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Cross-reactive “common” pneumococcal antigens offer an attractive alternative, or complement, to polysaccharides and polysaccharide-protein conjugate vaccines. These common antigens should be protective against strains of a wider range of capsular types than can be achieved with conjugate vaccines. Common protein antigens would be expected to be highly immunogenic in young children and should be able to be manufactured relatively inexpensively using recombinant techniques. It is hoped that these antigens will lead to a vaccine(s) that could have application worldwide, even in the poorest developing countries where the rates of fatal pneumococcal disease in children are the highest.

The capsule of Pasteurella multocida serotype A strain ATCC 11039 is composed of hyaluronic acid and is an important virulence factor. Repeated subculturing of certain capsular serotype A strains results in dissociation from a capsulated to a noncapsulated phenotype with a concomitant loss of virulence. Although noncapsulated variants have been thought to arise as a result of mutation, the molecular mechanisms underlying this event are unknown. In this study, we demonstrate that restoration of the capsulated phenotype occurs in vivo subsequent to intraperitoneal inoculation of BALB/c mice with a noncapsulated variant. Moreover, reverse transcription polymerase chain reaction analysis revealed the capsule locus to be under transcriptional control. Cloning and sequencing of a 290-bp fragment within the promoter containing intergenic region of the capsule locus of 11039/iso revealed no significant alterations occurred subsequent to subculturing. These results demonstrate that serotype A P. multocida strain ATCC 11039 regulates capsule expression in response to an unidentified environmental factor(s), thereby providing insights into the molecular mechanisms underlying colonial dissociation.

Serotype A extracellular polysaccharide produced by Pasteurella multocida is composed largely of hyaluronic acid and is an important virulence factor in avian pasteurellosis. Hyaluronidase, mechanical shearing, and serial subculturing were employed to decapsulate strains exhibiting disparate degrees of capsulation, thereby allowing an investigation of the role serotype A capsulation plays in outer cell envelope physiology and virulence of avian isolates. Experimental loss of capsular material occurred concomitantly with an overall increase in cell surface hydrophobicity values, which approached those of naturally-occurring noncapsulated strains. The electronegativity of capsulated cell surfaces was found to be significantly greater than that of both noncapsulated and experimentally decapsulated cells. Naturally-occurring noncapsulated variants were readily phagocytized by mouse peritoneal macrophages, whereas capsulated strains were not. A moderately capsulated variant was phagocytized less effectively than noncapsulated strains. Experimental decapsulation of capsulated variants did not significantly hinder their ability to resist phagocytosis. Moreover, small amounts of residual capsular material appeared to remain after experimental decapsulation as detected with the aid of light and transmission electron microscopy. These data suggest that while cell surface hydrophobicity, charge, and susceptibility to phagocytosis are influenced by the degree to which cells are capsulated with serotype A extracellular polysaccharide, other factors which are unaffected by experimental decapsulation may also be involved in protection from phagocytosis in capsulated avian isolates of P . multocida.

Nonspecific interactions related to physicochemical properties of bacterial cell surfaces, such as hydrophobicity and electrostatic charge, are known to have important roles in bacterium-host cell encounters. Streptococcus pneumoniae (pneumococcus) expresses multiple, surface-exposed, choline-binding proteins (CBPs) which have been associated with adhesion and virulence. The purpose of this study was to determine the contribution of CBPs to the surface characteristics of pneumococci and, consequently, to learn how CBPs may affect nonspecific interactions with host cells. Pneumococcal strains lacking CBPs were derived by adapting bacteria to a defined medium that substituted ethanolamine for choline. Such strains do not anchor CBPs to their surface. Cell surface hydrophobicity was tested for the wild-type and adapted strains by using a biphasic hydrocarbon adherence assay, and electrostatic charge was determined by zeta potential measurement. Adherence of pneumococci to human-derived cells was assessed by fluorescence-activated cell sorter analysis. Strains lacking both capsule and CBPs were significantly more hydrophobic than nonencapsulated strains with a normal complement of CBPs. The effect of CBPs on hydrophobicity was attenuated in the presence of capsule. Removal of CBPs conferred a greater electronegative net surface charge than that which cells with CBPs possessed, regardless of the presence of capsule. Strains that lack CBPs were poorly adherent to human monocyte-like cells when compared with wild-type bacteria with a full complement of CBPs. These results suggest that CBPs contribute significantly to the hydrophobic and electrostatic surface characteristics of pneumococci and may facilitate adherence to host cells partially through nonspecific, physicochemical interactions.

Pneumococcal surface protein A (PspA) is a pneumococcal virulence factor capable of eliciting protection against pneumococcal infection in mice. Previous studies have demonstrated that the protection is antibody mediated. Here we examined the ability ofpspAto elicit a protective immune response following genetic immunization of mice. Mice were immunized by intramuscular injections with a eukaryotic expression vector encoding the α-helical domain of PspA/Rx1. Immunization induced a PspA-specific serum antibody response, and immunized mice survived pneumococcal challenge. Survival and antibody responses occurred in a dose-dependent manner, the highest survival rates being seen with doses of 10 μg or greater. The ability of genetic immunization to elicit cross-protection was demonstrated by the survival of immunized mice challenged with pneumococcal strains differing in capsule and PspA types. Also, immunized mice were protected from intravenous and intratracheal challenges with pneumococci. Similar to the results seen with immunization with PspA, the survival of mice genetically immunized withpspAwas antibody mediated. There was no decline in the level of protection 7 months after immunization. These results support the use of genetic immunization to elicit protective immune responses against extracellular pathogens.

Eleven serotype A encapsulated and nonencapsulated strains of Pasteurella multocida were examined with regard to lipoprotein content. Relative amounts of an approximately 40-kDa lipoprotein (Plp-40) were found to correlate directly with the degree of encapsulation in that heavily encapsulated strains exhibited the greatest amounts, while nonencapsulated strains possessed little or no Plp-40.

Stability of microbial cultures during sampling and storage is a vital issue in various fields of medicine, biotechnology, food science, and forensics. We have developed a unique bacterial preservation process involving a non-toxic, water-soluble acacia gum polymer that eliminates the need for refrigerated storage of samples. The main goal of this study is to characterize the efficacy of acacia gum polymer for preservation of pathogenic bacteria (Bacillus anthracis and methicillin-resistant Staphylococcus aureus-MRSA) on different materials, used for swabbing and filtration: cotton, wool, polyester, rayon, charcoal cloth, and Whatman paper. Acacia gum polymer used for preservation of two pathogens has been shown to significantly protect bacteria during dehydration and storage in all tested samples at the range of temperatures (5-45°C for MRSA and 40-90°C for B. anthracis). Our results showed higher recovery as well as higher viability during the storage of both bacteria in all materials with acacia gum. Addition of acacia gum polymer to swabbing materials or filters will increase efficacy of sample collection and identification of pathogenic bacteria from locations such as hospitals or the environment. Proposed approach can also be used for long-term storage of culture collections, since acacia gum contributes to viability and stability of bacterial cultures.

Genome structural annotation, i.e., identification and demarcation of the boundaries for all the functional elements in a genome (e.g., genes, non-coding RNAs, proteins and regulatory elements), is a prerequisite for systems level analysis. Current genome annotation programs do not identify all of the functional elements of the genome, especially small non-coding RNAs (sRNAs). Whole genome transcriptome analysis is a complementary method to identify “novel” genes, small RNAs, regulatory regions, and operon structures, thus improving the structural annotation in bacteria. In particular, the identification of non-coding RNAs has revealed their widespread occurrence and functional importance in gene regulation, stress and virulence. However, very little is known about non-coding transcripts in Histophilus somni, one of the causative agents of Bovine Respiratory Disease (BRD) as well as bovine infertility, abortion, septicemia, arthritis, myocarditis, and thrombotic meningoencephalitis. In this study, we report a single nucleotide resolution transcriptome map of H. somni strain 2336 using RNA-Seq method. The RNA-Seq based transcriptome map identified 94 sRNAs in the H. somni genome of which 82 sRNAs were never predicted or reported in earlier studies. We also identified 38 novel potential protein coding open reading frames that were absent in the current genome annotation. The transcriptome map allowed the identification of 278 operon (total 730 genes) structures in the genome. When compared with the genome sequence of a non-virulent strain 129Pt, a disproportionate number of sRNAs (∼30%) were located in genomic region unique to strain 2336 (∼18% of the total genome). This observation suggests that a number of the newly identified sRNAs in strain 2336 may be involved in strain-specific adaptations.

Background Streptococcus pneumoniae (pneumococcus) causes respiratory and systemic infections that are a major public health problem worldwide. It has been postulated that pneumococci persist in vivo in biofilm communities. Methods In this study, we analyzed whether pneumococci form biofilms in vivo, and if so, whether biofilms correlated with bacterial persistence. Chinchillas were infected with S. pneumoniae TIGR4 and euthanized at varying times after infection, after which the superior ear bullae were excised and examined by culture and microscopy. Results Dense material, resembling the biofilms of other otitis media pathogens, was visible in the middle ear as late as 12 days after infection. Scanning electron microscopy revealed bacteria within an electron-dense matrix, similar to pneumococcal biofilms formed in vitro. Viability staining revealed groups of viable diplococci, as well as viable and nonviable host cells, attached to a fibrous matrix that was positive when stained with propidium iodide. Cryosections of biofilms were treated with polyclonal antibodies against the pneumococcal surface components pneumococcal surface protein A family 2, pneumococcal surface protein C, choline-binding protein, and neuraminidase, coupled with appropriate secondary antibody conjugates. Immunofluorescent staining showed the presence of pneumococcal communities within the material recovered from the middle ear chamber. Conclusions On the basis of these data, we conclude that pneumococci form biofilms in vivo and that this process may be intertwined with the formation of neutrophil extracellular traps. These findings provide new insights into the potential causes of antibiotic treatment failure and bacterial persistence in chronic pneumococcal otitis media.

Successful colonization of the upper respiratory tract by Streptococcus pneumoniae is an essential first step in the pathogenesis of pneumococcal disease. However, the bacterial and host factors that provoke the progression from asymptomatic colonization to invasive disease are yet to be fully defined. In this study, we investigated the effects of single and combined mutations in genes encoding pneumolysin (Ply), pneumococcal surface protein A (PspA), and pneumococcal surface protein C (PspC, also known as choline-binding protein A) on the pathogenicity of Streptococcus pneumoniae serotype 2 (D39) in mice. Following intranasal challenge with D39, stable colonization of the nasopharynx was maintained over a 7-day period at a level of approximately 10 5 bacteria per mouse. The abilities of the mutant deficient in PspA to colonize the nasopharynx and to cause lung infection and bacteremia were significantly reduced. Likewise, the PspC mutant and, to a lesser extent, the Ply mutant also had reduced abilities to colonize the nasopharynx. As expected, the double mutants colonized less well than the parent to various degrees and had difficulty translocating to the lungs and blood. A significant additive attenuation was observed for the double and triple mutants in pneumonia and systemic disease models. Surprisingly, the colonization profile of the derivative lacking all three proteins was similar to that of the wild type, indicating virulence gene compensation. These findings further demonstrate that the mechanism of pneumococcal pathogenesis is highly complex and multifactorial but ascribes a role for each of these virulence proteins, alone or in combination, in the process.

We report here that animals can be protected against lethal infection by Clostridium tetani cells and Bacillus anthracis spores following topical application of intact particles of live or γ-irradiated Escherichia coli vectors overproducing tetanus and anthrax antigens, respectively. Cutaneous γδT cells were rapidly recruited to the administration site. Live E. coli cells were not found in nonskin tissues after topical application, although fragments of E. coli DNA were disseminated transiently. Evidence suggested that intact E. coli particles in the outer layer of skin may be disrupted by a γδT-cell-mediated innate defense mechanism, followed by the presentation of E. coli ligand-adjuvanted intravector antigens to the immune system and rapid degradation of E. coli components. The nonreplicating E. coli vector overproducing an exogenous immunogen may foster the development of a new generation of vaccines that can be manufactured rapidly and administered noninvasively in a wide variety of disease settings.

Streptococcus pneumoniae cause considerable morbidity and mortality, with persistent neurological sequelae, particularly in young children and the elderly. It is widely assumed that carriage occurs through direct mucosal colonization from the environment whereas meningitis results from invasion from the blood. However, the results of published studies can be interpreted that pneumococci may enter the brain directly from the nasal cavity by axonal transport through olfactory nerves. This hypothesis is based on findings that ( i ) teichoic acid of the pneumococcal cell wall interact with gangliosides (GLS), ( ii ) the interaction of GLS with cholera toxin leads to axonal transport through the olfactory nerves into the brain, and ( iii ) viruses enter the brain through axonal transport into olfactory nerves. After nasal inoculation, we observe high numbers of pneumococci in nasal washes and the olfactory nerves and epithelium. Significant numbers of pneumococci also infected the olfactory bulbs, brain, and the trigeminal ganglia. The absence of bacteremia in this model makes it unlikely that the bacteria entered the brain from the blood stream. Recovery of colony-forming units from the brain, lungs, olfactory nerves, and epithelium and nasal washes was inhibited by incubating pneumococci with GLS before nasal inoculation. These findings, confirmed by PCR and immunohistochemistry, support a GLS-mediated process of infection and are consistent with pneumococci reaching the brain through retrograde axonal transport.

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