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

Abstract

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...

Toxins and Antibiotic Resistance in Staphylococcus aureus Isolated from a Major Hospital in Lebanon

“…The 16S – 23S ITS region was amplified using the following protocol: an aliquot of 2  μ L of DNA was added to 20  μ L of reaction mixture containing final concentrations of 1X Ampli Taq buffer, 2.5 mM MgCl 2 , 0.1 U of Ampli Taq gold polymerase (Applied Biosystems, Roche), 0.2 mM each deoxynucleoside triphosphate, 0.4  μ M staph ITS-F primer (5′-AGAGTTTGATCCTGGCTCAG-3′), 0.4  μ M staph ITS-R primer (5′-CAAGGCATCCACCGT-3′) [18]. The amplification was performed with an initial denaturation step for 12 min at 95°C followed by 30 cycles of 94°C for 30 sec, 42°C for 30 sec, 72°C for 1 min, and a final extension at 72°C for 10 min.…”

“…1  μ L of the enzyme was added to 12  μ L of the ITS PCR products, 16  μ L water, along with 2  μ L 10x Taq I buffer for 3 h at 65°C (Fermentas) [18]. Products were resolved on a 2% agarose gels in 1x tris-borate-EDTA buffer and were subsequently visualized by UV illumination after ethidium bromide staining.…”

Identification and characterization of a Streptomyces sp. isolate exhibiting activity against multidrug-resistant coagulase-negative Staphylococci

“…These are traditionally grouped into coagulase positive (CPS) and coagulase-negative Staphylococci (CNS) (Resch et al 2008). Main habitats of these bacteria are skin, skin glands and mucous membranes of humans and animals (Sudagidan et al 2005;Lis et al 2009;Koksal et al 2007). CNS have become the most frequently isolated organisms from bovine intra mammary infections in several countries and their prevalence is generally highest at calving (Katsuda et al 2005;Schultz et al 2009).…”

Identification of Staphylococcus spp. by PCR-Restriction Fragment Length Polymorphism Analysis of dnaJ Gene

“…Recently, PCR-RFLP analysis of the internal transcribed spacer of the 16S rRNA gene, the intergenic spacer of the 16S-23S rRNA gene, the tuf gene, the gap gene, and the groEL gene has been developed for the identification of Staphylococcus species; but the number of species and subspecies that could be identified did not exceed 25, and these were mainly limited to the most common human and animal staphylococcal pathogens (1,10,14,21,25). The novel method based on PCR-RFLP analysis of the dnaJ gene described here is able to increase considerably the list of species that can be precisely identified to as many as 41, including species associated with human and animal infections that could not be classified by PCR-RFLP analysis of other genes.…”

“…Due to the limited number of stable features that can be used for species discrimination, many taxa remain difficult to distinguish from one another and are misidentified by phenotypic tests (4). To solve these problems, restriction fragment length polymorphism (RFLP) analysis of PCR products and a number of PCR amplicon sequencing-based methods have been reported for use for the identification of staphylococci (1,2,5,10,11,12,13,14,17,18,21,22,23,24,25). This paper describes a sensitive and specific nucleic acidbased procedure that is able to differentiate 41 Staphylococcus species and subspecies, based on PCR-RFLP analysis of the dnaJ gene.…”