Typing of Staphylococcus Aureus Strains by PCR-amplification of Variable-Length 16S-23S rDNA Spacer Regions: Characterization of Spacer Sequences

Microbiology. 1995 May;141 ( Pt 5):1255-65. doi: 10.1099/13500872-141-5-1255.

Abstract

To develop a rapid and accurate method of typing large numbers of clinical isolates of Staphylococcus aureus, the spacer region C of the rRNA operon [1391-507 (16S-23S)] was enzymically amplified from 322 strains. When the products were separated by denaturing PAGE, 15 variable-length rrn alleles were demonstrated, ranging in size from 906 to 1223 bp. The variable-length HpaII-digested region C [(region E; 1446-196 (16S-23S)] amplification products were cloned into M13mp18RF to sequence separate variable-length alleles. A total of 17 region E inserts were sequenced, aligned and divided into nine alleles by length (938-1174) and sequence properties. The 16S-23S spacer rDNA varied in length (303-551 bp) and in properties; three alleles contained a tRNAIle gene alone, two alleles contained a tRNAIle and a tRNAAla gene, and four alleles lacked tRNA genes. The sequences of two alleles showed less than 1% variation when isolated from two or three S. aureus strains. The 48 penicillin- and methicillin-sensitive strains were divided into 26 ribotypes; in contrast, the 274 methicillin-resistant S. aureus (MRSA) strains were divided into nine ribotypes (A-I) with 97% typing as either ribotype A or B (rrnL was missing in B). The sequence conservation of the rrn operons argues for the use of the 16S-23S spacer region as a stable and direct indicator of the evolutionary divergence of S. aureus strains.

Publication types

  • Comparative Study

MeSH terms

  • Alleles
  • Base Sequence
  • DNA Primers
  • DNA, Ribosomal / chemistry
  • DNA, Ribosomal / genetics*
  • Genes, Bacterial
  • Humans
  • Methicillin Resistance / genetics
  • Molecular Sequence Data
  • Penicillin Resistance / genetics
  • Phylogeny*
  • Polymerase Chain Reaction / methods*
  • RNA, Ribosomal, 16S / genetics
  • RNA, Ribosomal, 23S / genetics
  • RNA, Transfer, Phe / genetics
  • Reproducibility of Results
  • Restriction Mapping
  • Sequence Homology, Nucleic Acid
  • Staphylococcus aureus / classification*
  • Staphylococcus aureus / genetics*
  • Staphylococcus aureus / isolation & purification

Substances

  • DNA Primers
  • DNA, Ribosomal
  • RNA, Ribosomal, 16S
  • RNA, Ribosomal, 23S
  • RNA, Transfer, Phe

Associated data

  • GENBANK/U11773
  • GENBANK/U11774
  • GENBANK/U11775
  • GENBANK/U11776
  • GENBANK/U11777
  • GENBANK/U11778
  • GENBANK/U11779
  • GENBANK/U11780
  • GENBANK/U11781
  • GENBANK/U11782
  • GENBANK/U11783
  • GENBANK/U11784
  • GENBANK/U11785
  • GENBANK/U11786
  • GENBANK/U11787
  • GENBANK/U11788
  • GENBANK/U11789