<i>rpoB</i>
            Gene Sequence-Based Identification of
            <i>Staphylococcu</i>
            s Species

Drancourt, Michel; Raoult, Didier

doi:10.1128/jcm.40.4.1333-1338.2002

Cited by 265 publications

(206 citation statements)

References 39 publications

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“…Species-level identification studies have often been based on sequencing of certain genes (Poyart et al, 2001;Drancourt & Raoult, 2002) and hybridization studies (Goh et al, 1997). In addition, PCR analyses of the length polymorphisms of the intergenic spacers residing between the 16S and 23S rRNA genes (Gurtler & Stanisich, 1996) or tRNA genes (Welsh & McClelland, 1992;Ehrenstein et al, 1996) have also been important targets for the species-level identification of bacteria.…”

Section: Resultsmentioning

confidence: 99%

Identification of staphylococci by 16S internal transcribed spacer rRNA gene restriction fragment length polymorphism

Sudağıdan

Yenidünya

Güneş

2005

Journal of Medical Microbiology

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The capacity of 16S internal transcribed spacer (16S-ITS) rRNA gene RFLP to differentiate 16 type strains and nine clinical isolates of staphylococci was evaluated. The 16S rRNA gene was amplified together with the ITS region and the amplification products were digested with TaqI restriction enzyme. Analysis of the 16S-ITS rRNA gene RFLP profiles differentiated each of the 16 type strains into distinct RFLP haplotypes. INTRODUCTIONThe habitat of staphylococci is skin, skin glands and mucous membranes of humans and many animals. The genus Staphylococcus includes both pathogenic and saprophytic strains that have been isolated from animal products such as meat and milk, and from environmental sources such as soil, sea water, fresh water, dust and air samples (Kloos et al., 1991).Coagulase-positive species such as Staphylococcus aureus are the cause of many types of infection (Forbes et al., 1998). During the last two decades, coagulase-negative staphylococci (CNS) have also emerged as pathogens causing medical-device-related infections (von Eiff et al., 2002). Because of the pathogenic potential of these bacteria, effective methods are required for their in vitro identification. Beside fatty acid analyses, many diagnostic tests rely on a miniaturized phenotypic characterization and a set of biochemical reactions. These methods enable the identification of S. aureus isolates, but often fail in the identification of CNS (Stoakes et al., 1994;Renneberg et al., 1995;Martineau et al., 1996). Analysis of specific regions of genomic DNA, on the other hand, has produced much more discriminative data. For example, several genomic targets have been effectively used for the identification of Staphylococcus species, including the 16S rRNA gene (Bialkowska-Hobrzanska et al., 1990;De Buyser et al., 1992), the tRNA gene intergenic spacer (Maes et al., 1997), the internal transcribed spacer (Couto et al., 2001), the heat-shock protein 60 (HSP60) gene (Goh et al., 1996), the chaperonin 60 gene (Goh et al., 1997), the femA gene (Vannuffel et al., 1999), the sodA gene (Poyart et al., 2001), the gap gene (Yugueros et al., 2000) and the nuc gene (Brakstad et al., 1992). Recently, an enterobacterial repetitive intergenic consensus PCR and BOX-PCR were also used in the identification of Staphylococcus epidermidis strains (Wieser & Busse, 2000). . The nine clinical isolates were S. capitis subsp. capitis (n ¼ 2), S. caprae (n ¼ 2), S. cohnii subsp. cohnii (n ¼ 1) and S. epidermidis (n ¼ 4). The clinical isolates were identified by basic phenotypic tests and the Staph ID 32 system (bioMérieux). All strains were cultured in tryptic soy broth (TSB; Merck) at 37 8C and stored in 25 % (v/v) glycerol at À80 8C.16S-ITS rRNA gene PCR. Genomic DNA was isolated as described by Arciola et al. (2001). Briefly, 100 ìl of overnight culture prepared in 5 ml TSB at 37 8C with shaking was pelleted by centrifugation. Cell pellets were resuspended in 45 ìl deionized water and 5 ìl lysostaphin solution (100 ìg ml À1 in dH 2 O; Sigma) and incubated for 10 min...

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Section: Resultsmentioning

confidence: 99%

Identification of staphylococci by 16S internal transcribed spacer rRNA gene restriction fragment length polymorphism

Sudağıdan

Yenidünya

Güneş

2005

Journal of Medical Microbiology

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“…In addition to the use of 16S rDNA sequencing for taxonomic purposes, subtle sequence differences in the 16S rDNA sequence were recently reported to be useful for species identification [34,39] and for subtyping and identifying hyper-virulent bacterial clones [5,31,33]. For the same purposes, the rpoB gene encoding the β-subunit of RNA polymerase has been studied for many bacterial species, such as Bacillus [24], Bartonella [37], Borrelia [28], Legionella [25], Staphylococcus [14], mycobacteria [22] and enteric bacteria [32]. The rpoB sequences of H. somni strains have been determined and analysed phylogenetically [1] although sequence stretches or cluster groups corresponding to the host-specific subgroups were not clear due to the use of limited number of bacterial strains.…”

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confidence: 99%

Differentiation between Bovine and Ovine Strains of Histophilus somni Based on the Sequences of 16S rDNA and rpoB Gene

Tanaka

Hoshinoo

Hoshino

et al. 2005

The Journal of Veterinary Medical Science

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ABSTRACT. Nucleotide sequences of 16S rDNA and rpoB gene of 25 bovine and 6 ovine Histophilus somni strains were determined to detect subtle differences between the host animal species. The 1465 nucleotide residues of the 16S rDNA exhibited levels of sequence similarities of 99.4% or more. The high sequence similarity of the 16S rDNA of recently described species H. somni was confirmed in the 31 strains from cattle and sheep. These results suggested that the intra-specific diversity of 16S rDNA was limited in bovine and ovine strains of H. somni. The specific association of strains was also observed in the 311 bp region of rpoB gene which sequence similarities were 98.6% or more. However, the phylogenetic tree analysis of the rpoB gene showed that the ovine strains appeared to form a subgroup recovered in 70% of the bootstrap trees. In the 311 bp region of the ovine strains, a HincII restriction endonuclease site was detected. The PCR-amplified rpoB DNA of 46 bovine and 20 ovine H. somni strains were examined for the digestion with HincII. As the results, 17 strains of ovine strains were cleaved by the enzyme but none of the bovine strains appeared to possess the restriction site. The restriction enzyme analysis of rpoB gene may be useful to differentiate ovine strains from bovine strains of H. somni. H. somni was a recently described bacterial species as a combination of "Haemophilus somnus", "Haemophilus agni" and "Histophilus ovis" [1]. The organisms can be pathogens responsible for substantial economic losses in cattle and sheep industries due to a variety of diseases, such as thrombotic meningoencephalitis, pneumonia, myocarditis, and reproductive disorders in cattle [17,18,27] as well as mastitis, septicemia, and reproductive disorders in sheep [4,18,38]. H. somni is also distributed in nasopharyngeal tracts and reproductive organs of healthy animals [8-11, 19, 20, 29, 41, 45]. Based on the profiles of outer membrane protein, a restriction endonuclease analysis, PCR-based fingerprinting analyses and a DNA reassociation analysis, it has been suggested that there are host-specific subgroups in the organisms [3,23,46,47]. Up to the date, however, no simple mean to differentiate between bovine and ovine strains of H. somni has been available. Such a method reflecting on the boundary can be a useful tool for understanding the epidemiology of the organisms. Based on the phylogenies of the 16S rDNAs, H. somni strains are clearly distinguished from the related taxa in the family Pasteurellaceae [1]. In addition to the use of 16S rDNA sequencing for taxonomic purposes, subtle sequence differences in the 16S rDNA sequence were recently reported to be useful for species identification [34,39] and for subtyping and identifying hyper-virulent bacterial clones [5,31,33]. For the same purposes, the rpoB gene encoding the β-subunit of RNA polymerase has been studied for many bacterial species, such as Bacillus [24] although sequence stretches or cluster groups corresponding to the host-specific subgroups were not clear d...

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“…In contrast to the probe hybridization technique and the RFLP approach, sequencing enables any isolate to be characterized, including new species by their phylogenetic relationships (Drancourt & Raoult 2002). And these authors concluded that the partial sequencing of the rpoB gene as a suitable new tool for the accurate identification of Staphylococcus isolates.…”

Section: Discussionmentioning

confidence: 99%

Comparison phenotypic and genotypic identification of Staphylococcus species isolated from bovine mastitis

et al. 2016

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In addition to Staphylococcus aureus nowadays other coagulase-positive staphylococci (CoPS) and coagulase-negative staphylococci (CoNS), earlier considered of minor importance, are now accepted as relevant pathogens for humans and animals. The involvement of these microorganisms in bovine mastitis etiology and the possibility their transmission through milk to humans justify the requirement of developing reliable methods for identification of the most frequent species among them. The purpose of this study was to compare the phenotypic techniques with the genotypic method carried out by sequencing of the rpoB gene in identification of several species of the genus Staphylococcus isolated from bovine mastitis. A total of 300 staphylococci isolates of bovine mastitis cases from several Brazilian dairy herds were studied by phenotypic and genotypic techniques, respectively: 150 CoPS and 150 CoNS strains. A total of 18 CoNS different species and 4 CoPS species were identified. Among the CoNS the following species were recognized: 48 (32%) Staphylococcus warneri, 22(15%) S. epidermidis, 20(13%) S. hyicus, 10(7%) S. xylosus, 7(5%) S. haemolyticus, 6(4%) S. simulans, 6(4%) S. schleiferi subsp schleiferi, 6(4%) S. hominis, 5(3%) S. pasteuri, 4(2.7%) S. cohnii, 3(2%) S. saprophyticus subsp. saprophyticus 3(2%) S. chromogenes 3(2%) S. sciuri, 2(1%) S. saccharolyticus, 2(1%) S. lugdunensi, 1(0,7%) S. auricularis, 1(70%) S. saprophyticus subsp. bovis, 1(0.7%) S. capitis. And among the 150 CoPS were identified respectively: 105 (70%) S. aureus, 21(14%), S. hyicus, 19(13%) S. intermedius e 5(3%) S. schleiferi subsp coagulans. Considering the 150 CoNS isolates, the identifications performed by phenotypic and genotypic tests presented 96.7% of concordance, kappa coefficient of agreement = 0.933, SE (standard error) of kappa=0.021 (95% confidence interval: 0.893 to 0.974), Pearson's correlation coefficient (r) = 0.9977, (confidence interval 95%: 0.9938 a 0.9992) and in relation to 150 CPS isolates it was detected an agreement of 98.7%, kappa = 0.960, SE of kappa = 0.016, (95% confidence interval: 0.929 to 0.992) Pearson's correlation coefficient (r) = 0.9994 (95% confidence interval: 0.9681 to 1.0000). The verified agreement strength between the identification methods can be considered as excellent. These results assure that according to laboratory resources any of them will be suitable to perform the staphylococci identification. Considerando-se os 150 SCN isolados, as identificações realizadas por testes fenotípicos e genotípicos apresentaram 96,7% de concordância, coeficiente de concordância kappa = 0,933, SE (erro padrão) de kappa = 0,021 (95% intervalo de confiança: 0,893-0,974), coeficiente de correlação de Pearson (r) = 0,9977, (intervalo de confiança de 95%: 0,9938 a 0,9992) e em relação a 150 SCP isolados foi observado uma concordância de 98,7%, kappa = 0,960, sE de kappa = 0,016, (95% de intervalo de confiança: 0,929 a 0,992) coeficiente de correlação de Pearson (r) = 0,9994 (95% intervalo de confiança: 0,9681-1,0000). A...

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rpoB Gene Sequence-Based Identification of Staphylococcu s Species

Cited by 265 publications

References 39 publications

Identification of staphylococci by 16S internal transcribed spacer rRNA gene restriction fragment length polymorphism

Identification of staphylococci by 16S internal transcribed spacer rRNA gene restriction fragment length polymorphism

Differentiation between Bovine and Ovine Strains of Histophilus somni Based on the Sequences of 16S rDNA and rpoB Gene

Comparison phenotypic and genotypic identification of Staphylococcus species isolated from bovine mastitis

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