DOCSLIB.ORG

Oligonucleotide Array-Based Detection and Genotyping of Mollicutes (Acholeplasma, Mycoplasma, and Ureaplasma)

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Oligonucleotide Array-Based Detection and Genotyping of Mollicutes (Acholeplasma, Mycoplasma, and Ureaplasma) J. Microbiol. Biotechnol. (2009), 19(3), 265–270 doi: 10.4014/jmb.0806.347 First published online 10 October 2008 Oligonucleotide Array-based Detection and Genotyping of Mollicutes (Acholeplasma, Mycoplasma, and Ureaplasma) Jang, Hyunjung1, Hyomyeung Kim1, Byeongchul Kang3, Cheolmin Kim1, 2, and Heekyung Park4* 1Institute for Genomic Medicine, GeneIn Co., Ltd, Busan 617-030, Korea 2Department of Biochemistry, Pusan National University Medical School, Busan 602-739, Korea 3Institute of Supportive Bioinformatics, Insilicogen Inc., Suwon 441-813, Korea 4PANAGENE Inc., 816 Tamnip-dong, Yuseong-gu, Daejeon 305-510, Korea Received: June 2, 2008 / Accepted: July 24, 2008 An oligonucleotide array was developed to detect and genotype Bacterial Strains and Preparation of Genomic DNAs mollicutes based on the internal transcribed spacer (ITS) Acholeplasma laidlawii ATCC 25937, Mycoplasma arginini sequence. The results of the assay were compared with ATCC 23838, Mycoplasma arthritidis ATCC 19611, those of a PCR-RFLP assay. The proposed oligonucleotide Mycoplasma bovis ATCC 27368, Mycoplasma cloacale array containing 5 genus- and 23 species-specific probes was ATCC 35276, Mycoplasma falconis ATCC 51372, Mycoplasma able to detect Mycoplasma species, including M. penetrans faucium ATCC 25293, Mycoplasma fermentans ATCC and M. spermatophilum, that were not detected by the PCR- 19989, Mycoplasma genitalium ATCC 33530, Mycoplasma RFLP assay. Therefore, the results demonstrated that hominis ATCC 23114, Mycoplasma hyorhinis ATCC 17981, the proposed oligonucleotide array was effective for the Mycoplasma hyosynoviae ATCC 25591, Mycoplasma detection and discrimination of 23 species, including an neurolyticum ATCC 19988, Mycoplasma opalescens ATCC acholeplasma, 21 mycoplasmas, and a ureaplasma, and 27921, Mycoplasma orale ATCC 23714, Mycoplasma showed promise as a countermeasure to ensure that biological penetrans ATCC 55252, Mycoplasma pirum ATCC 25960, products are safe and of good quality. Mycoplasma pneumoniae ATCC 15531, Mycoplasma Keywords: Oligonucleotide array, mollicute, internal transcribed primatum ATCC 15497, Mycoplasma salivarium ATCC 23064, spacer, genotyping Mycoplasma spermatophilum ATCC 49695, Mycoplasma synoviae ATCC 25204, and Ureaplasma urealyticum ATCC 27618 were all purchased from the American Type Culture Collection (ATCC), whereas the other bacteria, Bacillus The contamination of biological materials by mollicutes subtilis KCTC 1103, Lactobacillus casei KCTC 2180, (including Acholeplasma, Mycoplasma, and Ureaplasmas) Clostridium pasteurianum KCTC 1674, Escherichia coli can lead to unreliable experimental results and unsafe biological KCTC 1042, and Enterococcus faecalis KCTC 2011, were products. Mycoplasmas are phenotypically distinguished from obtained from the Korean Collection for Type Cultures other bacteria by their minute size and total lack of a cell (KCTC). The genomic DNAs were extracted using an wall. The methods for detecting pathogenic or contaminant InstaGene matrix kit (Bio-Rad Laboratories Inc., Hercules, mycoplasmas include microbiological cultures, immunological CA, U.S.A.) according to the manufacturer’s protocol. assays, DNA staining techniques, nucleic acid hybridization, and PCRs [4, 11]. Various laboratories have already developed PCR and Sequencing of ITS Regions species-specific PCR assays to detect mycoplasmas [12]. The primers used for the amplification of the ITS region of However, PCR assays can only detect a single or limited number the mycoplasmas (F2 and R2) and acholeplasmas (M78 and of mollicute species [5, 10]. Accordingly, this study analyzed R34) have already been described [10]. The PCR mixture a phylogeny tree of the ITS sequences of mollicutes and (50 µl) contained 1 U of Taq DNA polymerase, 1× Qiagen developed an oligonucleotide array that can genotype PCR buffer (Qiagen Inc., Valencia, CA, U.S.A.), and 20 pmol 23 mollicutes (an acholeplasma, 21 mycoplasmas, and a of each primer. The PCR conditions included 4 min at ureaplasma). 94oC; 30 cycles of 1 min at 94oC, 1 min at 55oC, and 1 min at 72oC; plus a further 10 min at 72oC. The PCR products *Corresponding author Phone: +82-42-936-9298; Fax: +82-42-861-9297; were sequenced after subcloning using a TA Cloning Kit E-mail: [email protected] (Promega, Madison, WI, U.S.A.), and compared with existing 266 Jang et al. sequences in the GenBank database. The ITSs of M. bovis, digestion with VspI (MBI Fermentas, Hanover, MD, U.S.A.), M. cloacale, M. falconis, M. faucium, M. spermatophilum, SspI and HindIII (New England BioLabs, Beverly, MA, and M. synoviae were newly sequenced in this study. U.S.A.), ClaI, DraI, and HinfI (DCC-BIONET, Korea), and the digested patterns determined by agarose gel electrophoresis. Phylogenetic Analysis The distances for each ITS were calculated using the PHYLIP Phylogenetic Relationships of Mycoplasmas Based on ITS package [2] and Kimura 2 parameter [4]. The phylogenetic Sequences trees were also constructed with the PHYLIP package using An acholeplasma, 21 mycoplasmas, and a ureaplasma were the neighbor-joining method [8]. The significance of the clustered in 5 groups based on the ITS sequences (Fig. 1). The resulting tree topologies was tested by performing a bootstrap overall ITS sequence similarity ranged from 32.0% to 99.0% analysis with resampling 1,000 times. (data not shown). The lowest similarity was 32.0% between M. hyorhinis and M. pirum, whereas the highest similarity was Oligonucleotide Array for Genotyping of Mollicutes 99% between M. arthritidis and M. faucium (data not shown). Genus- and species-specific probes were designed from the Therefore, these results indicated that the phylogenetic analysis conserved and polymorphic regions of the ITSs for using the ITS sequences was capable of distinguishing between Acholeplasma, Mycoplasma, and Ureaplasma, respectively. an acholeplasma, 21 mycoplasmas, and an ureaplasma, with The specificity of the probes was evaluated by testing using the exception of M. arthritidis and M. faucium. mollicutes and closely related bacteria (B. subtilis, Lb. casei, Cl. pasteurianum, E. coli, and En. faecalis). Genotyping of Mycoplasmas Using Oligonucletide Array: The fabrication, hybridization, and scanning processes of Specificity and Sensitivity the oligonucleotide array have been described previously [3, 6]. The ITS sequences of mollicutes contain both conserved and A schematic showing the layouts of the oligonucleotide polymorphic regions. Thus, the conserved regions in the ITS probes is shown in Figs. 2Y and 2Z. sequences of Acholeplasma and Mycoplasma were used to design several genus-specific probes. First, the optimal genus- PCR-Restriction Enzyme Analysis (PRA) specific probes were selected for 5 groups based on the ITS The unique restriction patterns predicted by the NEB cutter, sequences. The MP-CP probe was hybridized with the version 2.0 program (http://www.neb.com/rebase), were verified mycoplasmas in groups 1 and 3. The MP-CB probe was using the PCR products amplified with the F2 and R2 primers hybridized with the mycoplasmas in group 2. The MP-CE probe for the restriction enzyme analysis of the mollicutes. was hybridized with the mycoplasmas in groups 4 and 5. In Differentiation of the mollicute species was accomplished by addition, since there was no single genus-specific probe to Fig. 1. Phylogenic tree derived from ITS region sequences of mycoplasmas. The numbers on the tree are the percentages of occurrence in 1,000 bootstrapped trees; only values greater than 70% are shown. OLIGONUCLEOTIDE ARRAY TO DETECT MOLLICUTES 267 Fig. 2. Hybridization results for mycoplasma species when using an oligonucleotide array. A to W. Each species of mycoplasma exhibited a unique pattern of reactivity to each set of probes. X. Hybridization results for other pathogenic bacteria. Y. Image of QC probe before hybridization. Z. Layout of probes in the oligonucleotide array. The probes were patterned in duplicate. The probe names are as explained in Table 1. hybridize all the mycoplasmas, a combination of the four differentiation of an acholeplasma, 21 mycoplasmas, and a probes (MP-CP and MP-CB or MP-CP and MP-CE) was ureaplasma, yet was unable to differentiate M. arthritidis used to detect the mycoplasmas at the genus level. Finally, and M. faucium, owing to the 99% similarity of their the AP-CB probe was hybridized with the acholeplasma. respective ITS sequences. The oligonucleotide array also The presence of several polymorphic regions within the allowed for the identification of a number of mycoplasma ITS sequences enabled the design of individual species-specific species in one reaction. The genus-specific probes showed probes. The genus- and species-specific probes only detected a unique hybridization pattern with mycoplasmas (Figs. 2A their target, and there was no cross-hybridization with other to 2W) and a mismatch with other bacteria containing B. bacteria (Fig. 2). The oligonucleotide array allowed for the subtilis, Lb. casei, Cl. pasteurianum, E. coli, and En. faecalis, 268 Jang et al. Table 1. Probes designed in this study. Strain Probe Sequence Positionc a Acholeplasma AP-CB 5'-T15-AGTCTTTGAAAAGTAGATAAA-3' 134-154 d MP-CP 5'- T15-RWTCTTTVAAAACTRRATWN-3' 074-93 0 b d Mycoplasma MP-CB 5'- T15-GGKYAATTTGTTTWGAT-3' 028-44 0 e MP-CE 5'- T15-AATAAGTTACTAAGGGCTTAT-3' 201-221 M. arginini MP-arg 5'- T15-AGATTATATCATACAATAGA-3' 145-164 f M. arthritidis-faucium MP-arf 5'- T15-GAATACAAAATCAATACAATA-3' 146-166 M. cloacale MP-clo 5'-T15-AGTACAATTCTCACTGTTATG-3' 011-310
Load more

Recommended publications
  • Molecular Detection of Urogenital Mollicutes in Patients with Invasive Malignant Prostate Tumor Osama Mohammed Saed Abdul-Wahab1, Mishari H
    Abdul-Wahab et al. Infectious Agents and Cancer (2021) 16:6 https://doi.org/10.1186/s13027-021-00344-9 RESEARCH ARTICLE Open Access Molecular detection of urogenital mollicutes in patients with invasive malignant prostate tumor Osama Mohammed Saed Abdul-Wahab1, Mishari H. Al-Shyarba2, Boutheina Ben Abdelmoumen Mardassi3, Nessrine Sassi3, Majed Saad Shaya Al Fayi4, Hassan Otifi5, Abdullah Hassan Al Murea6, Béhija Mlik3 and Elhem Yacoub3* Abstract Background: The etiology of prostate cancer (PCa) is multiple and complex. Among the causes recently cited are chronic infections engendered by microorganisms that often go unnoticed. A typical illustration of such a case is infection due to mollicutes bacteria. Generally known by their lurking nature, urogenital mollicutes are the most incriminated in PCa. This study was thus carried out in an attempt to establish the presence of these mollicutes by PCR in biopsies of confirmed PCa patients and to evaluate their prevalence. Methods: A total of 105 Formalin-Fixed Paraffin-Embedded prostate tissues collected from 50 patients suffering from PCa and 55 with benign prostate hyperplasia were subjected to PCR amplification targeting species-specific genes of 5 urogenital mollicutes species, Mycoplasma genitalium, M. hominis, M. fermentans, Ureaplasma parvum, and U. urealyticum. PCR products were then sequenced to confirm species identification. Results significance was statistically assessed using Chi-square and Odds ratio tests. Results: PCR amplification showed no positive results for M. genitalium, M. hominis, and M. fermentans in all tested patients. Strikingly, Ureaplasma spp. were detected among 30% (15/50) of PCa patients. Nucleotide sequencing further confirmed the identified ureaplasma species, which were distributed as follows: 7 individuals with only U.
    [Show full text]
  • The Phylogenetic Composition and Structure of Soil Microbial Communities Shifts in Response to Elevated Carbon Dioxide
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by University of Minnesota Digital Conservancy The ISME Journal (2012) 6, 259–272 & 2012 International Society for Microbial Ecology All rights reserved 1751-7362/12 www.nature.com/ismej ORIGINAL ARTICLE The phylogenetic composition and structure of soil microbial communities shifts in response to elevated carbon dioxide Zhili He1, Yvette Piceno2, Ye Deng1, Meiying Xu1,3, Zhenmei Lu1,4, Todd DeSantis2, Gary Andersen2, Sarah E Hobbie5, Peter B Reich6 and Jizhong Zhou1,2 1Institute for Environmental Genomics, Department of Botany and Microbiology, University of Oklahoma, Norman, OK, USA; 2Ecology Department, Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA; 3Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou, China; 4College of Life Sciences, Zhejiang University, Hangzhou, China; 5Department of Ecology, Evolution, and Behavior, St Paul, MN, USA and 6Department of Forest Resources, University of Minnesota, St Paul, MN, USA One of the major factors associated with global change is the ever-increasing concentration of atmospheric CO2. Although the stimulating effects of elevated CO2 (eCO2) on plant growth and primary productivity have been established, its impacts on the diversity and function of soil microbial communities are poorly understood. In this study, phylogenetic microarrays (PhyloChip) were used to comprehensively survey the richness, composition and structure of soil microbial communities in a grassland experiment subjected to two CO2 conditions (ambient, 368 p.p.m., versus elevated, 560 p.p.m.) for 10 years. The richness based on the detected number of operational taxonomic units (OTUs) significantly decreased under eCO2.
    [Show full text]
  • Metabolic Network Percolation Quantifies Biosynthetic Capabilities
    RESEARCH ARTICLE Metabolic network percolation quantifies biosynthetic capabilities across the human oral microbiome David B Bernstein1,2, Floyd E Dewhirst3,4, Daniel Segre` 1,2,5,6,7* 1Department of Biomedical Engineering, Boston University, Boston, United States; 2Biological Design Center, Boston University, Boston, United States; 3The Forsyth Institute, Cambridge, United States; 4Harvard School of Dental Medicine, Boston, United States; 5Bioinformatics Program, Boston University, Boston, United States; 6Department of Biology, Boston University, Boston, United States; 7Department of Physics, Boston University, Boston, United States Abstract The biosynthetic capabilities of microbes underlie their growth and interactions, playing a prominent role in microbial community structure. For large, diverse microbial communities, prediction of these capabilities is limited by uncertainty about metabolic functions and environmental conditions. To address this challenge, we propose a probabilistic method, inspired by percolation theory, to computationally quantify how robustly a genome-derived metabolic network produces a given set of metabolites under an ensemble of variable environments. We used this method to compile an atlas of predicted biosynthetic capabilities for 97 metabolites across 456 human oral microbes. This atlas captures taxonomically-related trends in biomass composition, and makes it possible to estimate inter-microbial metabolic distances that correlate with microbial co-occurrences. We also found a distinct cluster of fastidious/uncultivated taxa, including several Saccharibacteria (TM7) species, characterized by their abundant metabolic deficiencies. By embracing uncertainty, our approach can be broadly applied to understanding metabolic interactions in complex microbial ecosystems. *For correspondence: DOI: https://doi.org/10.7554/eLife.39733.001 [email protected] Competing interests: The authors declare that no Introduction competing interests exist.
    [Show full text]
  • Ureaplasma Urealyticum and Ureaplasma Parvum
    The Journal of Molecular Diagnostics, Vol. 13, No. 2, March 2011 Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. DOI: 10.1016/j.jmoldx.2010.10.007 Modified Real-Time PCR for Detecting, Differentiating, and Quantifying Ureaplasma urealyticum and Ureaplasma parvum Ellen Vancutsem,* Oriane Soetens,* microbiological methods and are, therefore, usually re- Maria Breugelmans,† Walter Foulon,† ferred to as Ureaplasma species. They are found in the and Anne Naessens* lower genital tract of nearly 50% of pregnant women as part of the normal vaginal flora. However, in some cases, From the Departments of Microbiology and Infection Control* Ureaplasma species have interfered with normal fetal de- and Obstetrics,† Universitair Ziekenhuis Brussel, Brussels, velopment by causing an ascending infection.2–7 The Belgium reason for this infection is not fully understood but may be associated with the virulence of the microorganism, the host immune system, or local factors present in the lower We evaluated a previously described quantitative real- genital tract. Species differentiation might be important time PCR (qPCR) for quantifying and differentiating because previous studies2,8,9 suggest that nongonococ- Ureaplasma parvum and U. urealyticum. Because of cal urethritis and an adverse pregnancy outcome with nonspecific reactions with Staphylococcus aureus respect to birth weight, gestational age, and preterm DNA in the U. parvum PCR, we developed a modified delivery are implicated with the presence of U. urealyti- qPCR and designed new primers. These oligonucleo- cum and not with U. parvum. tides eradicated cross-reactions, indicating higher Because strains can only be differentiated with la- specificity.
    [Show full text]
  • Mycoplasma Pirum Sp. Nov. a Terminal Structured Mollicute from Cell Cultures
    INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, July 1985, p. 285-291 Vol. 35, No. 3 0020-7713/85/030285-07$02.00/0 Copyright 0 1985, International Union of Microbiological Societies Mycoplasma pirum sp. nov. a Terminal Structured Mollicute from Cell Cultures RICHARD A. DEL GIUDICE,'" JOSEPH G. TULLY,* DAVID L. ROSE,2 AND ROGER M. COLE3? Diagnostic Microbiology Laboratory, National Cancer Institute-Frederick Cancer Research Facility,' and Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases,2 Frederick, Maryland 21 701 and Laboratory of Streptococcal Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 202053 More than 200 mycoplasma isolates recovered from a variety of contaminated cell cultures from 1968 to the present were found to belong to a distinct serological group (serogroup 38). An analysis of representative strains of this collection showed that they possessed all of the characteristics of organisms belonging to the genus Mycoplasma. These organisms were capable of fermenting glucose and of hydrolyzing arginine, and they required cholesterol or serum for growth. These organisms were unusual in that they possessed an organized terminal structure or tip, a morphological structure which has been found in at least six other species in the genus Mycoplasma. These organisms were also serologically distinct from all previously established species in the genus Mycoplasma and the genus Acholeplasma. On the basis of these findings and other morphological, biological, and serological characteristics of the strains recovered, we propose that mycoplasmas with these properties belong to a new species, Mycoplasma pirum sp. nov. Strain HRC 70-159 (= ATCC 25960) is the type strain of this species.
    [Show full text]
  • Invivogen Insight Newsletter: Mycoplasma
    November/December 2005 An Insightful Look At InvivoGen’s Innovative Products Mycoplasma contamination remains a significant problem to Mycoplasma: The Insidious Invader of Cell Cultures the culture of mammalian cells. Mycoplasmas are the smallest and simplest and enzymatic procedures. However, none of these self-replicating organisms. Due to their seriously methods is 100% reliable. Direct growth methods are The detection of mycoplasma degraded genome they cannot perform many relatively sensitive to most species but the overall contamination is an important metabolic functions, such as cell wall production or procedure is lengthy (3 weeks), costly and less synthesis of nucleotides and amino acids. sensitive to noncultivable species. The PCR method, part of mycoplasma control and Mycoplasmas are strictly parasites. They parasitize a although rather fast and inexpensive, is limited by its should be an established wide range of organisms including humans, animals, sensitivity and the risk of positive and false negative method in every cell culture insects, and plants. results. Mycoplasma and Acholeplasma are Mollicutes, that InvivoGen has developed a new mycoplasma detection laboratory. InvivoGen is pleased comprise together more than 100 recognized species. method that promises to resolve these issues. This to introduce PlasmoTest™, a Among them, about 20 species have been described as method is based on the detection of mycoplasmas by contaminants of eukaryotic cell cultures. However engineered cells that express Toll-like receptor 2, a Mycoplasma detection kit based 5 species (Mycoplasma (M.) arginini, M. fermentans, pathogen recognition receptor that detects mycoplasmas. on a brand new technology. M. orale, M. hyorhinis and Acholeplasma laidlawii) PlasmoTest™, InvivoGen’s new mycoplasma detection are isolated in 90-95% of contaminated cell cultures1.
    [Show full text]
  • Comprehensive Analysis of Risk Factors for Periodontitis Focusing on the Saliva Microbiome and Polymorphism
    International Journal of Environmental Research and Public Health Article Comprehensive Analysis of Risk Factors for Periodontitis Focusing on the Saliva Microbiome and Polymorphism Naoki Toyama 1,* , Daisuke Ekuni 1 , Daisuke Matsui 2, Teruhide Koyama 2 , Masahiro Nakatochi 3, Yukihide Momozawa 4, Michiaki Kubo 4 and Manabu Morita 1 1 Department of Preventive Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan; [email protected] (D.E.); [email protected] (M.M.) 2 Department of Epidemiology for Community Health and Medicine, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566, Japan; [email protected] (D.M.); [email protected] (T.K.) 3 Public Health Informatics Unit, Department of Integrated Health Sciences, Nagoya University Graduate School of Medicine, Nagoya 461-8673, Japan; [email protected] 4 Laboratory for Genotyping Development, RIKEN Center for Integrative Medical Sciences, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama City 230-0045, Japan; [email protected] (Y.M.); [email protected] (M.K.) * Correspondence: [email protected]; Tel.: +81-86-235-6712 Abstract: Few studies have exhaustively assessed relationships among polymorphisms, the micro- biome, and periodontitis. The objective of the present study was to assess associations simultaneously among polymorphisms, the microbiome, and periodontitis. We used propensity score matching with a 1:1 ratio to select subjects, and then 22 individuals (mean age ± standard deviation, 60.7 ± 9.9 years) Citation: Toyama, N.; Ekuni, D.; were analyzed.
    [Show full text]
  • Genome Diversity of Spore-Forming Firmicutes MICHAEL Y
    Genome Diversity of Spore-Forming Firmicutes MICHAEL Y. GALPERIN National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894 ABSTRACT Formation of heat-resistant endospores is a specific Vibrio subtilis (and also Vibrio bacillus), Ferdinand Cohn property of the members of the phylum Firmicutes (low-G+C assigned it to the genus Bacillus and family Bacillaceae, Gram-positive bacteria). It is found in representatives of four specifically noting the existence of heat-sensitive vegeta- different classes of Firmicutes, Bacilli, Clostridia, Erysipelotrichia, tive cells and heat-resistant endospores (see reference 1). and Negativicutes, which all encode similar sets of core sporulation fi proteins. Each of these classes also includes non-spore-forming Soon after that, Robert Koch identi ed Bacillus anthracis organisms that sometimes belong to the same genus or even as the causative agent of anthrax in cattle and the species as their spore-forming relatives. This chapter reviews the endospores as a means of the propagation of this orga- diversity of the members of phylum Firmicutes, its current taxon- nism among its hosts. In subsequent studies, the ability to omy, and the status of genome-sequencing projects for various form endospores, the specific purple staining by crystal subgroups within the phylum. It also discusses the evolution of the violet-iodine (Gram-positive staining, reflecting the pres- Firmicutes from their apparently spore-forming common ancestor ence of a thick peptidoglycan layer and the absence of and the independent loss of sporulation genes in several different lineages (staphylococci, streptococci, listeria, lactobacilli, an outer membrane), and the relatively low (typically ruminococci) in the course of their adaptation to the saprophytic less than 50%) molar fraction of guanine and cytosine lifestyle in a nutrient-rich environment.
    [Show full text]
  • APUTS) Reporting Terminology and Codes Microbiology (V1.0
    AUSTRALIAN PATHOLOGY UNITS AND TERMINOLOGY (APUTS) Reporting Terminology and Codes Microbiology (v1.0) 1 12/02/2013 APUTS Report Information Model - Urine Microbiology Page 1 of 1 Specimen Type Specimen Macro Time Glucose Bilirubin Ketones Specific Gravity pH Chemistry Protein Urobilinogen Nitrites Haemoglobin Leucocyte Esterases White blood cell count Red blood cells Cells Epithelial cells Bacteria Microscopy Parasites Microorganisms Yeasts Casts Crystals Other elements Antibacterial Activity No growth Mixed growth Urine MCS No significant growth Klebsiella sp. Bacteria ESBL Klebsiella pneumoniae Identification Virus Fungi Growth of >10^8 org/L 10^7 to 10^8 organism/L of mixed Range or number Colony Count growth of 3 organisms 19090-0 Culture Organism 1 630-4 LOINC >10^8 organisms/L LOINC Significant growth e.g. Ampicillin 18864-9 LOINC Antibiotics Susceptibility Method Released/suppressed None Organism 2 Organism 3 Organism 4 None Consistent with UTI Probable contamination Growth unlikely to be significant Comment Please submit a repeat specimen for testing if clinically indicated Catheter comments Sterile pyuria Notification to infection control and public health departments PUTS Urine Microbiology Information Model v1.mmap - 12/02/2013 - Mindjet 12/02/2013 APUTS Report Terminology and Codes - Microbiology - Urine Page 1 of 3 RCPA Pathology Units and Terminology Standardisation Project - Terminology for Reporting Pathology: Microbiology : Urine Microbiology Report v1 LOINC LOINC LOINC LOINC LOINC LOINC LOINC Urine Microbiology Report
    [Show full text]
  • Moving Beyond Serovars
    ABSTRACT Title of Document: MOLECULAR AND BIOINFORMATICS APPROACHES TO REDEFINE OUR UNDERSTANDING OF UREAPLASMAS: MOVING BEYOND SEROVARS Vanya Paralanov, Doctor of Philosophy, 2014 Directed By: Prof. Jonathan Dinman, Cell Biology and Molecular Genetics, University of Maryland College Park Prof. John I. Glass, Synthetic Biology, J. Craig Venter Institute Ureaplasma parvum and Ureaplasma urealyticum are sexually transmitted, opportunistic pathogens of the human urogenital tract. There are 14 known serovars of the two species. For decades, it has been postulated that virulence is related to serotype specificity. Understanding of the role of ureaplasmas in human diseases has been thwarted due to two major barriers: (1) lack of suitable diagnostic tests and (2) lack of genetic manipulation tools for the creation of mutants to study the role of potential pathogenicity factors. To address the first barrier we developed real-time quantitative PCRs (RT-qPCR) for the reliable differentiation of the two species and 14 serovars. We typed 1,061 ureaplasma clinical isolates and observed about 40% of isolates to be genetic mosaics, arising from the recombination of multiple serovars. Furthermore, comparative genome analysis of the 14 serovars and 5 clinical isolates showed that the mba gene, used for serotyping ureaplasmas was part of a large, phase variable gene system, and some serovars shown to express different MBA proteins also encode mba genes associated with other serovars. Together these data suggests that differential pathogenicity and clinical outcome of an ureaplasmal infection is most likely due to the presence or absence of potential pathogenicity factors in individual ureaplasma clinical isolates and/or patient to patient differences in terms of autoimmunity and microbiome.
    [Show full text]
  • Rapid, Sensitive PCR-Based Detection of Mycoplasmas In
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1994, p. 953-959 Vol. 60, No. 3 0099-2240/94/$04.00 +0 Copyright © 1994, American Society for Microbiology Rapid, Sensitive PCR-Based Detection of Mycoplasmas in Simulated Samples of Animal Sera OLIVIER DUSSURGET AND DAISY ROULLAND-DUSSOIX* Laboratoire des Mycoplasnes, Institut Pasteur, 75724 Paris Cedex 15, Franice Received 2 August 1993/Accepted 17 December 1993 A fast and simple method to detect mycoplasmal contamination in simulated samples of animal sera by using a PCR was developed. The following five mycoplasma species that are major cell culture contaminants belonging to the class Mollicutes were investigated: Mycoplasma arginini, Acholeplasma laidlawii, Mycoplasma hyorhinis, Mycoplasma orale, and Mycoplasma fermentans. After a concentration step involving seeded sera, genus-specific primers were used to amplify a 717-bp DNA fragment within the 16S rRNA gene of mycoplasmas. In a second step, the universal PCR was followed by amplification of variable regions of the 16S rRNA gene by using species-specific primers, which allowed identification of contaminant mycoplasmas. With this method, 10 fg of purified DNA and 1 to 10 color-changing units of mycoplasmas could be detected. Since the sensitivity of the assay was increased 10-fold when the amplification products were hybridized with an internal mycoplasma-specific 32P-labelled oligonucleotide probe, a detection limit of 1 to 10 genome copies per PCR sample was obtained. This highly sensitive, specific, and simple assay may be a useful alternative to methods currently used to detect mycoplasmas in animal sera. Mycoplasmas (the trivial name for microorganisms belong- Like most cell cultures infected by mycoplasmas, serum infec- ing to the class Molliclutes) are the smallest self-replicating tions are rarely detected by visual inspection or light micros- bacteria.
    [Show full text]
  • Highly Conserved Gene and Its Use to Generate Species-Specific, Genus-Specific, Family- Specific, Group-Specific and Universal Nucleic Acid Probes for Microorganisms
    (19) TZZ ¥ T (11) EP 2 322 667 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 18.05.2011 Bulletin 2011/20 C12Q 1/68 (2006.01) C07K 14/00 (2006.01) C12N 15/63 (2006.01) C12N 5/10 (2006.01) (21) Application number: 10181529.8 (22) Date of filing: 28.09.2000 (84) Designated Contracting States: • Ouellette, Marc AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU Sillery, Québec G1S 3S1 (CA) MC NL PT SE • Picard, François J. Cap-Rouge, Québec G1Y 1A1 (CA) (30) Priority: 28.09.1999 CA 2283458 • Roy, Paul H. 19.05.2000 CA 2307010 Loretteville, Québec G2A 2X8 (CA) (62) Document number(s) of the earlier application(s) in (74) Representative: Pohlman, Sandra M. accordance with Art. 76 EPC: df-mp 00965686.9 / 1 246 935 Fünf Höfe Theatinerstrasse 16 (71) Applicant: Geneohm Sciences Canada, Inc. 80333 München (DE) Quebec City, Québec G1V 2K8 (CA) Remarks: (72) Inventors: •ThecompletedocumentincludingReferenceTables • Bergeron, Michel G. and the Sequence Listing can be downloaded from Québec, Québec G2K 1T8 (CA) the EPO website • Boissinot, Maurice •Claims filed after the date of filing of the application St-Augustin-de-Desmaures / after the date of receipt of the divisional application Québec G3A 2N2 (CA) (Rule 68(4) EPC). • Huletsky, Ann •This application was filed on 29-09-2009 as a Sillery, Québec G1S 4J3 (CA) divisional application to the application mentioned • Ménard, Christian under INID code 62. St-Lambert-de-Lévis Québec G0S 2W0 (CA) (54) Highly conserved gene and its use to generate species-specific, genus-specific, family- specific, group-specific and universal nucleic acid probes for microorganisms.
    [Show full text]