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PubMed2006

Identification of acquired DNA in Neisseria lactamica.

Anomalous DNA (aDNA) in prokaryotic genomes, identified by its aberrant nucleotide composition, generally represents horizontally acquired DNA. Previous studies showed that frequent DNA transfer occurs between commensal Neisseriae and Neisseria meningitidis. Currently, it is unknown whether aDNA regions are also transferred between these species. The genome of Neisseria lactamica strain 892586 was assessed by a strategy that enables the selective isolation of aDNA, using endonucleases with recognition sites that are overrepresented in aDNA. Of eight regions with aDNA, five displayed similarity to virulence-associated meningococcal sequences. Of three aDNA fragments with limited or no similarity to neisserial sequences, one encodes a novel putative autotransporter/adhesin. The remaining two fragments are adjacent in the N. lactamica genome, and encode a novel putative ATPase/subtilisin-like protease operon. A similar operon is present in the genomes of different respiratory tract pathogens. The identification of aDNA from N. lactamica with similarity to meningococcal aDNA shows that genetic exchange between the Neisseriae is not limited to the neisserial core genome. The discovery of aDNA in N. lactamica similar to a locus in other pathogens substantially expands the neisserial gene pool.

Adenosine Triphosphatases↗

PubMed2004

A novel genomics approach for the identification of drug targets in pathogens, with special reference to Pseudomonas aeruginosa.

Complete genome sequences of several pathogenic bacteria have been determined, and many more such projects are currently under way. While these data potentially contain all the determinants of host-pathogen interactions and possible drug targets, computational tools for selecting suitable candidates for further experimental analyses are currently limited. Detection of bacterial genes that are non-homologous to human genes, and are essential for the survival of the pathogen represents a promising means of identifying novel drug targets. We have used three-way genome comparisons to identify essential genes from Pseudomonas aeruginosa. Our approach identified 306 essential genes that may be considered as potential drug targets. The resultant analyses are in good agreement with the results of systematic gene deletion experiments. This approach enables rapid potential drug target identification, thereby greatly facilitating the search for new antibiotics. These results underscore the utility of large genomic databases for in silico systematic drug target identification in the post-genomic era.

Anti-Bacterial Agents↗

PubMed1997

Single-chain fusions of two unrelated homeodomain proteins trigger pathogenicity in Ustilago maydis.

Pathogenic and sexual development of the fungus Ustilago maydis, the causal agent of corn smut disease, is regulated by heterodimerization of two unrelated homeodomain proteins bE and bW, both encoded by the multi-allelic b mating-type locus. This complex can only be formed if the two proteins are derived from different alleles. The heterodimer is believed to function as a transcriptional regulator that binds to target sites upstream of developmentally regulated genes. We have synthesized a translational fusion in which bE is tethered to bW by a designed flexible kink region. U. maydis strains expressing this synthetic b-fusion become pathogenic for corn illustrating that the single-chain fusion substitutes for the active bE/bW heterodimer. Synthetic b-fusions in which bE and bW originate from the same allele as well as fusions deleted for the dimerization domains were shown to be active while both homeodomains were required for function. Such active fusion proteins are expected to be instrumental in the identification of pathogenicity genes.

Journal Article↗

PubMed1999

Interpretation of genetic test results for hereditary nonpolyposis colorectal cancer: implications for clinical predisposition testing.

CONTEXT: Genetic testing for cancer predisposition is evolving from purely research applications to affecting clinical management. OBJECTIVE: To determine how often genetic test results for hereditary nonpolyposis colorectal cancer (HNPCC) can be definitively interpreted and used to guide clinical management. DESIGN: Case-series study conducted in 1996 to 1998 in which a complete sequence analysis of hMSH2 and hMLH1 coding sequence and flanking intronic regions was performed. Mutations were categorized as protein truncating and missense. In the case of missense alterations, additional analyses were performed in an effort to assess pathogenicity. SETTING AND PARTICIPANTS: Families were identified by self-referral or health care provider referral to a cancer genetics program. Participants and kindreds were classified into 1 of 4 categories: (1) Amsterdam criteria for HNPCC, (2) modified Amsterdam criteria for HNPCC, (3) young age at onset, or (4) HNPCC-variant. In addition, each proband was classified according to the Bethesda guidelines for identification of individuals with HNPCC. MAIN OUTCOME MEASURE: Alterations of hMSH2 and hMLH1 genes. RESULTS: Twenty-seven alterations of hMSH2 and hMLH1 were found in 24 of 70 families (34.3%). Of these, deleterious mutations that could be used with confidence in clinical management were identified in 25.7% (18/70) of families. The rates of definitive results for families fulfilling Amsterdam criteria, modified Amsterdam criteria, young age at onset, HNPCC-variant, and Bethesda guidelines were 27 (39.3%), 13 (18.2%), 12 (16.7%), 11 (15.8%), and 21 (30.4%), respectively. The prevalence of missense mutations, genetic heterogeneity of the syndrome, and limited availability of validated functional assays present a challenge in the interpretation of genetic test results of HNPCC families. CONCLUSIONS: The identification of pathogenic mutations in a significant subset of families for whom the results may have marked clinical importance makes genetic testing an important option for HNPCC and HNPCC-like kindreds. However, for the majority of individuals in whom sequence analysis of hMSH2 and hMLH1 does not give a definitive result, intensive follow-up is still warranted.

Adaptor Proteins, Signal Transducing↗

PubMed1993

Elimination of contaminating DNA within polymerase chain reaction reagents: implications for a general approach to detection of uncultured pathogens.

Analysis based on comparisons of 16S rRNA sequences provides a rapid and reliable approach to identifying human pathogens. By directing oligonucleotide primers at sequences conserved throughout the eubacterial kingdom, bacterial 16S ribosomal DNA sequences of virtually any member of the eubacterial kingdom can be amplified by polymerase chain reaction and subsequently analyzed by sequence determination. Indeed, automated systems for broad-range amplification, sequencing, and data analysis are now feasible and may form the basis of the next generation of automated microbial identification systems. However, identification of pathogens by this strategy is hampered by the frequent contamination of reagents used for the amplification reaction, in particular Taq polymerase, with exogenous bacterial DNA. Here, we describe detailed investigations on the use of 8-methoxypsoralen and long-wave UV light to eliminate contaminating DNA in polymerase chain reaction reagents. The clinical utility of the developed procedure was demonstrated in a case of paucibacillary osteomyelitis, for which no specific bacterial agent had been cultured.

Bacteria↗

PubMed2006

Mitochondrial DNA mutation detection by electrospray mass spectrometry.

BACKGROUND: Mitochondrial DNA (mtDNA) mutations cause a large spectrum of clinically important neurodegenerative, neuromuscular, cardiovascular, and endocrine disorders. We describe the novel application of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) to the rapid and accurate identification of pathogenic mtDNA variants. METHODS: In a blinded study, we used ESI-FTICR MS to analyze 24 unrelated samples of total cellular DNA containing 12 mtDNA variants and compared the results with those obtained by conventional PCR-restriction fragment length polymorphism (PCR-RFLP) analysis and gel electrophoresis. RESULTS: From the 24-sample blinded panel, we correctly identified 12 of the samples as bearing an mtDNA variant and found the remaining 12 samples to have no pathogenic variants. The correlation coefficient between the 2 methods for mtDNA variant detection was 1.0; there were no false positives or false negatives in this sample set. In addition, the ESI-FTICR method identified 4 single-nucleotide polymorphisms (SNP) that had previously been missed by standard PCR-RFLP analysis. CONCLUSIONS: ESI-FTICR MS is a rapid, sensitive, and accurate method for the identification and quantification of mtDNA mutations and SNPs.

DNA, Mitochondrial↗

PubMed2006

Matrix-assisted laser desorption ionization-time of flight mass spectrometry for the discrimination of food-borne microorganisms.

A methodology based on matrix-assisted laser desorption ionization-time of flight mass spectrometry of intact bacterial cells was used for rapid discrimination of 24 bacterial species, and detailed analyses to identify Escherichia coli O157:H7 were carried out. Highly specific mass spectrometric profiles of pathogenic and nonpathogenic bacteria that are well-known major food contaminants were obtained, uploaded in a specific database, and made available on the Web. In order to standardize the analytical protocol, several experimental, sample preparation, and mass spectrometry parameters that can affect the reproducibility and accuracy of data were evaluated. Our results confirm the conclusion that this strategy is a powerful tool for rapid and accurate identification of bacterial species and that mass spectrometric methodologies could play an essential role in polyphasic approaches to the identification of pathogenic bacteria.

Bacteria↗

PubMed1991

Pathogenic and nonpathogenic Entamoeba histolytica: identification and molecular cloning of an iron-containing superoxide dismutase.

Superoxide dismutase (SOD) activity was determined in the cell lysate of the axenically cultured Entamoeba histolytica isolate HM-1:IMSS. Under anaerobic culture conditions, 18.7 (+/- 4.9) units SOD activity (mg protein)-1 were found. By inhibition studies the activity was attributed to an iron-containing type of SOD (FeSOD). Using degenerate oligonucleotide primers derived from regions highly conserved in prokaryotic FeSOD sequences, a genomic DNA fragment was amplified by the polymerase chain reaction. The fragment was used to isolate FeSOD specific cDNA clones from a pathogenic and a nonpathogenic E. histolytica isolate. A comparison of the 2 sequences revealed 5% nucleotide differences resulting in a single amino acid exchange. The primary structure showed the characteristics of an iron-containing type of SOD with a homology of approximately 55% with other FeSOD sequences. The enzyme was found to be encoded by single copy genes in both the pathogenic and the nonpathogenic E. histolytica, but restriction fragment lengths differed between the 2 groups. In 5 isolates studied, no correlation was found between pathogenic behavior of the amebae and the expression of FeSOD-related mRNA.

Amino Acid Sequence↗

PubMed2007

Conditional mutagenesis reveals immunological functions of widely expressed genes: activation thresholds, homeostatic mechanisms and disease models.

Evolutionarily conserved, widely expressed genes provide the functional backbone of most, if not all, cell types. Although mouse mutants created by germ line gene inactivation are instrumental in establishing the importance of such genes in vivo, distortion of embryonic development or multiple body systems often preclude detailed functional studies. To overcome this limitation, DNA recombination systems such as Cre/loxP of bacteriophage P1, have been adapted for use in mammalian cells. The mutagenic event is restricted to the tissue or cell type in question leaving other body systems undisturbed. Conditional inactivation of Csk or Socs3, for example, established their key role in the prevention of inappropriate inflammation, while unexpected immunoregulatory activities emerged from studies of the NF-kappaB and AP-1 pathways. Also, cell types responsible for protective or pathogenic TNFalpha production have been identified. Inactivation of immunoregulatory receptors in leukocyte subsets can provide robust experimental systems revealing the conceptual simplicity underlying the modulation of complex signaling pathways during homeostatic responses. As illustrated for TGF-beta receptor, such system-guided approaches can provide a comprehensive picture of the regulatory events driving in vivo phenotype and specific responses of primary cells. This in turn facilitates the identification of novel regulatory mechanisms, targets for therapeutic intervention and prediction of side effects. With the increasing evidence for a role of somatic mutations in a wider range of human diseases, conditional mouse models are set to play a continuing part in the identification of pathogenic mechanisms for restoration of normal cellular processes in diseases including cancer, inflammation and autoimmunity.

Animals↗

PubMed2001

Periodontal diseases: to protect or not to protect is the question?

For decades, investigations have identified local and systemic humoral immune responses to microorganisms comprising the supra- and subgingival biofilms in the oral cavity. Inflammation and tissue destruction in the periodontium are accompanied by alterations in the quantity, quality, and specificity of antibody. The conundrum in this scenario is the existence of a substantial plasma cell infiltrate at sites of periodontal lesions and a seemingly robust antibody response in the oral cavity and the serum, apparently coincident with progressing disease. Consequently, much effort has been expended to elucidate the critical characteristics of protective humoral responses and to develop strategies for enhancing these unique features. We and others have conducted studies attempting to distinguish disease susceptibility associated with: i) variations in response levels significantly increased to some species with disease, minimal response to others; ii) functional comparisons of antibody subclass differences, genetic regulation, and maturation of responses; iii) microbial and antigenic specificity of the antibody focus on specific pathogens and identification of selected antigens as targets for immunoprotection; and, iv) kinetics of responses during disease and therapeutic interventions linking immune changes with infection and as a measure of treatment success. This report summarizes varied research designs and results, to provide a profile of antibody in health, gingivitis, and periodontitis. These profiles may be used to provide a framework focusing on the humoral response to commensal microorganisms and likely pathogens, as they emerge in the biofilm--etiologic for or in response to disease processes. Models for antibody as a diagnostic adjunct and for predicting protective antibody responses are suggested. These concepts are likely relevant for considering vaccine approaches to periodontitis.

Antibodies, Bacterial↗

PubMed2026

Comparative evaluation of probe-capture and conventional metagenomic sequencing across multiple clinical sample types, with analysis of paired bronchoalveolar lavage fluid and blood samples.

Conventional metagenomic next-generation sequencing (mNGS) suffers from host nucleic acid interference and poor performance in low-biomass samples. Probe-capture metagenomic sequencing (PC-mNGS), which enriches microbial targets via hybridization probes, shows superior sensitivity but lacks systematic multi-sample evaluations. This study compared PC-mNGS and mNGS across diverse clinical specimens (bronchoalveolar lavage fluid [BALF], blood, cerebrospinal fluid [CSF]) and assessed the clinical utility of pathogen co-detection in paired BALF-blood samples from sepsis patients. A total of 282 samples (81 BALF, 141 blood, 25 CSF, 35 others) sequenced by both PC-mNGS and mNGS were analyzed. Additionally, 621 paired BALF-blood samples from sepsis patients with pulmonary infections were evaluated. PC-mNGS achieved higher pathogen detection rates (66.67% vs 57.10%, P = 0.000198) than mNGS, particularly in blood (66.67% vs 47.52%, P = 2.5 × 10⁻⁵). PC-mNGS detected more bacteria (19 species exclusive) and fungi (11 species exclusive) than mNGS. Viruses showed comparable detection. BALF and CSF exhibited high overall agreement (OPA: 96.30% and 88%, respectively), while blood had lower concordance (NPA: 54.05%, OPA: 70.92%). A total of 60.55% of BALF-positive samples (PC-mNGS) had co-detected pathogens in blood. Gram-negative bacteria (e.g., Klebsiella pneumoniae) and fungi (e.g., Candida albicans) showed higher blood co-detection rates than viruses. In this study, PC-mNGS detected more pathogens and showed a higher positivity rate than mNGS in blood samples. BALF sequencing data, particularly bacterial reads per million (RPM), may predict bloodstream co-detection, aiding in sepsis management. However, clinical validation and integration with traditional diagnostics are needed to confirm utility. This study highlights PC-mNGS as a promising tool for complex infections but underscores the need for rigorous multi-context validation.IMPORTANCEAccurate and rapid identification of pathogens is critical for effective treatment of severe infectious diseases, such as sepsis. This study demonstrates that probe-capture metagenomic sequencing (PC-mNGS) detected more pathogens in blood samples compared to conventional metagenomic sequencing, especially for bacterial and fungal infections. By analyzing paired lung and blood samples, we show that high pathogen levels in lung fluid may predict bloodstream infection, offering a potential early warning for clinicians. These findings support the use of PC-mNGS as a more sensitive diagnostic tool, which could lead to faster, more targeted therapies and better outcomes for patients with complex infections.

Humans↗

PubMed1994

[Diagnosis of tuberculosis with polymerase chain reaction].

A new simple method for the detection of pathogenic species Mycobacteriae in clinical samples by use of polymerase chain reaction (PCR) was described. This method is high sensitive, specific and fast. One pair of primers were synthetized for use in PCR with DNA-extracts and whole cells lysates from patient's sputum. Only strains belonging to species of the M. tuberculosis contained an amplifiable fragment of 240 base pairs. Using PCR we achieved the increasing of identification value pathogenic M. tuberculosis in sputum from 44.1% (Ziehl-Nielsen staining) and 55.9% (culturing) to 73.5%.

Base Sequence↗

PubMed1975

Identification of yeast phase of pathogenic fungi by the specificity of their aminopeptidase(s).

Specificity of aminopeptidase(s) was fluorimetrically determined in the yeast phase of Histoplasma capsulatum, H. duboisii, H. farciminosum, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Candida albicans and Crytococcus neoformans. After individually incubating each of 26 amino acid-beta-naphthylamides with each yeast, the amount of each amino acid-beta-naphthylamide hydrolyzed was determined by measuring the free naphythylamine. This resulted in a reproducible profile of the aminopeptidase(s) for each fungus when medium, growth time, size of inoculum and incubation period were standardized. This technique provided a rapid and specific means of identification and differentiation among these yeasts. Specific amino acids were identified from the profile of each yeast as those rapidly liberated by the aminopeptidase(s) of that yeast. These amino acids delineated the amino acid requirement for the normal growth of each yeast.

Aminopeptidases↗

PubMed1984

Influence of geographical factors in the distribution of pathogenic zymodemes of Entamoeba histolytica: identification of zymodeme XIV in India.

Stocks of Entamoeba histolytica isolated and maintained in a variety of culture media, both axenic and polyxenic, were compared with each other and with strains previously characterized. The present stocks were isolated from subjects living in various cities in India. Using the enzyme patterns of: E C 5319 glucose phosphate isomerase (GPI); E C 11140 L-malate: NADP+ oxidoreductase (oxaloacetate decarboxylating) (ME): E C 2751 phosphoglucomutase (PGM); and E C 2711 hexakinase (HK), developed after electrophoresis, three zymodemes were identified in 54 individual isolations of E. histolytica. Most importantly only one zymodeme associated with pathogenicity occurred, identified 28 times, among the collection, one of which was from a liver abscess.

Adult↗

PubMed1998

[Nucleotide sequence analysis of a species specific probe by an inserted fragment from recombinant plasmid pCX7 of L. interrogans sensu stricto serovar lai].

The etiological agents of leptospirosis are the pathogenic leptospires (L. interrogans sensu lato) which can be divided into 223 serovars organized into 23 serogroups. The serovar remains the basic taxon, but serotyping may now be accomplished and recognized by acceptable methods. Complementary molecular approaches are being used extensively to assess genetic relatedness amongst leptospires with restriction endonuclese analysis (REA), pulse field gel electrophoresis (PFGE) and DNA-DNA hybridization as well established tools. However, the method is cumbersome and unsuitable for routine application. To develop a sensitive and specific method for identification of pathogenic leptospires, a genomic library of L. interrogans sensu stricto serovar lai was constructed with the plasmid vector pUC9. A recombinant plasmid, designated pCX7 which has homologous fragment of pathogenic leptospires was screened from the bank. pCX7 could recognize pathogenic leptospiral DNA fragment 1.7 kb of strain 017 without cross hybridization to nonpathogenic leptospiral DNA. Inserted fragment of pCX7 DNA sequencing was performed by Dr. Yan Zhengxin (Max-Plank-Institut fur Biology, Tubingen, Germany). Insert fragment was cloned into pBluescript and sequenced by using ABI(Applied Bio. Systems, Model 373A). Nucleotide sequences were analyzed by Dr. Xiao Jianguo (Texas University Medical School and School of Public Health, Center for Infectious Diseases) using a suit of computer program (NIH). One open reading frame of 306 nucleotids were identified. There were identifiable initiation codons, terminators, pribnow box and sextama box within the sequenced regions. These results further confirmed that the little homology between L. interrogans sensu strito and L. borgpeterseni serovar javanica, L. inadai serovar ranarun and serovar manhao (L. genomospecies 2), L. biflexa serovar patoc, L. illini. pCX7 DNA probe could provide a base for identification and classification of leptospires.

DNA, Bacterial↗

PubMed2005

Polymerase chain reaction assay specific for pathogenic Leptospira based on the gene hap1 encoding the hemolysis-associated protein-1.

In this study, we used Southern hybridization of genomic DNA with the integral hap1 gene as a probe to show that this gene is only present in pathogenic Leptospira strains. We then selected PCR primers based on the hap1 gene, and tested them on several Leptospira strains and biological samples. Specific amplification was obtained for all pathogenic strains tested. Negative PCR results were observed with all saprophytic leptospire strains used as well as with other spirochetes and bacteria commonly found in biological samples. The results of direct PCR performed on biological samples, such as blood, urine or kidneys correlated with the results obtained with the classical Leptospira tests (culture and MAT). A PCR assay based on this gene would be a very useful tool for the rapid, sensitive and specific identification of pathogenic leptospires in samples for diagnosis or epidemiological survey.

Animals↗

PubMed1985

Genotypic approaches to the diagnosis of bacterial infections: plasmid analyses and gene probes.

Practical genetic approaches have been helpful in the diagnosis, epidemiology, and taxonomy of bacterial pathogens encountered in our laboratory at the Centers for Disease Control. There are many examples in which plasmid profiles have been used to define epidemic strains of enteric bacteria, staphylococci, pseudomonads, vibrios, and other pathogenic bacteria. Current methodologies should allow the microbiology laboratory to use plasmid profiles routinely and to identify plasmids associated with bacterial pathogenesis. Simplified DNA-DNA hybridization procedures have been used in our laboratory to survey or "probe' thousands of Escherichia coli colonies for the presence of enterotoxin genes, eliminating traditional tissue culture or animal assays. Research scientists continue to develop gene probes for a number of bacterial toxins and hemolysins and for the identification of pathogens such as legionellae and salmonellae. These and other probes as well as hybridization "kits' may be commercially available to diagnostic laboratories within the next few years.

Autoradiography↗

PubMed2002

[Microbiology--laboratory examinations for bacterias].

As it has been required to identify pathogenic microbes in shorter times, simple and rapid methods have been developed and used. Here, we summarized the present situation of rapid diagnostic testing in clinical microbiology in Japan, and also presented our results on PBP2' detection. The rapid test kits available in Japan for E. coli, Helicobacter pylori, Salmonella, Streptococcus and Staphylococcus aureus were described. Rapid examination methods are based mainly on immunologic reactions, which included slide agglutination using latex particle, immunochromatography and ELISA. Times required for the identification are 10 to 15 minutes. Moreover, rapid test kits employing PCR are also marketed. Further, we evaluated MRSA-LA "Seiken" which is a rapid detection kit for PBP2' produced by MRSA. The test was shown to be highly sensitive and specific. For the rapid identification of pathogenic microbes, simple and rapid test kits described here will be used more in clinical diagnosis.

Bacteria↗