Pneumococcal Vaccines.

Streptococcus pneumoniae is a Gram-Positive pathogen that is a major causative agent of pneumonia, otitis media, sepsis and meningitis across the world. The World Health Organization estimates that globally over 500,000 children are killed each year by this pathogen. Vaccines offer the best protection against S. pneumoniae infections. The current polysaccharide conjugate vaccines have been very effective in reducing rates of invasive pneumococcal disease caused by vaccine type strains. However, the effectiveness of these vaccines have been somewhat diminished by the increasing numbers of cases of invasive disease caused by non-vaccine type strains, a phenomenon known as serotype replacement. Since, there are currently at least 98 known serotypes of S. pneumoniae, it may become cumbersome and expensive to add many additional serotypes to the current 13-valent vaccine, to circumvent the effect of serotype replacement. Hence, alternative serotype independent strategies, such as vaccination with highly cross-reactive pneumococcal protein antigens, should continue to be investigated to address this problem. This chapter provides a comprehensive discussion of pneumococcal vaccines past and present, protein antigens that are currently under investigation as vaccine candidates, and other alternatives, such as the pneumococcal whole cell vaccine, that may be successful in reducing current rates of disease caused by S. pneumoniae.

Acanthamoeba encephalitis: A Case Report and Review of Therapy.

BACKGROUND: Acanthamoeba is a rare cause of encephalitis yet is associated with high mortality. Treatment protocols vary greatly and generally include combination therapy across a wide spectrum of antiinfective classes. CASE DESCRIPTION: A 63-year-old male who underwent renal transplantation presented 6 months after transplantation with depressed level of consciousness. Imaging of the head with computerized tomography showed an enhancing lesion suspicious for brain abscess. Biopsy of the lesion showed Acanthamoeba cysts. The patient was treated with sulfadiazine, fluconazole, flucytosine, azithromycin, and miltefosine but without success. We review recently published cases of Acanthamoeba encephalitis with an emphasis on treatment protocols and outcomes. CONCLUSION: Free-living protozoans such as Acanthamoeba are ubiquitous in the environment and should be suspected in immunosuppressed persons who present with central nervous system findings and brain abscess. Biopsy is critical to establish the etiology so that appropriate combination therapy can be deployed.

PspK of Streptococcus pneumoniae increases adherence to epithelial cells and enhances nasopharyngeal colonization.

Streptococcus pneumoniae (the pneumococcus) colonizes the human nasopharynx and can cause invasive disease aided by the pneumococcal capsule. Group II nontypeable S. pneumoniae (NTSp) lacks a polysaccharide capsule, and a subgroup of NTSp carriage isolates has been found to have a novel gene, pneumococcal surface protein K (pspK), which replaces the capsule locus. A recent rise in the number of NTSp isolates colonizing the human nasopharynx has been observed, but the colonization factors of NTSp have not been well studied. PspK has been shown to play a role in mouse colonization. We therefore examined PspK-mediated immune evasion along with adherence to host cells and colonization. PspK bound human secretory immunoglobulin A (sIgA) but not the complement regulator factor H and did not decrease C3b deposition on the pneumococcal surface. PspK increased binding of pneumococci to epithelial cells and enhanced pneumococcal colonization independently of the genetic background. Understanding how NTSp colonizes and survives within the nasopharynx is important due to the increase in NTSp carriage. Our data suggest that PspK may aid in the persistence of NTSp within the nasopharynx but is not involved in invasion.

Differential gene expression in Streptococcus pneumoniae in response to various iron sources.

Iron is a critical co-factor for several enzymes and is known to regulate gene expression in many pathogens. Streptococcus pneumoniae (pneumococcus) normally colonizes the upper respiratory mucosa, which is an iron-restricted environment. In contrast, during bacteremia available iron from heme and non-heme proteins potentially increases. In iron-depleted medium pneumococcal strain TIGR4 showed reduced growth, however, addition of several physiological iron sources restored growth. Gene expression of selected known and putative pneumococcal virulence factors was analyzed by quantitative RT-PCR in response to iron sources in vitro and during colonization, pneumonia, and bacteremia in a mouse model. Change in mRNA levels relative to transcription in iron-depleted medium was reported. In presence of iron sources, transcription of cps4A, zmpA, pavA, hemolysin and a putative exfoliative toxin was significantly increased, but nanB was suppressed. Hemoglobin at physiological concentration repressed ply and pspA expression. Ferritin, an acute phase protein, increased expression of an iron ABC transporter and repressed expression of a bacterial non-heme iron-containing ferritin. Transcription of cps4A, nanB, hemolysin, and a putative exfoliative toxin were significantly up-regulated during pneumonia and bacteremia, while mRNA of pavA and non-heme ferritin were expressed at higher levels during pneumonia and carriage. An iron ABC transporter was most up-regulated during bacteremia, while pspA and ply were expressed only in pneumonia. Transcription of zmpA was elevated during both pneumonia and bacteremia. These findings suggest that a subset of virulence genes in pneumococci is differentially regulated in response to the quantity and form of iron sources available in a host.

Immunization with polyamine transport protein PotD protects mice against systemic infection with Streptococcus pneumoniae.

The human pathogen Streptococcus pneumoniae contains genes for a putative polyamine ABC transporter which are organized in an operon and designated potABCD. Polyamine transport protein D (PotD) is an extracellular protein which binds polyamines and possibly other structurally related molecules. PotD has been shown to contribute to virulence in both a murine sepsis model and a pneumonia model with capsular type 3 pneumococci. The protective efficacy of recombinant PotD was evaluated by active immunization and intravenous challenge with capsular type 3 pneumococci in CBA/N mice. Immunized mice had 91.7% survival following lethal pneumococcal challenge, compared with 100% mortality in the control group. Immunized animals had high-titer anti-PotD antibodies following three immunizations with alum. Protection in a sepsis model was also seen after passive administration of rabbit antiserum raised against PotD (P < 0.004). These results suggest that antibodies to PotD confer protection against invasive pneumococcal disease and that this protein should be studied further as a potential vaccine candidate for protection against invasive pneumococcal infections.

Use of a putative transcriptional regulator gene as target for specific identification of Staphylococcus epidermidis.

AIMS: To investigate the use of a Staphylococcus epidermidis transcriptional regulator gene as target for species-specific determination. METHODS AND RESULTS: Staph. epidermidis genes encoding putative transcriptional regulators were retrieved from GenBank and those showing no homology with other bacterial sequences were selected. Of the four PCR primer sets analysed, the primers Serp0107F/R from serp0107 amplified a specific product of 581 bp from Staph. epidermidis DNA only, and they did not cross-react in PCR with nonepidermidis staphylococci and other common bacteria. CONCLUSION: Being uniquely present in Staph. epidermidis, putative transcriptional regulator gene serp0107 offers a valuable target for specific identification of Staph. epidermidis. SIGNIFICANCE AND IMPACT OF THE STUDY: As a member of a specialized gene group, putative transcriptional regulator gene serp0107 may be important to Staph. epidermidis adaptation to its niche environment. Further analysis of serp0107 and its related protein may help reveal new insights on the molecular regulation of Staph. epidermidis survival and virulence.

Involvement of potD in Streptococcus pneumoniae polyamine transport and pathogenesis.

Polyamines such as putrescine, spermidine, and cadaverine are small, polycationic molecules that are required for optimal growth in all cells. The intracellular concentrations of these molecules are maintained by de novo synthesis and transport pathways. The human pathogen Streptococcus pneumoniae possesses a putative polyamine transporter (pot) operon that consists of the four pot-specific genes potABCD. The studies presented here examined the involvement of potD in polyamine transport and in pneumococcal pathogenesis. A potD-deficient mutant was created in the mouse-virulent serotype 3 strain WU2 by insertion duplication mutagenesis. The growth of the WU2DeltapotD mutant was identical to that of the wild-type strain WU2 in vitro in rich media. However, WU2DeltapotD possessed severely delayed growth compared to wild-type WU2 in the presence of the polyamine biosynthesis inhibitors DFMO (alpha-dimethyl-fluoroornitithine) and MGBG [methylgloxal-bis (guanyl hydrazone)]. The mutant strain also showed a significant attenuation in virulence within murine models of systemic and pulmonary infection regardless of the inoculation route or location. These data suggest that potD is involved in pneumococcal polyamine transport and is important for pathogenesis within various infection models.

Utilization of putrescine by Streptococcus pneumoniae during growth in choline-limited medium.

Polyamines such as putrescine are small, ubiquitous polycationic molecules that are required for optimal growth of eukaryotic and prokaryotic cells. These molecules have diverse effects on cell physiology and their intracellular content is regulated by de novo synthesis and uptake from the environment. The studies presented here examined the structure of a putative polyamine transporter (Pot) operon in Streptococcus pneumoniae (pneumococcus) and growth of pneumococci in medium containing putrescine substituted for choline. RT-PCR experiments demonstrated that the four genes encoding the Pot system are co-transcribed with murB, a gene involved in an intermediary step of peptidoglycan synthesis. Pneumococci grown in chemically-defined media (CDM) containing putrescine without choline enter logarithmic phase growth after 36-48 hs. However, culture density at stationary phase eventually reaches that of choline-containing medium. Cells grown in CDM-putrescine formed abnormally elongated chains in which the daughter cells failed to separate and the choline-binding protein PspA was no longer cell-associated. Experiments with CDM containing radiolabeled putrescine demonstrated that pneumococci concentrate this polyamine in cell walls. These data suggest that pneumococci can replicate without choline if putrescine is available and this polyamine may substitute for aminoalcohols in the cell wall teichoic acids.

Pneumococcal surface protein A is expressed in vivo, and antibodies to PspA are effective for therapy in a murine model of pneumococcal sepsis.

Pneumococcal surface protein A (PspA) is an immunogenic protein expressed on the surface of all strains of Streptococcus pneumoniae (pneumococcus) and induces antibodies which protect against invasive infection in mice. Pneumococci used for infectious challenge in protection studies are typically collected from cultures grown in semisynthetic medium in vitro. The purpose of these studies is to confirm that PspA is expressed by pneumococci during growth in vivo at a level sufficient for antibodies to PspA to be protective. Mice were actively immunized with purified PspA or by passive transfer of monoclonal antibody (MAb) and challenged with a capsular type 3 strain in diluted whole blood from bacteremic mice. All were protected against challenge with 10 times the 50% lethal dose (LD(50)), and mice challenged with 1,000 times the LD(50) had increased survival compared with controls. Additionally, nonimmune mice treated with MAbs to PspA or PspA immune serum at 6 and 12 h after infection with 10 times the LD(50) also showed increased survival. Northern blot analysis of RNA from pneumococci grown either in vitro or in vivo showed similar levels of PspA mRNA. These results demonstrate that PspA is expressed in vivo in a mouse model and that immunization with PspA induces antibodies to an antigen which is expressed during the course of invasive infection. Immunotherapy with antibodies to PspA may have some utility in treating pneumococcal infections in humans.

Genetic immunization with the region encoding the alpha-helical domain of PspA elicits protective immunity against Streptococcus pneumoniae.

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 of pspA to elicit a protective immune response following genetic immunization of mice. Mice were immunized by intramuscular injections with a eukaryotic expression vector encoding the alpha-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 microg 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 with pspA was 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.

In vitro activity of four fluoroquinolones against clinical isolates of Pseudomonas aeruginosa determined by the E test.

Ciprofloxacin, levofloxacin, ofloxacin, and trovafloxacin were tested by the E-test against 100 clinical isolates of Pseudomonas aeruginosa. Ciprofloxacin was the most active of the tested agents with 82% of isolates having a MIC 8). Levofloxacin and trovafloxacin had nearly identical potency: 75% and 76% of the isolates were inhibited by 8 for levofloxacin; 0.19->8 for trovafloxacin). Ofloxacin was the least active of the four quinolones, with 43% of the isolates having a MIC >2 mg/l. All isolates resistant to ciprofloxacin were also resistant to the other agents, i.e. resistance to ciprofloxacin predicted resistance to all the quinolones tested in every case. This data demonstrates that fluoroquinolones are active agents against P. aeruginosa. In vitro susceptibility testing, however, is crucial to assess the resistance pattern in any specific location and for each individual agent.

Intranasal immunization of mice with a mixture of the pneumococcal proteins PsaA and PspA is highly protective against nasopharyngeal carriage of Streptococcus pneumoniae.

Acquisition of pneumococci is generally from carriers rather than from infected individuals. Therefore, to induce herd immunity against Streptococcus pneumoniae it will be necessary to elicit protection against carriage. Capsular polysaccharide-protein conjugates, PspA, and PsaA are known to elicit some protection against nasopharyngeal carriage of pneumococci but do not always completely eliminate carriage. In this study, we observed that PsaA elicited better protection than did PspA against carriage. Pneumolysin elicited no protection against carriage. Immunization with a mixture of PsaA and PspA elicited the best protection against carriage. These results indicate that PspA and PsaA may be useful for the elicitation of herd immunity in humans. As PspA and pneumolysin are known to elicit immunity to bacteremia and pneumonia, their inclusion in a mucosal vaccine may enable such a vaccine to prevent invasive disease as well as carriage.

Pneumococcal diversity: considerations for new vaccine strategies with emphasis on pneumococcal surface protein A (PspA).

Streptococcus pneumoniae is a problematic infectious agent, whose seriousness to human health has been underscored by the recent rise in the frequency of isolation of multidrug-resistant strains. Pneumococcal pneumonia in the elderly is common and often fatal. Young children in the developing world are at significant risk for fatal pneumococcal respiratory disease, while in the developed world otitis media in children results in substantial economic costs. Immunocompromised patients are extremely susceptible to pneumococcal infection. With 90 different capsular types thus far described, the diversity of pneumococci contributes to the challenges of preventing and treating S. pneumoniae infections. The current capsular polysaccharide vaccine is not recommended for use in children younger than 2 years and is not fully effective in the elderly. Therefore, innovative vaccine strategies to protect against this agent are needed. Given the immunogenic nature of S. pneumoniae proteins, these molecules are being investigated as potential vaccine candidates. Pneumococcal surface protein A (PspA) has been evaluated for its ability to elicit protection against S. pneumoniae infection in mouse models of systemic and local disease. This review focuses on immune system responsiveness to PspA and the ability of PspA to elicit cross-protection against heterologous strains. These parameters will be critical to the design of broadly protective pneumococcal vaccines.

Comparative in vitro activities of four new fluoroquinolones against Streptococcus pneumoniae determined by Etest.

Clinafloxacin, levofloxacin, sparfloxacin and trovafloxacin were tested by Etest against 188 Streptococcus pneumoniae isolates. Clinafloxacin and trovafloxacin were 2-4-fold more potent than sparfloxacin and 8-fold more than levofloxacin. Two isolates, both serotype 6, with high-level quinolone resistance (> or = 8 micrograms/ml) were detected. The Etest is a practical means for determining S. pneumoniae susceptibilities to new fluoroquinolones.

In vitro activities of oral antimicrobial agents against penicillin-resistant Streptococcus pneumoniae: implications for outpatient treatment.

We tested 83 penicillin-intermediate (Peni) and 50 penicillin-resistant (Penr) isolates of Streptococcus pneumoniae against eight oral antimicrobials. Clarithromycin's MICs (minimal inhibitory concentration) were generally the same or one to two dilutions less than those of azithromycin. Seventy-two percent of Peni isolates were susceptible to clarithromycin and azithromycin, in contrast to 42% and 40%, respectively, of Penr isolates. Cefuroxime activity exceeded that of cefprozil, which exceeded that of cefaclor, in Peni isolates. For all three cephalosporins, MICs of 90% of isolates tested were > or = 3 dilutions higher for Penr isolates than for Peni isolates. Percentages of Peni isolates susceptible to clindamycin and tetracycline were 92% and 83%, respectively, and 78% and 82% for Penr. Only 49% of Peni isolates and 4% of Penr isolates were susceptible to trimethoprim-sulfamethoxazole. Azithromycin, clarithromycin, cefuroxime, cefprozil, clindamycin, and tetracycline may be useful in treating infections caused by Peni S pneumoniae, but Penr isolates are frequently resistant to both old and newer agents.

Oligonucleotides identify conserved and variable regions of pspA and pspA-like sequences of Streptococcus pneumoniae.

Pneumococcal surface protein A (PspA) is an immunogenic surface protein of Streptococcus pneumoniae. PspA of S. pneumoniae strain Rx1 is a 65-kDa protein composed of an alpha-helical N-terminus of 288 amino acids followed by an 82-amino-acid proline-rich region, 10 repeats of 20 amino acids each, and a 17-amino-acid C-terminus. It has been demonstrated that the 3'-half of pspA is relatively conserved among unrelated pneumococcal isolates and the 5'-half of the gene is highly variable. Additionally, nearly all pneumococcal strains contain at least one other locus with sequence homology to pspA. In this study oligonucleotides derived from the DNA sequence of pspA of Rx1 were used both as hybridization probes and as primers in the polymerase chain reaction (PCR) to investigate genetic variation within domains of pspA and in the pspA-like sequences from 18 strains representing 12 capsule and 9 PspA serotypes. Sequences encoding the leader peptide, the proline-rich region, and the repeat region are highly conserved among pspA and pspA-like sequences. The alpha-helical coding domain is highly diverse among pspA and pspA-like sequences of different strains.

In vitro activities of cefepime versus cefotaxime and ceftriaxone against penicillin-resistant Streptococcus pneumoniae determined by Etest.

The Etest was used for determining in vitro susceptibilities of 144 unique clinical isolates of penicillin-intermediate and resistant Streptococcus pneumoniae to cefepime, cefotaxime, and ceftriaxone. MIC ranges were 0.12-8 mug/ml for cefepime and 0.06-16 mug/ml for cefotaxime and ceftriaxone. MICs for 50% of the isolates for the three agents were equivalent at 1 mug/ml, whereas MICs for 90% of the isolates were 2 mug/ml for cefotaxime and ceftriaxone, versus 4 mug/ml for cefepime. The Etest is a practical means for determining susceptibilities of S. pneumoniae to cefepime and other cephalosporins in diagnostic laboratories.

DNA polymorphisms and variant penicillin-binding proteins as evidence that relatively penicillin-resistant pneumococci in western Canada are clonally related.

Previous studies have suggested that relatively penicillin-resistant (RPR) capsular group 9L strains in western Canada may be clonally related. To test this hypothesis, restriction fragment length polymorphisms (RFLPs) were examined using DNA probes for pspA and a newly recognized pneumococcal genetic element, IS1167. Penicillin-binding proteins (PBPs) and PBP genes from representative strains were also studied. All RPR type 9L strains demonstrated an identical RFLP when probed with IS1167, and 12 of 14 RPR strains had the same RFLP when examined with pspA. Amplification of pspA by polymerase chain reaction and restriction endonuclease digestion showed that the 9L strains had common DNA fragments not identified in any of the penicillin-susceptible strains. The 9L strains apparently have a low-affinity PBP 2B distinct from those of other capsular types. These data derived from new genetic markers and PBP analysis strongly support a clonal origin of RPR type 9L pneumococci of western Canada.

PspA, a protection-eliciting pneumococcal protein: immunogenicity of isolated native PspA in mice.

PspA is a surface exposed virulence factor of S. pneumoniae that can elicit protective immunity to pneumococcal sepsis in mice. It can be released from pneumococci by washing them with a solution containing 2% choline chloride, by growing pneumococci in media containing 1.2% choline chloride, or by growing pneumococci in media in which the choline has been replaced by ethanolamine. Our results indicate that PspA is the major protection-eliciting antigen in each of these preparations. Two injections of < or = 1 microgram of native PspA purified by use of a choline-Sepharose column are highly immunogenic in BALB/c and CBA/N mice, and even in the absence of adjuvant can elicit protection against otherwise fatal sepsis with 100 times the LD50 of S. pneumoniae. Fragments comprising the N-terminal 115 and 245 amino acids of PspA were able to elicit protection but only in the presence of complete Freund's adjuvant (CFA). In the absence of CFA the 245 amino acid fragment was less than 1/100 as immunogenic as native PspA.