Presence of a novel DNA methylation enzyme in methicillin-resistant Staphylococcus aureus isolates associated with pig farming leads to uninterpretable results in standard pulsed-field gel electrophoresis analysis

Abstract

Genomic DNA from methicillin-resistant Staphylococcus aureus isolates recovered from pigs and their caretakers proved resistant to SmaI digestion, leading to uninterpretable results in standard pulsed-field gel electrophoresis. This is the result of a yet unknown restriction/methylation system in the genus Staphylococcus with the recognition sequence CCNGG.

FIG. 1.

PFGE analysis using restriction enzymes SmaI and XmaI. Whereas genomic DNA from the pig farming isolates is resistant to endonuclease treatment using SmaI, upon incubation with XmaI a typical PFGE banding pattern is obtained. The control strain yields identical banding patterns with both enzymes. Lower-intensity bands in the background may indicate that the DNA is only partially digested using XmaI.

FIG. 2.

Identification of methylated cytosine residues by sodium bisulfite sequencing. (A) Specific C residues in the target sequence are protected from sodium bisulfite conversion. The SmaI site in the target sequence is indicated. (B) Alignment of the original flanking sequences of nonconverted cytosines (indicated with an asterisk) revealing the consensus recognition sequence CC*NGG. (C) As it turns out, the SmaI site contains the consensus recognition site CC*NGG twice (overlined A and underlined and B) and, thus, is affected at both the second and third C residues.