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The sudden emergence and global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have greatly accelerated the adoption of novel vaccine strategies, which otherwise would have likely languished for years. In this light, vaccines for certain other pathogens could certainly benefit from reconsideration. One such pathogen is Streptococcus pneumoniae (pneumococcus), an encapsulated bacterium that can express >100 antigenically distinct serotypes. Current pneumococcal vaccines are based exclusively on capsular polysaccharide—either purified alone or conjugated to protein. Since the introduction of conjugate vaccines, the valence of pneumococcal vaccines has steadily increased, as has the associated complexity and cost of production. There are many pneumococcal proteins invariantly expressed across all serotypes, which have been shown to induce robust immune responses in animal models. These proteins could be readily produced using recombinant DNA technology or by mRNA technology currently used in SARS-CoV-2 vaccines. A door may be opening to new opportunities in affordable and broadly protective vaccines.

Infections due to Streptococcus pneumoniae, a commensal in the nasopharynx, still claim a significant number of lives worldwide. Genome plasticity, antibiotic resistance, and limited serotype coverage of the available polysaccharide-based conjugate vaccines confounds therapeutic interventions to limit the spread of this pathogen. Pathogenic mechanisms that allow successful adaption and persistence in the host could be potential innovative therapeutic targets. Polyamines are ubiquitous polycationic molecules that regulate many cellular processes. We previously reported that deletion of polyamine transport operon potABCD, which encodes a putrescine/spermidine transporter (ΔpotABCD), resulted in an unencapsulated attenuated phenotype. Here, we characterize the transcriptome, metabolome, and stress responses of polyamine transport-deficient S. pneumoniae. Compared with the wild-type strain, the expression of genes involved in oxidative stress responses and the nucleotide sugar metabolism was reduced, while expression of genes involved in the Leloir, tagatose, and pentose phosphate pathways was higher in ΔpotABCD. A metabolic shift towards the pentose phosphate pathway will limit the synthesis of precursors of capsule polysaccharides. Metabolomics results show reduced levels of glutathione and pyruvate in the mutant. Our results also show that the potABCD operon protects pneumococci against hydrogen peroxide and nitrosative stress. Our findings demonstrate the importance of polyamine transport in pneumococcal physiology that could impact in vivo fitness. Thus, polyamine transport in pneumococci represents a novel target for therapeutic interventions.

Polyamines such as putrescine, cadaverine, and spermidine are small cationic molecules that play significant roles in cellular processes, including bacterial stress responses and host–pathogen interactions. Streptococcus pneumoniae is an opportunistic human pathogen, which causes several diseases that account for significant morbidity and mortality worldwide. As it transits through different host niches, S. pneumoniae is exposed to and must adapt to different types of stress in the host microenvironment. We earlier reported that S. pneumoniae TIGR4, which harbors an isogenic deletion of an arginine decarboxylase (ΔspeA), an enzyme that catalyzes the synthesis of agmatine in the polyamine synthesis pathway, has a reduced capsule. Here, we report the impact of arginine decarboxylase deletion on pneumococcal stress responses. Our results show that ΔspeA is more susceptible to oxidative, nitrosative, and acid stress compared to the wild-type strain. Gene expression analysis by qRT-PCR indicates that thiol peroxidase, a scavenger of reactive oxygen species and aguA from the arginine deiminase system, could be important for peroxide stress responses in a polyamine-dependent manner. Our results also show that speA is essential for endogenous hydrogen peroxide and glutathione production in S. pneumoniae. Taken together, our findings demonstrate the critical role of arginine decarboxylase in pneumococcal stress responses that could impact adaptation and survival in the host.

Infections due to Streptococcus pneumoniae, a commensal in the nasopharynx, still claim a significant number of lives worldwide. Genetic plasticity, antibiotic resistance, limited serotype coverage of the available polysaccharide-based conjugate vaccines confounds therapeutic interventions. Pathogenic systems that allow successful adaption and persistence in the host could be potential innovative targets for mediations. Polyamines are ubiquitous polycationic molecules and regulate many cellular processes. We previously reported that deletion of potABCD, an operon that encodes a putrescine/spermidine transporter (∆potABCD), resulted in an un-encapsulated attenuated phenotype. Here we characterize the transcriptome, metabolome, and stress responses of S. pneumoniae that is dependent on the polyamine transporter. Expression of genes involved in oxidative stress responses and the central metabolism was reduced while that of genes involved in the Leloir, tagatose, and pentose phosphate pathways was increased in ΔpotABCD. Downregulation of genes of the central metabolism will reduce production of precursors of capsule polysaccharides. Metabolomics results show reduced glutathione and pyruvate levels in the mutant. We also show that the potABCD operon protects pneumococci against hydrogen peroxide and nitrosative stress. These results show the importance of the potABCD operon and polyamine transport in pneumococcal physiology and fitness that represents a novel target for therapeutic interventions.

Infections with Streptococcus pneumoniae (pneumococcus) are a major public health problem worldwide and cause significant morbidity and mortality. Despite their pathogenic potential, pneumococci are usually commensal inhabitants of the upper respiratory tract and colonize mucosal surfaces without symptoms. Infection of divergent sites follows colonization and is a function of both host and bacterial factors. The historical focus of vaccine development against pneumococcal infection has been serum antibody responses to polysaccharide capsule in an effort primarily to prevent invasive disease. Currently available vaccines are based on capsular polysaccharides and include only a small subset of the more than 90 serotypes identified. Because of the usual anatomical location of pneumococci in a host and the large diversity of capsular serotypes, pneumococcal vaccine efforts have been shifting to serotype-independent antigens delivered to mucosal surfaces to prevent the first essential step in pneumococcal infections. In this chapter, we review immune responses at the respiratory mucosa in response to both natural infection and experimental vaccines. Both antibody and Th17-biased CD4+ T cell responses appear to be involved in clearing pneumococci from mucosal surfaces. However, it is still not known what the optimal immune response induced by a mucosal vaccine would look like.

Objective: To review the current literature for the pathogenesis of mucormycosis, discuss diagnostic strategies, and evaluate the efficacy of polyenes, triazoles, and echinocandins as pharmacological treatment options. Data Sources: An electronic literature search was conducted in PubMed using the MESH terms Rhizopus, zygomycetes, zygomycosis, Mucorales and mucormycosis, with search terms amphotericin B, micafungin, anidulafungin, caspofungin, extended infusion amphotericin B, liposomal amphotericin B, combination therapy, triazole, posaconazole, isavuconazole, diagnosis, and clinical manifestations.Study Selection and Data Extraction: Studies written in the English language from January 1960 to March 2016 were considered for this review article. All search results were reviewed, and the relevance of each article was determined by the authors independently. Data Synthesis: Mucormycosis is a rare invasive fungal infection with an exceedingly high mortality and few therapeutic options. It has a distinct predilection for invasion of endothelial cells in the vascular system, which is likely important in dissemination of disease from a primary focus of infection. Six distinct clinical syndromes can occur in susceptible hosts, including rhino-orbital-cerebral, pulmonary, gastrointestinal, cutaneous, widely disseminated, and miscellaneous infection. Conclusion: Diagnosis of mucormycosis is typically difficult to make based on imaging studies, sputum culture, bronchoalveolar lavage culture, or needle aspirate. Surgical debridement prior to dissemination of infection improves clinical outcomes. Surgery combined with early, high-dose systemic antifungal therapy yields greater than a 1.5-fold increase in survival rates. The Mucorales are inherently resistant to most widely used antifungal agents. Amphotericin B is appropriate for empirical therapy, whereas posaconazole and isavuconazole are best reserved for de-escalation, refractory cases, or patients intolerant to amphotericin B.

Streptococcus pneumoniae (S. pneumoniae) is a major pathogen worldwide. The currently available polysaccharide-based vaccines significantly reduce morbidity and mortality. However, the inherent disadvantages of the currently available polysaccharide-based vaccines have motivated the search for other bacterial immunogens capable of eliciting a protective immune response against S. pneumoniae. Fructose-1,6-bisphosphate aldolase (FBA) is a glycolytic enzyme, which was found to localize to the bacterial surface, where it functions as an adhesin. Previously, immunizing mice with recombinant FBA (rFBA) in the presence of alum elicited a protective immune response against a lethal challenge with S. pneumoniae. Thus, the aim of the present study was to determine the cytokine responses that are indicative of protective immunity following immunization with rFBA. The protective effects against pneumococcal challenge in mice immunized with rFBA with complete Freund's adjuvant (CFA) in the initial immunization and with incomplete Freund's adjuvant (IFA) in booster immunizations surpassed the protective effects observed following immunization with either rFBA + alum or pVACfba. CD4+ T-cells obtained from the rFBA/CFA/IFA/IFA-immunized mice co-cultured with rFBA-pulsed antigen-presenting cells (APCs), exhibited a significantly greater proliferative ability than CD4+ T-cells obtained from the adjuvant-immunized mice co-cultured with rFBA‑pulsed APCs. The levels of the Th1-type cytokines, interferon (IFN)-γ, interleukin (IL)-2, tumor necrosis factor (TNF)-α and IL-12, the Th2-type cytokines, IL-4, IL-5 and IL-10, and the Th17-type cytokine, IL-17A, significantly increased within 72 h of the initiation of co-culture with CD4+ T-cells obtained from the rFBA‑immunized mice, in comparison with the co-cultures with CD4+ T-cells obtained from the adjuvant-immunized mice. Immunizing mice with rFBA resulted in an IgG1/IgG2 ratio of 41, indicating a Th2 response with substantial Th1 involvement. In addition, rabbit and mouse anti-rFBA antisera significantly protected the mice against a lethal S. pneumoniae challenge in comparison with preimmune sera. Our results emphasize the mixed involvement of the Th1, Th2 and Th17 arms of the immune system in response to immunization with pneumococcal rFBA, a potential vaccine candidate.

Epidemiologic links between chronic hepatitis C and herpes simplex type-2 infection have been suggested; however, type-specific tests for HSV-2 infection have not been validated in patients with chronic hepatitis C infection. The Focus HerpeSelect(®) HSV-2 IgG (Cypress, California) assay and the Biokit HSV-2 rapid assay (Biokit USA, Lexington, MA) were performed on serum samples obtained from 84 veterans with chronic hepatitis C who demonstrated a previously positive HSV-2 serologic test in their medical records. Using the Biokit HSV-2 as the comparator assay, the positive predictive value, and specificity for the HerpeSelect(®) HSV-2 assay were 62.1% (95%CI: 49.3-73.8) and 41.9% (95%CI: 27.0-57.9), respectively. Increasing the HerpeSelect(®) HSV-2 index value defining a positive test result from >1.1 to ≥2.89 increased the assay's specificity to 97.7% (95%CI: 87.7-99.6) and the positive predictive value to 94.1%(95%CI: 71.2-99.0). J. Med. Virol. 9999: 1-5, 2015. © 2015 Wiley Periodicals, Inc. In veterans with chronic hepatitis C infection, HerpeSelect(®) HSV-2 index values between 1.1 and 2.89 should be confirmed with an alternate test for HSV-2 infection.

Streptococcus pneumoniae is commonly found in the human nasopharynx and is the causative agent of multiple diseases. Since invasive pneumococcal infections are associated with encapsulated pneumococci, the capsular polysaccharide is the target of licensed pneumococcal vaccines. However, there is an increasing distribution of non-vaccine serotypes, as well as nonencapsulated S. pneumoniae (NESp). Both encapsulated and nonencapsulated pneumococci possess the polyamine oligo-transport operon (potABCD). Previous research has shown inactivation of the pot operon in encapsulated pneumococci alters protein expression and leads to a significant reduction in pneumococcal murine colonization, but the role of the pot operon in NESp is unknown. Here, we demonstrate deletion of potD from the NESp NCC1 strain MNZ67 does impact expression of the key proteins pneumolysin and PspK, but it does not inhibit murine colonization. Additionally, we show the absence of potD significantly increases biofilm production, both in vitro and in vivo. In a chinchilla model of otitis media (OM), the absence of potD does not significantly affect MNZ67 virulence, but it does significantly reduce the pathogenesis of the virulent encapsulated strain TIGR4 (serotype 4). Deletion of potD also significantly reduced persistence of TIGR4 in the lungs but increased persistence of PIP01 in the lungs. We conclude the pot operon is important for the regulation of protein expression and biofilm formation in both encapsulated and NCC1 nonencapsulated Streptococcus pneumoniae. However, in contrast to encapsulated pneumococcal strains, polyamine acquisition via the pot operon is not required for MNZ67 murine colonization, persistence in the lungs, or full virulence in a model of OM. Therefore, NESp virulence regulation needs to be further established to identify potential NESp therapeutic targets.

The arginine deiminase system (ADS) is associated with arginine catabolism and plays a role in virulence of several pathogenic bacteria. In Streptococcus pneumoniae , the ADS genes exist as a locus consisting of arcABCDT . A recent genome-wide mutagenesis approach revealed that both arcD and arcT are potentially essential in a chinchilla otitis media (OM) model. In the present study, we generated Δ arcD , Δ arcT , and Δ arcDT mutants by homologous recombination and evaluated their infectivity. Our results showed that only arcD , and not arcT , of an OM isolate is required during chinchilla middle ear infection. Additionally, D39 Δ arcD exhibited enhanced nasopharyngeal colonization and was attenuated in both mouse pneumonia and bacteremia models. In vitro , D39 Δ arcD displayed enhanced adherence to A549 epithelial cells and increased phagocytosis by J774A.1 macrophages compared to those with the parental strain. This mutant also exhibited an impaired capsule, as detected using electron microscopy, immunofluorescence, and a capsule assay. We demonstrated that the capsule defect in the D39 Δ arcD mutant may not be associated with a deficiency in arginine but rather is likely caused by a loss of interaction between the capsule and the transmembrane protein ArcD.

(See the Editorial Commentary by Lautenbach on pages 1266–9.) Escherichia coli causes diverse extraintestinal infections, including urinary tract infection, bacteremia, and meningitis, resulting in considerable morbidity, mortality, and increased costs [1]. Management of such infections is complicated by the rising prevalence of resistance to preferred antimicrobial agents such as trimethoprim-sulfamethoxazole (TMP-SMZ), fluoroquinolones (FQs), and extended-spectrum cephalosporins [1–4]. Contributing to this problem is E. coli sequence type 131 (ST131), a newly emerged, disseminated lineage of multidrug-resistant E. coli [2, 5–9]. In recent surveys ST131 has accounted for up to 10%–27% of the total clinical E. coli population in various locales, and for up to 52%–67% of all extended-spectrum β-lactamase (ESBL)–producing or FQ-resistant (FQ-R) E. coli [2, 10–12]. The Department of Veterans Affairs (VA) operates the largest integrated healthcare system in the United States. [13]. Escherichia coli infections, especially urinary tract infections, are quite common among veterans [14]. Based on associations of ST131 with older age and health care contact [15], and the high prevalence of these characteristics among veterans, we hypothesized that ST131 may contribute importantly to antimicrobial resistance in E. coli among veterans. Accordingly, we assessed the prevalence, geographic distribution, and contribution to antimicrobial resistance of E. coli ST131 among US veterans during 2010–2011 at multiple VA medical centers (VAMCs) across the United States, and explored associations of ST131 with source and fitness-promoting bacterial traits.

Streptococcus pneumoniae is the most common bacterial etiology of pneumococcal pneumonia in adults worldwide. Genomic plasticity, antibiotic resistance and extreme capsular antigenic variation complicates the design of effective therapeutic strategies. Polyamines are ubiquitous small cationic molecules necessary for full expression of pneumococcal virulence. Polyamine transport system is an attractive therapeutic target as it is highly conserved across pneumococcal serotypes. In this study, we compared an isogenic deletion strain of S. pneumoniae TIGR4 in polyamine transport operon (ΔpotABCD) with the wild type in a mouse model of pneumococcal pneumonia. Our results show that the wild type persists in mouse lung 24 h post infection while the mutant strain is cleared by host defense mechanisms. We show that intact potABCD is required for survival in the host by providing resistance to neutrophil killing. Comparative proteomics analysis of murine lungs infected with wild type and ΔpotABCD pneumococci identified expression of proteins that could confer protection to wild type strain and help establish infection. We identified ERM complex, PGLYRP1, PTPRC/CD45 and POSTN as new players in the pathogenesis of pneumococcal pneumonia. Additionally, we found that deficiency of polyamine transport leads to up regulation of the polyamine synthesis genes speE and cad in vitro.

Streptococcus pneumoniae remains an important human pathogen. For more than 100 years, there have been vaccine efforts to prevent pneumococcal infection. The pneumococcal conjugate vaccines have significantly reduced invasive disease. However, these vaccines have changed pneumococcal ecology within the human nasopharynx. We suggest that elimination of the pneumococcus from the human nasopharynx can have consequences that should be considered as the next generation of pneumococcal vaccines is developed.

In Streptococcus pneumonia, phosphoenolpyruvate protein phosphotransferase (PtsA) is an intracellular protein of the monosaccharide phosphotransferase systems. Biochemical and immunostaining methods were applied to show that PtsA also localizes to the bacterial cell-wall. Thus, it was suspected that PtsA has functions other than its main cytoplasmic enzymatic role. Indeed, recombinant PtsA and anti-rPtsA antiserum were shown to inhibit adhesion of S. pneumoniae to cultured human lung adenocarcinoma A549 cells. Screening of a combinatorial peptide library expressed in a filamentous phage with rPtsA identified epitopes that were capable of inhibiting S. pneumoniae adhesion to A549 cells. The insert peptides in the phages were sequenced, and homologous sequences were found in human BMPER, multimerin1, protocadherin19, integrinβ4, epsin1 and collagen type VIIα1 proteins, all of which can be found in A549 cells except the latter. Six peptides, synthesized according to the homologous sequences in the human proteins, specifically bound rPtsA in the micromolar range and significantly inhibited pneumococcal adhesion in vitro to lung- and tracheal-derived cell lines. In addition, the tested peptides inhibited lung colonization after intranasal inoculation of mice with S. pneumoniae. Immunization with rPtsA protected the mice against a sublethal intranasal and a lethal intravenous pneumococcal challenge. In addition, mouse anti rPtsA antiserum reduced bacterial virulence in the intravenous inoculation mouse model. These findings showed that the surface-localized PtsA functions as an adhesin, PtsA binding peptides derived from its putative target molecules can be considered for future development of therapeutics, and rPtsA should be regarded as a candidate for vaccine development.

Alcohol is the most frequently abused substance in the world. Both acute and chronic alcohol consumption have diverse and well-documented effects on the human immune system, leading to increased susceptibility to infections like bacterial pneumonia. Streptococcus pneumoniae is the most common bacterial etiology of community-acquired pneumonia worldwide. The frequency and severity of pneumococcal infections in individuals with a history of alcohol abuse is much higher than the general population. Despite this obvious epidemiological relevance, very few experimental studies have focused on the interaction of pneumococci with the immune system of a host acutely or chronically exposed to alcohol. Understanding these host-pathogen interactions is imperative for designing effective prophylactic and therapeutic interventions for such populations. Recent advances in pneumococcal research have greatly improved our understanding of pneumococcal pathogenesis and virulence mechanisms. Additionally, a large body of data is available on the effect of alcohol on the physiology of the lungs and the innate and adaptive immune system of the host. The purpose of this review is to integrate the available knowledge in these diverse areas of for a better understanding of the how the compromised immune system derived from alcohol exposure responds to pneumococcal infections.

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.

Streptococcus pneumoniae is an encapsulated pathogen that can cause invasive disease following colonization of the nasopharynx. Targeting colonization of mucosal surfaces may, therefore, be the best approach for vaccination to prevent pneumococcal invasive disease. Previous studies in our laboratory have shown that immunization with recombinant polyamine transport protein D (PotD) protects mice against systemic pneumococcal infections. In this study we investigated the efficacy of mucosal immunization with rPotD to protect against pneumococcal carriage and invasion in a murine model. Mice were intranasally immunized with either rPotD and cholera toxin B subunit (CTB) or CTB alone. Significantly less pneumococci were recovered from the nasopharynx of immunized mice compared to the control animals following intranasal challenge with either EF3030 (serotype 19F) ( P < 0.05) or an invasive serotype 4 isolate (TIGR4) ( P < 0.05). PotD immunized mice also had lesser bacteria in their sinus tissues ( P < 0.05), brains ( P < 0.05), lungs and olfactory bulbs following intranasal challenge with TIGR4. ELISA analysis demonstrated the presence of IgG antibodies to PotD in the serum and IgA antibodies in the saliva. These results indicate that mucosal immunization with PotD generates both mucosal and systemic immune responses and prevents establishment of nasopharyngeal carriage by multiple pneumococcal serotypes. Thus, PotD is a potentially important antigen for development of a pneumococcal protein vaccine.

Streptococcus pneumoniae has a potential ABC-type transporter (Pot) for extracellular polyamines. Polyamine transport protein D (PotD) is a membrane-associated, surface protein that putatively binds polyamines such as putrescine and spermidine. In this study we used quantitative PCR (qPCR) to analyze potD mRNA expression under physiologically relevant stress conditions in vitro, during in vivo infection, and in the presence of polyamines and choline. Expression of potD mRNA was elevated 2- and 4-fold when cells were grown at either 34 or 42 degrees C, respectively, in a choline restricted environment. Expression increased by 5- and 11-fold in response to oxidative stress in either low or high choline environments, respectively. Putrescine led to an increase in potD mRNA transcription, while choline and spermidine resulted in decreased gene expression. Transcription of potD in pneumococci harvested from blood of systemically infected mice was 43-fold higher compared to in vitro transcription levels. Flow cytometry analysis using PotD antiserum confirmed increased PotD expression on the pneumococcal surface. These results indicate that polyamines and polyamine transport systems potentially play an important role in Streptococcus pneumoniae pathogenesis, and may be important for bacterial response to temperature shock, oxidative stress, choline limitation and in vivo growth.

Understanding the growth of bacterial pathogens in a micronutrient restricted host environment can identify potential virulence proteins that help overcome this nutritional barrier to productive infection. In this study, we investigated the pneumococcal protein expression response to iron limitation using an in vitro model. We identified S. pneumoniae TIGR4 proteins by 2-D LC ESI MS/MS and determined significant changes in protein expression in response to iron restriction using computer-intensive random resampling methods. Differential protein expression was studied in the context of a S. pneumoniae TIGR4 protein interaction network using Pathway Studio. Our analysis showed that pneumococcal iron restriction response was marked by increased expression of known virulence factors like PsaA. It involved changes in the expression of stress response, and phase variation and biofilm formation proteins. The net effect of changes in all these biological processes could increase the virulence of S. pneumoniae TIGR4 during in vivo infection.

Polyamines are polycationic molecules with a hydrocarbon backbone and multiple amino groups. Descriptions of the physiological roles of polyamines have often been limited to their interaction with negatively charged nucleic acids. Of late, reports linking polyamines to microbial carcinogenesis, biofilm formation, escape from phagolysosomes, bacteriocin production, toxin activity and protection from oxidative and acid stress have been published, providing insights about their other important but lesser known functions. This review focuses on recently discovered novel functions of polyamines in microorganisms, with an emphasis on bacterial pathogens of humans.

Objectives: The aim of this study was to identify changes in the gene expression profile of Streptococcus pneumoniae in response to a subinhibitory concentration of penicillin in an effort to better understand mechanisms by which this organism copes with this stress. Methods: S. pneumoniae serotype 2 strain D39 was grown for 1 h in the presence or absence of penicillin at a concentration equivalent to half the MIC (0.03 mg/L). RNA was isolated and gene expression profiles were compared using DNA microarrays. Differential expression of select genes was confirmed by real-time RT‐PCR. Results: A total of 386 genes were found to be responsive to penicillin. Up-regulated genes included those of the ciaR‐ciaH operon, luxS, genes encoding cell envelope proteins and genes of the pst locus. Down-regulated genes included genes involved in competence, genes encoding capsular polysaccharide biosynthesis proteins, genes involved in fatty acid chain elongation and genes of the polyamine transporter operon. Conclusions: Altered expression of these genes reflects a protective response to perturbation of the bacterial cell wall by penicillin. Such genes may represent potential therapeutic targets for enhancing the activity of penicillin against this organism and provide insight into novel mechanisms of penicillin resistance.

Epidemiologic links between chronic hepatitis C and herpes simplex type-2 infection have been suggested; however, type-specific tests for HSV-2 infection have not been validated in patients with chronic hepatitis C infection. The Focus HerpeSelect(®) HSV-2 IgG (Cypress, California) assay and the Biokit HSV-2 rapid assay (Biokit USA, Lexington, MA) were performed on serum samples obtained from 84 veterans with chronic hepatitis C who demonstrated a previously positive HSV-2 serologic test in their medical records. Using the Biokit HSV-2 as the comparator assay, the positive predictive value, and specificity for the HerpeSelect(®) HSV-2 assay were 62.1% (95%CI: 49.3-73.8) and 41.9% (95%CI: 27.0-57.9), respectively. Increasing the HerpeSelect(®) HSV-2 index value defining a positive test result from1.1 to ≥2.89 increased the assay's specificity to 97.7% (95%CI: 87.7-99.6) and the positive predictive value to 94.1%(95%CI: 71.2-99.0). J. Med. Virol. 9999: 1-5, 2015. © 2015 Wiley Periodicals, Inc. In veterans with chronic hepatitis C infection, HerpeSelect(®) HSV-2 index values between 1.1 and 2.89 should be confirmed with an alternate test for HSV-2 infection.

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.

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 po lyamine t ransporter ( 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 WU2Δ potD mutant was identical to that of the wild-type strain WU2 in vitro in rich media. However, WU2Δ potD possessed severely delayed growth compared to wild-type WU2 in the presence of the polyamine biosynthesis inhibitors DFMO (α-dimethyl-fluroornitithine) 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.

Enterococcus faecalis, an opportunistic bacterial pathogen, has become prominent in recent decades due to its ability to develop resistance to various antibiotics. The ability to rapidly identify E. faecalis, followed by prompt treatment, is therefore vital for the control of this pathogen. In this report, upon comparative BLAST search of the E. faecalis genome against other bacterial DNA sequences at GenBank, an E. faecalis-specific gene Ef0027, that codes for a putative phosphosugar binding transcriptional regulator, was identified. Using PCR primers derived from this gene (i.e., Ef0027F/R), a specific DNA fragment of 518 bp was detected in E. faecalis strains only, and it was not found in other Enterococcus species and common bacteria. These results suggest that PCR amplification of the putative transcriptional regulator gene Ef0027 provides a useful means of identifying E. faecalis from other bacteria.

Streptococcus pyogenes (GAS) is a common bacterial pathogen that has emerged as an increasingly important health concern in many parts of the world. Although GAS may appear harmless in healthy individuals, the ability of this bacterium to take advantage of a weakened or compromised host defense system is extraordinary. Following the recent publication of the genome sequences of several S. pyogenes strains, we undertook an investigation of a specialized gene group in GAS that encodes transcriptional regulators. By screening S. pyogenes transcriptional regulator genes from the complete genome of M1 strain SF370 against other DNA sequences at GenBank by BLAST searches, we identified a gene (i.e., Spy1258) that is uniquely present in the bacterium. Application of PCR primers (spy1258F and spy1258R) derived from this gene facilitated amplification of a 407-bp DNA fragment from S. pyogenes only, but not from other species of the genus Streptococcus and common bacteria. Apart from offering an additional target for specific confirmation of GAS, further analysis of the putative transcriptional regulator gene Spy1258 and its related protein product may lead to new insights into the molecular mechanisms of S. pyogenes maintenance and pathogenicity.

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.

Infections caused by Streptococcus pneumoniae (pneumococcus) are a major cause of mortality throughout the world. This organism is primarily a commensal in the upper respiratory tract of humans, but can cause pneumonia in high-risk persons and disseminate from the lungs by invasion of the bloodstream. Currently, prevention of pneumococcal infections is by immunization with vaccines which contain capsular polysaccharides from the most common serotypes causing invasive disease. However, there are more than 90 antigenically distinct serotypes and there is concern that serotypes not included in the vaccines may become more prevalent in the face of continued use of polysaccharide vaccines. Also, certain high-risk groups have poor immunological responses to some of the polysaccharides in the vaccine formulations. Protein antigens that are conserved across all capsular serotypes would induce more effective and durable humoral immune responses and could potentially protect against all clinically relevant pneumococcal capsular types. This review provides a summary of work on pneumococcal proteins that are being investigated as components for future generations of improved pneumococcal vaccines.

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.

A case of vertebral osteomyelitis involving misidentification of Candida parapsilosis as C famata by the VITEK 2 compact is described. Species-specific primers were used in the polymerase chain reaction to correctly identify the clinical isolate. When uncommon species of Candida are reported using automated systems, heightened clinical suspicion is warranted.

Pneumococci covalently link phosphorylcholine to teichoic and lipoteichoic acids found in the peptidoglycan and cytoplasmic membrane, respectively. The functions of most of the choline-binding proteins (CBPs) are unknown, but a few have been studied in some depth and it is apparent that they have a role in pathogenesis and can be protective immunogens. The presence of choline-binding domains implies that any protein expressing these domains is secreted, since choline is a constituent of teichoic and lipoteichoic acids, which are cell surface polymers. The major pneumococcal autolysin LytA contains four to six choline-binding domains but is not found in eluates of pneumococcal strain Rx1 incubated with choline-containing buffers. Pneumococcal CBPs have been shown to play a role in pathogenesis in various murine models of disease. It is likely that there is some redundancy in the function of CBPs, and this, along with the large effect of the pneumococcal capsule, may explain why more is not known about the function of CBPs in pneumococcal disease. PspA and PspC are the two most well-characterized CBPs in terms of their biological functions and roles in disease. While these two proteins can be considered paralogs based on sequence homology, they make distinct contributions to pneumococcal virulence. The major autolysin of pneumococci, designated LytA, is known to be important in remodeling the cell wall of dividing pneumococci and is the common final point for many processes which lead to cell lysis.