The Alere StaphyType DNA microarray was employed using protocols and procedures
previously described in detail [29] (link); [ 96] (link). The DNA microarray covers 334 target sequences,
(approximately 170 distinct genes and their allelic variants) including species
markers, SCCmec, capsule and agr group typing
markers, resistance genes, exotoxins, and MSCRAMM genes. Primer and probe
sequences have been published previously [29] (link); [96] (link).
Target genes and information on primers and probes are provided in Supplemental
file
S1 .
MRSA were grown on Columbia blood agar and incubated overnight at 37°C.
Culture material was enzymatically lysed prior to DNA preparation using
commercially available spin columns (Qiagen, Hilden, Germany). Purified DNA
samples were used as templates in a linear primer elongation using one primer
per target. All targets were amplified simultaneously, and within this step,
biotin-16-dUTP was incorporated into the resulting amplicons. An alternate
protocol was used for a few isolates in which amplification and labelling were
directed by random primers [139] (link); [244] . As this protocol does not rely on conserved primer
binding sites, it proved to be useful for characterisation of unusual strains
which are not fully represented by the published genome sequences
(e.g., ST75 strains).
Amplicons obtained using either protocol were hybridised to the microarray
followed by washing and blocking steps, and the addition of
horseradish-peroxidase-streptavidin conjugate. After further incubation and
washing steps, hybridisations were visualised by using a precipitating dye. An
image of the microarray was taken and analysed using a designated reader and
software (ALERE Technologies GmbH, Jena, Germany). Normalised intensities of the
spots were calculated based their average intensities and on the local
background [96] (link). Results were regarded as negative if the normalised
intensity for a given probe was below 25% of the median value of species
markers (coa, eno, fnbA, gapA, katA, nuc, rrn, sarA sbi, spa,
vraS) and a biotin staining control. If the normalised intensity of
a given probe was higher than 50% of this breakpoint, it was interpreted
positive. If it was between 25% and 50%, the result was regarded
as ambiguous. For some markers, for which allelic variants were to be
discriminated (bbp, clfA, clfB and fnbB as
well as some set/ssl genes, isaB, mprF and
isdA), a different approach was used because these alleles
differed only in single nucleotides. Here, only the probe with the strongest
signal value was regarded as positive, provided that it exceeded the 50%
breakpoint. All others were regarded as ambiguous or, if below the 25%
breakpoint, as negative. This allowed an easy and clear distinction of clonal
complex-specific variants of these genes. Genes which are not present in all
tested isolates are labelled as rare, variable or common inFigures 2 and 3 as well as in Supplement S2 (see
legends).
The affiliation of isolates to clonal complexes (CCs) or sequence types (STs) as
defined by MLST [25] (link) was determined by an automated comparison of
hybridisation profiles to a collection of reference strains previously
characterised by MLST [29] (link); [96] (link). Analysis of hybridisation patterns cannot
discriminate sequence types which differ only in single point mutations
affecting MLST genes (e.g., ST5 and ST225, or ST59 and ST952).
However, there are also sequence types which originate from chromosomal
replacements as previously described [170] (link); [173] . As these events result in
different hybridisation patterns, such STs can be easily identified. MRSA
strains which belong to these sequence types will be described separately from
the parental CCs.
previously described in detail [29] (link); [ 96] (link). The DNA microarray covers 334 target sequences,
(approximately 170 distinct genes and their allelic variants) including species
markers, SCCmec, capsule and agr group typing
markers, resistance genes, exotoxins, and MSCRAMM genes. Primer and probe
sequences have been published previously [29] (link); [96] (link).
Target genes and information on primers and probes are provided in Supplemental
S1
MRSA were grown on Columbia blood agar and incubated overnight at 37°C.
Culture material was enzymatically lysed prior to DNA preparation using
commercially available spin columns (Qiagen, Hilden, Germany). Purified DNA
samples were used as templates in a linear primer elongation using one primer
per target. All targets were amplified simultaneously, and within this step,
biotin-16-dUTP was incorporated into the resulting amplicons. An alternate
protocol was used for a few isolates in which amplification and labelling were
directed by random primers [139] (link); [244] . As this protocol does not rely on conserved primer
binding sites, it proved to be useful for characterisation of unusual strains
which are not fully represented by the published genome sequences
(e.g., ST75 strains).
Amplicons obtained using either protocol were hybridised to the microarray
followed by washing and blocking steps, and the addition of
horseradish-peroxidase-streptavidin conjugate. After further incubation and
washing steps, hybridisations were visualised by using a precipitating dye. An
image of the microarray was taken and analysed using a designated reader and
software (ALERE Technologies GmbH, Jena, Germany). Normalised intensities of the
spots were calculated based their average intensities and on the local
background [96] (link). Results were regarded as negative if the normalised
intensity for a given probe was below 25% of the median value of species
markers (coa, eno, fnbA, gapA, katA, nuc, rrn, sarA sbi, spa,
vraS) and a biotin staining control. If the normalised intensity of
a given probe was higher than 50% of this breakpoint, it was interpreted
positive. If it was between 25% and 50%, the result was regarded
as ambiguous. For some markers, for which allelic variants were to be
discriminated (bbp, clfA, clfB and fnbB as
well as some set/ssl genes, isaB, mprF and
isdA), a different approach was used because these alleles
differed only in single nucleotides. Here, only the probe with the strongest
signal value was regarded as positive, provided that it exceeded the 50%
breakpoint. All others were regarded as ambiguous or, if below the 25%
breakpoint, as negative. This allowed an easy and clear distinction of clonal
complex-specific variants of these genes. Genes which are not present in all
tested isolates are labelled as rare, variable or common in
legends).
The affiliation of isolates to clonal complexes (CCs) or sequence types (STs) as
defined by MLST [25] (link) was determined by an automated comparison of
hybridisation profiles to a collection of reference strains previously
characterised by MLST [29] (link); [96] (link). Analysis of hybridisation patterns cannot
discriminate sequence types which differ only in single point mutations
affecting MLST genes (e.g., ST5 and ST225, or ST59 and ST952).
However, there are also sequence types which originate from chromosomal
replacements as previously described [170] (link); [173] . As these events result in
different hybridisation patterns, such STs can be easily identified. MRSA
strains which belong to these sequence types will be described separately from
the parental CCs.
Free full text: Click here
