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PubMed2004

The use of carbohydrate microarrays to study carbohydrate-cell interactions and to detect pathogens.

The use of carbohydrate microarrays to investigate the carbohydrate binding specificities of bacteria, to detect pathogens, and to screen antiadhesion therapeutics is reported. This system is ideal for whole-cell applications because microarrays present carbohydrate ligands in a manner that mimics interactions at cell-cell interfaces. Other advantages include assay miniaturization, since minimal amounts (approximately picomoles) of a ligand are required to observe binding, and high throughput, since thousands of compounds can be placed on an array and analyzed in parallel. Pathogen detection experiments can be completed in complex mixtures of cells or protein using the known carbohydrate binding epitopes of the pathogens in question. The nondestructive nature of the arrays allows the pathogen to be harvested and tested for antibacterial susceptibility. These investigations allow microarray-based screening of biological samples for contaminants and combinatorial libraries for antiadhesion therapeutics.

Bacteria↗
PubMed2005

Levels of complexity in pathogen recognition by C-type lectins.

In pathogen recognition by C-type lectins, several levels of complexity can be distinguished; these might modulate the immune response in different ways. Firstly, the pathogen-associated molecular pattern repertoire expressed at the microbial surface determines the interactions with specific receptors. Secondly, each immune cell type possesses a specific set of pathogen-recognition receptors. Thirdly, changes in the cell-surface distribution of C-type lectins regulate carbohydrate binding by modulating receptor affinity for different ligands. Crosstalk between these receptors results in a network of multimolecular complexes, adding a further level of complexity in pathogen recognition.

Animals↗
PubMed2004

Emerging waterborne pathogens: can we kill them all?

The rapid emergence of Cryptosporidium parvum and Escherichia coli 0157:H7 have created a threat to the drinking water industry and there is a growing need to develop a strategy for recognizing potential emerging waterborne pathogens. Globalization of trade, changing population demographics and changes in treatment technology have been driving factors in the emergence of these new pathogens. An understanding of disinfectant action and microbial resistance to treatment processes is needed to better identify those pathogens likely to be of greatest concern. Recent research on microbial resistance to treatment and disinfection demonstrates that the microbial surface structure and composition and the nature of the genome are key to determining the potential for waterborne transmission of emerging pathogens.

Adenoviridae↗
PubMed2006

Insect immunity shows specificity in protection upon secondary pathogen exposure.

Immunological memory in vertebrates, conferring lasting specific protection after an initial pathogen exposure, has implications for a broad spectrum of evolutionary, epidemiological, and medical phenomena . However, the existence of specificity in protection upon secondary pathogen exposure in invertebrates remains controversial . To separate this functional phenomenon from a particular mechanism, we refer to it as specific immune priming. We investigate the presence of specific immune priming in workers of the social insect Bombus terrestris. Using three bacterial pathogens, we test whether a prior homologous pathogen exposure gives a benefit in terms of long-term protection against a later challenge, over and above a heterologous combination. With a reciprocally designed initial and second-exposure protocol (i.e., all combinations of bacteria were tested), we demonstrate, even several weeks after the clearance of a first exposure, increased protection and narrow specificity upon secondary exposure. This demonstrates that the invertebrate immune system is functionally capable of unexpectedly specific and durable induced protection. Ultimately, despite general broad differences between vertebrates and invertebrates, the ability of both immune systems to show specificity in protection suggests that their immune defenses have found comparable solutions to similar selective pressures over evolutionary time.

Animals↗
PubMed2006

Expulsion of live pathogenic yeast by macrophages.

Phagocytic cells, such as neutrophils and macrophages, perform a critical role in protecting organisms from infection by engulfing and destroying invading microbes . Although some bacteria and fungi have evolved strategies to survive within a phagocyte after uptake, most of these pathogens must eventually kill the host cell if they are to escape and infect other tissues . However, we now demonstrate that the human fungal pathogen Cryptococcus neoformans is able to escape from within macrophages without killing the host cell by a novel expulsive mechanism. This process occurs in both murine J774 cells and primary human macrophages. It is extremely rapid and yet can occur many hours after phagocytosis of the pathogen. Expulsion occurs independently of the initial route of phagocytic uptake and does not require phagosome maturation . After the expulsive event, both the host macrophage and the expelled C. neoformans appear morphologically normal and continue to proliferate, suggesting that this process may represent an important mechanism by which pathogens are able to escape from phagocytic cells without triggering host cell death and thus inflammation .

Animals↗
PubMed2005

Antimicrobial susceptibilities of potential bacterial pathogens in adults with acute respiratory tract infections Prospective Epidemiological Network Investigating Community-acquired Infection SurveiLLance In Nagasaki (PENICILLIN) Study.

The prevalence and the antimicrobial susceptibilities of bacterial pathogens in acute upper respiratory tract infections (AURTIs) is not clear. We conducted a prospective community-based multicenter study in 45 centers across Nagasaki, Japan, between December 2001 and April 2002. We examined the nasopharyngeal or throat isolates in 930 adult outpatients with AURTIs. Potential bacterial pathogens, including Streptococcus pyogenes (from 6.8% patients), S. pneumoniae (5.3%), S. milleri group (2.0%), Staphylococcus aureus (26.0%), Moraxella catarrhalis (7.4%), Haemophilus influenzae (17.6%), and Klebsiella pneumoniae (1.2%), were isolated from 51.8% of the patients. Penicillin nonsusceptible phenotype accounted for 44.9% of S. pneumoniae isolates. Among Staphylococcus aureus isolates, 9.1% were methicillin-resistant. In H. influenzae isolates, 6.7% produced beta-lactamase and 23.2% were beta-lactamase-negative ampicillin-resistant. The results indicated that the potential bacterial pathogens were detected in the remarkable population of the adults with AURTIs and the current antimicrobial susceptibilities of such pathogens.

Acute Disease↗
PubMed2005

Antimicrobial activity of tigecycline tested against nosocomial bacterial pathogens from patients hospitalized in the intensive care unit.

The antimicrobial activity of tigecycline and selected antimicrobials was evaluated against bacterial pathogens isolated from patients hospitalized in intensive care units (ICUs) worldwide. A total of 9093 isolates were consecutively collected in >70 medical centers in North America (4157), South America (1830), Europe (3034), and the Asia-Australia (72) areas. The isolates were collected from the bloodstream (68.5%), respiratory tract (13.6%), skin/soft tissue (5.5%), and urinary tract (2.0%) infections in the 2000-2004 period, and susceptibility was tested by reference broth microdilution methods. The most frequently isolated pathogens were Staphylococcus aureus (32.1%), Enterococcus spp. (13.7%), coagulase-negative staphylococci (CoNS; 13.0%), Pseudomonas aeruginosa (8.4%), and Escherichia coli (7.9%). All Gram-positive pathogens (5665) were inhibited at < or =1 microg/mL of tigecycline. Resistance to oxacillin was detected in 43.5% of Staphylococcus aureus and in 85.0% of CoNS, and resistance to vancomycin was observed in 18.6% of enterococci. Tigecycline was very active against Enterobacteriaceae (1876 strains tested) with an MIC90 of < or =1 microg/mL, except for Serratia spp. (2 microg/mL). Extended-spectrum beta-lactamase (ESBL) phenotype was detected in 10% of E. coli and 31% of Klebsiella spp., whereas 28% of Enterobacter spp. were resistant to ceftazidime (AmpC enzyme production). These resistance phenotypes did not adversely affect tigecycline activity. Tigecycline and trimethoprim/sulfamethoxazole were the most active compounds against Stenotrophomonas maltophilia (MIC90, 2 and 1 microg/mL respectively). Tigecycline was also active against Acinetobacter spp. (MIC90, 1 microg/mL), but P. aeruginosa showed decreased susceptibility to tigecycline (MIC90, 16 microg/mL). In summary, isolates from ICU patients worldwide showed high rates of antimicrobial resistance. The most alarming problems detected were vancomycin resistance among enterococci, ESBL-mediated beta-lactam resistance and fluoroquinolone resistance among Enterobacteriaceae, and carbapenem resistance among P. aeruginosa and Acinetobacter spp. Tigecycline exhibited potent in vitro activity against most of clinically important pathogenic bacteria (except P. aeruginosa) isolated from ICU patients and may represent an excellent option for the treatment of infections in this clinical environment.

Anti-Bacterial Agents↗
PubMed2006

Ixodes ricinus: the potential of two-dimensional gel electrophoresis as a tool for studying host-vector-pathogen interactions.

Ixodes ricinus is a three-host tick, with three active instars. For moulting to occur the tick has to find a host where it can take a blood meal. Throughout feeding I. ricinus can be infected or infect the host with different pathogens, e.g., Tick-Borne Encephalitis virus or Borrelia burgdorferi. The host-vector-pathogen interaction is very complex, making a detailed study difficult. Here we analyse the potential of two-dimensional gel electrophoresis (2DE) to study the host-vector-pathogen interaction. We examined 20 nymphs, which as larvae parasitised either mouse or hen. After moulting, they were kept alive for up to 30 weeks, to analyse whether tick ageing influenced host determination, and for comparison of the 2D-gels. Even though the number of proteins in the gel decreased during ageing, some proteins of the host determination persisted for all 30 weeks. We also discovered persisting proteins in relation to nymphs. These findings showed that 2DE is suitable as a tool for studying host-vector-pathogen interactions.

Actins↗
PubMed2005

Interaction of pathogenic fungi with host cells: Molecular and cellular approaches.

This review provides an overview of several molecular and cellular approaches that are likely to supply insights into the host-fungus interaction. Fungi present intra- and/or extracellular host-parasite interfaces, the parasitism phenomenon being dependent on complementary surface molecules. The entry of the pathogen into the host cell is initiated by the fungus adhering to the cell surface, which generates an uptake signal that may induce its cytoplasmatic internalization. Furthermore, microbial pathogens use a variety of their surface molecules to bind to host extracellular matrix (ECM) components to establish an effective infection. On the other hand, integrins mediate the tight adhesion of cells to the ECM at sites referred to as focal adhesions and also play a role in cell signaling. The phosphorylation process is an important mechanism of cell signaling and regulation; it has been implicated recently in defense strategies against a variety of pathogens that alter host-signaling pathways in order to facilitate their invasion and survival within host cells. The study of signal transduction pathways in virulent fungi is especially important in view of their putative role in the regulation of pathogenicity. This review discusses fungal adherence, changes in cytoskeletal organization and signal transduction in relation to host-fungus interaction.

Cell Adhesion↗
PubMed2004

Age influences resistance of Caenorhabditis elegans to killing by pathogenic bacteria.

Caenorhabditis elegans has previously been proposed as an alternative host for models of infectious disease caused by human pathogens. When exposed to some human pathogenic bacteria, the life span of nematodes is significantly reduced. We have shown that mutations in the age-1, and/or age-2 genes of C. elegans, that normally enhance life expectancy, can also increase resistance to killing by the bacterial pathogens Pseudomonas aeruginosa, Salmonella enterica var. Typhimurium, Burkholderia cepacia or Yersinia pseudotuberculosis. We also found that the rate at which wild-type C. elegans was killed by the bacterial pathogens tested increased as nematodes aged. In the case of P. aeruginosa infection, the difference in life span of wild type and age-1 mutants of C. elegans was not due to differences in the level of bacterial colonisation of the gut.

Animals↗
PubMed2004

Comparative genomics identifies genes shared by distantly related insect-transmitted plant pathogenic mollicutes.

Phytoplasmas and spiroplasmas are distantly related insect-transmitted plant pathogens within the class Mollicutes. Genome sequencing projects of phytoplasma strain Aster Yellows-Witches' Broom (AY-WB) and Spiroplasma kunkelii are near completion. Complete genome sequences of seven obligate animal and human pathogenic mollicutes (Mycoplasma and Ureaplasma spp.), and OY phytoplasma have been reported. Putative ORFs predicted from the genome sequences of AY-WB and S. kunkelii were compared to those of the completed genomes. This resulted in identification of at least three ORFs present in AY-WB, OY and S. kunkelii but not in the obligate animal and human pathogenic mollicutes. Moreover, we identified ORFs that seemed more closely related between AY-WB and S. kunkelii than to their mycoplasma counterparts. Phylogenetic analyses using parsimony were employed to study the origin of these genes, resulting in identification of one gene that may have undergone horizontal gene transfer. The possible involvement of these genes in plant pathogenicity is discussed.

Animals↗
PubMed2004

Gene expression profile of the plant pathogen Xylella fastidiosa during biofilm formation in vitro.

A biofilm is a community of microorganisms attached to a solid surface. Cells within biofilms differ from planktonic cells, showing higher resistance to biocides, detergent, antibiotic treatments and host defense responses. Even though there are a number of gene expression studies in bacterial biofilm formation, limited information is available concerning plant pathogen. It was previously demonstrated that the plant pathogen Xylella fastidiosa could grow as a biofilm, a possibly important factor for its pathogenicity. In this study we utilized analysis of microarrays to specifically identify genes expressed in X. fastidiosa cells growing in a biofilm, when compared to planktonic cells. About half of the differentially expressed genes encode hypothetical proteins, reflecting the large number of ORFs with unknown functions in bacterial genomes. However, under the biofilm condition we observed an increase in the expression of some housekeeping genes responsible for metabolic functions. We also found a large number of genes from the pXF51 plasmid being differentially expressed. Some of the overexpressed genes in the biofilm condition encode proteins involved in attachment to surfaces. Other genes possibly confer advantages to the bacterium in the environment that it colonizes. This study demonstrates that the gene expression in the biofilm growth condition of the plant pathogen X. fastidiosa is quite similar to other characterized systems.

Biofilms↗
PubMed2005

Diverse pathogenicity of Burkholderia cepacia complex strains in the Caenorhabditis elegans host model.

A fast screening method was developed to assess the pathogenicity of a diverse collection of environmental and clinical Burkholderia cepacia complex isolates in the nematode Caenorhabditis elegans. The method was validated by comparison with the standard slow-killing assay. We observed that the pathogenicity of B. cepacia complex isolates in C. elegans was strain-dependent but species-independent. The wide range of observed pathogenic phenotypes agrees with the high degree of phenotypic variation among species of the B. cepacia complex and suggests that the taxonomic classification of a given strain within the complex cannot predict pathogenicity.

Animals↗
PubMed2004

Lessons from signature-tagged mutagenesis on the infectious mechanisms of pathogenic bacteria.

Studies on the genetic basis of bacterial pathogenicity have been undertaken for almost 30 years, but the development of new genetic tools in the past 10 years has considerably increased the number of identified virulence factors. Signature-tagged mutagenesis (STM) is one of the most powerful general genetic approaches, initially developed by David Holden and colleagues in 1995, which has now led to the identification of hundreds of new genes requested for virulence in a broad range of bacterial pathogens. We have chosen to present in this review, the most recent and/or most significant contributions to the understanding of the molecular mechanisms of bacterial pathogenicity among over 40 STM screens published to date. We will first briefly review the principle of the method and its major technical limitations. Then, selected studies will be discussed where genes implicated in various aspects of the infectious process have been identified (including tropism for specific host and/or particular tissues, interactions with host cells, mechanisms of survival and persistence within the host, and the crossing of the blood brain barrier). The examples chosen will cover intracellular as well as extracellular Gram-negative and Gram-positive pathogens.

Bacteria↗
PubMed2005

Analysis of expressed sequence tags from the wheat leaf blotch pathogen Mycosphaerella graminicola (anamorph Septoria tritici).

Mycosphaerella graminicola is a major fungal pathogen of wheat as the causal agent of Septoria leaf blotch disease. As a first step toward a greater understanding of the mechanism of host infection we have generated, sequenced, and analyzed three M. graminicola EST libraries from conditions predicted to resemble independent phases of the host infection process, including one library generated from the fungus during interaction with its host. A total of 5180 ESTs were sequenced and clustered into 886 contigs and 2039 singletons to give a set of 2925 unique sequences (unisequences). BLASTX analysis revealed 33% of the unknown M. graminicola unisequences to be orphans. Very limited inter-library overlap of expression was seen with the majority of unisequences (contigs and singletons) being library-specific. Analysis of EST redundancy between libraries demonstrated a significant difference in gene expression in the three conditions. Comparisons made against fully sequenced genomes revealed most M. graminicola sequences to be homologous to genes present in both pathogenic and non-pathogenic Ascomycete filamentous fungi. A range of sequences having significant homology to verified pathogenicity/virulence genes (HvPV-genes) of either plant or mammalian fungal and Oomycete pathogens were also identified (<1e-20). The generation of, and the diversity present within, this EST collection will facilitate future efforts aimed at a more detailed study of the transcriptome of the fungus during host infection.

Ascomycota↗
PubMed2006

Phase-specific gene expression underlying morphological adaptations of the dimorphic human pathogenic fungus, Coccidioides posadasii.

Coccidioides posadasii is a dimorphic fungal pathogen that grows as a filamentous saprobe in the soil and as endosporulating spherules within the host. To identify genes specific to the pathogenic phase of Co. posadasii, we carried out a large-scale study of gene expression in two isolates of the species. From the sequenced Co. posadasii genome, we chose 1,000 open reading frames to construct a 70-mer microarray. RNA was recovered from both isolates at three life-cycle phases: hyphae, presegmented spherules, and spherules releasing endospores. Comparative hybridizations were conducted in a circuit design, permitting comparison between both isolates at all three life-cycle phases, and among all life-cycle phases for each isolate. By using this approach, we identified 92 genes that were differentially expressed between pathogenic and saprobic phases in both fungal isolates, and 43 genes with consistent differential expression between the two parasitic developmental phases. Genes with elevated expression in the pathogenic phases of both isolates included a number of genes that were involved in the response to environmental stress as well as in the metabolism of lipids. The latter observation is in agreement with previous studies demonstrating that spherules contain a higher proportion of lipids than saprobic phase tissue. Intriguingly, we discovered statistically significant and divergent levels of gene expression between the two isolates profiled for 64 genes. The results suggest that incorporating more than one isolate in the experimental design offers a means of categorizing the large collection of candidate genes that transcriptional profiling typically identifies into those that are strain-specific and those that characterize the entire species.

Adaptation, Biological↗
PubMed2006

Deletion of the adenylate cyclase (sac1) gene affects multiple developmental pathways and pathogenicity in Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum, a broad host range plant pathogen, produces pigmented, multihyphal sclerotia that are capable of long-term survival. Under favorable conditions, sclerotia carpogenically germinate to give rise to apothecia and forcibly discharged ascospores which serve as the primary source of inoculum in the disease cycle. The molecular regulator(s) of sclerotial development in filamentous fungi are largely unknown; however, pharmacological data has revealed that cyclic AMP (cAMP) negatively regulates sclerotial biogenesis in S. sclerotiorum. Based on this observation, we analyzed the role of cAMP by deleting the single copy adenylate cyclase (AC) sac1 gene from S. sclerotiorum. In culture, cyclic AMP levels in the knock-out (KO1) strain were greatly reduced compared to wild type, the hyphal branching pattern was altered, microconidia (spermatia) were more abundant, and aberrant sclerotia were produced in a concentric pattern. The KO1 strain was pathogenic on mechanically wounded tissues; however, virulence was severely attenuated. The pathogenicity defect on unwounded leaves is attributed to the absence of infection cushions and the attenuated virulence on wounded leaves correlates with the slow growth rate observed in culture. This study presents the first description of an adenylate cyclase mutant that affects both pathogenicity and sclerotial development in a broad host range necrotroph.

Adenylyl Cyclases↗
PubMed2006

Effect of gamma-irradiation on pathogens inoculated into ready-to-use vegetables.

Three ready-to-use vegetables, cucumber, blanched and seasoned spinach, and seasoned burdock were selected and the effects of an irradiation treatment for eliminating pathogens were investigated. The pathogens tested were Salmonella Typhimurium, Escherichia coli, Staphylococcus aureus, and Listeria ivanovii. Inoculated viable cells of S. Typhimurium and L. ivanovii into cucumber and blanched and seasoned spinach were reduced about 4 decimal points by 2 kGy of irradiation and that of S. aureus inoculated into burdock showed about 4-decimal point reduction by 1 kGy. E. coli inoculated into burdock was not detected by 1 kGy. All the bacterial contents of test pathogens into the samples were reduced to below the limit of detection by 3 kGy irradiation. The range of the D10 value was 0.28-0.42 among the four pathogens. A Salmonella mutagenicity assay (Ames test) indicated that the 10 kGy-irradiated ready-to-use vegetables did not cause any increase. The studies indicated that a low-dose irradiation (3 kGy or less) can improve the microbial safety of ready-to-use vegetables.

Colony Count, Microbial↗