Rapid assignment of nucleotide sequence data to allele types for multi-locus sequence analysis (MLSA) of bacteria using an adapted database and modified alignment program

A novel database and modified alignment program is described which provides a fast and accurate procedure for assigning nucleotide sequences to allele types for multi-locus sequence analysis (MLSA). The database has between 40 and 160 alleles per organism including Neisseria meningitidis, Streptococcus pneumoniae, Staphylococcus aureus and Haemophilus influenzae. The database directly compares the query nucleotide sequence against all alleles within the database and this system reduces the time taken for the analysis of nucleotide sequence data and assignment of alleles for subsequent sequence analysis.     

Detection and discrimination of Mycoplasma pneumoniae and Mycoplasma genitalium by the in vitro DNA amplification.

By using the primers designed on the bases of the sequences of the 16S rRNA genes of Mycoplasma pneumoniae and Mycoplasma genitalium, respectively, specific and sensitive in vitro DNA amplification assay system for the detection and discrimination of these two mycoplasmas was established. The detection limit of the assay was 100 cells for M. pneumoniae and 1,000 cells for M. genitalium. Neither other human mycoplasmas nor oral bacteria existing in human saliva showed any cross-reactions with these primers.     

Usefulness of the BACTEC MYCO/F lytic system for detection of mycobacteremia in a clinical microbiology laboratory

278 BACTEC MYCO/F lytic system blood cultures for mycobacteria were evaluated between 1997 and 1999. Sixty of them were read as positive by the system, being considered 15 of them as false positives. Twenty-seven yielded mycobacterial growth (13 Mycobacterium avium from 3 patients and 14 Mycobacterium tuberculosis from 8 patients). Other bacteria isolated were coagulase-negative Staphylococcus (13 samples), Corynebacterium sp. (5 samples), Salmonella enteritidis (2 samples) and Klebsiella pneumoniae (1 sample). Five of these isolates were considered as true episodes of bacteremia. The average time for detection of mycobacteria was 12.6 days for M. avium and 26.4 days for M. tuberculosis. BACTEC MYCO/F lytic system is useful for detection of mycobacteremia in clinical microbiology laboratories.     

Detection of Mycoplasma pneumoniae in simulated and true clinical throat swab specimens by nanorod array-surface-enhanced Raman spectroscopy

The prokaryote Mycoplasma pneumoniae is a major cause of respiratory disease in humans, accounting for 20% of all community-acquired pneumonia and the leading cause of pneumonia in older children and young adults. The limitations of existing options for mycoplasma diagnosis highlight a critical need for a new detection platform with high sensitivity, specificity, and expediency. Here we evaluated silver nanorod arrays (NA) as a biosensing platform for detection and differentiation of M. pneumoniae in culture and in spiked and true clinical throat swab samples by surface-enhanced Raman spectroscopy (SERS). Three M. pneumoniae strains were reproducibly differentiated by NA-SERS with 95%-100% specificity and 94-100% sensitivity, and with a lower detection limit exceeding standard PCR. Analysis of throat swab samples spiked with M. pneumoniae yielded detection in a complex, clinically relevant background with >90% accuracy and high sensitivity. In addition, NA-SERS correctly classified with >97% accuracy, ten true clinical throat swab samples previously established by real-time PCR and culture to be positive or negative for M. pneumoniae. Our findings suggest that the unique biochemical specificity of Raman spectroscopy, combined with reproducible spectral enhancement by silver NA, holds great promise as a superior platform for rapid and sensitive detection and identification of M. pneumoniae, with potential for point-of-care application.     

Cyclodextrin-glycosyltransferase from Klebsiella pneumoniae M5a1: cloning, nucleotide sequence and expression

The structural gene encoding cyclodextrin-glycosyltransferase of Klebsiella pneumoniae strain M5a1 was cloned; it is expressed both in Escherichia coli and in K. pneumoniae and the gene product is secreted into the extracellular space. Determination of the nucleotide sequence revealed an open reading frame coding for a single polypeptide of 655 amino acid (aa) residues. The enzyme is synthesized as a precursor with an N-terminal signal peptide of 30 aa residues, which is proteolytically processed between two alanine residues during export. The primary structure of CGT bears homology with the sequences of amylases from both prokaryotic and eukaryotic origins.     

Polymerase chain reaction as a sensitive and rapid method for specific detection of Mycoplasma pneumoniae in clinical samples

The fast diagnosis of Mycoplasma primary atypical pneumonia is impaired by the lack of routinely available culture methods for isolation of Mycoplasma pneumoniae from clinical specimens. Likewise, serological methods commonly used for diagnosis are insensitive and non-specific. In this study, we have established and applied the polymerase chain reaction (PCR) technique to detect M. pneumoniae DNA in clinical samples originating from the respiratory tract. The PCR results were compared with those from culture and serology tests. To standardize the detection of M. pneumoniae by PCR, we first used DNA from culture grown organisms and clinical samples seeded with M. pneumoniae. PCR amplification was performed with M. pneumoniae-specific primers to amplify 144, 153 and 631 bp DNA fragments by using primer pairs MP5-1/MP5-2, P1-178/P1-331 and P1-178/P1-809, respectively. The amplification of the 631 bp DNA fragment was found to be most sensitive for the detection of M. pneumoniae. Using the most sensitive PCR, a total of 47 respiratory specimens from patients suspected of community acquired pneumonia were tested. While none of the specimens were positive for M. pneumoniae in culture, 6 specimens gave positive results by PCR. In 4 out of the 5 PCR positive samples tested serologically, the results were supported by elevated levels of anti-mycoplasma IgG/IgM/IgA. Thus, these results suggest that PCR is the most sensitive method to detect M. pneumoniae in clinical specimens.     

A latex agglutination test for the detection of Mycoplasma pneumoniae in respiratory exudates: a comparative study with a commercially available DNA-probe test.

We prepared polyclonal antibody specific to Mycoplasma pneumoniae. Using this antibody, we developed a latex agglutination test (LAT) for detecting the organism in respiratory exudates as rapid diagnosis of M. pneumoniae infection. Further, LAT was compared with DNA-probe test (DP) which was the only commercially available test for the rapid detection of the organism. In LAT, both M. pneumoniae and M. genitalium give positive agglutination, but the titer of M. genitalium was significantly lower than that of M. pneumoniae. The detection limit of LAT was 2 x 10(5) CFU/ml and that of DP was 5 x 10(4) CFU/ml in vitro. It was considered that target molecules in LAT were accumulated in the pharyngeal portion of the patients, because of their long half-life at 37 C. However, ribosomal RNA which was target molecule in DP was destroyed at 37 C much sooner, and the accumulation could not be expected. Actually, positive rate in LAT was higher than that in DP among clinical specimens in which M. pneumoniae was detected by culture method. The procedure of LAT is much easier and more rapid than that of DP in which radioactive isotope is required. LAT could be the choice of test for rapid diagnosis of M. pneumoniae infection.     

PCR-based detection of Mycoplasma species

In this study, we describe our newly-developed sensitive two-stage PCR procedure for the detection of 13 common mycoplasmal contaminants (M. arthritidis, M. bovis, M. fermentans, M. genitalium, M. hominis, M. hyorhinis, M. neurolyticum, M. orale, M. pirum, M. pneumoniae, M. pulmonis, M. salivarium, U. urealyticum). For primary amplification, the DNA regions encompassing the 16S and 23S rRNA genes of 13 species were targeted using general mycoplasma primers. The primary PCR products were then subjected to secondary nested PCR, using two different primer pair sets, designed via the multiple alignment of nucleotide sequences obtained from the 13 mycoplasmal species. The nested PCR, which generated DNA fragments of 165-353 bp, was found to be able to detect 1-2 copies of the target DNA, and evidenced no cross-reactivity with the genomic DNA of related microorganisms or of human cell lines, thereby confirming the sensitivity and specificity of the primers used. The identification of contaminated species was achieved via the performance of restriction fragment length polymorphism (RFLP) coupled with Sau3AI digestion. The results obtained in this study furnish evidence suggesting that the employed assay system constitutes an effective tool for the diagnosis of mycoplasmal contamination in cell culture systems.     

Characterization and nucleotide sequence of pqqE and pqqF in Methylobacterium extorquens AM1.

Methylobacterium extorquens AM1 pqqEF are genes required for synthesis of pyrroloquinoline quinone (PQQ). The nucleotide sequence of these genes indicates PqqE belongs to an endopeptidase family, including PqqF of Klebsiella pneumoniae, and M. extorquens AM1 PqqF has low identity with the same endopeptidase family. M. extorquens AM1 pqqE complemented a K. pneumoniae pqqF mutant.     

Use of PCR methods for detection of selected genes of Mycoplasma pneumoniae

The aim of this study was standardization of PCR for the detection of gene encoding the P1 protein, 16S rRNA and elongation factor Tu of M. pneumoniae. A total of 13 strains of M. pneumoniae, 28 strains of other mycoplasmas and 14 strains of different bacteria causing respiratory tract infections were tested. In all of tested M. pneumoniae strains the presence of the sought genes was confirmed. The specificity of DNA was confirmed by the restriction endonuclease analysis with enzymes Hind III, Alu I and Hha I. With none of primers specific for the M. pneumoniae genes amplification of DNA from other bacteria was noted. The PCR method with the selected primers allowed to detect from 10(2) to 10(4) cfu M. pneumoniae/ml suspended in broth. The obtained results indicate that the PCR method can be used for detection of M. pneumoniae genes. A very good sensitivity and specificity predestine++ PCR as a potential quick diagnostic method for identification of M. pneumoniae in clinical specimens.     

Repetitive DNA sequences in Mycoplasma pneumoniae.

Two types of different repetitive DNA sequences called RepMP1 and RepMP2 were identified in the genome of Mycoplasma pneumoniae. The number of these repeated elements, their nucleotide sequence and their localization on a physical map of the M. pneumoniae genome were determined. The results show that RepMP1 appears at least 10 times and RepMP2 at least 8 times in the genome. The repeated elements are dispersed on the chromosome and, in three cases, linked to each other by a homologous DNA sequence of 400 bp. The elements themselves are 300 bp (for RepMP1) and 150 bp (for RepMP2) long showing a high degree of homology. One copy of RepMP2 is a translated part of the gene for the major cytadhesin protein P1 which is responsible for the adsorption of M. pneumoniae to its host cell.     

Identification of a ribonuclease H gene in both Mycoplasma genitalium and Mycoplasma pneumoniae by a new method for exhaustive identification of ORFs in the complete genome sequences

Exhaustive identification of open reading frames in complete genome sequences is a difficult task. It is possible that important genes are missed. In our efforts to reanalyze the intergenic regions of Mycoplasma genitalium and Mycoplasma pneumoniae, we have newly identified a number of new open reading frames (ORFs) in both M. genitalium and M. pneumoniae. The most significant identification was that of a ribonuclease H enzyme in both species which until now has not been identified or assumed absent and interpreted as such. In this paper we discuss the biological importance of RNase H and its evolutionary implication. We also stress the usefulness of our method for identifying new ORFs by reanalyzing intergenic regions of existing ORFs in complete genome sequences.     

Multicenter evaluation of the novel enzyme immunoassay based on P1-enriched protein for the detection of Mycoplasma pneumoniae infection

The aim of the study was to evaluate new Mycoplasma pneumoniae IgG, IgA and IgM EIA methods based on the enrichment of P1-protein (ThermoLabsystems, Helsinki, Finland) (L) for the detection of acute infection. This evaluation was performed in two independent routine clinical microbiology laboratories. The first laboratory used samples preselected by IgG and IgM Platelia enzyme immunoassay (P) and the second used samples preseleced by Serion ELISA Classic M. pneumoniae IgG, IgM (V). The L method was also compared to the FDA approved method of ImmunoWell M. pneumoniae IgG and IgM (G). When the agreement of two methods was applied as a serologic criteria for an acute infection, the following ratios of acute to nonacute infection were calculated 32/86 (totally 118) in the first and 20/72 (totally 92) in the second laboratory. In the first laboratory, the corresponding ratios by methods were 35/83 (sensitivity 100%, specificity 96.5%), 31/87 (sensitivity 97%, specificity 100%), and 55/63 (sensitivity 100%, specificity 79%) for the L, P and G methods, respectively. In the second laboratory, the ratios were 21/71 (sensitivity 100%, specificity 99%), 16/76 (sensitivity 83%, specificity 100%), and 53/39 (sensitivity 100, specificity 69%) for the L, V and G methods, respectively. Taking into account that the tested sample material was preselected by the P and V methods, which may have introduced some bias in their favor, the newly developed L method utilizing P1-enriched protein was found reliable for serodiagnosis of acute M. pneumoniae infection. The method G was the least specific in detection of acute infection.     

Genetic relationships among mycoplasmas based on the 16S-23S rRNA spacer sequence

The nucleotide sequences of the spacer regions between the 16S and 23S rRNA genes of 20 Mycoplasma species were determined following amplification by PCR. Although the spacer regions lacked spacer tRNA genes, they contained the box B and box A sequences in this order from the 5' terminus. The sequence alignment indicated that the 20 species were divided into four clusters, the M. pneumoniae, M. hominis, M. hyorhinis and M. fermentans clusters, and a single floating species, M. hyopneumoniae.     

ERIC sequences: a novel family of repetitive elements in the genomes of Escherichia coli, Salmonella typhimurium and other enterobacteria

We describe a family of highly conserved, Enterobacterial Repetitive Intergenic Consensus (ERIC) sequences, 14 of which have been identified in Escherichia coli and Salmonella typhimurium and a further three in other enterobacterial species (Yersinia pseudotuberculosis, Klebsiella pneumoniae and Vibrio cholerae). ERIC sequences are 126 bp long and appear to be restricted to transcribed regions of the genome, either in intergenic regions of polycistronic operons or in untranslated regions upstream or downstream of open reading frames. ERIC sequences are highly conserved at the nucleotide sequence level but their chromosomal locations differ between species. Several features of ERIC sequences resemble those of REP sequences (Stern et al., 1984) although the nucleotide sequence is entirely different. The question of whether ERICs have a specific function, or represent a form of 'selfish' DNA, is discussed.     

Classification analysis of a latent dinucleotide periodicity of plant genomes

The information decomposition (ID) method has been used for searching dinucleotide periodicities, including latent ones, in plant genomes. In nucleotide sequences of genomes of various plants from the GenBank database, 14766 sequences with a periodicity of two nucleotides have been found. Classification of the periodicity matrices of the detected DNA sequences has yielded 141 classes of dinucleotide periodicity. Since ID does not detect periodicities with nucleotide deletions or insertions, modified profile analysis (MPA) has been applied to the obtained classes to reveal DNA sequences with dinucleotide periodicities containing nucleotide deletions and insertions. Combined use of ID and MPA has permitted the detection of 80 396 DNA sequences with dinucleotide periodicities in the genomes of various plants. The biological role of dinucleotide periodicity in the detected sequences is discussed.     

Single tube real time PCR for detection of Streptococcus pneumoniae, Mycoplasma pneumoniae, Chlamydophila pneumoniae and Legionella pneumophila from clinical samples of CAP

We designed a multiplex real time PCR for rapid, sensitive and specific detection of Streptococcus pneumoniae, Legionella pneumophila, Chlamydophila pneumoniae and Mycoplasma pneumoniae. The study cases consisted of 129 patients with community acquired pneumonia (CAP). Bacteriological techniques were implemented for detection of the cultivable organisms. DNA were extracted from sputa, throat swabs, bronchoalveolar lavages and tracheal aspirates and used as templates in real time PCR. The primers and probes were designed for cbpA (S. pneumoniae), p1adhesin (M. pneumoniae), mip (L. pneumophila) and ompA (C. pneumoniae). After optimization of real time PCR for every organism, the experiments were continued in multiplex in a single tube. Of 129 CAP specimens, the positive cultures included 14 (10.85%) for S. pneumoniae, 9 (6.98%) for L. pneumophila and 3 (2.33%) for M. pneumoniae. Four specimens (3.10%) were positive for C. pneumoniae by real time PCR. The sensitivity of our real time PCR was 100% for all selected bacteria. The specificity of the test was 98.26%, 98.34%, 100% and 100% for S. pneumoniae, L. pneumophila, M. pneumoniae and C. pneumoniae, respectively. This is the first report on the use of multiplex real time PCR for detection of CAP patients in the Middle East. The method covers more than 90% of the bacterial pathogens causing CAP.