- A novel comprehensive set of fungal Real time PCR assays (fuPCR) for the detection of fungi in immunocompromised haematological patients-A pilot study. [Journal Article]
- IJInt J Med Microbiol 2016 Oct 13
- Fungal infections are recognized in an increasing number of patients with immunological deficits and are associated with high rates of mortality (Brown et al., 2012a). In this pilot-study, a rapid Re...
Fungal infections are recognized in an increasing number of patients with immunological deficits and are associated with high rates of mortality (Brown et al., 2012a). In this pilot-study, a rapid Real time PCR (fuPCR) was designed for the detection and differentiation of fungal pathogens in clinical specimens of haematological patients. The fuPCR, targeting the internal transcribed spacer region 2 (ITS2) of rDNA region, is comprised of seven multiplex reactions, which were shown to be specific and sensitive for a comprehensive spectrum of clinically relevant fungal species. This was validated by testing respective fungal DNAs in each fuPCR reaction and 28 respiratory samples of fungal pneumonia-proven patients. Clinical sample sets of throat swab, EDTA-blood and blood sera from 50 patients with severe haematological malignancies, including haematopoietic stem cell transfer (HSCT), and samples from 30 healthy individuals were then analysed. In a first step, 198 samples of immunosuppressed patients were solely examined by fuPCR; and 50.8% (33/65) respiratory swabs, 4.8% (3/63) EDTA blood samples and 1.4% (1/70) blood serum samples were tested positive. In a second step, 56 respiratory samples of immunosuppressed patients and 30 of healthy individuals were simultaneously analysed by fuPCR and standard cultivation techniques. By both methods 30.4% (17/56) swabs of the immunocompromised patients were tested positive, 37.5% (21/56) were tested negative and 32.1% (18/56) were tested fuPCR positive and culture negative. In analysing the blood samples of the immunocompromised patients 5.4% (3/56) EDTA blood samples and 16.1% (9/56) sera samples were tested fuPCR-positive, whereas all samples of 30 healthy individuals with no signs of immunological deficits were tested negative by fuPCR. 38.9% (14/36) of the fungi detected in respiratory samples of the immunosuppressed patients, belonged to Candida spp., 47.2% (17/36) to Saccharomyces spp., 5.6% (2/36) to Cladosporium spp. and 8.3% (3/36) to Alternaria spp., whereas cultivation only identified Candida spp. (10/17) and Saccharomyces spp. (7/17). In this pilot study a novel fuPCR assay was developed and validated for the simultaneous and comprehensive detection of fungal pathogens in clinical respiratory specimens of haematological patients. Future work will focus on the validation of the blood-stream detected fungi in pathogenicity of these patients.
- Iron acquisition pathways and colonization of the inflamed intestine by Salmonella enterica serovar Typhimurium. [Journal Article]
- IJInt J Med Microbiol 2016 Oct 13
- Salmonella enterica serotype Typhimurium is able to expand in the lumen of the inflamed intestine through mechanisms that have not been fully resolved. Here we utilized streptomycin-pretreated mice a...
Salmonella enterica serotype Typhimurium is able to expand in the lumen of the inflamed intestine through mechanisms that have not been fully resolved. Here we utilized streptomycin-pretreated mice and dextran sodium sulfate (DSS)-treated mice to investigate how pathways for S. Typhimurium iron acquisition contribute to pathogen expansion in the inflamed intestine. Competitive infection with an iron uptake-proficient S. Typhimurium strain and mutant strains lacking tonB feoB, feoB, tonB or iroN in streptomycin pretreated mice demonstrated that ferric iron uptake requiring IroN and TonB conferred a fitness advantage during growth in the inflamed intestine. However, the fitness advantage conferred by ferrous iron uptake mechanisms was independent of inflammation and was only apparent in models where the normal microbiota composition had been disrupted by antibiotic treatment.
- Immunogencity of antigens from Mycobacterium tuberculosis self-assembled as particulate vaccines. [Journal Article]
- IJInt J Med Microbiol 2016 Oct 13
- Traditional approaches to vaccine development have failed to identify better vaccines to replace or supplement BCG for the control of tuberculosis (TB). Subunit vaccines offer a safer and more reprod...
Traditional approaches to vaccine development have failed to identify better vaccines to replace or supplement BCG for the control of tuberculosis (TB). Subunit vaccines offer a safer and more reproducible alternative for the prevention of diseases. In this study, the immunogenicity of bacterially derived polyester beads displaying three different Rv antigens of Mycobacterium tuberculosis was evaluated. Polyester beads displaying the antigens Rv1626, Rv2032, Rv1789, respectively, were produced in an endotoxin-free Escherichia coli strain. Beads were formulated with the adjuvant DDA and subcutaneously administered to C57BL/6 mice. Cytokine responses were evaluated by CBA and antibody responses by ELISA. Specificity of the IgG response was assessed by immunoblotting cell lysates of the vaccine production strains using sera from the vaccinated mice. Mice vaccinated with beads displaying Rv1626 had significantly greater IgG1 responses compared to mice vaccinated with Rv1789 beads and greater IgG2 responses than the group vaccinated with Rv2032 beads (p<0.05). Immunoblotting of antisera from these mice indicated the antibody responses were Rv1626 antigen-specific and there was no detectable immune response to the polyester component of the vaccine. Overall, this study suggested that selected TB antigens derived from reverse vaccinology approaches can be displayed on polyester beads to produce antigen-specific immune responses potentially relevant to the prevention of TB.
- The secretome of Acinetobacter baumannii ATCC 17978 type II secretion system reveals a novel plasmid encoded phospholipase that could be implicated in lung colonization. [Journal Article]
- IJInt J Med Microbiol 2016 Oct 1
- Acinetobacter baumannii infections are compounded with a striking lack of treatment options. In many Gram-negative bacteria, secreted proteins play an important early role in avoiding host defences. ...
Acinetobacter baumannii infections are compounded with a striking lack of treatment options. In many Gram-negative bacteria, secreted proteins play an important early role in avoiding host defences. Typically, these proteins are targeted to the external environment or into host cells using dedicated transport systems. Despite the fact that medically relevant species of Acinetobacter possess a type II secretion system (T2SS), only recently, its significance as an important pathway for delivering virulence factors has gained attention. Using in silico analysis to characterize the genetic determinants of the T2SS, which are found clustered in other organisms, in Acinetobacter species, they appear to have a unique genetic organization and are distributed throughout the genome. When compared to other T2SS orthologs, individual components of the T2SS apparatus showed the highest similarity to those of Pseudomonas aeruginosa. A mutant of Acinetobacter baumannii strain ATCC 17978 lacking the secretin component of the T2SS (ΔgspD), together with a trans-complemented mutant, were tested in a series of in vitro and in vivo assays to determine the role of T2SS in pathogenicity. The ΔgspD mutant displayed decreased lipolytic activity, associated with attenuated colonization ability in a murine pneumonia model. These phenotypes are linked to LipAN, a novel plasmid-encoded phospholipase, identified through mass spectroscopy as a T2SS substrate. Recombinant LipAN showed specific phospholipase activity in vitro. Proteomics on the T2-dependent secretome of ATCC 17978 strain revealed its potential dedication to the secretion of a number of lipolytic enzymes, among others which could contribute to its virulence. This study highlights the role of T2SS as an active contributor to the virulence of A. baumannii potentially through secretion of a newly identified phospholipase.
- Agents that activate the High Osmolarity Glycerol pathway as a means to combat pathogenic molds. [Journal Article]
- IJInt J Med Microbiol 2016 Sep 28
- Treatment of invasive fungal infections often fails due to the limited number of therapeutic options. In this study, we have analyzed the impact of agents activating the High Osmolarity Glycerol (HOG...
Treatment of invasive fungal infections often fails due to the limited number of therapeutic options. In this study, we have analyzed the impact of agents activating the High Osmolarity Glycerol (HOG) pathway on molds that cause infections in humans and livestock. We found that agents like fludioxonil and iprodione, have a clear anti-fungal activity against pathogenic Aspergillus, Lichtheimia, Rhizopus and Scedosporium species. Only A. terreus turned out to be resistant to fludioxonil, even though it is sensitive to iprodione and able to adapt to hyperosmotic conditions. Moreover, the A. terreus tcsC gene can fully complement an A. fumigatus ΔtcsC mutant, thereby also restoring its sensitivity to fludioxonil. The particular phenotype of A. terreus is therefore likely to be independent of its TcsC kinase. In a second part of this study, we further explored the impact of fludioxonil using A. fumigatus as a model organism. When applied in concentrations of 1-2μg/ml, fludioxonil causes an immediate growth arrest and, after longer exposure, a quantitative killing. Hyphae respond to fludioxonil by the formation of new septa and closure of nearly all septal pores. Mitosis occurs in all compartments and is accompanied by a re-localization of the NimA kinase to the cytoplasm. In the swollen compartments, the massive extension of the cell wall triggers a substantial reorganization resulting in an enhanced incorporation of chitin and, most strikingly, a massive loss of galactomannan. Hence, HOG-activating agents have dramatic cell biological consequences and may represent a valuable, future element in the armory that can be used to combat mold infections.
- High prevalence of nontoxigenic Clostridium difficile isolated from hospitalized and non-hospitalized individuals in rural Ghana. [Journal Article]
- IJInt J Med Microbiol 2016 Sep 24
- Since data about Clostridium difficile infection in sub-Saharan Africa are scarce, we determined its epidemiology and risk factors in a cross-sectional study in Eikwe, a rural community in Ghana. We ...
Since data about Clostridium difficile infection in sub-Saharan Africa are scarce, we determined its epidemiology and risk factors in a cross-sectional study in Eikwe, a rural community in Ghana. We tested stool samples from 176 hospitalized patients with diarrhoea and from 131 asymptomatic non-hospitalized individuals for C. difficile and some other enteric pathogens. The overall prevalence rate of C. difficile was 4.9% with ribotype 084 being predominant. With 75% of the isolates, a high rate of nontoxigenic strains was present in symptomatic patients, most of whom had no other identified enteric pathogens. All strains were susceptible against metronidazole and vancomycin, respectively. Data on lifestyle and medical history showed that age <5years (p=0.004), and use of ceftriaxone (p=0.023) were the most important risk factors for C. difficile carriage status. Although our data suggest that C. difficile is currently not a major cause of diarrhoea in this setting, the epidemiology of C. difficile in sub-Saharan Africa awaits further investigation.
- Role of a sensor histidine kinase ChiS of Vibrio cholerae in pathogenesis. [Journal Article]
- IJInt J Med Microbiol 2016 Sep 19
- Vibrio cholera survival in an aquatic environment depends on chitin utilization pathway that requires two factors, chitin binding protein and chitinases. The chitinases and the chitin utilization pat...
Vibrio cholera survival in an aquatic environment depends on chitin utilization pathway that requires two factors, chitin binding protein and chitinases. The chitinases and the chitin utilization pathway are regulated by a two-component sensor histidine kinase ChiS in V. cholerae. In recent studies these two factors are also shown to be involved in V. cholerae pathogenesis. However, the role played by their upstream regulator ChiS in pathogenesis is yet to be known. In this study, we investigated the activation of ChiS in presence of mucin and its functional role in pathogenesis. We found ChiS is activated in mucin supplemented media. The isogenic chiS mutant (ChiS(-)) showed less growth compared to the wild type strain (ChiS(+)) in the presence of mucin supplemented media. The ChiS(-) strain also showed highly retarded motility as well as mucin layer penetration in vitro. Our result also showed that ChiS was important for adherence and survival in HT-29 cell. These observations indicate that ChiS is activated in presence of intestinal mucin and subsequently switch on the chitin utilization pathway. In animal models, our results also supported the in vitro observation. We found reduced fluid accumulation and colonization during infection with ChiS(-) strain. We also found ChiS(-) mutant with reduced expression of ctxA, toxT and tcpA. The cumulative effect of these events made V. cholerae ChiS(-) strain hypovirulent. Hence, we propose that ChiS plays a vital role in V. cholerae pathogenesis.
- Staphylococcus aureus PSM peptides induce tolerogenic dendritic cells upon treatment with ligands of extracellular and intracellular TLRs. [Journal Article]
- IJInt J Med Microbiol 2016 Sep 3
- Dendritic cells (DCs) are key players of the immune system and thus a target for immune evasion by pathogens. We recently showed that the virulence factor phenol-soluble modulin (PSM) produced by com...
Dendritic cells (DCs) are key players of the immune system and thus a target for immune evasion by pathogens. We recently showed that the virulence factor phenol-soluble modulin (PSM) produced by community-associated methicillin-resistant Staphylococcus aureus strains induces tolerogenic DCs upon Toll-like receptor (TLR) 2 activation via the p38-CREB-IL-10 pathway. Here, we addressed the question whether this tolerogenic phenotype of DCs induced by PSMs is specific for TLR2 activation. Therefore, bone marrow-derived DCs were treated with various ligands for extracellular and intracellular TLRs simultaneously with PSMα3. We show that PSMα3 modulates antigen uptake, maturation and cytokine production of DCs activated by TLR1/2, TLR2/6, TLR4, TLR7, and TLR9. Pre-incubation of DCs with a p38 MAP kinase inhibitor prevented the PSMα3-induced IL-10 secretion, as well as MHC class II up-regulation upon TLR activation. In consequence, the tolerogenic DCs induced by PSMα3 in response to several TLR ligands promoted priming of regulatory T cells. Thus, PSMs could be useful as inducers of tolerogenic DCs upon TLR ligand stimulation for therapeutic applications.
- Potential of combination therapy of endolysin MR-10 and minocycline in treating MRSA induced systemic and localized burn wound infections in mice. [Journal Article]
- IJInt J Med Microbiol 2016 Sep 1
- MRSA is the predominant pathogen responsible for fatal burn wound infection in patients. Antibiotic resistance and its ability to form biofilms on the surface of burn wounds limit the use of antibiot...
MRSA is the predominant pathogen responsible for fatal burn wound infection in patients. Antibiotic resistance and its ability to form biofilms on the surface of burn wounds limit the use of antibiotics to contain this pathogen. The results of present study have shown that single dose of combination therapy of endolysin MR-10 (50μg/s.c) and minocycline (50mg/kg/orally) resulted in 100% survival of group of mice with systemic MRSA infection. Maximum reduction in bacterial load in various organs was observed in the group that received combination therapy. In comparison to control, a significant reduction (p<0.01) of 4.82, 1.81, 1.51, 1.2 logs was observed in skin, blood, liver and spleen respectively, by 3rd day post infection. As a result of which, all organs became sterile thereby protecting mice from mortality. Histopathological analysis corroborated our findings showing no signs of inflammation and bacterial infection in the group that received combination therapy. Treatment of localized burn wound infection with combination therapy resulted in early resolution of infection followed by fast healing. The group that received combination therapy showed complete resolution of infection in less than 10days. Moreover, the skin samples obtained from animals treated with combination therapy showed no myeloperoxidase (MPO) activity on 10th day post treatment. In combination therapy group, ∼98% wound contraction was observed by 12th day followed by complete closure of wound within ∼14days. The histopathological analysis showed no signs of inflammation and infection. Collagen staining revealed early signs of re-epithelization of epidermis and signs of collagen regeneration in-group that received combination therapy. Hence, this study suggests that the combined therapy of endolysin MR-10 and minocycline is a better option in controlling burn wound infections.
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- Interactions of surface-displayed glycolytic enzymes of Mycoplasma pneumoniae with components of the human extracellular matrix. [Journal Article]
- IJInt J Med Microbiol 2016 Sep 3
- Mycoplasma pneumoniae is a major cause of community-acquired respiratory infections worldwide. Due to the strongly reduced genome, the number of virulence factors expressed by this cell wall-less pat...
Mycoplasma pneumoniae is a major cause of community-acquired respiratory infections worldwide. Due to the strongly reduced genome, the number of virulence factors expressed by this cell wall-less pathogen is limited. To further understand the processes during host colonization, we investigated the interactions of the previously confirmed surface-located glycolytic enzymes of M. pneumoniae (pyruvate dehydrogenase A-C [PdhA-C], glyceraldehyde-3-phosphate dehydrogenase [GapA], lactate dehydrogenase [Ldh], phosphoglycerate mutase [Pgm], pyruvate kinase [Pyk] and transketolase [Tkt]) to the human extracellular matrix (ECM) proteins fibrinogen (Fn), fibronectin (Fc), lactoferrin (Lf), laminin (Ln) and vitronectin (Vc), respectively. Concentration-dependent interactions between Fn and Vc and all eight recombinant proteins derived from glycolytic enzymes, between Ln and PdhB-C, GapA, Ldh, Pgm, Pyk and Tkt, between Lf and PdhA-C, GapA and Pyk, and between Fc and PdhC and GapA were demonstrated. In most cases, these associations are significantly influenced by ionic forces and by polyclonal sera against recombinant proteins. In immunoblotting, the complex of human plasminogen, activator (tissue-type or urokinase plasminogen activator) and glycolytic enzyme was not able to degrade Fc, Lf and Ln, respectively. In contrast, degradation of Vc was confirmed in the presence of all eight enzymes tested. Our data suggest that the multifaceted associations of surface-localized glycolytic enzymes play a potential role in the adhesion and invasion processes during infection of human respiratory mucosa by M. pneumoniae.