Τίτλος:
Harmonization of pulsed-field gel electrophoresis protocols for
epidemiological typing of strains of methicillin-resistant
Staphylococcus aureus: a single approach developed by consensus in 10
European laboratories and its application for tracing the spread of
related strains
Γλώσσες Τεκμηρίου:
Αγγλικά
Περίληψη:
Pulsed-field gel electrophoresis (PFGE) is the most common genotypic
method used in reference and clinical laboratories for typing
methicillin-resistant Staphylococcus aureus (MRSA). Many different
protocols have been developed in laboratories that have extensive
experience with the technique and have established national databases.
However, the comparabilities of the different European PFGE protocols
for MRSA and of the various national MRSA clones themselves had not been
addressed until now. This multinational European Union (EU) project has
established for the first time a European database of representative
epidemic MRSA (EMRSA) strains and has compared them by using a new
“harmonized” PFGE protocol developed by a consensus approach that
has demonstrated sufficient reproducibility to allow the successful
comparison of pulsed-field gels between laboratories and the tracking of
strains around the EU. In-house protocols from 10 laboratories in eight
European countries were compared by each center with a “gold
standard” or initial harmonized protocol in which many of the
parameters had been standardized. The group found that it was not
important to standardize some elements of the protocol, such as the type
of agarose, DNA block preparation, and plug digestion. Other elements
were shown to be critical, namely, a standard gel volume and
concentration of agarose, the DNA concentration in the plug, the ionic
strength and volume of running buffer used, the running temperature, the
voltage, and the switching times of electrophoresis. A new harmonized
protocol was agreed on, further modified in a pilot study in two
laboratories, and finally tested by all others. Seven laboratories’ gels
were found to be of sufficiently good quality to allow comparison of the
strains by using a computer software program, while two gels could not
be analyzed because of inadequate destaining and DNA overloading.
Good-quality gels and inclusion of an internal quality control strain
are essential before attempting intercenter PFGE comparisons. A number
of clonally related strains have been shown to be present in multiple
countries throughout Europe. The well-known Iberian clone has been
demonstrated in Belgium, Finland, France, Germany, and Spain (and from
the wider HARMONY collection in Portugal, Slovenia, and Sweden). Strains
from the United Kingdom (EMRSA-15 and -16) have been identified in
several other countries, and other clonally related strains have also
been identified. This highlights the need for closer international
collaboration to monitor the spread of current epidemic strains as well
as the emergence of new ones.
Συγγραφείς:
Murchan, S
Kaufmann, ME
Deplano, A
de Ryck, R
Struelens,
M
Zinn, CE
Fussing, V
Salmenlinna, S
Vuopio-Varkila, J
and El Solh, N
Cuny, C
Witte, W
Tassios, PT
Legakis, N
and van Leeuwen, W
van Belkum, A
Vindel, A
Laconcha, I and
Garaizar, J
Haeggman, S
Olsson-Liljequist, B
Ransjo, U and
Coombes, G
Cookson, B
Περιοδικό:
Journal of Clinical Microbiology
Εκδότης:
AMER SOC MICROBIOLOGY
DOI:
10.1128/JCM.41.4.1574-1585.2003
