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PubMed1990

The agent of bacillary angiomatosis. An approach to the identification of uncultured pathogens.

BACKGROUND: Bacillary angiomatosis is an infectious disease causing proliferation of small blood vessels in the skin and visceral organs of patients with human immunodeficiency virus infection and other immunocompromised hosts. The agent is often visualized in tissue sections of lesions with Warthin-Starry staining, but the bacillus has not been successfully cultured or identified. This bacillus may also cause cat scratch disease. METHODS: In attempting to identify this organism, we used the polymerase chain reaction. We used oligonucleotide primers complementary to the 16S ribosomal RNA genes of eubacteria to amplify 16S ribosomal gene fragments directly from tissue samples of bacillary angiomatosis. The DNA sequence of these fragments was determined and analyzed for phylogenetic relatedness to other known organisms. Normal tissues were studied in parallel. RESULTS: Tissue from three unrelated patients with bacillary angiomatosis yielded a unique 16S gene sequence. A sequence obtained from a fourth patient with bacillary angiomatosis differed from the sequence found in the other three patients at only 4 of 241 base positions. No related 16S gene fragment was detected in the normal tissues. These 16S sequences associated with bacillary angiomatosis belong to a previously uncharacterized microorganism, most closely related to Rochalimaea quintana. CONCLUSIONS: The cause of bacillary angiomatosis is a previously uncharacterized rickettsia-like organism, closely related to R. quintana. This method for the identification of an uncultured pathogen may be applicable to other infectious diseases of unknown cause.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Comparison of phenotypic and genotypic techniques for identification of unusual aerobic pathogenic gram-negative bacilli.

Rapid and accurate identification of bacterial pathogens is a fundamental goal of clinical microbiology, but one that is difficult or impossible for many slow-growing and fastidious organisms. We used identification systems based on cellular fatty acid profiles (Sherlock; MIDI, Inc., Newark, Del.), carbon source utilization (Microlog; Biolog, Inc., Hayward, Calif.), and 16S rRNA gene sequence (MicroSeq; Perkin-Elmer Applied Biosystems Division, Foster City, Calif.) to evaluate 72 unusual aerobic gram-negative bacilli isolated from clinical specimens at the Mayo Clinic. Compared to lengthy conventional methods, Sherlock, Microlog, and MicroSeq were able to identify 56 of 72 (77.8%), 63 of 72 (87.5%), and 70 of 72 (97.2%) isolates to the genus level (P = 0.002) and 44 to 65 (67.7%), 55 of 65 (84.6%), and 58 of 65 (89.2%) isolates to the species level (P = 0.005), respectively. Four Acinetobacter and three Bordetella isolates which could not be identified to the species level by conventional methods were identified by MicroSeq. In comparison to the full 16S rDNA sequences, the first 527 bp provided identical genus information for all 72 isolates and identical species information for 67 (93.1%) isolates. These data show that MicroSeq provides rapid, unambiguous identification of clinical bacterial isolates. The improved turnaround time provided by genotypic identification systems may translate into improved clinical outcomes.

Carbon↗
PubMedDate not supplied

Availability of laboratory testing services for identification of periodontal pathogens in dental plaque.

Microbiologic monitoring of key subgingival plaque organisms has been proposed to enhance management of some patients with destructive forms of periodontal disease. However, isolation and identification of many of these species is available only through selected microbiology laboratories possessing special expertise with periodontal microorganisms. The purpose of this study was to determine the availability of periodontal microbiology laboratory services in the U.S. and Canada that identify suspected periodontal pathogens for dentists in clinical practice. A survey questionnaire was mailed to all U.S. and Canadian dental schools. Six laboratories that process plaque samples for dentists in private clinical practice were identified. All of the testing services examine plaque specimens for Actinobacillus actinomycetemcomitans and Bacteroides gingivalis. Four of the laboratories used bacterial culturing to characterize a wide range of plaque species and to determine antibiotic sensitivity of isolates. Two other laboratories use indirect immunofluorescence microscopy or DNA probes to screen for three putative periodontal pathogens. All but one of the testing services analyze plaque samples that are collected by private practitioners in their offices and forwarded to the laboratory through next-day delivery services or regular mail. The availability of these adjunctive diagnostic services may enhance the ability of dentists in clinical practice to apply recent advances in knowledge on periodontal microbiology into routine preventive and therapeutic patient care.

Bacteria↗
PubMed2001

Genetic variability of the postharvest pathogen Gilbertella persicaria: identification of randomly amplified polymorphic DNA (RAPD) markers correlating with (+) and (-) mating types.

Random amplified polymorphic DNA (RAPD) and isoenzyme polymorphisms among 16 isolates of the postharvest pathogen Gilbertella persicaria were examined. Six different 10-bp primers were used to determine the extent of intraspecific genetic variability. Nine composite amplification types were identified. RAPD markers were obtained which correlated with the mating types of the G. persicaria isolates. The variability of the isoenzyme patterns was very low and no correlation was found between the isoenzyme markers and the mating abilities. When 80 single carbon substrates were tested in utilization assays, most of them were utilized uniformly by the 16 G. persicaria strains. However, some compounds elicited differences between the isolates representing the two mating types. Beta-alanine (0.2%) has little effect on the germination of the sporangiospores of the (+) isolates, but inhibited the germination of (-) sporangiospores. Glycerol-1-monoacetate supported the growth of both mating types, but at concentrations higher than 4% this was accompanied with a compact (colonial) growth for plus mating type isolates only.

Carbon↗
PubMed2007

Use of robust-long serial analysis of gene expression to identify novel fungal and plant genes involved in host-pathogen interactions.

Identification of important transcripts from fungal pathogens and host plants is indispensable for full understanding the molecular events occurring during fungal-plant interactions. Recently, we developed an improved LongSAGE method called robust-long serial analysis of gene expression (RL-SAGE) for deep transcriptome analysis of fungal and plant genomes. Using this method, we made 10 RL-SAGE libraries from two plant species (Oryza sativa and Zea maize) and one fungal pathogen (Magnaporthe grisea). Many of the transcripts identified from these libraries were novel in comparison with their corresponding EST collections. Bioinformatic tools and databases for analyzing the RL-SAGE data were developed. Our results demonstrate that RL-SAGE is an effective approach for large-scale identification of expressed genes in fungal and plant genomes.

Base Sequence↗
PubMed2004

Identification of novel pathogenicity genes by PCR signature-tagged mutagenesis and related technologies.

Microbial pathogens possess a repertoire of virulence determinants that make unique contributions to bacterial fitness during infection. In this chapter, we focus on the recent progress and adaptations of signature-tagged mutagenesis (STM) by PCR instead of hybridization. This is a PCR-based STM mutation-based screening method using a population of bacterial mutants for the simultaneous identification of multiple virulence genes in microbial pathogens by negative selection. Modifications of STM developed in our laboratory have been applied to Pseudomonas aeruginosa PAO1. Screening of a collection of 6912 STM mutants in the rat chronic lung model of infection identified 214 P. aeruginosa STM mutants defective in virulence. For further studies, and to illustrate better the strategies that need to be utilized, we present detailed analysis of nine selected STM mutants. The data obtained indicate that in vivo, defects in virulence give a wide variety of phenotypes: defects in known virulence factors have been found, thereby validating the method; defects have also been found in orthologs with predicted functions, and in some genes whose functions cannot be predicted from databases. A general strategy and a simple scenario is discussed using the nine STM mutants selected for further characterization. PCR-based STM represent a genomics-based method for in vivo high-throughput screening of new virulence factors.

Bacteria↗
PubMed2006

Towards the definition of pathogenic microbe.

Identification and typing of spoilage and pathogenic microorganisms have become major objectives over the past decade in microbiology. In food, strain typing is necessary to ensure food safety and for linking cases of foodborne infections to suspected items. Recent advances in molecular biology have resulted in the development of numerous DNA-based methods for discrimination among bacterial strains. Here, we present the use of Simple Sequence Repeats (SSR, or Microsatellites) for bacterial typing. SSRs are a class of short DNA sequence motifs that are tandemly repeated at a specific locus. Computer-based screen of the complete genomic DNA sequences of various prokaryotes showed the existence of tens of thousands well distributed SSR tracts. Mono Nucleotides Repeats (MNRs) are the majority of SSR tracts in bacteria, therefore selected MNR loci were analyzed for variation among strains belonging to three bacterial species: Escherichia coli, Listeria monocytogenes and Vibrio cholerae. High levels of polymorphism in the number of repeats was observed. The finding that most of the MNR tracts are variable in bacterial genomes, but stable at the strain level, allows the use of MNRs for bacterial strains identification. The variation in MNR tracts enables the separation between virulent and non-virulent strain groups and further discriminates among bacterial isolates, in the three tested bacterial species. The uncovered MNR polymorphism is important as a genome-wide source of variation, both in practical applications (e.g. rapid strain identification) and in evolutionary studies. This multi-locus MNR strategy could be applied for high throughput bacterial typing by assigning an "identity number" for each strain based on MNR variations. The developed typing technology should include the fingerprint database for large bacterial strain collections and a high throughput scanner. This accurate and rapid tool can have a major role in decreasing the incidences of food-related outbreaks and will contribute to limit epidemics.

Bacterial Typing Techniques↗
PubMed1996

Detection and identification of human pathogenic Leishmania and Trypanosoma species by hybridization of PCR-amplified mini-exon repeats.

A single pair of PCR primers within a conserved region of the mini-exon repeat was used to amplify the repeats from 10 species of pathogenic Leishmania belonging to four major clinical groups and also from three species of Trypanosoma. Oligonucleotide hybridization probes for the detection and identification of the PCR-amplified repeats were constructed from alignments of mini-exon intron and intergenic sequences. The probes generated from mini-exon intergenic regions of the L. (V.) braziliensis, L. (L.) donovani, and L. (L.) mexicana species hybridized specifically to their cognate groups without discriminating between the species within the groups. The probes for L. (L.) major and L. (L.) aethiopica were species-specific, while the L. (L.) tropica probe also hybridized with the L. (L.) aethiopica mini-exon repeat. The mini-exon intron-derived probes for T. cruzi, T. rangeli, and T. brucei were species-specific. This method involving the detection of specific PCR-amplified products produced using a single primer set represents a novel sensitive and specific assay for multiple trypanosomatid species and groups.

Animals↗
PubMed1975

A simple carbohydrate fermentation test for identification of the pathogenic Neisseria.

The carbohydrate fermentation test in cystine-Trypticase agar-tubed medium was compared with the Minitek system with carbohydrate-impregnated paper disks in Müeller-Hinton broth for identification of Neisseria gonorrhoeae and N. meningitidis. There was 100% agreement between the methods for confirmation of N. meningitidis. The paper disk method confirmed 98% of the N. gonorrhoeae isolates; the cystine-Trypticase agar method confirmed 96%. Reactions with the paper disk method could be read in 4 h.

Bacteriological Techniques↗
PubMed2005

[Laboratory methods for detection and identification of biological pathogens].

Laboratory detection and recognition methods of infectious diseases agents have developed markedly in recent years, following the proliferation of nucleic acid and immuno-based detection technologies. The present review summarizes the state of the art in current biorecognition methods: antigenic identification, genetic identification such as PCR, RFLP and FISH, protemics and mass spectrometry. For each method we have specified the technology and qualification required, time to result, specifity and sensitivity, while emphasizing the advantages and disadvantages of using each method for the detection of a given pathogen. Nucleic acid-based detection is more specific and sensitive than immunological-based detection, while the latter is simpler and expected to further development with the improvements in the affinity, specifity and mass production of new immunoglobulins. Protein-based detection methods have an advantage comparing to nucleic acid identification: the presence of the protein approves that the tested gene is functional. Mass spectrometry enables simultaneous detections of multiple proteins and thus holds a promise for new technical developments with a vast array of applications. Most physicians do not practice biodetection technologies in their every day routine, but encounter those terms in their clinical and academic work. The review aims to display basic information in this field in order to enable a common language with basic science specialists.

Bacteria↗
PubMed2005

Use of oligonucleotide array for identification of six foodborne pathogens and Pseudomonas aeruginosa grown on selective media.

Identification of presumptive foodborne pathogens grown on selective media may take one to several days and requires a different battery of biochemical tests for each microorganism. A molecular identification method was developed in which universal primers were used to amplify the 16S to 23S rDNA intergenic spacer of target microorganisms, and PCR products were hybridized to a panel of species-specific oligonucleotides that were immobilized on a nylon membrane. The seven target microorganisms were Bacillus cereus, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella, Staphylococcus aureus, and Vibrio parahaemolyticus. After testing a large collection of target bacteria (29 to 51 strains) and nontarget bacteria (> 500 strains), the performances (sensitivity and specificity) of the oligonucleotide array were as follows: B. cereus (100 and 77%), E. coli (100 and 100%), L. monocytogenes (100 and 90%), P. aeruginosa (100 and 100%), Salmonella (100 and 100%), S. aureus (100 and 100%), and V. parahaemolyticus (100 and 94.2%). Other species in the B. cereus group cross-hybridized to the probes used for identification of B. cereus, and positive results should be confirmed by additional morphological observation of colonies. Listeria innocua cross-reacted with probes used to identify L. monocytogenes, but a simple hemolysis test was used to differentiate the two species. Some strains of Vibrio harveyi and Vibrio mimicus cross-hybridized with probes used for identification of V. parahaemolyticus and caused false-positive reactions. The advantage of the array is that a common protocol was used to identify the seven target microorganisms and multiple different microorganisms could be simultaneously identified on a single array.

Bacillus cereus↗
PubMed2002

Comparison of three chromogenic agar plates for isolation and identification of urinary tract pathogens.

OBJECTIVE: To comparatively assess the performance of three chromogenic agar plates, CPS ID2, Chromogenic UTI, and USA, for the detection and enumeration of all urinary tract pathogens and the direct identification of Escherichia coli, Proteus mirabilis and Enterococcus spp. METHODS: Two hundred and forty-three urine specimens prospectively collected from hospitalized patients were randomly inoculated in parallel on the three media. RESULTS: Of the 243 urine specimens, 235 yielded positive cultures, of which 151 were pure cultures and 84 were mixed cultures. CPS ID2, Chromogenic UTI and USA agar gave detection rates of 99.1%, 97.1% and 96.6%, respectively. The main difference in non-detection between CPS ID2 agar and the two new media concerned Staphylococcus spp. strains. Based on the total number of strains detected (n = 348), the total identification rates of E. coli, P. mirabilis and Enterococcus spp. on CPS ID2 agar, Chromogenic UTI agar and USA agar were 60.3%, 61.2% and 59.2%, respectively. CONCLUSION: The detection rates and identification rates of the three media were very close and only minor differences were noted. The lower detection rates for Chromogenic UTI and USA were mainly due to their lesser ability to support growth of Staphylococcus spp.

Agar↗
PubMed2006

Rapid identification of fungal pathogens in BacT/ALERT, BACTEC, and BBL MGIT media using polymerase chain reaction and DNA sequencing of the internal transcribed spacer regions.

We report a direct polymerase chain reaction/sequence (d-PCRS)-based method for the rapid identification of clinically significant fungi from 5 different types of commercial broth enrichment media inoculated with clinical specimens. Media including BacT/ALERT FA (BioMérieux, Marcy l'Etoile, France) (n = 87), BACTEC Plus Aerobic/F (Becton Dickinson, Microbiology Systems, Sparks, MD) (n = 16), BACTEC Peds Plus/F (Becton Dickinson) (n = 15), BACTEC Lytic/10 Anaerobic/F (Becton Dickinson) (n = 11) bottles, and BBL MGIT (Becton Dickinson) (n = 11) were inoculated with specimens from 138 patients. A universal DNA extraction method was used combining a novel pretreatment step to remove PCR inhibitors with a column-based DNA extraction kit. Target sequences in the noncoding internal transcribed spacer regions of the rRNA gene were amplified by PCR and sequenced using a rapid (24 h) automated capillary electrophoresis system. Using sequence alignment software, fungi were identified by sequence similarity with sequences derived from isolates identified by upper-level reference laboratories or isolates defined as ex-type strains. We identified Candida albicans (n = 14), Candida parapsilosis (n = 8), Candida glabrata (n = 7), Candida krusei (n = 2), Scedosporium prolificans (n = 4), and 1 each of Candida orthopsilosis, Candida dubliniensis, Candida kefyr, Candida tropicalis, Candida guilliermondii, Saccharomyces cerevisiae, Cryptococcus neoformans, Aspergillus fumigatus, Histoplasma capsulatum, and Malassezia pachydermatis by d-PCRS analysis. All d-PCRS identifications from positive broths were in agreement with the final species identification of the isolates grown from subculture. Earlier identification of fungi using d-PCRS may facilitate prompt and more appropriate antifungal therapy.

Costs and Cost Analysis↗
PubMed2003

DNA-based methodologies for rapid detection, quantification, and species- or strain-level identification of respiratory pathogens (Mycobacteria and Pseudomonads) in metalworking fluids.

Mycobacteria and pseudomonads occurring in modern metalworking fluids (MWF) have been implicated in occupational health hazards as causal agents for hypersensitivity pneumonitis (HP) and other respiratory illnesses in machine workers exposed to these fluids and their aerosols. Unlike the conventional cultural and biochemical methods, which are often slow and ambiguous and detect only culturable cells, DNA-based methods offer a time-saving alternative for reliable detection and identification of both culturable and nonculturable bacteria in MWF and for selective quantification of individual genera of pathogens of interest in these fluids. This is the first report on DNA-based direct detection of mycobacteria and pseudomonads in MWF without culturing. Genus-specific PCR approach was successfully applied for screening of field MWF samples originating from different industrial users for detection of mycobacteria or pseudomonads including both culturable and nonculturable cells. PCR in combination with amplicon DNA sequencing led to the identification of Mycobacterium chelonae, Pseudomonas nitroreducens, and an undefined Pseudomonas species from these fluids. Genome fingerprinting by pulsed-field gel electrophoresis (PFGE) on Mycobacterium isolates further showed that the isolates represented three strains of M. chelonae although the possibility of one of the strains being clonal with M. immunogenum cannot be excluded. In parallel efforts, a quantitative competitive PCR method developed based on the Pseudomonas-specific PCR was applied to quantify total P. fluorescens cells in contaminated metalworking fluid and MWF aerosol without culturing. The DNA-based protocols developed in this study will allow rapid screening of field MWF samples for the presence of both culturable and nonculturable cells and thus facilitate effective fluid management and timely exposure assessment.

DNA Fingerprinting↗
PubMedDate not supplied

[Identification and differentiation of pathogenic Burkholderia].

In this review modern methods for the identification and differential diagnostics of the causative agents of glanders and melioidosis, recently included into the genus Burkholderia, are presented. The known phenotypic signs and genetic markers permitting the identification of two pathogenic microorganisms on the definite taxonomic level are described.

Animals↗
PubMed1996

Sequence-based identification of microbial pathogens: a reconsideration of Koch's postulates.

Over 100 years ago, Robert Koch introduced his ideas about how to prove a causal relationship between a microorganism and a disease. Koch's postulates created a scientific standard for causal evidence that established the credibility of microbes as pathogens and led to the development of modern microbiology. In more recent times, Koch's postulates have evolved to accommodate a broader understanding of the host-parasite relationship as well as experimental advances. Techniques such as in situ hybridization, PCR, and representational difference analysis reveal previously uncharacterized, fastidious or uncultivated, microbial pathogens that resist the application of Koch's original postulates, but they also provide new approaches for proving disease causation. In particular, the increasing reliance on sequence-based methods for microbial identification requires a reassessment of the original postulates and the rationale that guided Koch and later revisionists. Recent investigations of Whipple's disease, human ehrlichiosis, hepatitis C, hantavirus pulmonary syndrome, and Kaposi's sarcoma illustrate some of these issues. A set of molecular guidelines for establishing disease causation with sequence-based technology is proposed, and the importance of the scientific concordance of evidence in supporting causal associations is emphasized.

Base Sequence↗
PubMed1999

Comparison of the API Candida system with the AUXACOLOR system for identification of common yeast pathogens.

Two commercial systems for the identification of yeasts were evaluated by using 159 clinical isolates that had also been identified by conventional biochemical and morphological methods. The API Candida system correctly identified 146 isolates (91.8%), and the AUXACOLOR system correctly identified 145 isolates (91.2%). However, of the 146 isolates identified by the API Candida system, 23 required supplemental biochemical tests or morphological assessment to obtain the correct identification. The AUXACOLOR system gave no identification in 13 cases (8.2%), while the API Candida system gave an unreadable profile in only one case. Incorrect identifications were more common with the API Candida system (12 isolates; 7.5%) than with the AUXACOLOR system (1 isolate; 0.6%).

Automation↗
PubMed1985

Rapid identification of periodontal pathogens in subgingival dental plaque. Comparison of indirect immunofluorescence microscopy with bacterial culture for detection of Bacteroides gingivalis.

A large body of research implicates Bacteroides gingivalis in the etiology of adult periodontitis, however, the application of this information to clinical diagnosis and treatment has been hampered by the need for a simple, rapid, and reliable means of detecting this microorganism. In the present study, indirect immunofluorescence microscopy using species specific, polyclonal antisera and a monoclonal antibody was evaluated in the clinical identification and quantitation of B. gingivalis in human subgingival dental plaque. One hundred and twenty subgingival plaque samples were obtained from predetermined sites by means of sterile paper points from 20 human subjects including 10 adult periodontitis patients and 10 periodontally normal subjects. The proportions of cultivable B. gingivalis in each sample were determined following anaerobic culture on nonselective blood agar media and selective media containing kanamycin. These results were then compared to quantitative estimates of B. gingivalis by indirect immunofluorescent microscopic evaluation of heat-fixed plaque smears. Using both immunofluorescence microscopy and bacterial culture, the present study confirms the importance of B. gingivalis in adult periodontitis previously described by culture. The organism was cultivable from 70% of the adult periodontitis patients but not from any of the normal adults. In contrast, indirect immunofluorescence microscopy detected the organism in up to 40% of the subgingival sites in 100% subgingival sites in 100% of the adult periodontitis patients as well as four sites in the periodontally normal subjects. The sensitivity of indirect immunofluorescence microscopy compared to culture ranged from 91 to 100% while the specificity varied from 87 to 89%.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗