Examining the Distribution and Impact of Single-Nucleotide Polymorphisms in the Capsular Locus of Streptococcus pneumoniae Serotype 19A.

Streptococcus pneumoniae serotype 19A prevalence has increased after the implementation of the PCV7 and PCV10 vaccines. In this study, we have provided, with high accuracy, the genetic diversity of the 19A serotype in a cohort of Dutch invasive pneumococcal disease patients and asymptomatic carriers obtained in the period from 2004 to 2016. The whole genomes of the 338 pneumococcal isolates in this cohort were sequenced and their capsule (cps) loci compared to examine their diversity and determine the impact on the production of capsular polysaccharide (CPS) sugar precursors and CPS shedding. We discovered 79 types with a unique cps locus sequence. Most variation was observed in the rmlB and rmlD genes of the TDP-Rha synthesis pathway and in the wzg gene, which is of unknown function. Interestingly, gene variation in the cps locus was conserved in multiple alleles. Using RmlB and RmlD protein models, we predict that enzymatic function is not affected by the single-nucleotide polymorphisms as identified. To determine if RmlB and RmlD function was affected, we analyzed nucleotide sugar levels using ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS). CPS precursors differed between 19A cps locus subtypes, including TDP-Rha, but no clear correlation was observed. Also, significant differences in multiple nucleotide sugar levels were observed between phylogenetically branched groups. Because of indications of a role for Wzg in capsule shedding, we analyzed if this was affected. No clear indication of a direct role in shedding was found. We thus describe genotypic variety in rmlB, rmlD, and wzg in serotype 19A in the Netherlands, for which we have not discovered an associated phenotype.

Detection and epidemiology of carbapenemase producing Enterobacteriaceae in the Netherlands in 2013-2014.

Laboratory detection of carbapenemase-producing Enterobacteriaceae (CPE) is complicated. Screening with MIC values below clinical breakpoints followed by genotypic confirmation is recommended. We evaluated the application of recommended CPE screening and confirmation methods and provide an overview of CPE epidemiology in E. coli and K. pneumoniae in the Netherlands. Data on E. coli and K. pneumoniae isolates with elevated meropenem (>0.25 mg/L) and/or imipenem (>1 mg/L) MIC values in 2013-2014 were selected from the Infectious Disease Surveillance Information System for Antibiotic Resistance. Laboratories were requested to provide additional results of any confirmatory testing performed. Confirmation of elevated carbapenem MIC values using gradient testing was performed in 59.8 % of eligible isolates. Confirmatory testing showed elevated MIC values in 8 % of E. coli and 32 % of K. pneumoniae isolates. The overall proportion of confirmed non-susceptible E. coli and K. pneumoniae was 0.01 % and 0.16 %, respectively. Genotypic confirmation was performed in 61.0 % of isolates with confirmed elevated carbapenem MIC values. A carbapenemase gene was identified in 47 % of E. coli and 65 % of K. pneumoniae isolates. OXA-48, NDM and KPC were the most frequently found carbapenemase genes. The majority (62 %) of CPE isolates was detected through targeted screening. CPE are a rare finding in the Netherlands. Adherence to the national guideline is suboptimal and differs between laboratories, implying a risk of inadequate CPE detection. Since accurate identification of CPE is the first step in prevention of CPE spread, successful implementation of guidelines for testing and reporting of CPE is essential.

Variation at the capsule locus, cps, of mistyped and non-typable Streptococcus pneumoniae isolates.

The capsule polysaccharide locus (cps) is the site of the capsule biosynthesis gene cluster in encapsulated Streptococcus pneumoniae. A set of pneumococcal samples and non-pneumococcal streptococci from Denmark, the Gambia, the Netherlands, Thailand, the UK and the USA were sequenced at the cps locus to elucidate serologically mistyped or non-typable isolates. We identified a novel serotype 33B/33C mosaic capsule cluster and previously unseen serotype 22F capsule genes, disrupted and deleted cps clusters, the presence of aliB and nspA genes that are unrelated to capsule production, and similar genes in the non-pneumococcal samples. These data provide greater understanding of diversity at a locus which is crucial to the antigenic diversity of the pathogen and current vaccine strategies.

Two DHH subfamily 1 proteins contribute to pneumococcal virulence and confer protection against pneumococcal disease.

Streptococcus pneumoniae is an important human bacterial pathogen, causing such infections as pneumonia, meningitis, septicemia, and otitis media. Current capsular polysaccharide-based conjugate vaccines protect against a fraction of the over 90 serotypes known, whereas vaccines based on conserved pneumococcal proteins are considered promising broad-range alternatives. The pneumococcal genome encodes two conserved proteins of an as yet unknown function, SP1298 and SP2205, classified as DHH (Asp-His-His) subfamily 1 proteins. Here we examined their contribution to pneumococcal pathogenesis using single and double knockout mutants in three different strains: D39, TIGR4, and BHN100. Mutants lacking both SP1298 and SP2205 were severely impaired in adherence to human epithelial Detroit 562 cells. Importantly, the attenuated phenotypes were restored upon genetic complementation of the deleted genes. Single and mixed mouse models of colonization, otitis media, pneumonia, and bacteremia showed that bacterial loads in the nasopharynx, middle ears, lungs, and blood of mice infected with the mutants were significantly reduced from those of wild-type-infected mice, with an apparent additive effect upon deletion of both genes. Minor strain-specific phenotypes were observed, i.e., deletion of SP1298 affected host-cell adherence in BHN100 only, and deletion of SP2205 significantly attenuated virulence in lungs and blood in D39 and BHN100 but not TIGR4. Finally, subcutaneous vaccination with a combination of both DHH subfamily 1 proteins conferred protection to nasopharynx, lungs, and blood of mice infected with TIGR4. We conclude that SP1298 and SP2205 play a significant role at several stages of pneumococcal infection, and importantly, these proteins are potential candidates for a multicomponent protein vaccine.

Genome-wide identification of Streptococcus pneumoniae genes essential for bacterial replication during experimental meningitis.

Meningitis is the most serious of invasive infections caused by the Gram-positive bacterium Streptococcus pneumoniae. Vaccines protect only against a limited number of serotypes, and evolving bacterial resistance to antimicrobials impedes treatment. Further insight into the molecular pathogenesis of invasive pneumococcal disease is required in order to enable the development of new or adjunctive treatments and/or pneumococcal vaccines that are efficient across serotypes. We applied genomic array footprinting (GAF) in the search for S. pneumoniae genes that are essential during experimental meningitis. A total of 6,000 independent TIGR4 marinerT7 transposon mutants distributed over four libraries were injected intracisternally into rabbits, and cerebrospinal fluid (CSF) was collected after 3, 9, and 15 h. Microarray analysis of mutant-specific probes from CSF samples and inocula identified 82 and 11 genes mutants of which had become attenuated or enriched, respectively, during infection. The results point to essential roles for capsular polysaccharides, nutrient uptake, and amino acid biosynthesis in bacterial replication during experimental meningitis. The GAF phenotype of a subset of identified targets was followed up by detailed studies of directed mutants in competitive and noncompetitive infection models of experimental rat meningitis. It appeared that adenylosuccinate synthetase, flavodoxin, and LivJ, the substrate binding protein of a branched-chain amino acid ABC transporter, are relevant as targets for future therapy and prevention of pneumococcal meningitis, since their mutants were attenuated in both models of infection as well as in competitive growth in human cerebrospinal fluid in vitro.

Development of a non-invasive murine infection model for acute otitis media.

Otitis media (OM) is one of the most frequent diseases in childhood, and Streptococcus pneumoniae is among the main causative bacterial agents. Since current experimental models used to study the bacterial pathogenesis of OM have several limitations, such as the invasiveness of the experimental procedures, we developed a non-invasive murine OM model. In our model, adapted from a previously developed rat OM model, a pressure cabin is used in which a 40 kPa pressure increase is applied to translocate pneumococci from the nasopharyngeal cavity into both mouse middle ears. Wild-type pneumococci were found to persist in the middle ear cavity for 144 h after infection, with a maximum bacterial load at 96 h. Inflammation was confirmed at 96 and 144 h post-infection by IL-1beta and TNF-alpha cytokine analysis and histopathology. Subsequently, we investigated the contribution of two surface-associated pneumococcal proteins, the streptococcal lipoprotein rotamase A (SlrA) and the putative proteinase maturation protein A (PpmA), to experimental OM in our model. Pneumococci lacking the slrA gene, but not those lacking the ppmA gene, were significantly reduced in virulence in the OM model. Importantly, pneumococci lacking both genes were significantly more attenuated than the DeltaslrA single mutant. This additive effect suggests that SlrA and PpmA exert complementary functions during experimental OM. In conclusion, we have developed a highly reproducible and non-invasive murine infection model for pneumococcal OM using a pressure cabin, which is very suitable to study pneumococcal pathogenesis and virulence in vivo.

Surface-associated lipoprotein PpmA of Streptococcus pneumoniae is involved in colonization in a strain-specific manner.

Streptococcus pneumoniae produces two surface-associated lipoproteins that share homology with two distinct families of peptidyl-prolyl isomerases (PPIases), the streptococcal lipoprotein rotamase A (SlrA) and the putative proteinase maturation protein A (PpmA). Previously, we have demonstrated that SlrA has PPIase activity, and that the enzyme plays a role in pneumococcal virulence. Here, we investigated the contribution of PpmA to pneumococcal pathogenesis. Pneumococcal mutants of D39 and TIGR4 lacking the gene encoding PpmA were less capable of persisting in the nasopharynx of mice, demonstrating the contribution of PpmA to pneumococcal colonization. This observation was partially confirmed in vitro, as the pneumococcal mutants NCTC10319DeltappmA and TIGR4DeltacpsDeltappmA, but not D39DeltacpsDeltappmA, were impaired in adherence to Detroit 562 pharyngeal cells. This suggests that the contribution of PpmA to pneumococcal colonization is not solely the result of its role in adherence to epithelial cells. Deficiency in PpmA did not result in reduced binding to various extracellular matrix and serum proteins. Similar to SlrA, we observed that PpmA was involved in immune evasion. Uptake of PpmA-deficient D39Deltacps and NCTC10319 by human polymorphonuclear leukocytes was significantly enhanced compared to the isogenic wild-types. In addition, ingestion of D39DeltappmA, but not that of either NCTC10319DeltappmA or TIGR4DeltappmA, by murine macrophage cell line J774 was also enhanced, whereas intracellular killing remained unaffected. We conclude that PpmA contributes to the early stages of infection, i.e. colonization. The contribution of PpmA to virulence can be explained by its strain-specific role in adherence to epithelial cells and contribution to the evasion of phagocytosis.

Species- and strain-specific control of a complex, flexible regulon by Bordetella BvgAS.

The Bordetella master virulence regulatory system, BvgAS, controls a spectrum of gene expression states, including the virulent Bvg(+) phase, the avirulent Bvg(-) phase, and at least one Bvg-intermediate (Bvg(i)) phase. We set out to define the species- and strain-specific features of this regulon based on global gene expression profiling. Rather than functioning as a switch, Bvg controls a remarkable continuum of gene expression states, with hundreds of genes maximally expressed in intermediate phases between the Bvg(+) and Bvg(-) poles. Comparative analysis of Bvg regulation in B. pertussis and B. bronchiseptica revealed a relatively conserved Bvg(+) phase transcriptional program and identified previously uncharacterized candidate virulence factors. In contrast, control of Bvg(-)- and Bvg(i)-phase genes diverged substantially between species; regulation of metabolic, transporter, and motility loci indicated an increased capacity in B. bronchiseptica, compared to B. pertussis, for ex vivo adaptation. Strain comparisons also demonstrated variation in gene expression patterns within species. Among the genes with the greatest variability in patterns of expression, predicted promoter sequences were nearly identical. Our data suggest that the complement of transcriptional regulators is largely responsible for transcriptional diversity. In support of this hypothesis, many putative transcriptional regulators that were Bvg regulated in B. bronchiseptica were deleted, inactivated, or unregulated by BvgAS in B. pertussis. We propose the concept of a "flexible regulon." This flexible regulon may prove to be important for pathogen evolution and the diversification of host range specificity.

Diversity in the Bordetella virulence regulon: transcriptional control of a Bvg-intermediate phase gene.

The BvgAS signal transduction system controls the expression of at least three distinct phenotypic phases that lie along a continuum of gene expression states. The Bvg+ phase is characterized by the expression of adhesins and toxins, whereas the Bvg- phase is characterized by motility in Bordetella bronchiseptica and the expression of vrg loci in Bordetella pertussis. The Bvg-intermediate (Bvgi) phase is characterized by the absence of Bvg-repressed phenotypes, the expression of some, but not all, Bvg-activated virulence factors and the presence of a recently discovered set of antigens and phenotypes that are unique to this phase. We report here the transcriptional regulation of bipA, the first-identified Bvgi phase gene. We have mapped the bipA promoter and identified numerous BvgA binding sites in the transcriptional control region. Based on these data, we present a model in which phase-dependent expression of bipA results from the spatial distribution and relative affinities of multiple BvgA binding sites relative to the start site of transcription.

Multilocus sequence typing system for Campylobacter jejuni.

The gram-negative bacterium Campylobacter jejuni has extensive reservoirs in livestock and the environment and is a frequent cause of gastroenteritis in humans. To date, the lack of (i) methods suitable for population genetic analysis and (ii) a universally accepted nomenclature has hindered studies of the epidemiology and population biology of this organism. Here, a multilocus sequence typing (MLST) system for this organism is described, which exploits the genetic variation present in seven housekeeping loci to determine the genetic relationships among isolates. The MLST system was established using 194 C. jejuni isolates of diverse origins, from humans, animals, and the environment. The allelic profiles, or sequence types (STs), of these isolates were deposited on the Internet (http://mlst.zoo.ox.ac.uk), forming a virtual isolate collection which could be continually expanded. These data indicated that C. jejuni is genetically diverse, with a weakly clonal population structure, and that intra- and interspecies horizontal genetic exchange was common. Of the 155 STs observed, 51 (26% of the isolate collection) were unique, with the remainder of the collection being categorized into 11 lineages or clonal complexes of related STs with between 2 and 56 members. In some cases membership in a given lineage or ST correlated with the possession of a particular Penner HS serotype. Application of this approach to further isolate collections will enable an integrated global picture of C. jejuni epidemiology to be established and will permit more detailed studies of the population genetics of this organism.

Genesis of BRO beta-lactamase-producing Moraxella catarrhalis: evidence for transformation-mediated horizontal transfer.

The dramatic rise in BRO-producing M. catarrhalis strains observed in the last decades is without precedence. The aim of this study was to elucidate the events that led to the emergence of BRO-1 and BRO-2 beta-lactamases. Previously, we showed bro1 and bro2 to be >99% identical. Data presented here suggested that bro2 was acquired by a fortuitous event and inserted between M. catarrhalis genes orf1 and orf3. Subsequently, bro1 evolved from bro2. Promoter-up mutations increased fitness of bro2, explaining its present predominance. The highly conserved nature of bro compared with orf1 and orf3 suggested that acquisition has occurred relatively recently. The random distribution of bro among M. catarrhalis fingerprint types indicated that bro has spread by horizontal transfer. Sequence analysis revealed that 80-200 bp is generally cotransferred with bro, serving as regions of homology that target bro to the same chromosomal locus. A region of 160 bases upstream of bro1 lacked polymorphism, indicating it was derived from the original strain that acquired bro2. We observed that bro was readily transferred by transformation between M. catarrhalis strains in vitro, suggesting a mechanism by which bro has disseminated. In conclusion, we have been able to reconstruct the steps that led to the emergence of BRO-producing M. catarrhalis.

Analysis of Moraxella catarrhalis by DNA typing: evidence for a distinct subpopulation associated with virulence traits.

Two DNA typing methods, probe-generated restriction fragment length polymorphism analysis and single-adapter amplified fragment length polymorphism analysis, were used to study the genetic relationships among 90 Moraxella catarrhalis strains. Both methods were found to be highly concordant, generating a dendrogram with 2 main branches. The division of the M. catarrhalis population into 2 subspecies was supported by analysis of the 16S rRNA sequences. Both beta-lactamase-positive and beta-lactamase-negative strains were found in all main branches, suggesting horizontal transfer of the beta-lactamase gene. In contrast, 2 virulence traits, complement resistance and adherence to epithelial cells, were strongly associated with 1 of the 2 subspecies. The branch depth suggested that complement-resistant adherent strains diverged from a common ancestor more recently than did complement-sensitive nonadherent strains. These findings suggest the existence of subpopulations of M. catarrhalis that differ in virulence, and they may have implications for vaccine development.

Moraxella (Branhamella) catarrhalis BRO beta-lactamase: a lipoprotein of gram-positive origin?

In the past 20 years, BRO beta-lactamase-producing Moraxella catarrhalis strains have emerged. We show that BRO is expressed as a 33-kDa lipoprotein associated with the inner leaflet of the outer membrane. To our knowledge, this is the first description of a lipidated beta-lactamase in a gram-negative species.

Molecular characterization of the BRO beta-lactamase of Moraxella (Branhamella) catarrhalis.

A rapid increase in the prevalence of beta-lactamase-producing Moraxella (Branhamella) catarrhalis strains has been noticed during the last decades. Today, more than 80% of strains isolated worldwide produce beta-lactamase. To investigate beta-lactamase(s) of M. catarrhalis at the molecular level, the BRO-1 beta-lactamase gene (bla) was isolated as part of a 4,223-bp HindIII fragment. Sequence analysis indicated that bla encodes a polypeptide of 314 amino acid residues. Insertional inactivation of bla in M. catarrhalis resulted in complete abrogation of beta-lactamase production and ampicillin resistance, demonstrating that bla is solely responsible for beta-lactam resistance. Comparison with other beta-lactamases suggested that M. catarrhalis beta-lactamase is a unique enzyme with conserved residues at the active sites. The presence of a signal sequence for lipoproteins suggested that it is lipid modified at its N terminus. In keeping with this assumption was the observation that 10% of beta-lactamase activity was found in the membrane compartment of M. catarrhalis. M. catarrhalis strains produce two types of beta-lactamase, BRO-1 and BRO-2, which differ in their isoelectric points. The BRO-1 and BRO-2 genes from two ATCC strains of M. catarrhalis were sequenced, and only one amino acid difference was found between the predicted products. However, there was a 21-bp deletion in the promoter region of the BRO-2 gene, possibly explaining the lower level of production of BRO-2. The G + C content of bla (31%) was significantly lower than those of the flanking genes (47 and 50%), and the overall G + C content of the M. catarrhalis genome (41%). These results indicate that bla was acquired by horizontal gene transfer from another, still unknown species.

Protective effects of orally administered, Klebsiella-containing bacterial lysates in mice.

The efficacy, as oral vaccines, of hepta- and mono-valent, Klebsiella-containing bacterial lysates and a number of control preparations was tested in mice. The preparations were administered during two periods of four days each, interrupted by an interval of 3 days. Fourteen days after the first dose, the animals were challenged either intraperitoneally (i.p.; peritonitis/sepsis model) or intranasally (i.n.; pneumonia model). Animals treated with low doses of Klebsiella lysate, in the form of either a 7-valent lysate or a Klebsiella monolysate, showed enhanced survival in both the peritonitis/sepsis and the pneumonia models. Hexa- and tetra-valent preparations without Klebsiella were not protective in the models tested. Furthermore, it was found that the protection is accompanied by priming for Klebsiella-specific IgG responsiveness (probably at the T cell level) and by significant IgA anti-Klebsiella serum antibody levels in about one third of the animals. The oral efficacy of Klebsiella-containing lysates suggests the presence of an adjacent component that directs Klebsiella antigen(s) to follow a selective intestinal pathway which renders them immunogenic. The identity of this component is under investigation.

Antibodies against phosphatidylinositol and inositol monophosphate specifically inhibit tumour necrosis factor induction by malaria exoantigens.

The active component of the exoantigens of malarial parasites which stimulates macrophages to secrete tumour necrosis factor (TNF) has been shown to depend upon a phospholipid, the activity of which was blocked by phosphatidylinositol (PI) and inositol monophosphate (IMP) in competitive inhibition studies. Antisera made against the exoantigens of Plasmodium yoelii, which inhibited their induction of TNF, were found by an ELISA assay to contain antibody against several other phospholipids. However, the inhibitory antibody was removed specifically by adsorption with liposomes containing PI, but not other phospholipids. Furthermore, PI was the only phospholipid in non-liposomal form which induced the production of inhibitory antisera. Mice immunized with IMP, but not inositol, also produced inhibitory antisera. When incorporated into liposomes several other phospholipids did give rise to inhibitory antibodies but, in contrast to the antisera against parasite exoantigens, PI and IMP, the inhibitory activity was removed by adsorption with heterologous phospholipid liposomes, suggesting that it was directed against a common determinant, presumably the phosphate ester head group. Inhibitory antibodies in the antisera tested were predominantly IgM and titres were not increased after repeated injections. Antisera raised against PI, IMP or the cross-reacting phospholipid liposomes also inhibited TNF secretion by macrophages stimulated by exoantigens of the human parasites P. falciparum and P. vivax, but not by bacterial lipopolysaccharide. These findings confirm our conclusion that exoantigens from these different species contain phosphate bound to inositol in their TNF-inducing moiety.

Rapid isolation of human complement component C9 to verify the specificity of a haemolytic C9 microassay.

A sensitive, haemolytic microassay of human complement component C9 was developed. The assay is based on the principle of reactive (C5b6-initiated) haemolysis and uses commercially available C9-depleted serum as reagent for C9. The specificity of the assay was verified by rapid, activity-guided isolation of the haemolytic component from human serum using high-performance liquid chromatography (HPLC) on a system for fast protein liquid chromatography. This isolation yielded a single component with characteristics of C9. The results suggest that rapid, activity-guided isolation as a new application of HPLC can be a useful tool to demonstrate the specificity of a functional assay.

Initial characterization of the four promoters of the human insulin-like growth factor II gene.

The human insulin-like growth factor II (IGF-II) gene contains four promoters (P1-P4), which are expressed in a tissue-specific and development-dependent way. Analysis of IGF-II mRNAs in different tissues has revealed that promoters P3 and P4 are expressed in all fetal and in nonhepatic adult tissues. In adult liver, however, the promoters P2, P3 and P4 are completely shut off and another promoter, P1, is activated. To obtain more insight in the mechanisms involved in the regulation of IGF-II gene expression we have performed an initial characterization of the IGF-II promoters employing transient expression of IGF-II promoter constructs in Hep3B and HeLa cells. These studies have revealed that promoters P1, P3 and P4 are active in both cell lines tested, while no activity of promoter P2 could be detected. Employing gel retardation and DNaseI footprint analysis we have identified in the three IGF-II promoters a number of elements which are bound by nuclear proteins.