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Haemophilus influenzae causes common and sometimes severe adult and pediatric disease including chronic obstructive respiratory disease, otitis media and infections of the central nervous system. Serotype b strains, with a b-type capsule, have been the historical cause of invasive disease, and the introduction of a serotype b-specific vaccine has led to their decline. However, unencapsulated or non-b-type H. influenzae infections are not prevented by the vaccine and appear to be increasing in frequency. Here we report two pediatric cases of severe central nervous system H. influenzae infection presenting to the same hospital in San Diego, California during the same week in January 2016. Due to good vaccine coverage in this part of the world, H. influenzae cases are normally rare and seeing two cases in the same week was unexpected. We thus suspected a recent transmission chain, and possible local outbreak. To test this hypothesis, we isolated and sequenced whole genomes from each patient and placed them in a phylogenetic tree spanning the known diversity of H. influenzae. Surprisingly, we found that the two isolates (SD2016_1 and SD2016_2) belonged to distantly related lineages, suggesting two independent transmission events and ruling out a local outbreak. Despite being distantly related, the two isolates belong to two different lineages that have exchanged capsule loci in the recent past. Therefore, as in other bacterial pathogens, capsule switching by horizontal gene transfer may be an important evolutionary mechanism of vaccine evasion in H. influenzae.

Endothelial cell (EC) metabolism is thought to be one of the driving forces for angiogenesis. Here we report the identification of the hexosamine D-mannosamine (ManN) as an EC mitogen and survival factor for bovine and human microvascular EC, with an additivity with VEGF. ManN inhibits glycosylation in ECs and induces significant changes in N-glycan and O-glycan profiles. We further demonstrate that ManN and two N-glycosylation inhibitors stimulate EC proliferation via both JNK activation and the unfolded protein response caused by ER stress. ManN results in enhanced angiogenesis in a mouse skin injury model. ManN also promotes angiogenesis in a mouse hindlimb ischemia model, with accelerated limb blood flow recovery compared to controls. In addition, intraocular injection of ManN induces retinal neovascularization. Therefore, activation of stress pathways following inhibition of protein glycosylation can promote EC proliferation and angiogenesis and may represent a therapeutic strategy for treatment of ischemic disorders.

Haemophilus influenzae causes common and sometimes severe adult and pediatric disease including chronic obstructive respiratory disease, otitis media and infections of the central nervous system. Serotype b strains, with a b-type capsule, have been the historical cause of invasive disease, and the introduction of a serotype b-specific vaccine has led to their decline. However, unencapsulated or non-b-type H. influenzae infections are not prevented by the vaccine and appear to be increasing in frequency. Here we report two pediatric cases of severe central nervous system H. influenzae infection presenting to the same hospital in San Diego, California during the same week in January 2016. Due to good vaccine coverage in this part of the world, H. influenzae cases are normally rare and seeing two cases in the same week was unexpected. We thus suspected a recent transmission chain, and possible local outbreak. To test this hypothesis, we isolated and sequenced whole genomes from each patient and placed them in a phylogenetic tree spanning the known diversity of H. influenzae. Surprisingly, we found that the two isolates (SD2016_1 and SD2016_2) belonged to distantly related lineages, suggesting two independent transmission events and ruling out a local outbreak. Despite being distantly related, the two isolates belong to two different lineages that have exchanged capsule loci in the recent past. Therefore, as in other bacterial pathogens, capsule switching by horizontal gene transfer may be an important evolutionary mechanism of vaccine evasion in H. influenzae.

Soft-rot Enterobacteriaceae (SRE), typified by Pectobacterium and Dickeya genera, are phytopathogenic bacteria inflicting soft-rot disease in crops worldwide. By combining genomic information from 100 SRE with whole-transcriptome data sets, we identified novel genomic and transcriptional associations among key pathogenicity themes in this group. Comparative genomics revealed solid linkage between the type I secretion system (T1SS) and the carotovoricin bacteriophage (Ctv) conserved in 96.7% of Pectobacterium genomes. Moreover, their coactivation during infection indicates a novel functional association involving T1SS and Ctv. Another bacteriophage-borne genomic region, mostly confined to less than 10% of Pectobacterium strains, was found, presumably comprising a novel lineage-specific prophage in the genus. We also detected the transcriptional coregulation of a previously predicted toxin/immunity pair (WHH and SMI1_KNR4 families), along with the type VI secretion system (T6SS), which includes hcp and/or vgrG genes, suggesting a role in disease development as T6SS-dependent effectors. Further, we showed that another predicted T6SS-dependent endonuclease (AHH family) exhibited toxicity in ectopic expression assays, indicating antibacterial activity. Additionally, we report the striking conservation of the group 4 capsule (GFC) cluster in 100 SRE strains which consistently features adjacently conserved serotype-specific gene arrays comprising a previously unknown organization in GFC clusters. Also, extensive sequence variations found in gfcA orthologs suggest a serotype-specific role in the GfcABCD machinery. Importance: Despite the considerable loss inflicted on important crops yearly by Pectobacterium and Dickeya diseases, investigations on key virulence and interbacterial competition assets relying on extensive comparative genomics are still surprisingly lacking for these genera. Such approaches become more powerful over time, underpinned by the growing amount of ge

Haemophilus influenzae type b (Hib) affects 337,000 Indian children every year. A vaccine against Hib was introduced in 2011 as part of the pentavalent vaccine and scaled up nationwide. This study investigated the associations between Hib vaccination and child anthropometry, cognition, and schooling outcomes in India. We used longitudinal survey data and employed propensity score matching to control for observed systematic differences between children who reported receipt or nonreceipt of Hib vaccine before age 6 years (n = 1824). Z-scores of height-for-age (HAZ) and BMI-for-age (BMIZ), percentage scores of English, mathematics, reading, and Peabody Picture Vocabulary tests, and attained schooling grade of children were examined. Hib-vaccinated children had 0.25 higher HAZ, scored 4.09 percentage points (pp) higher on the English test and 4.78 pp higher on the mathematics test, and attained 0.16 more schooling grades than Hib-unvaccinated children at age 11-12 years. At age 14-15 years, they had 0.18 higher HAZ, scored 3.63 pp higher on the reading test and 3.22 pp higher on the mathematics test, and attained 0.15 more schooling grades than Hib-unvaccinated children. The findings indicate potential long-term health, cognitive, and schooling benefits of the Hib vaccine, subject to the effect of unobserved confounding factors.

Francisella tularensis is the causative agent of tularemia and a Tier I bioterrorism agent. In the 1900s, several vaccines were developed against tularemia including the killed "Foshay" vaccine, subunit vaccines comprising F. tularensis protein(s) or lipoproteins(s) in an adjuvant formulation, and the F. tularensis Live Vaccine Strain (LVS); none were licensed in the U.S.A. or European Union. The LVS vaccine retains toxicity in humans and animals-especially mice-but has demonstrated efficacy in humans, and thus serves as the current gold standard for vaccine efficacy studies. The U.S.A. 2001 anthrax bioterrorism attack spawned renewed interest in vaccines against potential biowarfare agents including F. tularensis. Since live attenuated-but not killed or subunit-vaccines have shown promising efficacy and since vaccine efficacy against respiratory challenge with less virulent subspecies holarctica or F. novicida, or against non-respiratory challenge with virulent subsp. tularensis (Type A) does not reliably predict vaccine efficacy against respiratory challenge with virulent subsp. tularensis, the route of transmission and species of greatest concern in a bioterrorist attack, in this review, we focus on live attenuated tularemia vaccine candidates tested against respiratory challenge with virulent Type A strains, including homologous vaccines derived from mutants of subsp. holarctica, F. novicida, and subsp. tularensis, and heterologous vaccines developed using viral or bacterial vectors to express F. tularensis immunoprotective antigens. We compare the virulence and efficacy of these vaccine candidates with that of LVS and discuss factors that can significantly impact the development and evaluation of live attenuated tularemia vaccines. Several vaccines meet what we would consider the minimum criteria for vaccines to go forward into clinical development-safety greater than LVS and efficacy at least as great as LVS, and of these, several meet the higher standard of h

Group A Streptococcus (GAS) commonly infects human skin and occasionally causes severe and life-threatening invasive diseases. The hyaluronan (HA) capsule of GAS has been proposed to protect GAS from host defense by mimicking endogenous HA, a large and abundant glycosaminoglycan in the skin. However, HA is degraded during tissue injury, and the functions of short-chain HA that is generated during infection have not been studied. To examine the impact of the molecular mass of HA on GAS infection, we established infection models in vitro and in vivo in which the size of HA was defined by enzymatic digestion or custom synthesis. We discovered that conversion of high molecular mass HA to low molecular mass HA facilitated GAS phagocytosis by macrophages and limited the severity of infection in mice. In contrast, native high molecular mass HA significantly impaired internalization by macrophages and increased GAS survival in murine blood. Thus, our data demonstrate that GAS virulence can be influenced by the size of HA derived from both the bacterium and host and suggest that high molecular mass HA facilitates GAS deep tissue infections, whereas the generation of short-chain HA can be protective.

The pathogen Campylobacter jejuni is the principal cause of bacterial food-borne infections. The mechanism(s) that contribute to bacterial survival and disease are still poorly understood. In other bacterial species, type VI secretion systems (T6SS) are increasingly recognized to contribute to bacterial pathogenesis by toxic effects on host cells or competing bacterial species. Here we report the presence of a functional Type VI secretion system in C. jejuni. Proteome and genetic analyses revealed that C. jejuni strain 108 contains a 17-kb T6SS gene cluster consisting of 13 T6SS-conserved genes, including the T6SS hallmark genes hcp and vgrG. The cluster lacks an ortholog of the ClpV ATPase considered important for T6SS function. The sequence and organization of the C. jejuni T6SS genes resemble those of the T6SS located on the HHGI1 pathogenicity island of Helicobacter hepaticus. The C. jejuni T6SS is integrated into the earlier acquired Campylobacter integrated element CJIE3 and is present in about 10% of C. jejuni isolates including several isolates derived from patients with the rare clinical feature of C. jejuni bacteremia. Targeted mutagenesis of C. jejuni T6SS genes revealed T6SS-dependent secretion of the Hcp needle protein into the culture supernatant. Infection assays provided evidence that the C. jejuni T6SS confers contact-dependent cytotoxicity towards red blood cells but not macrophages. This trait was observed only in a capsule-deficient bacterial phenotype. The unique C. jejuni T6SS phenotype of capsule-sensitive contact-mediated hemolysis represents a novel evolutionary pathway of T6SS in bacteria and expands the repertoire of virulence properties associated with T6SS.

The presence of pXO1- and/or pXO2-like plasmid(s) in clinical isolates of B. cereus (sensu stricto) and in strains of the biopesticide Bacillus thuringiensis have been reported recently, and the pXO2-like plasmid pBT9727 was found conjugative as well as another pXO2-like plasmid pAW63. In this study, a total of 1,000 B. cereus group isolates were analyzed for the presence of pXO1- and pXO2-like replicons and for pXO2-related conjugative modules. The occurrence frequency of pXO1- and pXO2-like replicons in random environmental samples was ca. 6.6% and 7.7%, respectively, and ca. 1.54% of the strains were positive for pXO2-like transfer module genes. Only the strains harboring a pXO2-like replicon also contained the corresponding transfer genes. For the strains which contained pXO1- and/or pXO2-like replicon(s), large plasmid(s) with a size similar to that of pXO1-like and/or pXO2-like plasmids were also observed, but none of these isolates were found to carry the B. anthracis toxin or capsule virulence genes. Furthermore, 17 out of 22 pXO2-like plasmids containing the transfer modules displayed the capability of self-transfer and of mobilizing small plasmids. No pXO1-, or pXO2-like plasmid lacking the cognate transfer modules has been found to have transfer potential. Among the strains possessing the putative pXO2-like conjugative apparatus, variations on the presence of the group II introns B.th.I.1 and B.th.I.2 were observed, suggesting an important flexibility of the conjugation modules and their regulation. There was no consistent correlation between pXO2-like repA dendrogram and presence/absence of tra region, or between virB4 dendrogram and transfer capabilities. Discrepancies between pXO2-like repA and virB4 dendrograms were also observed, indicating that the evolution of pXO2 is an active process.

Several circulating Bacillus anthracis strains isolated in Italy and belonging to the A1.a cluster, genotype 3 (A1.a-3) are genotypically indistinguishable from Carbosap, a live attenuated vaccine strain, containing both pXO1 and pXO2 plasmids. The genotype was assessed by using eight-locus multilocus variable-number tandem repeat analysis. We describe here the use of a ninth locus able to explore variability among strains that have the same genotype. It is important to be able to genotype the wild isolate of B. anthracis strains from outbreaks of anthrax in areas where Carbosap vaccination of cattle and sheep is common practice. A total of 27 representative field strains isolated in Italy and four vaccinal strains, namely, Carbosap, Sterne, Pasteur I, and Pasteur II, were characterized by a ninth marker, called pXO2-A. Twenty-three field strains were genotype 3 and therefore identical to Carbosap. The marker was in the pXO2 plasmid and is based on the polymorphism of the already-known VX2-3 locus. Detection was obtained by PCR with fluorescence-labeled forward primers in order to produce appropriate fragments for capillary electrophoresis with an ABI 310 genetic analyzer. Genetic relationships showed heterogeneity in all of the examined samples. Interestingly, with respect to genotype 3, samples grouped into eight different subtypes, A to H, and the subtype G, had only two samples indistinguishable from Carbosap. The results of the present study confirm the validity of a hierarchical progressive protocol for discrimination among closely related isolates.

Serotype-specific mortality from invasive Streptococcus pneumoniae disease revisited.Martens P, Worm SW, Lundgren B, Konradsen HB, Benfield T. Department of Infectious Diseases 144, Hvidovre University Hospital, DK-2650 Hvidovre, Denmark. pernillemartens@yahoo.com BACKGROUND: Invasive infection with Streptococcus pneumoniae (pneumococci) causes significant morbidity and mortality. Case series and experimental data have shown that the capsular serotype is involved in the pathogenesis and a determinant of disease outcome. METHODS: Retrospective review of 464 cases of invasive disease among adults diagnosed between 1990 and 2001. Multivariate Cox proportional hazard analysis. RESULTS: After adjustment for other markers of disease severity, we found that infection with serotype 3 was associated with an increased relative risk (RR) of death of 2.54 (95% confidence interval (CI): 1.22-5.27), whereas infection with serotype 1 was associated with a decreased risk of death (RR 0.23 (95% CI, 0.06-0.97)). Additionally, older age, relative leucopenia and relative hypothermia were independent predictors of mortality. CONCLUSION: Our study shows that capsular serotypes independently influenced the outcome from invasive pneumococcal disease. The limitations of the current polysaccharide pneumococcal vaccine warrant the development of alternative vaccines. We suggest that the virulence of pneumococcal serotypes should be considered in the design of novel vaccines.

Serotype-specific mortality from invasive Streptococcus pneumoniae disease revisited.Martens P, Worm SW, Lundgren B, Konradsen HB, Benfield T. Department of Infectious Diseases 144, Hvidovre University Hospital, DK-2650 Hvidovre, Denmark. pernillemartens@yahoo.com BACKGROUND: Invasive infection with Streptococcus pneumoniae (pneumococci) causes significant morbidity and mortality. Case series and experimental data have shown that the capsular serotype is involved in the pathogenesis and a determinant of disease outcome. METHODS: Retrospective review of 464 cases of invasive disease among adults diagnosed between 1990 and 2001. Multivariate Cox proportional hazard analysis. RESULTS: After adjustment for other markers of disease severity, we found that infection with serotype 3 was associated with an increased relative risk (RR) of death of 2.54 (95% confidence interval (CI): 1.22-5.27), whereas infection with serotype 1 was associated with a decreased risk of death (RR 0.23 (95% CI, 0.06-0.97)). Additionally, older age, relative leucopenia and relative hypothermia were independent predictors of mortality. CONCLUSION: Our study shows that capsular serotypes independently influenced the outcome from invasive pneumococcal disease. The limitations of the current polysaccharide pneumococcal vaccine warrant the development of alternative vaccines. We suggest that the virulence of pneumococcal serotypes should be considered in the design of novel vaccines.

Middle ears of male Sprague-Dawley rats were injected with suspensions of thirteen Haemophilus influenzae strains of different sero- and biotypes and at various concentrations. Systemic and local changes were monitored by clinical observations, otomicroscopy, and analysis of bacterial samples from blood and middle ears. Two patterns of response were recognized, a nontypeable and an encapsulated pattern. The nontypeable H. influenzae middle ear infection required a high bacterial dose and was well past its peak 8 days after challenge, when the encapsulated H. influenzae otitis media was still purulent. The most severe infections were caused by H. influenzae type b strains. The overall mortality rate was zero and the animals recovered without permanent deterioration or otomicroscopically discernable changes. The results of this study show the rat to be a suitable animal model for the study of H. influenzae otitis media.