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PubMed1994

Sequence determination of rRNA genes of pathogenic Vibrio species and whole-cell identification of Vibrio vulnificus with rRNA-targeted oligonucleotide probes.

A comparative analysis of seven new 16S rRNA gene sequences of pathogenic Vibrio species with previously published vibrio sequences confirmed that Vibrio vulnificus represents a group that is not closely related to the core organisms of the genus Vibrio. In addition, we found that V. vulnificus, Listonella (Vibrio) anguillarum and Vibrio diazotrophicus branch off separately from the core group. A comparison of the 16S rRNA gene sequences of V. vulnificus strains belonging to biotypes 1 and 2 revealed that the sequences of all but four biotype 1 strains were identical to each other but slightly different (17 bases) from the sequences of the rest of the V. vulnificus strains investigated. In addition, the sequences of variable regions of the 23S rRNA genes of Vibrio fluvialis, Vibrio furnissii, Vibrio harveyi, Vibrio cholerae, and V. vulnificus C7184 and TW1 were determined, aligned, and compared with all available bacterial 23S rRNA sequences in order to search for specific target sites. As a result, four oligonucleotide probes specific for V. vulnificus were synthesized, and the specificities of these probes were evaluated by dot blot hybridization to membrane-bound RNAs from 21 V. vulnificus strains, 13 strains belonging to other Vibrio species, 61 strains belonging to species that are members of the alpha, beta, and gamma subclasses of the Proteobacteria, and 3 eucaryotic microorganisms. Two probes hybridized with all of the V. vulnificus strains tested, and the other two probes distinguished V. vulnificus biotype 1 strains from all other organisms. In situ identification of V. vulnificus by using tetramethylrhodamine- or fluorescein-labelled oligonucleotides is now possible.

Bacterial Typing Techniques↗
PubMed1994

A Coxiella burnetii gene encodes a sensor-like protein.

Two-component regulatory systems play important roles in the adaptive responses of many bacteria to environmental changes. The sensor proteins of these systems are highly conserved near their C-termini. We exploited this feature to isolate a gene encoding a putative sensor component from the obligate intracellular rickettsial parasite Coxiella burnetii (Cb). Using degenerate primers and the polymerase chain reaction (PCR), we isolated a DNA fragment from a genomic library of Cb containing an open reading frame (ORF), sufficient to encode a 48-kDa protein. Sequence comparison revealed that the deduced protein shared high homology to members of the bacterial sensor protein family, particularly at three conserved regions of the C terminus. When the Cb sensor-like gene was cloned into a high-copy-number vector and introduced into an E. coli strain (phoM, phoR), the mutant expressed low levels of alkaline phosphatase activity, suggesting that the gene functioned as a sensor protein in E. coli. Consequently, we designated this gene qrsA (for Q fever agent regulatory sensor-like gene). Because two-component regulatory systems have been implicated in a variety of cellular processes, including virulence determinants in some pathogenic bacteria, the identification of qrsA in Cb may shed light on how the pathogen adapts to extracellular changes during infection, as it proliferates in the phagolysosome.

Amino Acid Sequence↗
PubMedDate not supplied

The central role of Fas-ligand cell signaling in inflammatory lung diseases.

Following inflammation and injury in the lung, loss of epithelial cell precursors could determine the balance between tissue regeneration and fibrosis. This review discusses evidence that proapoptotic Fas-Fas ligand (FasL) signaling plays a central role in pulmonary inflammation, injury and fibrosis. FasL signaling induces inflammatory apoptosis in epithelial cells and alveolar macrophages, with concomitant IL-1 beta and chemokine release, leading to neutrophil infiltration. FasL signaling plays a critical role in models of acute lung injury, idiopathic pulmonary fibrosis and silicosis; blockade of Fas-FasL interactions either prevents or attenuates pulmonary inflammation and fibrosis. Serologic and immunohistochemical studies in patients support a major pathogenic role of Fas and FasL molecules in inflammatory lung diseases. Identification of the pathogenic role of FasL could facilitate the discovery of more effective treatments for currently untreatable inflammatory lung diseases.

Acute Disease↗
PubMed2006

[Status of the clinical laboratory in the mandatory postgraduate medical training system: from the medical technologists' view (2)].

When the new system of mandatory postgraduate medical training was introduced, we developed a new training program in our clinical laboratory. We will describe this training program in the microbiology laboratory and our experience. We have had 45 min practice of basic microbiology tests, including Gram staining and microscopic examination, for first-grade postgraduate doctors since 2004, and during this practice we emphasize the importance of quality in the clinical specimens collected for cultures and at the same time, explain how to read microbiological test results. Although we had prepared a kit for the Gram-staining procedure to be used any time they want, it was very disappointing that no doctors used this kit to diagnose an infectious disease. In 2005, four 2nd grade doctors selected the training course in our clinical laboratory for 2 months and 3 of them have finished. In the microbiological laboratory we were in charge of the program for 1 or 2 weeks, which includes the isolation and identification of bacterial pathogens from clinical samples and their susceptibility tests, and the isolation, identification and nucleic acid amplification test for Mycobacterium tuberculosis. All of these trainees acquired the basic knowledge and skills required for microbiological diagnosis. This activity gave us a great opportunity to understand what they do not know and what they want to know. We now have some useful suggestions to develop a good education system for medical microbiology for postgraduate doctors.

Education, Medical, Graduate↗
PubMed2005

A differential medium for the isolation and rapid identification of a plant soft rot pathogen, Erwinia chrysanthemi.

A medium was developed for the isolation and differentiation of Erwinia chrysanthemi from other Erwinia spp. based on the production of blue-pigmented indigoidine. The medium, named NGM, consists of nutrient agar supplemented with 1% glycerol, that induces pigment production, and 2 mM MnCl2*4H2O, that further enhances color development. More than fifty E. chrysanthemi strains from six different plant hosts were tested. All tested strains of E. chrysanthemi grew well on the NGM medium, developing dark brownish to blue colonies easily distinguishable from other Erwinia spp. The results indicate that pigment production on the NGM medium is a very stable property and can be used as a phenotypic property to differentiate E. chrysanthemi from other Erwinia spp. In addition, a specific oligonucleotide primer set was designed for the detection of indC, which is involved in indigoidine biosynthesis. All E. chrysanthemi strains tested contained indC as determined by PCR amplification. No amplification was observed with other Erwinia spp. Thus, pigment production of E. chrysanthemi on the NGM medium is consistent with the existence of indC. The NGM medium was used to isolate and identify the causal agent of soft rot lesions of diseased Phalaenopsis orchids from three orchid cultivation areas in Taiwan. The causal agents of Phalaenopsis soft rot were all identified as E. chrysanthemi. The results indicate that the NGM medium is efficient in isolation and identification of E. chrysanthemi from plants with soft rot symptoms and can also be used for epidemiological studies.

Culture Media↗
PubMed2006

Enhanced identification of viral and atypical bacterial pathogens in lower respiratory tract samples with nucleic acid amplification tests.

The advantages of nucleic acid amplification tests (NAT) over conventional methods for the detection of pathogens in lower respiratory tract samples have not been established. NAT for respiratory pathogens were performed on 439 endotracheal tube (ETT) and bronchoalveolar lavage (BAL) samples. A potential pathogen was detected in 87 samples. Of 22 samples that tested positive by conventional methods, 15 tested positive for the same pathogen by NAT, 1 tested positive for a different pathogen, 2 had co-infections identified only by NAT, and 4 tested negative by NAT. An additional 73 pathogens were detected by NAT in 65 samples including 30 pathogens that were missed by conventional methods (19 adenovirus, 6 respiratory syncytial virus, 3 parainfluenza virus 1-4, 2 influenza A), 41 pathogens not routinely identified by conventional methods in most laboratories (23 rhinovirus, 8 human coronavirus OC43, 5 human metapneumovirus (hMPV), 2 human coronavirus 229E, 2 human coronavirus NL63, 1 Chlamydophila pneumoniae) and 2 pathogens from samples where no respiratory virus testing was requested (1 influenza A, 1 parainfluenza virus). Four of 52 patients who had multiple BAL samples submitted on the same day had negative and positive results by NAT on different samples. NAT improves detection of potential pathogens from ETT and BAL samples.

Adolescent↗
PubMed2006

Identification and characterization of non-pathogenic Fusarium oxysporum capable of increasing and decreasing Fusarium wilt severity.

Fusarium wilt of banana is a potentially devastating disease throughout the world. Options for control of the causal organism, Fusarium oxysporum f.sp. cubense (Foc) are limited. Suppressive soil sites have previously been identified where, despite the presence of Foc, Fusarium wilt does not develop. In order to understand some aspects of this disease suppression, endophytic Fusarium oxysporum isolates were obtained from banana roots. These isolates were genetically characterized and compared with an isolate of Fusarium oxysporum previously identified as being capable of suppressing Fusarium wilt of banana in glasshouse trials. Three additional isolates were selected for glasshouse trials to assess suppression of Fusarium wilt in two different cultivars of banana, Cavendish and Lady Finger. One isolate (BRIP 29089) was identified as a potential biocontrol organism, reducing the disease severity of Fusarium wilt in Lady Finger and Cavendish cultivars. Interestingly, one isolate (BRIP 45952) increased Fusarium wilt disease severity on Cavendish. The implications of an isolate of Fusarium oxysporum, non-pathogenic on banana, increasing disease severity and the potential role of non-pathogenic isolates of Fusarium oxysporum in disease complexes are discussed.

Fusarium↗
PubMed2003

Design and development of a DNA array for rapid detection and identification of multiple tomato vascular wilt pathogens.

Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici, and Verticillium wilt, caused by either Verticillium albo-atrum or Verticillium dahliae, are devastating diseases of tomato (Lycopersicon esculentum) found worldwide. Monitoring is the cornerstone of integrated pest management of any disease. The lack of rapid, accurate, and reliable means by which plant pathogens can be detected and identified is one of the main limitations in integrated disease management. In this paper, we describe the development of a molecular detection system, based on DNA array technology, for rapid and efficient detection of these vascular wilt pathogens. We show the utility of this array for the sensitive detection of these pathogens from complex substrates like soil, plant tissues and irrigation water, and samples that are collected by tomato growers in their greenhouses.

DNA, Bacterial↗
PubMed2005

Isolation, identification and antifungal susceptibility of lemon pathogenic and non pathogenic fungi.

Numerous species of filamentous fungi were isolated from lemon on different plantations in the province of Tucuman, Argentina. The techniques suggested by the Subcommittee of Antifungal Susceptibility of the National Committee for Clinical Laboratory Standards, (USA) were adapted. The effect of three different concentrations of the fungicides imazalil, guazatine, SOPP and thiabendazole on the fungi Fusarium oxysporum, Fusarium moniliforme, Aspergillus niger, Aspergillus flavus, Aspergillus clavatus, Geotrichum candidum, Rhizopus sp, Penicillium sp, Penicillium digitatum and Mucor sp were studied. All the tested strains were resistant to thiabendazole. We assayed a mixture of SOPP (5%), guazatine (350 ppm) and imazalil (100 ppm), which showed a synergic effect on Rhizopus sp. Mucor sp was the only fungus resistant to the four fungicides tested as well as to the above mentioned mixture.

Aspergillus↗
PubMed2005

Identification of trypanothione from the human pathogen Entamoeba histolytica by mass spectrometry and chemical analysis.

In this paper, we present definitive data to show, from ESI (electrospray ionization) studies, that the thiol-bimane compound isolated and purified from Entamoeba histolytica trophozoites, corresponds unequivocally to the structure of trypanothione. Trypanothione disulphide was shown to have a molecular ion of m/z 722. It was further demonstrated by MALDI-TOF (matrix-assisted laser desorption ionization-time-of-flight) MS that this thiol compound also corresponds to the characteristic monoprotonated ion of trypanothione-(bimane)(2), which has a molecular ion of m/z 1103.95. The ion pattern of the thiol-bimane compound prepared from the commercial trypanothione standard is identical with the E. histolytica thiol-bimane compound. After HPLC separation, chemical amino acid analysis by dabsylation and dansylation of the thiol bimane compound from Entamoeba showed the presence of the following trypanothione components: glutamic acid, cysteic acid, glycine and spermidine. We can conclude from these highly reliable MS experiments and chemical analyses that E. histolytica contains the thiol compound trypanothione, which was previously thought to occur only in trypanosomatids.

Animals↗
PubMed2001

The relevance of evolutionary genetics for identification of Trichinella sp. and other pathogens at the strain, subspecies and species levels.

In the specific field of Trichinella research, the impact of evolutionary methods has been until now limited. This is all the more distressing, since this group of parasites is characterized by hard taxonomical problems. Moreover, for the epidemiological tracking of Trichinella populations, high-discriminating genetic typing, conveniently analyzed by population genetic methods, could be of considerable help. With no additional efforts, many molecular data are already available and could be used for retrospective analyses able to solve many taxonomical problems. It is desirable that coming studies are specifically designed according to evolutionary genetics principles and the question under study (sample size, selection of the appropriate marker).

Animals↗
PubMed1998

Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms.

Traditional and molecular typing schemes for the characterization of pathogenic microorganisms are poorly portable because they index variation that is difficult to compare among laboratories. To overcome these problems, we propose multilocus sequence typing (MLST), which exploits the unambiguous nature and electronic portability of nucleotide sequence data for the characterization of microorganisms. To evaluate MLST, we determined the sequences of approximately 470-bp fragments from 11 housekeeping genes in a reference set of 107 isolates of Neisseria meningitidis from invasive disease and healthy carriers. For each locus, alleles were assigned arbitrary numbers and dendrograms were constructed from the pairwise differences in multilocus allelic profiles by cluster analysis. The strain associations obtained were consistent with clonal groupings previously determined by multilocus enzyme electrophoresis. A subset of six gene fragments was chosen that retained the resolution and congruence achieved by using all 11 loci. Most isolates from hyper-virulent lineages of serogroups A, B, and C meningococci were identical for all loci or differed from the majority type at only a single locus. MLST using six loci therefore reliably identified the major meningococcal lineages associated with invasive disease. MLST can be applied to almost all bacterial species and other haploid organisms, including those that are difficult to cultivate. The overwhelming advantage of MLST over other molecular typing methods is that sequence data are truly portable between laboratories, permitting one expanding global database per species to be placed on a World-Wide Web site, thus enabling exchange of molecular typing data for global epidemiology via the Internet.

Bacterial Typing Techniques↗
PubMedDate not supplied

Identification and DNA sequence of a pathogenicity gene of Xanthomonas campestris pv. campestris.

A region of Xanthomonas campestris pv. campestris DNA containing at least two pathogenicity genes was identified. Mutants in one gene were clearly reduced in pathogenicity while mutants in the other were only moderately reduced. Both classes of mutants were prototrophic and motile, and had wild-type levels of extracellular enzymes and extracellular polysaccharide. They also grew in vitro and in planta at the same rate as the wild type. Experiments involving one of the clear pathogenicity mutants indicated that the recovery of mutant cells from turnip seedlings 24 hr after inoculation was lower than for the wild type. This may be due to cell death as a result of action by some preformed or induced plant factor. From DNA sequencing an open reading frame was identified that encompassed the site of the mutations giving a clear reduction in pathogenicity. The predicted protein sequence had no homology with other proteins in the computer data base.

Amino Acid Sequence↗
PubMed2001

Identification of a genetic determinant of pathogenicity in chicken anaemia virus.

The molecular basis of pathogenicity of the chicken anaemia virus (CAV) needs to be clarified in order to develop a safe, live virus vaccine. In this study, several high- and low-pathogenic infectious DNA clones were obtained from field virus samples after 12 or 38 passages in MDCC-MSB1 cells. The high-pathogenic clones induced a low haematocrit, low weight gain and high mortality. Nucleotide sequence analyses identified one amino acid, at residue 394 of the VP1 capsid protein, as a major determinant of pathogenicity. To determine the role of this amino acid in pathogenicity, chimeric infectious DNA clones and point-mutated clones were used for chicken pathogenicity tests. These analyses clearly demonstrated that residue 394 of VP1 was crucial for the pathogenicity of CAV; all of the cloned viruses with glutamine at this position were highly pathogenic, whereas those with histidine had low pathogenicity. Low-pathogenic CAV, based on an infectious DNA clone, is a candidate for a genetically homogeneous and stable CAV live vaccine.

Amino Acid Sequence↗
PubMed2003

Identification of a novel antigen of pathogenic Leptospira spp. that reacted with convalescent mice sera.

The virulence of leptospires isolated from human patients against C3H/HeJ mice was investigated. Infection with clinical isolates from patients with severe leptospirosis was lethal to C3H/HeJ mice, suggesting that C3H/HeJ mice are suitable as an acute lethal model of severe leptospirosis. Using this model, a novel antigen of pathogenic Leptospira spp. (named LAg42), which reacted with convalescent mice sera, was identified. LAg42 is a 42 kDa inner-membrane protein and its immunogenic region is located in the C-terminal region. The gene for LAg42 is conserved among pathogenic leptospires but not among non-pathogenic leptospires, which suggests its involvement in virulence.

Animals↗
PubMed2000

Identification of a gene within a pathogenicity island of enterotoxigenic Escherichia coli H10407 required for maximal secretion of the heat-labile enterotoxin.

Studies of the pathogenesis of enterotoxigenic Escherichia coli (ETEC) have largely centered on extrachromosomal determinants of virulence, in particular the plasmid-encoded heat-labile (LT) and heat-stable enterotoxins and the colonization factor antigens. ETEC causes illnesses that range from mild diarrhea to severe cholera-like disease. These differences in disease severity are not readily accounted for by our current understanding of ETEC pathogenesis. Here we demonstrate that Tia, a putative adhesin of ETEC H10407, is encoded on a large chromosomal element of approximately 46 kb that shares multiple features with previously described E. coli pathogenicity islands. Further analysis of the region downstream from tia revealed the presence of several candidate open reading frames (ORFs) in the same transcriptional orientation as tia. The putative proteins encoded by these ORFs bear multiple motifs associated with bacterial secretion apparatuses. An in-frame deletion in one candidate gene identified here as leoA (labile enterotoxin output) resulted in marked diminution of secretion of the LT enterotoxin and lack of fluid accumulation in a rabbit ileal loop model of infection. Although previous studies have suggested that E. coli lacks the capacity to secrete LT, our studies show that maximal release of LT from the periplasm of H10407 is dependent on one or more elements encoded on a pathogenicity island.

Adhesins, Bacterial↗