ASSESSMENT OF RESOLUTION AND INTERCENTER REPRODUCIBILITY OF RESULTS OF GENOTYPING STAPHYLOCOCCUS-AUREUS BY PULSED-FIELD GEL-ELECTROPHORESISOF SMAI MACRORESTRICTION FRAGMENTS - A MULTICENTER STUDY

Twenty well characterized isolates of methicillin-resistant Staphyloco ccus aureus were used to study the optimal resolution and interlaborat ory reproducibility of pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments. Five identical isolates tone PFGE type), 5 isolates that produced related PFGE subtypes, and 10 isolates with un ique PFGE patterns were analyzed blindly in 12 different laboratories by inhouse protocols. In several laboratories a standardized PFGE prot ocol with a commercial kit was applied successfully as well. Eight of the centers correctly identified the genetic homogeneity of the identi cal isolates by both the in-house and standard protocols. Four of 12 l aboratories failed to produce interpretable data by the standardized p rotocol, due to technical problems (primarily plug preparation). With the five related isolates, five of eight participants identified the s ame subtype interrelationships with both in-house and standard protoco ls. However, two participants identified multiple strain types in this group or classified some of the isolates as unrelated isolates rather than as subtypes. The remaining laboratory failed to distinguish diff erences between some of the related isolates by utilizing both the in- house and standardized protocols. There were large differences in the relative genome lengths of the isolates as calculated on the basis of the gel pictures. By visual inspection, the numbers of restriction fra gments and overall banding pattern similarity in the three groups of i solates showed interlaboratory concordance, but centralized computer a nalysis of data from four laboratories yielded percent similarity valu es of only 85% for the group of identical isolates. The differences be tween the data sets obtained with in-house and standardized protocols could be the experimental parameters which differed with respect to th e brand of equipment used, imaging software, running time (20 to 48 h) , and pulsing conditions. In conclusion, it appears that the standardi zation of PFGE depends on controlling a variety of experimental intric acies, as is the case with other bacterial typing procedures.