On the nature and use of randomly amplified DNA from Staphylococcus aureus

Abstract

Various DNA-based methods have been introduced to genetically type Staphylococcus aureus strains but not a single technique is universally applicable. In order to search for DNA probes suitable for differentiating strains, randomly amplified polymorphic DNA patterns were generated for 243 S. aureus strains and a single isolate of Staphylococcus intermedius. All fingerprints were examined for unique amplicons, and the nature of 42 of these DNA fragments was investigated. Partial DNA sequences were determined, and several homologies were discovered with known S. aureus sequences (plasmid pSH6 DNA with insertion sequences, agrA and agrB sequences, hld genes, the gene for 23S rRNA, the lysyl tRNA synthetase gene, and the threonyl tRNA synthetase gene) and with genes from other species (Haemophilus influenzae bexA and Bacillus subtilis spoF and ctrA). Thirty fragments were of previously unknown origin. In Southern blots containing EcoRI-digested DNA from S. aureus strains and the S. intermedius strain, nine probes demonstrated the capacity to differentiate strains on the basis of the presence or absence of the sequence element in the staphylococcal genome involved. The remainder of the probes displayed restriction fragment length polymorphisms (n ‫؍‬ 12), hybridized in a homogeneously positive fashion (n ‫؍‬ 13) or hybridized only with their source strains (n ‫؍‬ 8) (four of the latter were specific to S. intermedius). Three of the nine strain-specific probes were overlapping, and two of the others were found to display a high level of inconsistency among epidemiologically related strains. Thus, five strain-specific probes remained that, in a 5-digit typing system, accurately distinguished epidemiologically related and unrelated strains of S. aureus. We conclude that application of strain-specific DNA probes, selected on the basis of differing randomly amplified polymorphic DNA patterns, promises to become a technically simple, robust, and reproducible tool that may significantly facilitate the study of the epidemiology of S. aureus infections.

Assessment of Resolution and Intercenter Reproducibility of Results of Genotyping Staphylococcus aureus by Pulsed-Field Gel Electrophoresis of Sma I Macrorestriction Fragments: a Multicenter Study

“…For the purpose of additional comparison, bacteriophage-typing and antimicrobial susceptibility testing were performed for all isolates of the test panel. The isolates were typed by arbitrary primed PCR (AP-PCR), binary typing, and target 916-Shine-Dalgarno PCR (tar 916-shida PCR) as described previously (3,16,18).…”

Contaminations of laboratory surfaces with Staphylococcus aureus are affected by the carrier status of laboratory staff

“…Three different primers were used to amplify polymorphic regions of the S. aureus genome in three independent standard PCRs as described elsewhere (van Belkum et al 1995;van Leeuwen et al 1996): 5 ng (in the AP-1 and AP-7 reaction) or 25 ng (in the ERIC-2 reaction) of genomic DNA from overnight cultures were used as templates together with 1· PCR Gold buffer (Applied Biosystems), 2AE5 mmol l )1 MgCl 2 , 0AE2 units AmpliTaq Gold Ò DNA Polymerase (Applied Biosystems) 0AE2 mmol l )1 dNTPs and 1 lmol l )1 Primer (AP-1, AP-7 or ERIC-2) in a 50-ll reaction. Forty cycles of 1 min 94°C, 1 min 40°C, 2 min 72°C were run and the amplified fragments were separated on a ethidium bromidestained 1AE5% agarose gel and analysed under UV light.…”

Dissemination of a single clone of methicillin-resistant Staphylococcus aureus among Turkish hospitals

“…Although there appeared to be no 100% overlap, the arbitrarily primed PCR B type seemed to be linked to a B ribotype. Probing of genomic DNA with DNA probes capable of differentiating among the MRSA strains (21) fully corroborated the arbitrarily primed PCR and ribotyping data.…”

“…Hybridization studies with isolate-specific DNA probes. Differentiation of staphylococcal strains can be based upon their reactivities toward isolate-specific DNA probes (21). Southern blots of purified staphylococcal DNA were prepared as de-scribed above (see ribotyping section).…”