Development and Evaluation of a Quality-Controlled Ribosomal Sequence Database for 16S Ribosomal DNA-Based Identification of Staphylococcus Species

Abstract

To establish an improved ribosomal gene sequence database as part of the Ribosomal Differentiation of Microorganisms (RIDOM) project and to overcome the drawbacks of phenotypic identification systems and publicly accessible sequence databases, both strands of the 5 end of the 16S ribosomal DNA (rDNA) of 81 type and reference strains comprising all validly described staphylococcal (sub)species were sequenced. Assuming a normal distribution for pairwise distances of all unique staphylococcal sequences and choosing a reporting criterion of >98.7% similarity for a "distinct species," a statistical error probability of 1.0% was calculated. To evaluate this database, a 16S rDNA fragment (corresponding to Escherichia coli positions 54 to 510) of 55 clinical Staphylococcus isolates (including those of the small-colony variant phenotype) were sequenced and analyzed by the RIDOM approach. Of these isolates, 54 (98.2%) had a similarity score above the proposed threshold using RIDOM; 48 (87.3%) of the sequences gave a perfect match, whereas 83.6% were found by searching National Center for Biotechnology Information (NCBI) database entries. In contrast to RIDOM, which showed four ambiguities at the species level (mainly concerning Staphylococcus intermedius versus Staphylococcus delphini), the NCBI database search yielded 18 taxon-related ambiguities and showed numerous matches exhibiting redundant or unspecified entries. Comparing molecular results with those of biochemical procedures, ID 32 Staph (bioMérieux, Marcy I'Etoile, France) and VITEK 2 (bioMérieux) failed to identify 13 (23.6%) and 19 (34.5%) isolates, respectively, due to incorrect identification and/or categorization below acceptable values. In contrast to phenotypic methods and the NCBI database, the novel high-quality RIDOM sequence database provides excellent identification of staphylococci, including rarely isolated species and phenotypic variants.The emerging role of coagulase-negative staphylococci (CoNS), along with Staphylococcus aureus, in connection with the expanding number of staphylococcal (sub)species described necessitates their comprehensive and accurate identification. A variety of methods have been proposed for the identification of staphylococcal species, including conventional identification schemes based on publications of Kloos and Schleifer (18) and commercial identification tests. In general, methods based on phenotypic characteristics are hampered by the fact that they are dependent on the expression of metabolic activities and/or morphological features. Furthermore, commercial systems often offer two or more suggestions for identification with a comparable safety level.Nowadays, with the advent of molecular biology-based techniques, investigations based on comparative DNA sequence analysis of the genes of conserved macromolecules have become commonplace in microbiology as a tool for classification of microbial organisms. The most useful and extensively investigated taxonomic marker molecules are the larger rRNAs and their corresponding ...

Isolamento de Streptococcus agalactiae em diferentes órgãos de tilápias do nilo (Oreochromis niloticus) criadas em tanques-rede

“…Umas dessas técnicas é o sequenciamento do gene do RNA ribossomal 16S, amplamente utilizado com finalidade taxonômica e filogenética e que consiste na análise comparativa da sequência de determinados genes de macromoléculas conservadas como o RNA ribossomal (BECKER et al, 2004). As sequências encontradas são comparadas com aquelas depositadas em bancos de dados, como o National Center for Biotechnology Information (NCBI) (LANGE et al, 2011).…”

Species Identification and Strain Typing of Staphylococcus agnetis and Staphylococcus hyicus Isolates from Bovine Milk by Use of a Novel Multiplex PCR Assay and Pulsed-Field Gel Electrophoresis

“…Analysis of the 16S rRNA gene sequence is the most commonly used method for the identification and classification of bacteria (15). However, the usefulness of 16S rRNA gene sequencing is limited when applied to certain staphylococcal species because of the high degree of similarity between closely related species (15,16). For example, 16S rRNA gene sequences of S. agnetis isolates were found to exhibit 99.7% identity to S. hyicus ATCC 11249 T and 99.1% identity to S. chromogenes ATCC 43764 (10).…”

Identification of Coagulase-Negative Staphylococci by SodA Gene Sequence Analysis

“…Although the API Staph ID test was more reliable than the BD Phoenix Automated Microbiology system, the genotypic means via 16S rRNA, tuf and sodA gene sequencing method was found to be a more superior identification method for CoNS isolates (Heikens et al, 2005). Similarly, the 5' end of the 16S rDNA of 81 type and reference strains of staphylococcal were sequenced and the results revealed four ambiguities as compared to 13 (23.6%) and 19 (34.5%) isolates misidentified by ID 32 Staph and VITEK 2 (Becker et al, 2004).…”

“…Analysis of 16S rRNA gene used to be the most common method used for bacteria classification and identification. However, the use of this gene is limited due to high degree of sequence similarity between closely related species (Becker et al, 2004;Goh et al, 1996;Gribaldo et al, 1997;Kwok et al, 1999). Hence, other highly conserved sequence has been exploited which includes sodA (Poyart et al, 2001), gap (Bergeron et al, 2011;Ghebremedhin et al, 2008), hsp60 (Ghebremedhin et al, 2008;Kwok et al, 1999), rpoB (Drancourt and Raoult, 2002;Mellmann et al, 2006), tuf (Heikens et al, 2005) and dnaJ (Shah et al, 2007) gene sequencing methods.…”