Molecular Typing of Methicillin-ResistantStaphylococcus aureusby Pulsed-Field Gel Electrophoresis: Comparison of Results Obtained in a Multilaboratory Effort Using Identical Protocols and MRSA Strains

Abstract

Pulsed-field gel electrophoresis (PFGE) has become the gold standard of molecular methods in epidemiological investigations. In spite of its high resolving power, use of the method has been hampered by inadequate laboratory-to-laboratory reproducibility. In the project described here we have addressed this problem by organizing a multilaboratory effort in which the same bacterial strains (subtype variants of the Iberian and Brazilian methicillin-resistant Staphylococcus aureus--MRSA--clones) were analyzed by twenty investigators in thirteen different laboratories according to an indentical protocol, which is reproduced here in detail. PFGE patterns obtained were analyzed at a central laboratory in order to identify specific technical problems that produced substandard macrorestriction patterns. The results including the specific technical problems and their most likely causes are described in this communication. Also listed are seven major epidemic clones of MRSA which have been characterized by molecular fingerprinting techniques and the prototypes of which have been deposited at the American Type Culture Collection, from where they will be available for interested investigators for the purpose of typing MRSA isolates. It is hoped that this communication will contribute to the improvement of the reproducibility and technical/aesthetic quality of PFGE analysis.

High prevalence of ST121 in community-associated methicillin-susceptible Staphylococcus aureus lineages responsible for skin and soft tissue infections in Portuguese children

“…1) were performed by the semi-automatic VITEK2 system (bioMérieux, SA, France) and the disk diffusion method for clindamycin [11]. All isolates were tested by polymerase chain reaction (PCR) for the presence of mecA [12] and were also characterized by pulsed-field gel electrophoresis (PFGE) [13]. The resulting SmaI patterns were analyzed by both visual inspection and automatically with BioNumerics software version 4.61 (Applied Maths, Sint-Martens-Latem, Belgium) [14,15].…”

Comparison of Multilocus Sequence Typing and Pulsed-Field Gel Electrophoresis as Tools for Typing Staphylococcus aureus Isolates in a Microepidemiological Setting

“…In principle, the results generated should lead to good reproducibility and to comparability between centers, although this has yet to be formally demonstrated. This contrasts with PFGE, which has been shown to be poorly reproducible between centers (4,6,15,19), leading to considerable effort to introduce standardized protocols to improve reproducibility and technical quality (3,15). However, MLST is expensive and technically demanding, and it is important to establish whether it represents a clear advantage in terms of discrimination.…”

Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus in Finland

“…However, the majority of these isolates showed no resistance to methicillin. The isolates which were resistant to methicillin had a further copy of mecA, identical to that of MRSA isolates (46,309).…”

“…In addition, international comparison of MRSA strains has become increasingly relevant because of the pandemic spread of MRSA strains. Despite established interpretation criteria (262), availability of sophisticated software packages, and several inter-laboratory standardization initiatives for PFGE (46,281), inter-laboratory reproducibility is difficult to achieve. Although software which transform bands mathematically to numerical values do exist, PFGE and other banding pattern based methods are prone to bias due to manual selection of bands.…”