Comparison of genotypic and phenotypic methods for species-level identification of clinical isolates of coagulase-negative staphylococci

Abstract

To compare commonly used phenotypic methods with genotypic identification methods 47 clinical isolates of coagulase-negative staphylococci (CONS), 10 CONS ATCC strains, and a Staphylococcus aureus clinical isolate were identified using the API Staph ID test, BD Phoenix Automated Microbiology System, and 16S rRNA gene and tuf gene sequencing. When necessary part of the sodA gene was sequenced for definitive identification. The results show that tuf gene sequencing is the best method for identification of CONS, but the API Staph ID test is a reasonably reliable phenotypic alternative. The performance of the BD Phoenix Automated Microbiology System for identification of CONS is poor. The present study also showed that although genotypic methods are clearly superior to phenotypic identifications, a drawback of sequence-based genotypic methods may be a lack of quality of deposited sequences in data banks. In particular, 16S rRNA gene sequencing suffers from the lack of high quality among sequences deposited in GenBank. Furthermore, genotypic identification based on 16S rRNA sequences has limited discriminating power for closely related Staphylococcus species. We propose partial sequencing of the tuf gene as a reliable and reproducible method for identification of CONS species.

FIG. 1.

Neighbor-joining tree based on 16S rRNA sequences, showing the phylogenetic relationships among the 58 Staphylococcus isolates included in this study. The value on each branch is the occurrence of the branching order in 1,000 bootstrapped trees. The scale bar represents 1% differences in nucleotide sequences. a, S1717, S2114, S2518, U2081, U477, ATCC 35661; b, 91-960, 95-1922, 95-1930, 95-1953, 95-2003, 95-2071, 97-110, 97-137, 97-141, 97-174, 97-178, 97-186, 97-192, 97-200, 99-410, 99-419, S2320, U77, U78, U225, U302, U472, U500, U921, U1100, U1571, U1622, U1731, U1839, U1988, U2075, ATCC 49134, ATCC 14990, ATCC 12228; c, 97-101, 99-418, 99-434, 99-436, U208, U922.

FIG. 2.

Neighbor-joining tree based on tuf sequences, showing the phylogenetic relationships among the 58 Staphylococcus isolates included in this study. The value on each branch is the occurrence of the branching order in 1,000 bootstrapped trees. The scale bar represents 1% differences in nucleotide sequences. a, 97-137, 97-186, 97-200, 99-410, U1731; b, 91-960, 95-1922, 95-1930, 95-1953, 95-2003, 95-2071, 97-110, 97-141, 97-174, 97-178, 97-192, 99-419, S2320, U77, U78, U225, U302, U472, U500, U921, U1100, U1571, U1622, U1839, U1988, U2075, ATCC 49134, ATCC 14990, ATCC 12228; c, S1717, S2114, S2518, U2081, U477, ATCC 35661; d, 97-101, 99-418, 99-434, 99-436.