Prevalence of the erm(T) Gene in Clinical Isolates of Erythromycin-Resistant Group D Streptococcus and Enterococcus

Group D streptococci (GDS) include members of the Streptococcus bovis group. Proposed nomenclature changes for the S. bovis group would establish several new species, including Streptococcus gallolyticus subsp. gallolyticus, S. gallolyticus subsp. pasteurianus, Streptococcus infantarius subsp. infantarius, and Streptococcus lutetiensis (7, 19, 21). GDS are commensal organisms of the human gut and have been found in 14% of fecal samples from normal controls (6). GDS bacteremia is often associated with underlying gastrointestinal diseases, such as colon cancer (9, 15). Enterococcus faecalis and Enterococcus faecium are found in the feces of most adults but can cause infections such as bacteremia and endocarditis (14). GDS, E. faecalis, and E. faecium can have erythromycin (ERY) resistance rates as high as 75% (11, 20, 23).

ERY ribosomal methylases (coded for by erm genes) methylate the bacterial ribosome, impairing the binding of macrolide, lincosamide, and streptogramin B antibiotics and resulting in resistance (MLS_B phenotype) (10). Another common ERY resistance mechanism is macrolide efflux, coded for by mef genes, which are common in streptococci (17). mef genes cause resistance to 14- and 15-membered macrolides (such as ERY) but not 16-membered macrolides, lincosamides, and streptogramin B (M phenotype).

Recently, we reported high levels of ERY (54%) and clindamycin (CLI) (33%) resistance in clinical group B Streptococcus (GBS) isolates (4). Sixty-one of 66 (92%) MLS_B GBS isolates were shown to contain erm(A) or erm(B), but 5 MLS_Berm(A)- and erm(B)-negative GBS isolates and 1 erm(B)-positive isolate were found to contain the erm(T) gene (5). Since the erm(T) gene has been found in MLS_BS. bovis isolates in Asia (12, 23), we investigated the prevalence of the erm(T) gene in clinical isolates of GDS. In addition, since E. faecalis and E. faecium can be highly resistant to ERY, we also studied the prevalence of erm(T) in these organisms.

(This work was presented in part at the 107th General Meeting of the American Society for Microbiology, Toronto, Canada, 21 to 25 May 2007.)

One hundred twenty-seven clinical GDS isolates were evaluated. Thirty-three GDS isolates were collected from northeast Ohio and 12 from Chicago. Twenty-nine additional ERY-resistant GDS isolates were obtained from the U.S. SENTRY program from a collection of 82 GDS isolates. GDS were originally isolated from blood, urine, skin, and soft tissue samples. Isolates were identified by the laboratories of origin as members of the S. bovis group or simply GDS. Forty-one E. faecalis and 35 E. faecium blood isolates collected at Summa Health System were also studied. Identifications were performed using standard laboratory methods.

Disk diffusion testing for ERY and CLI resistance in GDS from northeast Ohio and Chicago and 29 ERY-resistant SENTRY isolates was performed and interpreted according to the CLSI (3). ERY and CLI disks were placed 15 mm apart edge to edge (D test) to detect inducible CLI resistance in GDS (26). Additionally, E. faecalis and E. faecium isolates were tested for susceptibility to ERY by disk diffusion. MIC testing of selected isolates was done by Etest according to the manufacturer's guidelines (AB Biodisk, Solna, Sweden).

Isolates were tested for macrolide resistance genes erm(A), erm(B), erm(C), erm(T), mef(A), and mef(E) by PCR as previously described (4, 5). erm(T) PCR products from seven erm(T)-positive GDS isolates and four erm(T)-positive E. faecium isolates were DNA sequenced by Sequetech (Mountain View, CA) as previously described (5). DNA database searches and DNA sequence identity analysis used Basic Local Alignment Tool (BLASTn) (1).

Forty-four of 127 (35%) GDS isolates had the MLS_B phenotype, while 4 had the M phenotype (Table 1). Twenty-seven E. faecalis and 31 E. faecium isolates were ERY resistant (Table 2).

erm(T) was the predominant macrolide resistance gene found among the ERY-resistant GDS (Table 1). Of the 44 MLS_B GDS, 36 (82%) contained erm(T), while 8 (18%) contained erm(B). Thirty-three of the 36 (92%) erm(T)-positive isolates were inducibly CLI resistant (iMLS_B phenotype), while 3 (8%) were constitutively CLI resistant (cMLS_B phenotype), one of which also contained erm(B). Two GDS isolates with the M phenotype contained the efflux gene mef(A), and two contained mef(E) (Table 1). The erm(A) and erm(C) genes were not found in this collection of GDS.

Twenty-four ERY/CLI-susceptible GDS isolates were tested for the presence of erm(A), erm(B), erm(C), erm(T), mef(A), and mef(E). One ERY/CLI-susceptible isolate (ERY MIC, 0.064 μg/ml; CLI MIC, 0.125 μg/ml) was found to contain the erm(T) gene; all others were negative for the genes tested.

erm(T) was found along with erm(B) in only four ERY-resistant E. faecium isolates (Table 2). The ERY resistance genes erm(A), erm(C), mef(A), and mef(E) were not found in E. faecalis or E. faecium isolates. Although erm(B) has often been found in ERY-resistant enterococci, efflux genes mef(A/E) and msr(C) have also been reported (13, 16). Our enterococcal isolates were negative for mef(A/E), and msr(C) was not examined in this study. There may be other not-yet-characterized macrolide resistance mechanisms in enterococci, as recently suggested (8).

The erm(T) gene PCR product was sequenced from seven erm(T)-positive GDS isolates. Six GDS isolates had the MLS_B phenotype (ERY MIC, ≥256 μg/ml), and the seventh was the ERY/CLI-susceptible but erm(T)-positive isolate. In addition, the four erm(T) gene PCR products from E. faecium were sequenced. All 11 erm(T) PCR products had 99 to 100% sequence identity with the previously published erm(T) sequence from a clinical Streptococcus gallolyticus subsp. pasteurianus (Streptococcus bovis) isolate (GenBank database accession number AY894138; http://www.ncbi.nlm.nih.gov/Genbank/index.html ) (23, 24). The single ERY/CLI-susceptible GDS isolate which tested positive for erm(T) had 100% DNA sequence identity with the published erm(T) gene sequence mentioned above. ERY-susceptible erm-positive isolates are uncommon. There may be a mutation in the portion of the gene not sequenced in our study or in the promoter region of the gene. This is presently under study in our laboratory.

Studies in Europe found the erm(B) gene in ERY-resistant clinical isolates of S. bovis (11, 18). However, in two Asian studies, the erm(T) gene was found in 46% and 37% of ERY-resistant clinical S. bovis isolates, respectively (12, 23); the remaining isolates contained erm(B). In the present study of 44 MLS_B U.S. GDS isolates, 36 (82%) contained the erm(T) gene and 8 (18%) contained erm(B).

The erm(T) gene (originally called ermGT) was first found in a poultry isolate of Lactobacillus reuteri in 1994. The erm(T) gene was shown to have 81% nucleotide sequence identity with the erm(C) gene (22). In 2001, an erm(T) gene discovered in a swine feces isolate, Lactobacillus strain 121B, showed 99% DNA sequence identity with the erm(T) gene found in Lactobacillus reuteri (25). Also in 2001, an erm(T) gene was found in MLS_B clinical S. bovis isolates in Taiwan (23). This gene demonstrated 98.5% nucleotide sequence identity with the erm(T) gene found in L. reuteri (22, 23). Recent studies involving erm genes in livestock manure and manure management systems found erm(T) to be the second most abundant erm gene found in swine manure (2).

Given the high prevalence of erm(T) in MLS_B GDS in the United States, this organism may represent a major reservoir for the transfer of erm(T) to other human enteric commensals and pathogens such as GBS and enterococci. To our knowledge, this is the first report of the erm(T) gene in U.S. GDS and the first report of erm(T) in enterococci.

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