected Gln-extension of a classical Ser-His-Asp triad, embedded in an $α$/$β$-hydrolase fold., Published by Nature Publishing Group UK, [London]

Selection pressures exerted on Staphylococcus aureus by host factors during infection may lead to the emergence of regulatory phenotypes better adapted to the infection site. Traits convenient for persistence may be fixed by mutation thus turning these mutants into microevolution endpoints. The feasibility that stable, non-encapsulated S. aureus mutants can regain expression of key virulence factors for survival in the bloodstream was investigated. S. aureus agr mutant HU-14 (IS256 insertion in agrC) from a patient with chronic osteomyelitis was passed through the bloodstream using a bacteriemia mouse model and derivative P3.1 was obtained. Although IS256 remained inserted in agrC, P3.1 regained production of capsular polysaccharide type 5 (CP5) and staphyloxanthin. Furthermore, P3.1 expressed higher levels of asp23/SigB when compared with parental strain HU-14. Strain P3.1 displayed decreased osteoclastogenesis capacity, thus indicating decreased adaptability to bone compared with strain HU-14 and exhibited a trend to be more virulent than parental strain HU-14. Strain P3.1 exhibited the loss of one IS256 copy, which was originally located in the HU-14 noncoding region between dnaG (DNA primase) and rpoD (sigA). This loss may be associated with the observed phenotype change but the mechanism remains unknown. In conclusion, S. aureus organisms that escape the infected bone may recover the expression of key virulence factors through a rapid microevolution pathway involving SigB regulation of key virulence factors. Fil: Suligoy Lozano, Carlos Mauricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina Fil: Díaz, Rocío E.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina Fil: Gehrke, Ana-katharina Elsa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Maimónides. Área de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; Argentina Fil: Ring, Natalie. University of Edinburgh; Reino Unido Fil: Yebra, Gonzalo. University of Edinburgh; Reino Unido Fil: Alves, Joana. University of Edinburgh; Reino Unido Fil: Gómez, Marisa Ileana. Universidad Maimónides. Área de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; Argentina Fil: Wendler, Sindy. Universitätsklinikum Jena Und Medizinische Fakultät; Alemania Fil: Fitzgerald, J. Ross. University of Edinburgh; Reino Unido Fil: Tuchscherr, Lorena. Jena University Hospital; Alemania Fil: Löffler, Bettina. Jena University Hospital; Alemania Fil: Sordelli, Daniel Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina Fil: Noto Llana, Mariangeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina Fil: Buzzola, Fernanda Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina

The purpose of this study was to investigate H. influenzae epidemiology in the Republic of Ireland. We performed serotyping, multi-locus sequence typing (MLST) and susceptibility testing on H. influenzae isolates received by the Irish Meningitis and Sepsis Reference Laboratory from 2010 to 2018. Three hundred sixty-seven invasive and 41 non-invasive infection (NII) isolates were received. Invasive isolates were mostly recovered from paediatric (21%) and elderly (42%) populations. Invasive disease was more prevalent in females of childbearing age (72%) compared with males the same age (28%). Non-typeable H. influenzae (NTHi) predominated among invasive (83%) and NII (95%). Invasive Hib disease isolates were infrequent (4%, n = 15). Among invasive disease, Hif was the commonest encapsulated serotype (10%, n = 37), and the only encapsulated serotype detected in NII (5%, 2/41). The first PCR-confirmed serotypes d and a in Ireland were characterised among invasive disease in 2017 and 2018, respectively. MLST revealed a diverse NTHi population, while encapsulated serotypes were clonal. Sequence type (ST) 103 (n = 14) occurred exclusively in invasive NTHi disease. Ampicillin resistance (AmpR) was 18% among invasive isolates and 22% in NII. β-Lactamase production was the main source of ampicillin resistance in invasive and NII isolates. We detected β-lactamase negative ampicillin resistance (BLNAR) among invasive isolates. We report an NTHi fluoroquinolone-resistant clone: ST1524 among invasive (n = 2) and NII isolates (n = 2). The Hib vaccine has positively impacted on Hib disease in Ireland, given the low frequency of Hib. The dominance of NTHi, emergence of serotypes a and d and BLNAR suggest a changing H. influenzae epidemiology in Ireland.

To report the first case of a lung abscess caused by Neisseria meningitidis (Nm) and to genetically characterize the rare underlying capsule switching event. The strain (PT NmX) was subjected to whole genome sequencing, and a comparative gene-by-gene analysis was performed based on 1605 N. meningitidis core loci that constitute the MLST core-genome scheme (cgMLST) V1.0. All ~ 9,600 genomes available on Neisseria PubMLST (until 30th November 2019) from all serogroups were used to better identify the genome make-up of the PT NmX strain. This strain was found to be highly divergent from other NmX reported worldwide and to belong to a new sequence type (ST-14273), with the finetype X: P1.19,15-1:F5-2. Moreover, it revealed a closer genetic proximity to strains from serogroup B than to other serogroups, suggesting a genome backbone associated with serogroup B, while it presents a capsule synthesis region derived from a NmX strain. We describe a new hybrid NmB/X isolate from a noninvasive meningococcal infection, causing lung abscess. Despite capsular switching events involving serogroup X are rare, it may lead to the emergence of pathogenic potential. Studies should continue to better understand the molecular basis underlying Neisseria strains' ability to spread to body compartments other than the tissues for which their tropism is already known. This work was supported by GenomePT (ref. POCI-01-0145-FEDER-022184) from Fundação para a Ciência e Tecnologia, Portugal and by National Institute of Health Ricardo Jorge, Lisbon, Portugal. info:eu-repo/semantics/publishedVersion

A variety of cell surface structures dictate interactions between bacteria and their environment, including their viruses (bacteriophages). Members of the human gut Bacteroidetes characteristically produce several phase-variable capsular polysaccharides (CPSs), but their contributions to bacteriophage interactions are unknown. To begin to understand how CPSs have an impact on Bacteroides–phage interactions, we isolated 71 Bacteroides thetaiotaomicron-infecting bacteriophages from two locations in the United States. Using B. thetaiotaomicron strains that express defined subsets of CPSs, we show that CPSs dictate host tropism for these phages and that expression of non-permissive CPS variants is selected under phage predation, enabling survival. In the absence of CPSs, B. thetaiotaomicron escapes bacteriophage predation by altering expression of eight distinct phase-variable lipoproteins. When constitutively expressed, one of these lipoproteins promotes resistance to multiple bacteriophages. Our results reveal important roles for Bacteroides CPSs and other cell surface structures that allow these bacteria to persist under bacteriophage predation, and hold important implications for using bacteriophages therapeutically to target gut symbionts. Isolation of phages associated with the gut commensal Bacteroides thetaiotaomicron reveals a link between cell surface structures, including phase-variable capsular polysaccharides, lipoproteins and S-layer proteins, and susceptibility to phage infection.

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.

Hyaluronan is widely used in cosmetics and pharmaceutics. Development of robust and safe cell factories and cultivation approaches to efficiently produce hyaluronan is of many interests. Here, we describe the metabolic engineering of Corynebacterium glutamicum and application of a fermentation strategy to manufacture hyaluronan with different molecular weights. C. glutamicum is engineered by combinatorial overexpression of type I hyaluronan synthase, enzymes of intermediate metabolic pathways and attenuation of extracellular polysaccharide biosynthesis. The engineered strain produces 34.2 g L−1 hyaluronan in fed-batch cultures. We find secreted hyaluronan encapsulates C. glutamicum, changes its cell morphology and inhibits metabolism. Disruption of the encapsulation with leech hyaluronidase restores metabolism and leads to hyper hyaluronan productions of 74.1 g L−1. Meanwhile, the molecular weight of hyaluronan is also highly tunable. These results demonstrate combinatorial optimization of cell factories and the extracellular environment is efficacious and likely applicable for the production of other biopolymers., Bioproduction of hyaluronan needs increases in yield and greater diversity of the molecular weights. Here, the author increases hyaluronan production and diversifies the molecular weights through engineering the hyaluronan biosynthesis pathway and disruption of Corynebacterium glutamicum encapsulation caused by secreted hyaluronan.

This paper describes the predicted structure for the cps loci involved in capsule biosynthesis for Streptococcus parauberis serotypes III, IV, and V. Based on the specific serotype regions I, II, and III, a multiplex PCR protocol (mPCR) was designed to differentiate the main serotypes causing fish diseases. A real-time PCR method (qPCR) is also described to identify S. parauberis of serotype III in bacterial cultures and fish tissues. In silico and in vitro analyses revealed that both methods have a 100% specificity. The mPCR assay was optimized for the detection of S. parauberis strains of subtypes Ia (amplicon size 213 bp), subtypes Ib and Ic (both amplicon size 303 bp), serotype II (amplicon size 403 bp), and serotype III (amplicon size 130 bp) from bacterial cultures. The qPCR assay was optimized for the identification and quantification of S. parauberis serotype III strains in bacterial cultures and fish tissues. This assay achieved a sensitivity of 2.67 × 102 gene copies (equivalent to 3.8 × 10−9 ng/μl) using pure bacterial cultures of S. parauberis serotype III and 1.76 × 102 gene copies in fish tissues experimentally and naturally infected with S. parauberis of the serotype III. The specificity and sensitivity of the protocols described in this study suggest that these methods could be used for diagnostic and/or epidemiological purposes in clinical diagnostic laboratories. Key Points • Structure of loci cps for S. parauberis of serotypes III, IV and V was described. • mPCR to differentiate S. parauberis serotypes causing disease in fish was optimized. • qPCR assay to quantify strains of S. parauberis serotype III in fish tissues. Electronic supplementary material The online version of this article (10.1007/s00253-020-10683-z) contains supplementary material, which is available to authorized users.

Background Polysaccharide conjugate vaccines (PCVs) target the pneumococcal capsular types that most commonly cause fatal pneumonia and sepsis. Because these types were eliminated by the vaccines, it became apparent that in immunized populations, most invasive pneumococcal diseases, including bacteremia, sepsis and complicated pneumonia, were greatly reduced. However, the protective effects of PCVs against another invasive disease, meningitis, has shown much less or no decrease in disease incidence. Methods References were identified through searches of PubMed for articles published from January 1930 to the present by use of specific search terms. Relevant articles were also identified through searches in Google and Google Scholar. Relevant references cited in those articles were also reviewed. Results Even in the presence of the PCVs, meningitis rates in children have been reported globally to be as high as 13 per 100,000 annually. Widespread use of vaccines resulted in the emergence of a broad diversity of replacement non-PCV type strains. These strains generally failed to cause sepsis, but caused meningitis of comparable severity and levels similar to, or in excess of, prior pneumococcal meningitis rates. This is probably because these non-PCV type strains do not survive well in the blood, therefore possibly entering the brain through nonhematogenous routes. Conclusions Because virtually all cases of pneumococcal meningitis lead to either permanent neurologic sequelae or death, it would be well worth the effort to develop a new vaccine capable of preventing pneumococcal meningitis regardless of capsular type. Such a vaccine would need to protect against colonization with most, if not all, pneumococci.

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

Background Klebsiella pneumoniae is a public health concern because of its ability to develop multidrug resistance and hypervirulent genotypes, of those capsular types K1 and K2 cause community and nosocomial life-threatening infections. This study aimed to determine the antibiotic susceptibility patterns and genotypic traits of a collection of Klebsiella spp. isolates. Furthermore, the clonal relatedness of blaNDM producing strains was investigated. Methods During a 19-months surveillance study, 122 Klebsiella spp. isolates were cultured from extraintestinal specimens of patients admitted to the tertiary referral hospital in Semnan, Iran. Isolates were identified using biochemical tests and subjected to determination of phylogroups, capsular types and virulence/resistance genes content. Hypervirulent K. pneumoniae (hvKp) strains were detected genotypically, and Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR fingerprinting was used to determine the clonality of blaNDM producing strains. Results Multidrug resistant phenotype was detected in 75 (61.5%) isolates and amikacin was found as the most potent antibiotic with the susceptibility rate of 85.2%. The carbapenemase genes were detected in 45 (36.8%) strains, including 21 (17.2%) blaOXA-48, 7 (5.6%) blaNDM-1, 14 (11.4%) blaNDM-1/OXA-48 and 3 (2.4%) blaIMP- carrying strains, while 55 (45.08%) isolates showed carbapenem resistant phenotype. The first blaNDM-1 carrying strain was cultured from a sputum specimen on March 2015, while the last positive one was recovered from blood culture on September 2016. Most of the isolates (80.3%) belonged to phylogroup I, and blaNDM-1 was identified among all three phylogroups. The ERIC-PCR clustered the 101 blaNDM negative and 21 blaNDM-1 positive isolates into 25 and five clusters, respectively, and the latter group belonged to clonal complex 147 (CC147). One K1 and 15 K2 blaNDM-1 negative isolates were detected, of those three strains were identified as hvKp. Five K2 positive strains, including four blaOXA-48 producer and one hvKp sequence type 86 (ST86) were carbapenem resistant. Among carbapenem resistant isolates, CC147 strains harboured higher rates of siderophores iutA and ybtS. Conclusion The present findings showed a hospital circulation of CC147 blaNDM-1 or blaNDM-1/OXA-48 producing strains, disseminated in different wards. The hvKp/ST86 strain expressing K2 capsular type and carbapenem resistant phenotype wasn’t reported from Iran so far. So, it seems that we must be aware of the emergence and spread of new K. pneumoniae clones associated with resistant and hypermucoviscous phenotypes.

Capsular polysaccharide (CPS), isolated from Acinetobacter baumannii LUH5549 carrying the KL32 capsule biosynthesis gene cluster, was studied by sugar analysis, Smith degradation, and one- and two-dimensional 1H and 13C NMR spectroscopy. The K32 CPS was found to be composed of branched pentasaccharide repeats (K units) containing two residues of β-D-GalpNAc and one residue of β-D-GlcpA (β-D-glucuronic acid) in the main chain and one residue each of β-D-Glcp and α-D-GlcpNAc in the disaccharide side chain. Consistent with the established CPS structure, the KL32 gene cluster includes genes for a UDP-glucose 6-dehydrogenase (Ugd3) responsible for D-GlcA synthesis and four glycosyltransferases that were assigned to specific linkages. Genes encoding an acetyltransferase and an unknown protein product were not involved in CPS biosynthesis. Whilst the KL32 gene cluster has previously been found in the global clone 2 (GC2) lineage, LUH5549 belongs to the sequence type ST354, thus demonstrating horizontal gene transfer between these lineages.

The K17 capsular polysaccharide (CPS) produced by Acinetobacter baumannii G7, which carries the KL17 configuration at the capsule biosynthesis locus, was isolated and studied by chemical methods along with one- and two-dimensional 1H and 13C NMR spectroscopy. Selective cleavage of the glycosidic linkage of a 2,4-diacetamido-2,4,6-trideoxy- d -glucose ( d -QuiNAc4NAc) residue by (i) trifluoroacetic acid solvolysis or (ii) alkaline β-elimination (NaOH-NaBH4) of the 4-linked D-alanine amide of a 2-acetamido-2-deoxy- d -galacturonic acid residue ( d -GalNAcA6DAla) yielded trisaccharides that were isolated by Fractogel TSK HW-40 gel-permeation chromatography and identified by using NMR spectroscopy and high-resolution electrospray ionization mass spectrometry. The following structure was established for the trisaccharide repeat (K unit) of the CPS: →4)- α -d -GalpNAcA6 d Ala-(1 → 4)- α -d -GalpNAcA-(1 → 3)- β- d -QuipNAc4NAc-(1 → . The presence of the itrA1 gene coding for the initial glycosylphosphotransferase in the KL17 gene cluster established the first sugar of the K unit as d -QuipNAc4NAc. KL17 includes genes for three transferases that had been annotated previously as glycosyltransferases (Gtrs). As only two Gtrs are required for the K17 structure and one d -GalpNAcA residue is modified by a d -alanine amide, these assignments were re-assessed. One transferase was found to belong to the ATPgrasp_TupA protein family that includes d -alanine- d -alanine ligases, and thus was renamed Alt1 ( al anine t ransferase). Alt1 represents a novel family that amidate the carboxyl group of d -GalpNAcA or d -GalpA.

Acinetobacter baumannii emerged as one of the most important pathogens that causes nosocomial infections due to its increased multidrug resistance. Identifying capsular epidemiology in A. baumannii can aid in the development of effective treatments and preventive measures against this emerging pathogen. Here we established a wzc-based method, and combined it with wzy-PCR to determine capsular types of A. baumannii causing nosocomial bacteraemia collected at two medical centres in Taiwan from 2015 to 2017. Among the 237 patients with A. baumannii bacteraemia, 98 (41.4%) isolates were resistant to carbapenems. Four prevalent capsular types (KL2, KL10, KL22, and KL52) accounted for 84.7% of carbapenem-resistant A. baumannii (CRAB) and 12.2% of non-CRAB. The rate of pneumonia, intensive care unit admission, APACHE II score, and Pitt bacteraemia score were higher in patients with KL2/10/22/52 infection than in those with non-KL2/10/22/52 infection. Patients with KL2/10/22/52 infection and patients with CRAB infection have a higher cumulative incidence of attributable and all-cause in-hospital 30-day mortality. On multivariate analysis, appropriate empirical antimicrobial therapy within 24 h was associated with a lower risk of 30-day attributable mortality in the KL2/10/22/52 isolates (odds ratio = 0.19, 95% CI: 0.06–0.66, p = 0.008) but not in non-KL2/10/22/52 isolates. Early recognition of carbapenem resistance-associated capsular types may help clinicians to promptly implement appropriate antimicrobial therapy for improving the outcomes in patients with CRAB bacteraemia.

Pneumococcal capsular polysaccharide (PCP) is the major virulence determinant of Streptococcus pneumoniae (pneumococcus). Strains devoid of the capsule are avirulent or highly attenuated. PCP is present in soluble form and on pneumococci in infected individuals. The present study was undertaken to study the interaction of PCP from serotype 1 (PCP1) with immune cells, and its proinflammatory, immunomodulatory and antigenic properties. Binding of PCP1 to the surface of immune cells led to proinflammatory cytokine production which was not cell line or cytokine restricted. HEK293T transfectants expressing TLR1 and TLR2 produced IL-8 upon stimulation with PCP1, untransfected cells did not do so. PCP1 failed to induce TNF-α production from RAW264.7 cells when pre-incubated with a TLR2 blocking antibody. The surface binding of PCP1 was abrogated in the presence of TLR2 blocking antibody. PCP1 failed to bind TLR2 deficient RAW264.7 cells and induce TNF-α production. Unlike PCP1, alkali-treated PCP1 failed to stimulate RAW264.7 cells to produce TNF-α indicating the importance of alkali-sensitive moieties like O-acetyl groups. Alkali-treated PCP1 elicited lower anti-PCP1 antibody response. Mice experiments suggested that alkali-sensitive groups are significant target of protective antibodies in PCP1 immunized mice. Our findings demonstrate that PCP1 is an important modulator of immune response against pneumococci.

Comparative Study Haemophilus influenzae is an opportunistic pathogen adapted to the human respiratory tract. Non-typeable H. influenzae are highly heterogeneous, but few studies have analysed the genomic variability of capsulated strains. This study aims to examine the genetic diversity of 37 serotype f isolates from the Netherlands, Portugal, and Spain, and to compare all capsulated genomes available on public databases. Serotype f isolates belonged to CC124 and shared few single nucleotide polymorphisms (SNPs) (n = 10,999), but a high core genome (> 80%). Three main clades were identified by the presence of 75, 60 and 41 exclusive genes for each clade, respectively. Multi-locus sequence type analysis of all capsulated genomes revealed a reduced number of clonal complexes associated with each serotype. Pangenome analysis showed a large pool of genes (n = 6360), many of which were accessory genome (n = 5323). Phylogenetic analysis revealed that serotypes a, b, and f had greater diversity. The total number of SNPs in serotype f was significantly lower than in serotypes a, b, and e (p < 0.0001), indicating low variability within the serotype f clonal complexes. Capsulated H. influenzae are genetically homogeneous, with few lineages in each serotype. Serotype f has high genetic stability regardless of time and country of isolation. Tis study was funded by Instituto de Salud Carlos III (ISCIII) through the Projects from the Fondo de Investigaciones Sanitarias “PI16/00977” to SM, and CIBER de Enfermedades Respiratorias (CIBERES–CB06/06/0037), co-funded by the European Regional Development Fund/European Social Fund (ERDF/ESF, “Investing in your future”), and CERCA Programme/Generalitat de Catalunya for institutional support. Bioinformatic analysis was supported by an Amazon Web Services (AWS) research grant to SM. AC was supported by FPU grant “FPU16/02202” (Formación de Profesorado Universitario, Ministerio de Educación, Spain), and SM was supported by Miguel Servet contract “CP19/00096” (ISCIII). info:eu-repo/semantics/publishedVersion

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.

Background and purpose Nox4 is the major isoform of NADPH oxidase found in the kidney and contributes to the pathogenesis of diabetic nephropathy. However, the molecular mechanisms of increased Nox4 expression induced by hyperglycaemia remain to be elucidated. Here, the role of the connexin32-Nox4 signalling axis in diabetic nephropathy and its related mechanisms were investigated. Experimental approach Diabetes was induced in mice by low-dose streptozotocin (STZ) combined with a high-fat diet. Effects of connexin32 on Nox4 expression and on renal function and fibrosis in STZ-induced diabetic mice were investigated using adenovirus-overexpressing connexin32 and connexin32-deficient mice. Interactions between connexin32 and Nox4 were analysed by co-immunoprecipitation and immunofluorescence assays. Key results Connexin32 was down-regulated in the kidneys of STZ-induced diabetic mice. Overexpression of connexin32 reduced expression of Nox4 and improved renal function and fibrosis in diabetic mice, whereas connexin32 deficiency had opposite effects. Down-regulation of fibronectin expression by connexin32 was not dependent on gap junctional intercellular communication involving connexin32. Connexin32 interacted with Nox4 and reduced the generation of hydrogen peroxide, leading to the down-regulation of fibronectin expression. Mechanistically, connexin32 decreased Nox4 expression by promoting its K48-linked polyubiquitination. Interestingly, Smurf1 overexpression inhibited K48-linked polyubiquitination of Nox4. Furthermore, connexin32 interacted with Smurf1 and inhibited its expression. Conclusion and implications Connexin32 ameliorated renal fibrosis in diabetic mice by promoting K48-linked Nox4 polyubiquitination and degradation via inhibition of Smurf1 expression. Targeting the connexin32-Nox4 signalling axis may contribute to the development of novel treatments for diabetic nephropathy.

Pasteurella multocida possesses a polysaccharide capsule composed of a viscous surface layer that acts as a critical structural component and virulence factor. Capsular polysaccharides are structurally similar to vertebrate glycosaminoglycans, providing an immunological mechanism for bacterial molecular mimicry, resistance to phagocytosis, and immune evasion during the infection process. In recent years, a series of important research advances have been made in understanding the biosynthesis and regulatory aspects of the P. multocida capsule. This review systematically examines the serogroups, polysaccharide composition and structures, biosynthetic loci and functions, biosynthesis pathways, and expression regulation mechanisms of the P. multocida capsule, supplying a theoretical basis for the molecular pathogenesis of the P. multocida capsule and the future development of capsular polysaccharide vaccines.

To support vaccine decision-making we estimated from the societal perspective the potential health impact and costs averted through immunization with three vaccines - Haemophilus influenzae type b (Hib), pneumococcal conjugate vaccine (PCV) and rotavirus vaccine (RVV).Based on variability in disease burden, strength of health system and economic status, we selected four states in India: Bihar, New Delhi, Maharashtra and Tamil Nadu. We used secondary data sources to estimate the number of under-5 deaths averted from Hib, pneumococcus and rotavirus in each state and back-calculated the total cases averted. We synthesized available data to estimate the disease burden, treatment cost, caretaker productivity loss and vaccine coverage in each state. A Delphi Survey and roundtable among Indian experts was conducted to reach consensus on model inputs.By scaling up coverage of Hib, PCV and RVV, India could save over US$1 billion (uncertainty range: US$0.9-US$2.4 billion) in economic benefits and avert more than 90,000 needless child deaths each year. An estimated US$1 billion (US$0.9-US$2 billion) or 88% of the total amount of cost savings would be attributable to lost productivity due to premature pneumococcal death. Another US$112.8 million (US$105-297 million), or 10% of the total cost would be accounted by costs related to loss of productivity due to disability as a result of these diseases. Treatment costs of Hib, pneumococcal disease and rotavirus gastroenteritis, would account for US$8.4 million (US$4-12 million) or1% of the total costs of these diseases. Finally, caretaker productivity loss from seeking care would represent US$1.5 million (US$ 1-4.9 million). Cost savings varied by vaccine, coverage scenarios and states.Hib, PCV and RVV vaccine introduction in India can result in immediate benefits to the government and households in terms of savings.

Background K1 capsular polysaccharide (CPS)-associated Klebsiella pneumoniae is the primary cause of pyogenic liver abscesses (PLA) in Asia. Patients with PLA often have serious complications, ultimately leading to a mortality of ~ 5%. This K1 CPS has been reported as a promising target for development of glycoconjugate vaccines against K. pneumoniae infection. The pyruvylation and O-acetylation modifications on the K1 CPS are essential to the immune response induced by the CPS. To date, however, obtaining the fragments of K1 CPS that contain the pyruvylation and O-acetylation for generating glycoconjugate vaccines still remains a challenge. Methods We analyzed the digested CPS products with NMR spectroscopy and mass spectrometry to reveal a bacteriophage-derived polysaccharide depolymerase specific to K1 CPS. The biochemical and biophysical properties of the enzyme were characterized and its crystal structures containing bound CPS products were determined. We also performed site-directed mutagenesis, enzyme kinetic analysis, phage absorption and infectivity studies, and treatment of the K. pneumoniae-infected mice with the wild-type and mutant enzymes. Results We found a bacteriophage-derived polysaccharide lyase that depolymerizes the K1 CPS into fragments of 1–3 repeating trisaccharide units with the retention of the pyruvylation and O-acetylation, and thus the important antigenic determinants of intact K1 CPS. We also determined the 1.46-Å-resolution, product-bound crystal structure of the enzyme, revealing two distinct carbohydrate-binding sites in a trimeric β-helix architecture, which provide the first direct evidence for a second, non-catalytic, carbohydrate-binding site in bacteriophage-derived polysaccharide depolymerases. We demonstrate the tight interaction between the pyruvate moiety of K1 CPS and the enzyme in this second carbohydrate-binding site to be crucial to CPS depolymerization of the enzyme as well as phage absorption and infectivity. We also demonstrate that the enzyme is capable of protecting mice from K1 K. pneumoniae infection, even against a high challenge dose. Conclusions Our results provide insights into how the enzyme recognizes and depolymerizes the K1 CPS, and demonstrate the potential use of the protein not only as a therapeutic agent against K. pneumoniae, but also as a tool to prepare structurally-defined oligosaccharides for the generation of glycoconjugate vaccines against infections caused by this organism.

Escherichia coliandSalmonellaisolates produce a range of different polysaccharide structures that play important roles in their biology.E. coliisolates often possess capsular polysaccharides (K antigens), which form a surface structural layer. These possess a wide range of repeat-unit structures.

Anthrax is considered one of the most dangerous bioweapon agents, and concern about multidrug-resistant strains has led to the development of alternative therapeutic approaches that target the antiphagocytic capsule, an essential virulence determinant of

Polysaccharide capsule is the main virulence factor ofK.pneumoniae, a major pathogen of bloodstream infections in humans. While more than 80 capsular serotypes have been identified inK.pneumoniae, only several serotypes are frequently identified in invasive infections. It is documented that the capsule enhances bacterial resistance to phagocytosis, antimicrobial peptides and complement deposition underin vitroconditions. However, the precise role of the capsule in the process ofK.pneumoniaebloodstream infections remains to be elucidated. Here we show that the capsule promotesK.pneumoniaesurvival in the bloodstream by protecting bacteria from being captured by liver resident macrophage Kupffer cells (KCs). Our real-timein vivoimaging revealed that blood-borne acapsularK.pneumoniaemutant is rapidly captured and killed by KCs in the liver sinusoids of mice, whereas, to various extents, encapsulated strains bypass the anti-bacterial machinery in a serotype-dependent manner. Using capsule switched strains, we show that certain high-virulence (HV) capsular serotypes completely block KC’s capture, whereas the low-virulence (LV) counterparts confer partial protection against KC’s capture. Moreover, KC’s capture of the LVK.pneumoniaecould bein vivoneutralized by free capsular polysaccharides of homologous but not heterologous serotypes, indicating that KCs specifically recognize the LV capsules. Finally, immunization with inactivatedK.pneumoniaeenables KCs to capture the HVK.pneumoniae. Together, our findings have uncovered that KCs are the major target cells ofK.pneumoniaecapsule to promote bacterial survival and virulence, which can be reversed by vaccination.

MOP (Multidrug/Oligosaccharidyl-lipid/Polysaccharide) family transporters are found in almost all life forms. They are responsible for transporting lipid-linked precursors across the cell membrane to support the synthesis of various glycoconjugates. While significant progress has been made in elucidating their transport mechanism, how these transporters select their substrates remains unclear. Here, we systematically tested the MOP transporters in the Streptococcus pneumoniae capsule pathway for their ability to translocate noncognate capsule precursors. Sequence similarity cannot predict whether these transporters are interchangeable. We showed that subtle changes in the central aqueous cavity of the transporter are sufficient to accommodate a different cargo. These changes can occur naturally, suggesting a potential mechanism of expanding substrate selectivity. A directed evolution experiment was performed to identify gain-of-function variants that translocate a noncognate cargo. Coupled with a high-throughput mutagenesis and sequencing (Mut-seq) experiment, residues that are functionally important for the capsule transporter were revealed. Lastly, we showed that the expression of a flippase that can transport unfinished precursors resulted in an increased susceptibility to bacitracin and mild cell shape defects, which may be a driving force to maintain transporter specificity. IMPORTANCE All licensed pneumococcal vaccines target the capsular polysaccharide (CPS). This layer is highly variable and is important for virulence in many bacterial pathogens. Most of the CPSs are produced by the Wzx/Wzy mechanism. In this pathway, CPS repeating units are synthesized in the cytoplasm, which must be flipped across the cytoplasmic membrane before polymerization. This step is mediated by the widely conserved MOP (Multidrug/Oligosaccharidyl-lipid/Polysaccharide) family transporters. Here, we systematically evaluated the interchangeability of these transporters and identified the residues important for substrate specificity and function. Understanding how CPS is synthesized will inform glycoengineering, vaccine development, and antimicrobial discovery.