The possibility of intragenic heterogeneity between copies of the long intergenic (1 63-235 rDNA) spacer region (LISR) was investigated by specific amplification of this region from 21 Enterococcus faecalis isolates. Three copies of the LlSR (rrnA, B and C) were demonstrated by hybridization of the LlSR to genomic DNA cleaved with I-Ceul and Smal. When the LISR amplicon was digested with Tsp5091, two known nucleotide substitutions were detected, one 4 nt upstream from the 5' end of the tRNAaIa gene (allele rrnB has the TspSOSl site and rrnA and Cdo not) and the other 22 nt downstream from the 3' end of the tRNAa'* gene (rmC has the Tsp5091 site). Sequence differences at these sites were detected at the allelic level (alleles rrnA, B and C) and different combinations of these alleles were designated lsp Types. Using densitometry to analyse bands from electrophoresis gels, the intra-isolate ratios of the separate alleles (rmA:rmB:rmC) were determined in each Tsp Type: I (0:3:0), II (l:2:U), 111 (2:O:I)' IV (3:0:0)# V (2:1:0) and VI (1:l:l). Sequence variation between the three copies of the LlSR was confirmed by the detection of at least five other intra-isolate nucleotide substitutions using heteroduplex analysis by conformation-sensitive gel electrophoresis (CSGE) that were not detected by Tsp5091 cleavage. Perpendicular denaturing gradient gel electrophoresis was capable of resolving homoduplexes; six to seven out of a possible nine curves were obtained in some isolates. In the isolate where seven curves were obtained one or more further nucleotide substitutions, not detected by TspSO9l cleavage or CSGE, were detected. On the basis of LlSR sequence heterogeneity, isolates were categorized into homogeneous (only one allele sequence present) and heterogeneous (two or three allele sequences present). The transition between homogeneous and heterogeneous LlSRs may be useful in studying evolutionary mechanisms between E. faecalis isolates.

Two colonial variants of Staphylococcus epidernidis were isolated from the valvular tissue of a patient with native valve endocarditis. In addition to differing in colonial morphology, the two variants differed in hemolysis on blood-containing media, in adherence capacity, and in the expression of certain enzymes. Under suitable conditions, both variants were themselves capable of phenotypic variation, although they differed in the rate at which variants were generated. The variants yielded identical profiles on restriction endonuclease analysis of plasmid DNA and pulsed-field gel electrophoresis of whole-cell DNA. This report suggests a possible role for phenotypic variation in coagulase-negative staphylococcal virulence. Congo red agar would be an excellent medium for studying the contribution of variation to the virulence of these organisms.

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Between 1991 and 1995, an apparent high rate of Staphylococcus warneri bacteremias at the Royal Children's Hospital, Melbourne, Victoria, Australia, raised the possibility of a virulent nosocomial strain. In a retrospective review of 30 S. warneri bacteremias in children, organisms were viable and verified in 22 episodes, 12 representing significant bacteremias. Of these 12 episodes, 2 pairs shared chromosomal DNA pulsed-field gel electrophoresis patterns in unconnected patients, dispelling concerns about a single virulent strain.