Abstract
Among 48 erythromycin-resistant group D streptococci (GDS), 36 had the erm(T) resistance gene. erm(T) was also found in 4 of 31 erythromycin-resistant Enterococcus faecium isolates. This is the first report of the erm(T) gene in U.S. GDS isolates and the first report of the erm(T) gene in enterococci.
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 (MLSB 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%) MLSB GBS isolates were shown to contain erm(A) or erm(B), but 5 MLSB erm(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 MLSB S. 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 MLSB phenotype, while 4 had the M phenotype (Table 1). Twenty-seven E. faecalis and 31 E. faecium isolates were ERY resistant (Table 2).
TABLE 1.
Distribution of ERY resistance phenotypes and genes in 48 ERY-resistant clinical GDS isolates
| Resistance phenotype | No. of isolates | No. (%) of isolates with ERY resistance gene(s)
|
|||||
|---|---|---|---|---|---|---|---|
| erm(B) | erm(T) | erm(B) and erm(T) | mef(A) | mef(E) | Negative | ||
| iMLSB | 33 | 33 (69) | |||||
| cMLSB | 11 | 7 (15) | 2 (4) | 1 (2) | 1 (2) | ||
| M | 4 | 2 (4) | 2 (4) | ||||
| Total | 48 | 7 (15) | 35 (73) | 1 (2) | 2 (4) | 2 (4) | 1 (2) |
TABLE 2.
Distribution of ERY resistance phenotypes and genes among 41 clinical Enterococcus faecalis and 35 clinical Enterococcus faecium isolates
| Species (no. of isolates) | ERY resistance phenotype | No. of isolates | No. (%) of isolates with ERY resistance gene(s)
|
||
|---|---|---|---|---|---|
| erm(B) | erm(B) and erm(T) | Negative | |||
| Enterococcus faecalis (41) | Resistant | 27 | 24 (59) | 3 (7) | |
| Intermediate | 13 | 13 (32) | |||
| Susceptible | 1 | 1 (2) | |||
| Enterococcus faecium (35) | Resistant | 31 | 25 (71) | 4 (11) | 2 (6) |
| Susceptible | 4 | 4 (11) | |||
erm(T) was the predominant macrolide resistance gene found among the ERY-resistant GDS (Table 1). Of the 44 MLSB 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 (iMLSB phenotype), while 3 (8%) were constitutively CLI resistant (cMLSB 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 MLSB 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 MLSB 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 MLSB 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 MLSB 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.
Acknowledgments
This work is supported by a research grant from Merck & Co., Inc., and by the Summa Health System Foundation.
We thank Gerri Hall, Tom Thomson, and JMI Laboratories for the contribution of GDS isolates.
Footnotes
Published ahead of print on 19 February 2008.
REFERENCES
- 1.Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Chen, J., Z. Yu, F. C. Michel, T. Wittum, and M. Morrison. 2007. Development and application of real-time PCR assays for quantification of erm genes conferring resistance to macrolides-lincosamides-streptogramin B in livestock manure and manure management systems. Appl. Environ. Microbiol. 73:4407-4416. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Clinical and Laboratory Standards Institute. 2006. Performance standards for antimicrobial susceptibility testing; 16th informational supplement. CLSI document M100-S16. Clinical and Laboratory Standards Institute, Wayne, PA.
- 4.DiPersio, L. P., and J. R. DiPersio. 2006. High rates of erythromycin and clindamycin resistance among OBGYN isolates of group B Streptococcus. Diagn. Microbiol. Infect. Dis. 54:79-82. [DOI] [PubMed] [Google Scholar]
- 5.DiPersio, L. P., and J. R. DiPersio. 2007. Identification of an erm(T) gene in strains of inducibly clindamycin-resistant group B Streptococcus. Diagn. Microbiol. Infect. Dis. 57:189-193. [DOI] [PubMed] [Google Scholar]
- 6.Dubrow, R., S. Edberg, E. Wikfors, D. Callan, F. Troncale, R. Vender, M. Brand, and R. Yapp. 1991. Fecal carriage of Streptococcus bovis and colorectal adenomas. Gastroenterology 101:721-725. [DOI] [PubMed] [Google Scholar]
- 7.Facklam, R. 2002. What happened to the streptococci: overview of taxonomic and nomenclature changes. Clin. Microbiol. Rev. 15:613-630. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Hayes, J. R., D. D. Wagner, L. L. English, L. E. Carr, and S. W. Joseph. 2005. Distribution of streptogramin resistance determinants among Enterococcus faecium from a poultry production environment of the USA. J. Antimicrob. Chemother. 55:123-126. [DOI] [PubMed] [Google Scholar]
- 9.Klein, R. S., M. T. Catalano, S. C. Edberg, J. I. Casey, and N. H. Steigbigel. 1979. Streptococcus bovis septicemia and carcinoma of the colon. Ann. Intern. Med. 91:560-562. [DOI] [PubMed] [Google Scholar]
- 10.Leclercq, R. 2002. Mechanisms of resistance to macrolides and lincosamides: nature of the resistance elements and their clinical implications. Clin. Infect. Dis. 34:482-492. [DOI] [PubMed] [Google Scholar]
- 11.Leclercq, R., C. Huet, M. Picherot, P. Trieu-Cuot, and C. Poyart. 2005. Genetic basis of antibiotic resistance in clinical isolates of Streptococcus gallolyticus (Streptococcus bovis). Antimicrob. Agents Chemother. 49:1646-1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Lee, R. A., P. C. Y. Woo, A. P. C. To, S. K. P. Lau, S. S. Y. Wong, and K. Y. Yuen. 2003. Geographical difference of disease association in Streptococcus bovis bacteremia. J. Med. Microbiol. 52:903-908. [DOI] [PubMed] [Google Scholar]
- 13.Luna, V. A., P. Coates, E. A. Eady, J. H. Cove, T. T. H. Nguyen, and M. C. Roberts. 1999. A variety of Gram-positive bacteria carry mobile mef genes. J. Antimicrob. Chemother. 44:19-25. [DOI] [PubMed] [Google Scholar]
- 14.Murray, B. E. 1990. The life and times of the enterococcus. Clin. Microbiol. Rev. 3:46-65. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Murray, H. W., and R. B. Roberts. 1978. Streptococcus bovis bacteremia and underlying gastrointestinal disease. Arch. Intern. Med. 138:1097-1099. [PubMed] [Google Scholar]
- 16.Portillo, A., F. Ruiz-Larrea, M. Zarazaga, A. Alonso, J. L. Martinez, and C. Torres. 2000. Macrolide resistance genes in Enterococcus spp. Antimicrob. Agents Chemother. 44:967-971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Roberts, M. C., J. Sutcliffe, P. Courvalin, L. B. Jensen, J. Rood, and H. Seppala. 1999. Nomenclature for macrolide and macrolide-lincosamide-streptogramin B resistance determinants. Antimicrob. Agents Chemother. 43:2823-2830. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Rodríguez-Avial, I., C. Rodriguez-Avial, E. Culebras, and J. J. Picazo. 2005. In vitro activity of telithromycin against viridans group streptococci and Streptococcus bovis isolated from blood: antimicrobial susceptibility patterns in different groups of species. Antimicrob. Agents Chemother. 49:820-823. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Schlegel, L., F. Grimont, E. Ageron, P. A. D. Grimont, and A. Bouvet. 2003. Reappraisal of the taxonomy of the Streptococcus bovis/Streptococcus equinus complex and related species: description of Streptococcus gallolyticus subsp. gallolyticus subsp. nov., S. gallolyticus subsp. macedonicus subsp. nov. and S. gallolyticus subsp. pasteurianus subsp. nov. Int. J. Syst. Evol. Microbiol. 53:631-645. [DOI] [PubMed] [Google Scholar]
- 20.Schouten, M. A., A. Voss, J. A. A. Hoogkamp-Korstanje, and the European VRE Study Group. 1999. Antimicrobial susceptibility patterns of enterococci causing infections in Europe. Antimicrob. Agents Chemother. 43:2542-2546. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Tafe, L. J., and K. L. Ruoff. 2007. Streptococcus bovis: answers and questions. Clin. Microbiol. Newsl. 29:49-55. [Google Scholar]
- 22.Tannock, G. W., J. B. Luchansky, L. Miller, H. Connell, S. Thode-Andersen, A. A. Mercer, and T. R. Klaenhammer. 1994. Molecular characterization of a plasmid-borne (pGT633) erythromycin resistance determinant (ermGT) from Lactobacillus reuteri 100-63. Plasmid 31:60-71. [DOI] [PubMed] [Google Scholar]
- 23.Teng, L. J., P. R. Hsueh, S. W. Ho, and K. T. Luh. 2001. High prevalence of inducible erythromycin resistance among Streptococcus bovis isolates in Taiwan. Antimicrob. Agents Chemother. 45:3362-3365. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Tsai, J. C., P. R. Hsueh, H. J. Chen, S. P. Tseng, P. Y. Chen, and L. J. Teng. 2005. The erm(T) gene is flanked by IS1216V in inducible erythromycin-resistant Streptococcus gallolyticus subsp. pasteurianus. Antimicrob. Agents Chemother. 49:4347-4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Whitehead, T. R., and M. A. Cotta. 2001. Sequence analyses of a broad host-range plasmid containing ermT from a tylosin-resistant Lactobacillus sp. isolated from swine feces. Curr. Microbiol. 43:17-20. [DOI] [PubMed] [Google Scholar]
- 26.York, M. K., L. Gibbs, F. Perdreau-Remington, and G. F. Brooks. 1999. Characterization of the antimicrobial resistance in Streptococcus pyogenes isolates from the San Francisco Bay area of Northern California. J. Clin. Microbiol. 37:1727-1731. [DOI] [PMC free article] [PubMed] [Google Scholar]