MAJOR METALLOPROTEASE GENE OF SERRATIA-MARCESCENS IS CONSERVED AND PROVIDES A MOLECULAR TYPING METHOD TO DIFFERENTIATE CLINICAL ISOLATES

The occurrence and heterogeneity of the 51-kDa major metalloprotease g ene from Serratia marcescens which is thought to be involved in pathog enesis was examined using polymerase chain reaction (PCR) and restrict ion endonuclease analysis (REA). Ten clinical isolates of S. marcescen s associated with a previously characterized outbreak and 80 epidemiol ogically unrelated isolates were investigated. In all isolates, a 1.5- kbp DNA fragment was amplified from the metalloprotease gene. REA of t he amplified DNA showed 3 restriction fragment length polymorphism (RF LP) patterns using HincII and 5 patterns using RsaI. Molecular subtypi ng with the protease gene was performed on 20 epidemiologically unrela ted isolates and 10 outbreak-associated isolates. Size-separated genom ic DNA fragments from HincII or RsaI enzyme digestions were hybridized with the 1.5-kbp metalloprotease gene probe by the method of Southern . Analysis of combined hybridization profiles of all 30 isolates produ ced 23 different protease genotypes. All 20 epidemiologically unrelate d isolates had different profiles, whereas 8 of 10 outbreak-associated isolates showed identical profiles. Discriminatory power of protease genotyping method was compared with the established molecular subtypin g method of ribotyping using a gene probe for Escherichia coli 16S rRN A. Results showed agreement for 20 of the 23 genotypes by both methods . Two other epidemiologically unrelated isolates that displayed the sa me ribotype were differentiated by protease genotyping. Molecular subt yping using the major metalloprotease gene of S. marcescens reported h ere provided differentiation of clinically related and unrelated isola tes and could be used in epidemiological investigations to increase th e discriminatory power of ribotyping.