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PubMed1997

Identification and characterization of cell wall-cell division gene clusters in pathogenic gram-positive cocci.

Clusters of peptidoglycan biosynthesis and cell division genes (DCW genes) were identified and sequenced in two gram-positive cocci, Staphylococcus aureus and Enterococcus faecalis. The results indicated some similarities in organization compared with previously reported bacterial DCW gene clusters, including the presence of penicillin-binding proteins at the left ends and ftsA and ftsZ cell division genes at the right ends of the clusters. However, there were also some important differences, including the absence of several genes, the comparative sizes of the div1B and ftsQ genes, and a wide range of amino acid sequence similarities when the genes of the gram-positive cocci were translated and compared to bacterial homologs.

Bacterial Proteins↗

PubMed1997

Identification of gene loci controlling pectate lyase production and soft-rot pathogenicity in Pseudomonas marginalis.

Pseudomonas marginalis is an important postharvest pathogen capable of causing soft rot in a wide variety of harvested fruits and vegetables. Following transposon mutagenesis, we isolated two groups of P. marginalis CY091 mutants deficient in production of pectate lyase (Pel) and soft-rot pathogenicity in plants. The first group, designated Pel-, was caused by the insertion of Tn5 into a pel structural gene, and the second group, designated LemA-, was caused by the insertion of Tn5 into a regulatory locus corresponding to the lemA gene previously identified in other Gram-negative bacteria. The LemA- mutants also exhibited alteration in colony morphology and showed deficiency in production of protease (Prt). A cosmid clone pCIC carrying the P. marginalis lemA gene was isolated and characterized. pCIC was capable of restoring Pel production and soft-rot pathogenicity in LemA- mutants of P. marginalis and Pseudomonas viridiflava, indicating that the function of lemA gene in these two pseudomonads was similar and interchangeable. Using MudI-mediated mutagenesis, we isolated a third group of P. marginalis mutants deficient in production of Pel, Prt, and soft-rot pathogenicity. Mutants in this group (designated GacA-1) contained an insertion of MudI in a locus corresponding to the gacA gene of P. viridiflava. Like LemA- mutants, GacA- mutants also exhibited alteration in colony morphology and showed deficiency in production of Pel and Prt. However, GacA- mutants produced much lower levels of levan and fluorescent pyoverdine siderophore than the wild type and LemA- mutants. These results provide the first genetic evidence that P. marginalis produces a single alkaline Pel for maceration of plant tissue and demonstrate that production of Pel, Prt, levan, and pyoverdin by this bacterium is mediated by the two-component lemA/gacA gene system.

Bacterial Proteins↗

PubMed2002

Sequencing the Aspergillus fumigatus genome.

Aspergillus fumigatus is the most common mould pathogen of human beings and unusually causes both invasive disease in immunocompromised patients and allergic disease in patients with atopic immune systems. 4% of patients dying in modern European teaching hospitals have invasive aspergillosis and it is the leading infectious cause of death in leukaemia and bone marrow transplant patients. Until 2001, only two licensed antifungal drugs were available to treat aspergillosis-amphotericin B and itraconazole. Its 28-30Mb genome is being sequenced in an international collaboration, with the Wellcome Trust Sanger Institute (UK) and The Institute for Genomic Research (TIGR, USA) as the two main centres. A whole-genome shotgun approach was adopted and initiated in 2001 with an expected completion date in 2003. The complete sequence will permit identification of pathways specific to pathogenic Aspergillus species, help identify new targets for antifungal drugs, and enable investigations into the basic biology of fungi. Numerous secondary metabolic pathways with biotechnological applications and pharmacological properties are found in the Aspergilli and the genome sequence will facilitate research in this area.

Amphotericin B↗

PubMed2001

Microarray analysis of microbial virulence factors.

Hybridization with oligonucleotide microchips (microarrays) was used for discrimination among strains of Escherichia coli and other pathogenic enteric bacteria harboring various virulence factors. Oligonucleotide microchips are miniature arrays of gene-specific oligonucleotide probes immobilized on a glass surface. The combination of this technique with the amplification of genetic material by PCR is a powerful tool for the detection of and simultaneous discrimination among food-borne human pathogens. The presence of six genes (eaeA, slt-I, slt-II, fliC, rfbE, and ipaH) encoding bacterial antigenic determinants and virulence factors of bacterial strains was monitored by multiplex PCR followed by hybridization of the denatured PCR product to the gene-specific oligonucleotides on the microchip. The assay was able to detect these virulence factors in 15 Salmonella, Shigella, and E. coli strains. The results of the chip analysis were confirmed by hybridization of radiolabeled gene-specific probes to genomic DNA from bacterial colonies. In contrast, gel electrophoretic analysis of the multiplex PCR products used for the microarray analysis produced ambiguous results due to the presence of unexpected and uncharacterized bands. Our results suggest that microarray analysis of microbial virulence factors might be very useful for automated identification and characterization of bacterial pathogens.

Bacterial Proteins↗

PubMed2006

Identification of differentially regulated proteins in response to a compatible interaction between the pathogen Fusarium graminearum and its host, Triticum aestivum.

Using proteomic analyses, a study was carried out aimed at understanding the molecular mechanism of interaction between Fusarium graminearum and Triticum aestivum. Wheat spikelets were inoculated with H2O and conidia spores of F. graminearum. Proteins were extracted from spikelets harvested at three time points: 1, 2 and 3 days post inoculation. About 1380 protein spots were displayed on 2-D gels stained with Sypro Ruby. In total, 41 proteins were detected to be differentially regulated due to F. graminearum infection, and were analyzed with LC-MS/MS for their identification. The proteins involved in the antioxidant and jasmonic acid signaling pathways, pathogenesis-related response, amino acid synthesis and nitrogen metabolism were up-regulated, while those related to photosynthesis were less abundant following F. graminearum infection. The DNA-damage inducible protein was found to be induced and glycosylated in F. graminearum-infected spikelets. Using TargetP program, seven of the identified wheat proteins were predicted to be located in the chloroplast, implying that the chloroplast is the organelle mostly affected by F. graminearum infection. Eight identified fungal proteins possess possible functions such as antioxidant and acquiring carbon from wheat through glycolysis in a compatible interaction between F. graminearum and wheat.

Chloroplasts↗

PubMed2003

Characterization of strains of Vibrio splendidus and V. tapetis isolated from corkwing wrasse Symphodus melops suffering vibriosis.

Two vibrio bacteria pathogenic to the corkwing wrasse Symphodus melops were isolated. Vibriosis-inducing strain LP1 was isolated as the dominanting bacterium in kidney samples of dead and moribund wrasse from a population suffering vibriosis and high daily mortality in 1998 on the Norwegian west coast. The other vibriosis-inducing strain, LP2, was isolated from wrasse captured the following year. Re-infection experiments have confirmed that these strains cause vibriosis in corkwing wrasse. Both strains were typical vibrios sharing the traits of fermentative Gram-negative curved rods with motility and a positive oxidase reaction. Detailed biochemical and genetic characterisation revealed a close affiliation to known species of the marine environment. The first isolate, LP1, is a form of the ubiquitous seawater organism Vibrio splendidus, while the second isolate, LP2, is closely related to V. tapetis (previously only known as the brown ring disease agent in clams). Identification of the new wrasse pathogens V. splendidus LP1 and V. tapetis LP2 is facilitated by break points observed in this study.

Animals↗

PubMed1991

The relationship of microbial pathogens to acute infectious diarrhoea of childhood.

Bacterial, viral and parasitic enteric pathogens were detected in 692 of 916 children below 36 months of age with acute diarrhoea and in 289 of 587 matched controls. The rates of identification of only four groups of pathogens, rotavirus, Shigellae, Salmonella typhimurium and enterotoxigenic E. coli, were significantly higher in the patients. The prevalence of a variety of other enteric pathogens was similar in controls of patients. Shigellosis had a characteristic clinical profile but none of the other agents could be suspected on clinical grounds. The high prevalence of pathogens in controls suggested that the population may be partially protected against a variety of enteric pathogens and that final common pathways leading to diarrhoea may be activated by changes in the microbial ecology of the gut lumen.

Acute Disease↗

PubMed2003

Detection of Tritrichomonas foetus by polymerase chain reaction in cultured isolates, cervicovaginal mucus, and formalin-fixed tissues from infected heifers and fetuses.

A rapid, reliable polymerase chain reaction (PCR) assay, originally developed for definitive laboratory identification of the bovine venereal pathogen Tritrichomonas foetus from cultures of male reproductive tract fluids, was used for testing the following: 1) cultured, geographically disparate trichomonad isolates, 2) formalin-fixed tissues from infected heifers and naturally infected fetuses, and 3) cervicovaginal mucus (CVM) from experimentally infected females. In 12 of 12 Western Hemisphere isolates of pathogenic T. foetus (isolated from outbreaks of clinical trichomoniasis or from screening surveys) and in 1 of 1 American Type Culture Collection strain of Tritrichomonas suis, PCR yielded a positive result, i.e., a 347-base pair amplicon in the 5.8S ribosomal RNA and internal transcribed spacer (5.8S-ITS) region of the genome, whereas cultures of Trichomonas vaginalis and Trichomonas gallinae did not produce a PCR product. The PCR assay was also positive in formalin-fixed, paraffin-embedded endometrial samples from 4 of 4 experimentally infected heifers, as well as in archived tissues from 2 of 2 T. foetus-infected aborted bovine fetuses that were submitted to the diagnostic laboratory from a natural outbreak. It was negative in fixed, embedded uterine tissues of 2 of 2 uninfected virgin heifers used as negative controls and in archived fixed gut tissue of a T. gallinae-infected pigeon. In another experiment, CVM aspirated from 4 of 4 experimentally infected heifers in the fifth or sixth postinfection week yielded a positive PCR product of the expected size, whereas CVM from 2 of 2 controls were PCR negative. Pending validation in larger clinical studies, the PCR assay for the 5.8S-ITS coding region of the T. foetus genome offers the prospect of definitive identification of this agent directly from CVM or from formalin-fixed tissues or when false-positive culture results are suspected.

Abortion, Veterinary↗

PubMed2005

Use of fatty acid profiles to identify food-borne bacterial pathogens and aerobic endospore-forming bacilli.

Capillary gas chromatography (GC) with flame ionization detection was used to determine the cellular fatty acid profiles of various food-borne microbial pathogens and to compare the fatty acid profiles of spores and vegetative cells of the same endospore-forming bacilli. Fifteen bacteria, representing eight genera (Staphylococcus, Listeria, Bacillus, Yersinia, Salmonella, Shigella, Escherichia, and Vibrio) and 11 species were used to compare the extracted fatty acid methyl esters (FAMEs). Endospore-forming bacilli were processed to obtain pure spores and whole cell FAMEs for GC analysis. A data set for each bacterial agent was prepared using fatty acid profiles from five replicates prepared on different days. The results showed that these fatty acid intensity profiles were unique for each of the 11 species and that they could be used as a fingerprint for the organisms. The cellular fatty acid profiles for Bacillus anthracis and Bacillus cereus show that there are two branched chain fatty acids, iso 17:1 omega10c and 17:1 anteiso, which are unique in these species. Iso 17:1 omega10c is present in B. cereus vegetative cells and spores but is not observed in B. anthracis. The 17:1 anteiso fatty acid is present in B. anthracis cells but not in B. cereus cells. Fatty acids 16:0 2OH and 17:0 iso 3OH are present in B. anthracis and B. cereus spores but not in the vegetative cells. In summary, analysis of FAMEs from bacteria and spores can provide a sensitive procedure for the identification of food-borne pathogens.

Bacillus anthracis↗

PubMed1998

Chlamydia pneumoniae and occlusive vascular disease: identification and characterization.

Chlamydia pneumoniae, a respiratory pathogen, has been associated with occlusive vascular disease, including atherosclerosis and intimal hyperplasia, through seroepidemiologic studies. Furthermore, using immunohistochemistry (IHC), polymerase chain reaction (PCR), transmission electron microscopy (TEM), and in situ hybridization, this association has been reconfirmed by detecting this organism in atherosclerotic vascular tissue. This review summarizes and critically analyzes these findings and also discusses various mechanisms of how Chlamydia pneumoniae could be involved in the pathogenesis of occlusive vascular disease. Although more studies are needed to reproduce these results and, possibly, uncover a mechanism, the current literature fails to include detailed methodologies for studying Chlamydia pneumoniae. Therefore, to provide a general standard, we have also outlined specific protocols for IHC, PCR, and TEM. These protocols incorporate essential components from various studies and are presented in a concise and easily adaptable format.

Arterial Occlusive Diseases↗

PubMed2006

Extraintestinal isolates of Escherichia coli: identification and prospects for vaccine development.

Extraintestinal pathogenic Escherichia coli (ExPEC) cause a wide variety of infections that are responsible for significant morbidity, mortality and costs to our healthcare system. Thereby, the development of an efficacious ExPEC vaccine will minimize disease and may be cost-effective in selected patient groups. Surface polysaccharides, such as capsule, have been traditional targets for vaccine development. Considering that significant antigenic heterogeneity exists among surface polysaccharides present in various ExPEC strains, their use as vaccine candidates will be challenging. Therefore, alternative vaccine candidates/approaches are being identified and evaluated and are discussed in this review. The authors envision that an efficacious ExPEC vaccine will consist of either a polyvalent subunit vaccine or a genetically engineered killed whole-cell vaccine.

Animals↗

PubMedDate not supplied

The Amazonia variant of Vibrio cholerae: molecular identification and study of virulence genes.

The pathogenic O1 Amazonia variant of Vibrio cholerae has been shown previously to have a cytotoxin acting on cultured Vero and Y-1 cells, and to lack important virulence factors such as the cholera toxin (Coelho et al. 1995a). This study extends the molecular analysis of the Amazonia strains, detecting the presence of the toxR gene, with a very similar sequence to that of the E1 Tor and classical biotypes. The outer membrane proteins are analyzed, detecting a variation among the group of Amazonia strains, with three different patterns found. As a by-product of this work a polymerase chain reaction fragment was sequenced, reading part of the sequence of the Lon protease of the Amazonia strains. This gene was not previously described in V. cholerae, but its sequence is present in the TIGR database specific for this species.

ATP-Dependent Proteases↗

PubMed2004

Wegener's granulomatosis is associated with organ-specific antiendothelial cell antibodies.

BACKGROUND: Antiendothelial cell antibodies (AECA), usually detected using human umbilical vein endothelial cells (HUVEC), are frequently observed in systemic vasculitis, but their pathogenic role is unclear. Heterogeneity of endothelial cells necessitates use of clinically relevant endothelial cells for elucidation of the role of AECA in systemic vasculitis involving small blood vessels of specific organs. METHODS: Human endothelial cells were isolated from normal tissue specimens from the nose, kidney, lung, liver, and umbilical vein. Using flow cytometry, AECA were detected against both unstimulated and cytokine-stimulated [tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma)] endothelial cells. Functional capacity of AECA was determined by complement fixation assay. Sera from patients with Wegener's granulomatosis (16), limited Wegener's granulomatosis (8), renal limited disease (4), microscopic polyangiitis (MPA) (5), rheumatoid arthritis (10), and systemic lupus erythematosus (SLE) (9), and from healthy controls (20) were analyzed. RESULTS: Compared with controls (1) Wegener's granulomatosis is significantly associated with noncytotoxic AECA that selectively bind surface antigens on unstimulated nasal, kidney, and lung endothelial cells; (2) binding of Wegener's granulomatosis AECA to kidney and nasal endothelial cells in particular was lost upon treatment with IFN-gamma and TNF-alpha; (3) the two cytokines per se were cytotoxic (30%) to nasal and lung endothelial cells and lysis was further increased (60%) by addition of systemic vasculitis serum; and (4) Wegener's granulomatosis serum caused agglutination of cytokine-stimulated nasal endothelial cells. CONCLUSION: Based on these findings we suggest that AECA may be one factor involved in the initiation of Wegener's granulomatosis. Antigen identification and elucidation of the pathogenic roles of AECA and inflammatory cytokines in systemic vasculitis using these cells will be particularly important.

Autoantibodies↗

PubMed2000

Differentiation of clinical Helicobacter pullorum isolates from related Helicobacter and Campylobacter species.

BACKGROUND: Helicobacter pullorum, first detected in the liver and intestinal contents of poultry, was defined as a new species in 1994. This organism has since been isolated from humans with gastroenteritis. Phenotypic as well as genotypic methods have been used to identify H. pullorum associated with cases of human disease. MATERIALS AND METHODS: Clinical isolates were submitted for identification to the National Laboratory for Enteric Pathogens by Provincial Public Health Laboratories within Canada. Phenotypic characterization was conducted using a variety of growth and biochemical tests including oxidase, catalase, indoxyl acetate, H2S production in triple sugar iron (TSI) agar, antimicrobial susceptibility testing, and fatty acid analysis. Genotypic identification was performed using a polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) analysis of a 1-kb fragment of the Helicobacter 16S rRNA gene. RESULTS: During the last 7 years (1993-1999) a total of 11 isolates of H. pullorum were detected from patients with gastroenteritis for inclusion in this study. Typically, these isolates were oxidase and catalase positive, produced optimal growth at 42 degrees C, and produced H2S in TSI. Of these 11 isolates, 1 showed DNase activity, while another did not produce H2S in TSI, and only 2 showed tolerance to 1% bile. Antimicrobial susceptibility assays indicated that 6 of the 11 strains were resistant to nalidixic acid. The fatty acid profiles of the isolates were similar to each other and provided a distinguishing profile from the other related species. Genetically identical and distinct species-specific restriction fragment-length polymorphism (RFLP) patterns were produced using the restriction enzymes Bsr I and Dde I. CONCLUSION: Phenotypic and genotypic procedures were used to identify H. pullorum. Interspecies phenotypic variability was apparent and supported the use of a polyphasic approach for identification. Similarities to the more prominent human pathogens Campylobacter coli and C. lari were also noted. The use of a combination of phenotypic and, in particular, genotypic markers for H. pullorum should prove valuable both for epidemiological investigations and for the diagnosis of disease related to this emerging human pathogen.

Campylobacter↗

PubMed2001

New molecular marker for Trypanosoma (Duttonella) vivax identification.

Trypanosoma vivax is a widespread hemoparasite in tropical areas and is pathogenic to ruminant domestic livestock as well as wild ruminants. The accurate identification of parasites in both hosts and vectors is crucial for epidemiological studies and disease control programs. We describe here the development of molecular markers specific for T. vivax identification. These markers were used to identify mouthpart infections in field-collected tsetse flies from Cameroon. The markers target the genomic sequence of a species-specific antigen from the bloodstream stages. No cross amplification with other trypanosome species was observed, which makes the markers a reliable tool to detect T. vivax infections, both in hosts and vectors. The PCR-amplified sequence contains a (CA)(n) microsatellite repeat for which 11 different alleles were identified. This microsatellite, which showed high polymorphism, provides a suitable marker for population genetic studies.

Animals↗

PubMed1995

Current research on Sarcocystis species of domestic animals.

The genus Sarcocystis is composed of about 130 species of heteroxenous cyst-forming coccidia with differences in life cycle and pathogenicity. Pathogenic Sarcocystis spp. can cause disease in their intermediate hosts, in particular in ruminants. Research on Sarcocystis infections has been impeded by several facets of the parasites. Intermediate as well as definitive hosts can be parasitized by several different species with similarities in biology and morphology. Antigen preparations derived from pathogenic Sarcocystis spp. are highly cross-reactive with antibodies directed against non-pathogenic species. As a consequence, none of the currently available immunological tests is species-specific and can differentiate between pathogenic and non-pathogenic Sarcocystis spp. Over the last decade, new techniques in immunology, protein chemistry and molecular biology have facilitated more advanced studies on the molecular composition and molecular biology of Sarcocystis spp. in various laboratories. The development of species-specific monoclonal antibodies and analyses of the molecular composition of some life-cycle stages of Sarcocystis spp. of cattle and sheep showed that species-specific proteins and antigens exist in these species, although they are not highly abundant. In addition, comparisons of rRNA genes of different Sarcocystis spp. identified unique sequences in the rRNA of pathogenic Sarcocystis spp. that are suitable targets for species-specific identification. Thus, tools have become available that facilitate the development of methods for species-specific identification and differentiation of Sarcocystis spp. as well as the identification and study of molecules that are associated with pathogenicity of some of these parasites.

Animals↗

PubMed2003

A comprehensive guide to antibody and T-cell responses in type 1 diabetes.

Type 1 diabetes (T1D) is an organ-specific autoimmune disease in which the insulin-producing beta cells in the pancreatic islets are selectively eliminated. T cells specific for beta-cell antigens are the mediators of this precise cellular destruction. However, antibodies to beta-cell proteins are also generated and may be used for predicting disease in at-risk populations. Over the past two decades, numerous beta-cell proteins and lipids have been implicated as autoantigens in patients or in non-obese diabetic (NOD) mice, a well-studied animal model of T1D. Here, we present a review of these antigens, accompanied by their T-cell epitopes, where known, and a discussion of our current understanding of why particular self-proteins become disease-inciting antigens. Although two dozen beta-cell antigens have been identified to date, few of these have been confirmed to be recognized by pathogenic T cells early in the disease process. Further identification and characterization of initiating beta-cell antigens targeted by pathogenic T cells should be a priority for future studies.

Animals↗

PubMed2006

HOOF prints: Brucella strain typing by PCR amplification of multilocus tandem-repeat polymorphisms.

A critical component of limiting bacterial disease outbreaks is the tracing of the infection to the index source, which can be facilitated by using a highly discriminating bacterial identification system that will reliably identify genetically related bacterial populations. For pathogenic bacteria with highly conserved genomes, such as the zoonotic pathogen Brucella, finding distinguishing markers or traits for strain identification is challenging. This chapter describes a relatively new procedure for identifying Brucella strains. The procedure, which is called "HOOF prints" (hypervariable octameic oligonucleotide fingerprints), is based on high levels of polymorphism observed at several genomic loci in the Brucella genomes that contain small tandemly repeated deoxyribonucleic sequences. The technique described is designed for medium- to high-throughput analyses. However, the method described can be modified to characterize fewer samples.

Animals↗