Use of groESL as a target for identification of Abiotrophia, Granulicatella, and Gemella species

Abstract

We determined the groESL sequences of three species of nutritionally variant streptococci (Abiotrophia defectiva, Granulicatella adiacens, and Granulicatella elegans) and three Gemella species (Gemella morbillorum, Gemella haemolysans, and Gemella sanguinis). The nucleotide sequence similarities between the groES and groEL genes of the above genera were 41.7 to 85.9% and 63.7 to 84.3%, respectively. The intraspecies similarities of groESL sequences for the isolates of Abiotrophia and Granulicatella species were 94.4 to 97.8% for groES and 94.0 to 98.2% for groEL. For Ge. morbillorum and Ge. sanguinis, all strains showed the same groESL spacer length (8 bp), and sequence identities within species were >97.8% for groES and >96.1% for groEL. However, higher intraspecies heterogeneity was observed in Ge. haemolysans. Phylogenetic analysis of groEL sequences separated the 6 isolates of Ge. haemolysans into two subgroups. Among these isolates, three isolates with the same groESL spacer region length (45 bp) clustered together but were distant from the ATCC reference strain (with a spacer length of 8 bp). The remaining three isolates, with a spacer length of 50 or 8 bp, clustered together. Although 16S rRNA gene sequence analysis did not provide enough discrimination for the 6 Ge. haemolysans isolates, rpoB gene sequence analysis supported the subgrouping. Based on the obtained groESL sequences, we developed a multiplex PCR that enables simple, rapid, and accurate identification of Abiotrophia, Granulicatella, and Gemella at the genus level. This assay would be helpful for identifying these fastidious and slow-growing organisms in clinical laboratories.

FIG. 1.

Phylogenetic tree based on partial groEL nucleotide sequences (nt 1 to 816). The phylogenetic tree was generated using the unrooted neighbor-joining method in the MEGA4 package. The numbers at the nodes are confidence levels, expressed as percentages of occurrence in 500 bootstrapped resamplings. The scale bar indicates the evolutionary distance between sequences, as determined by measuring the lengths of the horizontal lines connecting two organisms. The GenBank accession number for each species' groEL genes is shown after the species name. Sequences determined in this study are shown in bold.

FIG. 2.

Multiplex PCR analysis. (A) Multiplex PCR amplification of genomic DNAs of Abiotrophia, Granulicatella, and Gemella species. Lanes M, DNA size markers (100-bp ladder; Invitrogen); lane 1, A. defectiva ATCC 49176; lane 2, A. defectiva NTUH_9251; lane 3, Gr. adiacens ATCC 49175; lane 4, Gr. adiacens NTUH_8436; lane 5, Gr. elegans ATCC 700633; lane 6, Gr. elegans NTUH_4128; lane 7, Ge. morbillorum ATCC 27824; lane 8, Ge. morbillorum NTUH_2065; lane 9, Ge. haemolysans ATCC 10379; lanes 10 to 12, Ge. haemolysans NTUH_1465, -2196, and -4957; lane 13, Ge. sanguinis ATCC 700632; lane 14, Ge. sanguinis NTUH_2000; and lane 15, distilled water as a negative control. (B) Specificity of multiplex PCR tested with 14 other clinically relevant Gram-positive cocci. Lane 1, A. defectiva ATCC 49176; lane 2, Gr. adiacens ATCC 49175; lane 3, Ge. morbillorum ATCC 27824; lane 4, Streptococcus constellatus ATCC 27823; lane 5, Streptococcus intermedius ATCC 27335; lane 6, Streptococcus anginosus ATCC 33397; lane 7, Streptococcus gordonii CAPQ-2; lane 8, Streptococcus sanguinis ATCC 10556; lane 9, Streptococcus oralis ATCC 35037; lane 10, Streptococcus pneumoniae ATCC 49619; lane 11, Streptococcus mitis ATCC 49456; lane 12, Streptococcus pyogenes ATCC 19615; lane 13, Streptococcus agalactiae ATCC 13813; lane 14, Streptococcus salivarius ATCC 7073; lane 15, Staphylococcus aureus ATCC 29213; lane 16, Enterococcus faecium ATCC 19434; and lane 17, Enterococcus faecalis ATCC 19433.