Sequencing and staphylococci identification.The emerging clinical importance of staphylococcal infections Staphylococcal Infections Definition Staphylococcal (staph) infections are communicable conditions caused by certain bacteria and generally characterized by the formation of abscesses. prompted us to establish a reference database for partial RNA polymerase RNA polymerase n. A polymerase that catalyzes the synthesis of RNA from a DNA or RNA template. B (rpoB; nucleotides 1444-1928) gene sequences from type strains of all staphylococcal staphylococcal pertaining to Staphylococcus spp. staphylococcal clumping test used as a means of measuring the quantity of fibrinogen-split products in a sample of blood. species and subspecies subspecies, also called race, a genetically distinct geographical subunit of a species. See also classification. . This database correctly identified 55 clinical staphylococcal isolates; all were correctly identified at the species level. At the subspecies level, rpoB misidentified only 2 isolates. ********** The emerging clinical importance of Staphylococcus aureus Staphylococcus au·re·us n. A bacterium that causes furunculosis, pyemia, osteomyelitis, suppuration of wounds, and food poisoning. Staphylococcus aureus Staphylococcus pyogenes and coagulase-negative staphylococci staph·y·lo·coc·cus n. pl. staph·y·lo·coc·ci A spherical gram-positive parasitic bacterium of the genus Staphylococcus, usually occurring in grapelike clusters and causing boils, septicemia, and other infections. (1) in connection with the expanding number of staphylococcal subspecies described requires accurate identification to the subspecies level. Currently, the genus Staphylococcus Noun 1. genus Staphylococcus - includes many pathogenic species bacteria genus - a genus of bacteria family Micrococcaceae, Micrococcaceae - spherical or elliptical usually aerobic eubacteria that produce yellow or orange or red pigment; includes is divided into 36 species and 21 subspecies. Staphylococcal subspecies not included in the databases of commercial identification systems, as well as phenotypic variants (e.g., small-colony variants), are often misidentified (2). We recently described the usefulness of genotypic identification of staphylococcal subspecies by using partial 16S rDNA sequences in comparison with phenotypic tests (3). However, the partial 16S rDNA sequences used were not discriminative dis·crim·i·na·tive adj. 1. Drawing distinctions. 2. Marked by or showing prejudice: discriminative hiring practices. enough to differentiate all staphylococcal subspecies. When searching for a molecular target for discrimination of staphylococci, several genes have been evaluated, e.g., heat shock protein heat shock protein n. Any of a group of cellular proteins that are produced under conditions of heat stress and help to stabilize other cellular proteins exposed to high temperatures. 60 (hsp60) (4), superoxide dismutase superoxide dismutase n. An enzyme that catalyzes the decomposition of a superoxide into hydrogen peroxide and oxygen. superoxide dismutase A (sodA) (5), and RNA polymerase B (rpoB) (6). However, these studies concentrated only on a limited number of staphylococcal species. Therefore, a complete reference database of partial rpoB gene sequences from type strains (n = 47) and other culture collection strains, including all validly described staphylococcal subspecies, was created for this study. This reference database was then evaluated with clinical isolates. Results were compared with those previously obtained by 16S rDNA sequencing and conventional phenotypic tests. The Study We analyzed 82 type and other culture collection strains encompassing all validly described staphylococcal species (n = 38) and subspecies (n = 21; according to according to prep. 1. As stated or indicated by; on the authority of: according to historians. 2. In keeping with: according to instructions. 3. the current List of Bacterial Names with Standing in Nomenclature, updated May 14, 2005) (7). Two strains of the recently proposed candidate species S. pettenkoferi (8) were added to complete the rpoB sequence reference database. Using this database, we analyzed 55 clinical staphylococcal isolates collected from human (n = 52) and animal (S. intermedius, n = 2; S. felis, n = 1) specimens; 6 of the human isolates exhibited the small-colony variant (SCV SCV Santa Clarita Valley (California) SCV Sons of Confederate Veterans SCV Santa Clara Vanguard SCV Singapore Cable Vision SCV Special Category Visa (Australia) SCV StarHub Cable Vision ) phenotype. This strain collection was previously analyzed by the API ID 32 Staph staph n. Staphylococcus. staph adj. and VITEK 2 systems (both obtained from bioM6rieux, Marcy l'Etoile, France), partial 16S rDNA sequencing, chemotaxonomy chemotaxonomy Method of biological classification based on similarities in the structure of certain compounds among the organisms being classified. Advocates argue that, because proteins are more closely controlled by genes and less subject to natural selection than are , and riboprinting to determine species designation (3). The thermal cycling condition to amplify the partial rpoB gene (899 bp) was 35 cycles of denaturation denaturation, term used to describe the loss of native, higher-order structure of protein molecules in solution. Most globular proteins exhibit complicated three-dimensional folding described as secondary, tertiary, and quarternary structures. at 94[degrees]C for 45 s (300 s for the first cycle), annealing annealing (ənēl`ĭng), process in which glass, metals, and other materials are treated to render them less brittle and more workable. (60 s at 52[degrees]C), and extension (90 s at 72[degrees]C, 600 s for the last cycle). The Staphylococcus-specific primers used for amplification and sequencing of rpoB are shown in Table 1. Sequencing reactions were performed in a total volume of 10 [micro]L containing 0.5 [micro]L premix premix a finite mixture of nutritional supplements such as minerals and vitamins, usually combined with a carrier and ready for mixing with a total ration. (ABI Abi (ā`bī) [short for Abijah], in the Bible, King Hezekiah's mother. (Application Binary Interface) A specification for a specific hardware platform combined with the operating system. Prism BigDye Terminator v3.0 Ready Reaction Cycle Sequencing Kit, Applied Biosystems Applied Biosystems, Inc. (formerly NASDAQ: ABIO) is the original name of a pioneer biotechnology company founded in 1981 in Foster City, California, among the Silicon Valley cities of the southern San Francisco Bay Area. , Darmstadt, Germany), 1.8 [micro]L 400 mmol/L Tris-HCl, 10 mmol/L Mg[Cl.sub.2], 10 pmol sequencing primer, and 2 [micro]L polymerase chain reaction polymerase chain reaction (pŏl`ĭmərās') (PCR), laboratory process in which a particular DNA segment from a mixture of DNA chains is rapidly replicated, producing a large, readily analyzed sample of a piece of DNA; the process is product. The sequencing products were purified by using the Centri-Sep Spin Columns (Princeton Separations, Adelphia, NJ, USA) and analyzed with the ABI Prism 3100 Avant Genetic Analyzer (Applied Biosystems) according to the manufacturer's instructions. For further analysis, nucleotides 1444-1928 (corresponding to S. aureus The aureus (pl. aurei) was a gold coin of ancient Rome valued at 25 silver denarii. The aureus was regularly issued from the 1st century BC to the beginning of the 4th century AD, when it was replaced by the solidus. rpoB gene positions of the GenBank accession no. X64172) of the rpoB gene were used. The sequences were analyzed by using Ridom TraceEditPro version 1.0 software (Ridom GmbH, Wurzburg, Germany). Staphylococcal partial rpoB reference sequences determined in this study were deposited in GenBank under accession nos. DQ120729-DQ120752. Partial rpoB sequences were determined for 82 culture collection strains and 55 clinical isolates. All staphylococcal type strains were distinguishable by rpoB; the only exception was the S. equorum subspecies that shared the same sequence (online Appendix Figure, available from http://www.cdc.gov/ncidod/EID/vol12no02/05-0962_G.htm). The mean pairwise distance of all type and other culture collection strains exhibiting a unique rpoB sequence (n = 68) was 13.7% (range 0%-21.4%) and the standard deviation In statistics, the average amount a number varies from the average number in a series of numbers. (statistics) standard deviation - (SD) A measure of the range of values in a set of numbers. was 3.3%. When assuming a normal distribution for the distances and choosing a reporting criterion [greater than or equal to] 94.0%, the similarity for a distinct species correlates with a statistical error probability of 1.0% (9). The definitive identification of 55 clinical isolates and the rpoB gene sequence similarity search results are shown in Table 2. At the species level, the correct species designation for all 55 clinical isolates was made by rpoB sequence similarity search (sequence similarity [greater than or equal to] 94.0%). Of 21 clinical isolates belonging to species currently divided into subspecies, 17 isolates were correctly identified to the subspecies level. Subspecies identification for isolates M26 and M53 was unsuccessful by rpoB or partial 16S rDNA sequencing, riboprinting, and chemotaxonomy (data not shown). Only isolates M20 and M39 were misidentified by rpoB sequencing as S. saprophyticus subsp, saprophyticus instead of subsp. bovis. Conclusions Our previous study demonstrated the superiority of sequence-based methods over phenotypic approaches using the API ID 32 Staph and VITEK 2 systems (3). The advantage of a sequence-based method became most evident when differentiating isolates with the SCV phenotype, in which the API ID 32 Staph and VITEK 2 systems misidentified 2 and 4 isolates, respectively. When both sequence-based approaches used were compared, rpoB sequencing was superior to partial 16S rDNA identification. Although the 16S rDNA procedure differentiated 50 (90.9%) of all tested clinical isolates at species level, rpoB identified 100%. Therefore, if an unknown organism needs to be identified, 16S rDNA sequencing is the method of choice because of the availability of universal primers (10). However, if the genus is already known, the rpoB method should be used. Compared with other published molecular probes Molecular Probes is a biotechnology company located in Eugene, Oregon specializing in fluorescence. The company was founded in 1975 by Richard and Rosaria Haugland in their kitchen in Minnesota, then moved briefly to Texas and finally to Oregon in the early 1980s. , rpoB showed the highest discriminatory power, e.g., hsp60 and sodA sequencing did not differentiate subspecies of S. carnosus, S. cohnii, S. hominis, S. schleiferi, or S. succinus (4, 5). In a previous study, rpoB sequence-based identification of Staphylococcus staphylococcus (stăf'ələkŏk`əs), any of the pathogenic bacteria, parasitic to humans, that belong to the genus Staphylococcus. The spherical bacterial cells (cocci) typically occur in irregular clusters [Gr. species has been reported (6). However, a limited number of taxa taxa: see taxon. were included, and the primers used were not appropriate to detect all staphylococcal subspecies. Sequencing of rpoB was also used to identify other bacterial species (11,12). A higher discrimination with rpoB sequencing compared with 16S rDNA sequencing has been demonstrated for the genera Corynebacterium Corynebacterium /Co·ry·ne·bac·te·ri·um/ (-bak-ter´e-um) a genus of bacteria including C. ac´nes, a species present in acne lesions, C. diphthe´riae, the etiologic agent of diphtheria, C. (13) and Bacillus bacillus (bəsĭl`əs), any rod-shaped bacterium or, more particularly, a rod-shaped bacterium of the genus Bacillus. Some bacterium in the genus cause disease, for example B. (14). DNA sequencing DNA sequencing The determination of the sequence of nucleotides in a sample of DNA. is a rapid alternative to biochemical and other phenotypic procedures for the differentiation of bacterial pathogens because of its decreased costs and increased automation (15). Thus, rpoB is a useful molecular target for differentiating staphylococcal isolates to the species and subspecies level. References (1.) von Eiff C, Peters G, Heilmann C. Pathogenesis of infections due to coagulase-negative staphylococci. Lancet Infect Dis. 2002;2:677-85. (2.) Seifert H, Wisplinghoff H, Schnabel P, von Eiff C. Small colony variants of Staphylococcus aureus and pacemaker-related infection. Emerg Infect Dis. 2003;9:1316-8. (3.) Becker K, Harmsen D, Mellmann A, Meier C, Schumann P, Peters G, et al. Development and evaluation of a quality-controlled ribosomal sequence database for 16S ribosomal DNA-based identification of Staphylococcus species. J Clin Microbiol. 2004;42:4988-95. (4.) Kwok AY, Su SC, Reynolds RP, Bay SJ, Av-Gay Y, Dovichi NJ, et al. Species identification and phylogenetic phy·lo·ge·net·ic adj. 1. Of or relating to phylogeny or phylogenetics. 2. Relating to or based on evolutionary development or history. relationships based on partial HSP60 gene sequences within the genus Staphylococcus. Int J Syst Bacteriol. 1999;49:1181-92. (5.) Poyart C, Quesne G, Boumaila C, Trieu-Cuot R Rapid and accurate species-level identification of coagulase-negative staphylococci by using the sodA gene as a target. J Clin Microbiol. 2001;39:4296-301. (6.) Drancourt M, Raoult D. rpoB gene sequence-based identification of Staphylococcus species. J Clin Microbiol. 2002;40:1333-8. (7.) Euzeby JP. List of bacterial names with standing in nomenclature: a folder available on the internet. Int J Syst Bacteriol. 1997;47:590-2. (8.) Trulzsch K, Rinder H, Trcek J, Bader L, Wilhelm U, Heesemann J. 'Staphylococcus pettenkoferi' a novel staphylococcal species isolated from clinical specimens. Diagn Microbiol Infect Dis. 2002;43:175-82. (9.) Harmsen D, Karch H. 16S rDNA for diagnosing pathogens: a living tree. ASM (1) (Association for Systems Management) An international membership organization based in Cleveland, Ohio. Founded in 1947 and disbanded in 1996, it sponsored conferences in all phases of administrative systems and management. News. 2004;70:19-24. (10.) Clarridge JE. Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology Clinical microbiology The adaptation of microbiological techniques to the study of the etiological agents of infectious disease. Clinical microbiologists determine the nature of infectious disease and test the ability of various antibiotics to inhibit or kill and infectious diseases infectious diseases: see communicable diseases. . Clin Microbiol Rev. 2004;17:840-62. (11.) Drancourt M, Roux Roux , Pierre Paul Émile 1853-1933. French bacteriologist. His work with the diphtheria bacillus led to the development of antitoxins to neutralize pathogenic toxins. V, Fournier P, Raoult D. rpoB gene sequence-based identification of aerobic gram-positive cocci cocci /coc·ci/ (kok´si) plural of coccus. cocci [L.] plural of coccus. of the genera Streptococcus streptococcus (strĕp'təkŏk`əs), any of a group of gram-positive bacteria, genus Streptococcus, some of which cause disease. , Enterococcus enterococcus /en·tero·coc·cus/ (en?ter-o-kok´us) pl. enterococ´ci an organism belonging to the genus Enterococcus. Enterococcus /En·tero·coc·cus/ ( , Gemella, Abiotrophia, and Granulicatella. J Clin Microbiol. 2004;42:497-504. (12.) Mollet C, Drancourt M, Raoult D. rpoB sequence analysis as a novel basis for bacterial identification. Mol Microbiol. 1997;26:1005-11. (13.) Khamis A, Raoult D, La Scola B. Comparison between rpoB and 16S rRNA gene sequencing for molecular identification of 168 clinical isolates of Corynebacterium. J Clin Microbiol. 2005;43:1934-6. (14.) Blackwood KS, Turenne CY, Hannsen D, Kabani AM. Reassessment of sequence-based targets for identification of Bacillus species. J Clin Microbiol. 2004;42:1626-30. (15.) Cook VJ, Turenne CY, Wolfe J, Pauls R, Kabani A. Conventional methods versus 16S ribosomal DNA Not to be confused with Reformed Druids of North America. Ribosomal DNA (rDNA) are sequences encoding ribosomal RNA. These sequences regulate amplification and transcription initiation and contain transcribed and nontranscribed spacer segments. sequencing for identification of nontuberculous mycobacteria Nontuberculous mycobacteria (NTM), or atypical mycobacteria or mycobacteria other than tuberculosis (MOTT), are mycobacteria which do not cause tuberculosis or Hansen's disease (leprosy). : cost analysis. J Clin Microbiol. 2003;41:1010-5. Alexander Mellmann, * Karsten Becker, * Christof von Eiff, * Ursula Keckevoet,* Peter Schumann Peter Schumann (b. 1934) is the founder and director of the Bread & Puppet Theater. Born in Silesia, he was a sculptor and dancer in Germany before moving to the United States in 1961. , ([dagger]) and Dag Dag(h)da great god of Celts; father of Danu. [Celtic Myth.: Parrinder, 68; Jobes, 405] See : Fatherhood Dag (h)da god of abundance, war, healing. [Celtic Myth. Harmsen * * University Hospital Munster, Munster, Germany; and ([dagger]) Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany Address for correspondence: Alexander Mellmann, Institut fur Hygiene, Universitatsklinikum Munster, Robert-Koch-Strasse 41, D-48149 Munster, Germany; fax: 49-251-83-55688; email: mellmann@unimuenster.de Use of trade names is for identification only and does not imply endorsement by the Public Health Service or by the U.S. Department of Health and Human Services Noun 1. Department of Health and Human Services - the United States federal department that administers all federal programs dealing with health and welfare; created in 1979 Health and Human Services, HHS . Dr Mellmann is a consultant for medical microbiology Medical microbiology is a branch of microbiology which deals with the study of microorganisms including bacteria, viruses, fungi and parasites which are of medical importance and are capable of causing diseases in human beings. , hygiene, and infectious diseases at the University Hospital Manster. His professional interests include molecular identification and epidemiology of bacterial pathogens.
Table 1. Primers used for amplification and partial sequencing of the
partial staphylococcal RNA polymerase B (rpoB) gene
Primer sequence
Primer Application (5' [right arrow] 3')
Staph rpoB 1418f * Amplification CAA TTC ATG GAC CAA GC
and sequencing
Staph rpoB 3554r Amplification CCG TCC CAA GTC ATG
AAA C
Staph rpoB 1975r * Sequencing GCI ACI TGI TCC ATA
CCT GT
Staph rpoB 1876r * ([dagger]) Sequencing GAG TCA TCI TTY TCT
AAG AAT GG
Annealing
temperature
Primer ([degrees]C) Reference
Staph rpoB 1418f * 52 Modified from 7
Staph rpoB 3554r 52 7
Staph rpoB 1975r * 52 Modified from 7
Staph rpoB 1876r * ([dagger]) 52 This study
* Primers are numbered from the 3' end of the primer on the forward
strand of Staphylococcus aureus (GenBank accession no. X64172).
([dagger]) Primer was used for sequencing when primer Staph rpoB 1975r
did not work.
Table 2. Identification of 55 clinical staphylococcal isolates by using
RNA polymerase B (rpoB) gene sequencing
Strain rpoB gene (% similarity *)
M01 Staphylococcus arlettae (100.0)
M02 S. aureus subsp. aureus (100.0)
M03 S. aureus subsp. aureus (100.0)
M04 S. aureus subsp. aureus (99.8)
M05 ([double dagger]) S. aureus subsp. aureus (99.8)
M06 S. aureus subsp. aureus (100.0)
M07 ([double dagger]) S. aureus subsp. aureus (100.0)
M08 S. haemolyticus (94.0)
M09 S. epidermidis (100.0)
M10 S. capitis subsp. capitis (100.0)
M11 S. epidermidis (100.0)
M12 ([double dagger]) S. epidermidis (100.0)
M13 ([double dagger]) S. capitis subsp. capitis (99.8)
M14 S. caprae (99.8)
M15 S. caprae (99.8)
M16 S. chromogenes (100.0)
M17 S. cohnii subsp. cohnii (99.8)
M18 S. cohnii subsp. cohnii (99.8)
M20 S. saprophyticus subsp. saprophyticus (100.0)
M21 S. epidermidis (99.0)
M22 S. epidermidis (100.0)
M23 ([double dagger]) S. epidermidis (100.0)
M24 S. epidermidis (100.0)
M25 ([double dagger]) S. epidermidis (100.0)
M26 S. equorum subsp. equorum (100.0); S. Equorum
subsp. linens (100.0)
M27 S. felis (99.8)
M28 S. haemolyticus (100.0)
M29 S. haemolyticus (99.8)
M30 S. epidermidis (100.0)
M31 S. epidermidis (100.0)
M32 S. hyicus (100.0)
M33 S. intermedius (100.0)
M34 S. intermedius (100.0)
M35 S. intermedius (100.0)
M36 S. xylosus (100.0)
M37 S. lugdunensis (100.0)
M38 S. lugdunensis (100.0)
M39 S. saprophyticus subsp. saprophyticus (100.0)
M40 S. aureus subsp. aureus (100.0)
M41 S. schleiferi subsp. schleiferi (100.0)
M42 S. schleiferi subsp. schleiferi (100.0)
M43 S. sciuri subsp. sciuri (99.8)
M44 S. sciuri subsp. sciuri (99.8)
M45 S. sciuri subsp. sciuri (100.0)
M46 S. simulans (100.0)
M47 S. hominis subsp. novobiosepticus (99.6)
M48 S. felis (99.8)
M49 S. felis (99.8)
M50 S. warneri (95.9)
M51 S. warneri (95.3)
M52 S. warn eri (96.0)
M53 S. equorum subsp. equorum (99.8); S. Equorum
subsp. linens (99.8)
M54 S. xylosus (99.0)
M55 S. xylosus (97.1)
M56 S. xylosus (98.6)
Strain Definitive identification ([dagger])
M01 S. arlettae
M02 S. aureus subsp. aureus
M03 S. aureus subsp. aureus
M04 S. aureus subsp. aureus
M05 ([double dagger]) S. aureus subsp. aureus
M06 S. aureus subsp. aureus
M07 ([double dagger]) S. aureus subsp. aureus
M08 S. haemolyticus
M09 S. epidermidis
M10 S. capitis subsp. capitis
M11 S. epidermidis
M12 ([double dagger]) S. epidermidis
M13 ([double dagger]) S. capitis subsp. capitis
M14 S. caprae
M15 S. caprae
M16 S. chromogenes
M17 S. cohnii subsp. cohnii
M18 S. cohnii subsp. cohnii
M20 S. saprophyticus subsp. bovis
M21 S. epidermidis
M22 S. epidermidis
M23 ([double dagger]) S. epidermidis
M24 S. epidermidis
M25 ([double dagger]) S. epidermidis
M26 S. equorum; subspecies not known
M27 S. felis
M28 S. haemolyticus
M29 S. haemolyticus
M30 S. epidermidis
M31 S. epidermidis
M32 S. hyicus
M33 S. intermedius
M34 S. intermedius
M35 S. intermedius
M36 S. xylosus
M37 S. lugdunensis
M38 S. lugdunensis
M39 S. saprophyticus subsp. bovis
M40 S. aureus subsp. aureus
M41 S. schleiferi subsp. schleiferi
M42 S. schleiferi subsp. schleiferi
M43 S. sciuri subsp. sciuri
M44 S. sciuri subsp. sciuri
M45 S. sciuri subsp. sciuri
M46 S. simulans
M47 S. hominis subsp. novobiosepticus
M48 S. felis
M49 S. felis
M50 S. warneri
M51 S. warneri
M52 S. warneri
M53 S. equorum; subspecies not known
M54 S. xylosus
M55 S. xylosus
M56 S. xylosus
* Similarity in comparison with the reference database.
([dagger]) By phenotypic and genotypic methods as previously
published (4).
([double dagger]) Isolate exhibiting the small colony variant
phenotype.
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