Abstract
A DNA microarray was developed to detect bacterial genes conferring resistance to macrolides and related antibiotics. A database containing 65 nonredundant genes selected from publicly available DNA sequences was constructed and used to design 100 oligonucleotide probes that could specifically detect and discriminate all 65 genes. Probes were spotted on a glass slide, and the array was reacted with DNA templates extracted from 20 reference strains of eight different bacterial species (Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus haemolyticus, Escherichia coli, and Bacteroides fragilis) known to harbor 29 different macrolide resistance genes. Hybridization results showed that probes reacted with, and only with, the expected DNA templates and allowed discovery of three unexpected genes, including msr(SA) in B. fragilis, an efflux gene that has not yet been described for gram-negative bacteria.
Resistance to macrolides and related antibiotics (macrolides- lincosamides-streptogramins [MLS]) is of great concern because these drugs are commonly used to treat many different infectious syndromes and because this resistance is spreading among gram-positive and gram-negative bacteria, including strains isolated from life-threatening infections such as pneumonia, sepsis, endocarditis, and meningitis. Different classes of genes coding for MLS resistance have been described, and their nucleotide sequences are available in public databases (22). Although macrolide resistance is present worldwide, patterns and mechanisms of resistance may vary widely in different geographic areas, leading to different therapeutic strategies for infective syndromes, such as community-acquired pneumonia (15, 16, 19).
Detection of single bacterial genes (e.g., antibiotic resistance genes or species-specific genes) in diagnostics and in epidemiological studies is typically carried out by PCR, whereas DNA microarrays have been developed to perform a large number of different hybridization experiments simultaneously on a single membrane or glass substrate. They are well-suited to comprehensively investigate and quantitatively compare the expression levels of a large number of genes, but they can also be easily used in qualitative studies to detect selected DNA sequences (7, 8, 21). To assist epidemiological studies on the genetics of macrolide resistance in clinical isolates, a method based on DNA microarrays was developed to comprehensively assess the presence of MLS genes in bacterial genomes.
MATERIALS AND METHODS
Database construction and probe design.
The sequences of MLS resistance genes were retrieved from public databases and comparatively analyzed to avoid redundancy. The file containing the selected sequences in multi-FASTA format (http://www.compbio.ox.ac.uk/faq/format_examples.shtml) was used to generate a database to be searched by Array Designer 2.0 software (Premier Biosoft, Palo Alto, CA). Probes, 40 to 60 nucleotides in size, with a melting temperature of 83 ± 1°C, were designed to specifically target each gene of the database. Oligonucleotide probes generated by the software were checked for homology to unrelated sequences present in public databases, and, when possible, two probes for each gene were designed for the array.
Construction of microarray slides.
Oligonucleotide probes were synthesized by MWG Biotech (Munich, Germany), with a C6 amino linker to allow better binding to the slide. Epoxy-modified glass slides (Pan-Epoxy slides; MWG Biotech) and a four-head pin ring spotting apparatus (GMS 417 arrayer; Genetics MicroSystems, Woburn, MA) were used. Probes were spotted in at least three replicates at a concentration of 30 pmol/μl in 20% dimethyl sulfoxide and 0.1% Tween 20. Resulting spots had a diameter of 80 to 120 μm.
Template DNA extraction, labeling, and hybridization.
Genomic DNA was extracted from a 10-ml bacterial culture harvested in exponential phase, according to a published protocol (4). For staphylococci, 20 U of lysostaphin was added to the lysis solution. One microgram of template DNA, in a reaction volume of 25 μl, was labeled with the fluorescent cytosine analog Cy5 (Amersham Biosciences, Piscataway, NJ) by random priming using 40 U of Klenow DNA polymerase, with a Cy5/dCTP ratio of 1. Ten microliters of the labeled DNA was brought to a volume of 14 μl in hybridization buffer (3× SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 30 mM HEPES, pH 8, 0.3% sodium dodecyl sulfate, 5× Denhardt's solution), containing tRNA of Saccharomyces cerevisiae (Sigma, St. Louis, Mo.) at 1.5 mg per ml. After 2 min of denaturation at 100°C and 10 min at room temperature, the 14-μl mix was layered on the slide and hybridized for 1 h at 55°C. Slides were washed twice for 5 min in 2× SSC-0.1% sodium dodecyl sulfate at 65°C and then twice for 5 min in 1× SSC at room temperature and twice for 5 min in 0.2× SSC.
Data analysis.
Microarray slides were read using a GMS 428 array scanner (Genetics MicroSystems, Woburn, MA). Data were acquired using GenePix Pro 5.0 software (Axon Instruments, Union City, CA) and managed with Microsoft Excel. For each spot, median pixel intensity was assessed, and background signal was subtracted. To control for congruity of results obtained with replicate spots of a probe, the mean fluorescence intensity and the standard deviation (intraprobe standard deviation) was calculated for each probe. If, for a probe, the intraprobe standard deviation was higher than the mean fluorescence intensity, hybridization results were considered negative. The standard deviation of the mean fluorescence intensity of all probes of the microarray was also calculated. A probe was considered positive when its fluorescence intensity was higher than the mean fluorescence intensity of all probes plus 1 standard deviation.
Bacterial strains.
We hybridized total DNA from 20 bacterial strains carrying reference MLS resistance genes (Table 1).
TABLE 1.
Bacterial strains
| Strain (plasmid[s]) | Gene(s) | Source (reference) |
|---|---|---|
| Streptococcus pyogenes A200 | erm(TR) | H. Seppala (25) |
| Staphylococcus aureus N315 (pN315) | erm(A) | T. Ito (14) |
| S. aureus BM4611 | erm(C)a, lnu(A)b | P. Courvalin (6) |
| S. aureus BM3093 (pIP680) | vat(A), vgb(A), vga(A) | N. El Solh (12) |
| S. aureus BM12392 (pIP1714) | vgb(B), vat(C) | N. El Solh (2) |
| S. aureus BM12235 (pIP1633) | vga(B), vat(B), vga(A)va | N. El Solh (1) |
| Staphylococcus haemolyticus BM4610 (pIP855) | lnu(A)c | P. Courvalin (5) |
| Enterococcus faecium A41 | vat(E-3), erm(B) | N. Woodford (26) |
| E. faecium UW1965 | vat(E), erm(B) | G. Werner (27) |
| Enterococcus faecalis JH2-2 (pAM401) | erm(B), lsa | This laboratory (28) |
| E. faecalis JH2-2 (pAMβ1) | erm(B), lsa | This laboratory (18) |
| Escherichia coli DH1 (pVA891) | erm(B), mac(A), mac(B) | This laboratory (17) |
| E. coli DH5α (pTZ3519) | mph(A), mac(A), mac(B) | N. Noguchi (20) |
| E. coli DB10 (pAT421) | vat(D), mph(A), mac(A), mac(B) | P. Courvalin (24) |
| E. coli BM2506 (pTZ3721 and pTZ3723) | mph(B), erm(B), mac(A), mac(B) | P. Courvalin (13) |
| E. coli BM2570 (pIP1527) | ere(B), erm(B), mac(A), mac(B) | P. Courvalin (3) |
| E. coli BM8463 (pIP1810) | vga(A)v, mac(A), mac(B) | N. El Solh (11) |
| Bacteroides fragilis V503 (pVA503) | erm(FU), msr(SA)a | M. C. Halula (10) |
| Streptococcus pneumoniae PN150 | mef(E), mel | A. Pantosti (9) |
| S. pneumoniae MF4 | mef(A), msr(D) | This laboratory (23) |
RESULTS AND DISCUSSION
Probes for macrolide resistance genes.
A database which included 65 nonredundant macrolide resistance genes published in GenBank was selected (Tables 2 to 4). Genes were identified by accession number, since in some cases two or more genes with different sequences share the same name. One hundred oligonucleotide probes were designed and spotted on the microarray slide to allow differential detection of the 65 selected MLS genes. Probes for ribosomal methylation genes and their positions in the coding sequence are reported in Table 2, probes for efflux genes in Table 3, and probes for genes coding for esterases, nucleotidyltransferases, phosphotransferases, acetyltransferases, and hydrolases in Table 4.
TABLE 2.
Probes for ribosomal methylation genes
| Gene | GenBank accession no. | Probe | Position (nucleotides) |
|---|---|---|---|
| erm(A) | AP003129 | 013 | 56198-56252 |
| 014a | 56494-56547 | ||
| erm(B) | Y00116 | 017 | 362-405 |
| 123 | 615-662 | ||
| erm(C) | Y17294 | 019 | 818-877 |
| erm(C) | Y09003 | 020 | 546-606 |
| erm(33) | AJ313523 | 021b | 286-348 |
| erm(D) | M29832 | 095 | 1062-1107 |
| erm(D) | M77505 | 088 | 1009-1072 |
| erm(F) | M14730 | 152 | 585-634 |
| erm(FU) | M62487 | 091 | 754-796 |
| 092 | 910-959 | ||
| erm(G) | M15332 | 089 | 793-857 |
| erm(GM) | AB014481 | 090 | 662-725 |
| erm(H) | M16503 | 077 | 525-560 |
| 078 | 365-399 | ||
| erm(K) | AB024564 | 085 | 1296-1339 |
| 086 | 1103-1146 | ||
| erm(K) | M77505 | 087 | 1371-1419 |
| erm(M) | AF462611 | 083 | 217-251 |
| 084 | 892-927 | ||
| erm(Q) | L22689 | 082 | 626-687 |
| erm(T) | AF310974 | 081 | 1419-1480 |
| erm(TR) | AF002716 | 015 | 368-417 |
| 016 | 684-740 | ||
| erm(X) | AF411029 | 079 | 1731-1776 |
| erm(X) | AF338706 | 080 | 1293-1336 |
| erm(34) | AY234334 | 148 | 913-949 |
| 147 | 818-863 | ||
| erm(35) | AF319779 | 094 | 271-334 |
| erm(38) | AY154657 | 134 | 199-233 |
| 135 | 136-174 |
TABLE 4.
Probes for genes coding for esterases, nucleotidyltransferases, phosphotransferases, acetyltransferases, and hydrolases
| Gene product | Gene | GenBank accession no. | Probe | Position (nucleotides) |
|---|---|---|---|---|
| Esterase | ere(A) | AY183453 | 098 | 3173-3216 |
| 099 | 3049-3091 | |||
| ere(A-2) | AF099140 | 096 | 1362-1406 | |
| 097 | 177-223 | |||
| ere(B) | X03988 | 022 | 772-827 | |
| Nucleotidyltransferase | lnu(A) | J03947 | 069 | 939-987 |
| lnu(A) | M14039 | 072 | 457-510 | |
| lnu(B) | AJ238249 | 067 | 281-324 | |
| lnu(B)-like | AJ293027 | 065 | 5830-5770 | |
| 066 | 5501-5448 | |||
| Phosphotransferase | mph(A) | U36578 | 143 | 1004-1042 |
| 144 | 1117-1151 | |||
| mph(B) | D85892 | 063 | 1685-1729 | |
| 064 | 2019-2064 | |||
| mph(C) | AB013298 | 059 | 2497-2554 | |
| 060 | 2514-2556 | |||
| mph(C) | AF167161 | 061 | 5883-5925 | |
| 062 | 5866-5923 | |||
| Acetyltransferase | vat(A) | L07778 | 052 | 634-680 |
| vat(B) | U19459 | 050 | 408-459 | |
| 051 | 260-317 | |||
| vat(C) | AF015628 | 048 | 1661-1703 | |
| 049 | 1595-1641 | |||
| vat(D) | L12033 | 046 | 563-614 | |
| 047 | 362-420 | |||
| vat(E) | AF139725 | 045a | 430-476 | |
| 044 | 74-120 | |||
| vat(E-3) | AY008284 | 042 | 7-52 | |
| Hydrolase | vgb(A) | M20129 | 128 | 1221-1277 |
| 127 | 899-950 | |||
| vgb(B) | AF015628 | 035 | 908-953 | |
| 034 | 1016-1068 |
Reacts also with vat(E-3) (AY008284).
TABLE 3.
Probes for efflux genes
| Gene | GenBank accession no. | Probe | Position (nucleotides) |
|---|---|---|---|
| mef(A) | AF227520 | 008 | 4168-4221 |
| 010 | 4205-4251 | ||
| mef(E) | AF376746 | 001a | 1561-1609 |
| 012 | 2265-2319 | ||
| msr(D) | AF227520 | 027b | 5416-5460 |
| mel | AF376746 | 028 | 2829-2877 |
| msr(SA) | AB013298 | 031c | 1530-1582 |
| 138 | 513-574 | ||
| msr(A) | AF167161 | 142 | 4228-4293 |
| msr(A) | X52085 | 141 | 471-536 |
| lmr(A) | X59926 | 075 | 318-352 |
| 076 | 1208-1244 | ||
| car(A) | M80346 | 100 | 424-462 |
| lmr(C) | X79146 | 073 | 33240-33206 |
| mac(A) | AE016758 | 114 | 72526-72565 |
| mac(A) | AE009478 | 115 | 3649-3690 |
| 116 | 4344-4383 | ||
| mac(A) | AE016866 | 117 | 46881-46920 |
| 118 | 47318-47357 | ||
| mac(B) | AB071146 | 119 | 1615-1656 |
| 120 | 835-877 | ||
| mac(B) | AE016866 | 125 | 48789-48828 |
| 126 | 49664-49704 | ||
| mre(A) | U92073 | 103 | 304-349 |
| 104 | 696-741 | ||
| ole(C)-orf5 | AL939112 | 057 | 2835-2871 |
| 058 | 2486-2520 | ||
| tlr(C) | M57437 | 056 | 277-311 |
| var(M) | AB035547 | 054 | 2690-2724 |
| 055 | 2840-2874 | ||
| vga(A) | M90056 | 040 | 1712-1764 |
| 041 | 1637-1693 | ||
| vga(A)v | AF186237 | 039 | 5242-5293 |
| vga(B) | U82085 | 036 | 1547-1604 |
| 037 | 1943-2006 | ||
| lsa | AE016955 | 130 | 196532-196585 |
Microarray hybridization.
Microarray slides were tested by hybridizing DNA templates extracted from 20 strains belonging to eight different species and known to harbor 29 different MLS genes (Table 1). All of the probes designed to be specific for the 29 MLS genes reacted with the predicted DNA templates, allowing validation of a total of 48 probes (Table 5). Three unexpected results were also obtained: (i) the DNA of Bacteroides fragilis V503 reacted with probe msrSA-31, (ii) the DNA of Staphylococcus aureus BM12235 reacted with probe vgaAv-39, and (iii) the DNA of S. aureus BM4611 reacted with probes ermC-19 and ermC-20 (Table 5).
TABLE 5.
Hybridization resultsa
| Organism | Strain (plasmid[s]) | Positive probe(s)b |
|---|---|---|
| Bacteroides fragilis | V503 (pVA503) | ermFU-91, ermFU-92, msrSA-31* |
| Escherichia coli | BM8463 (pIP1810) | vgaAv-39, macA-114, macB-119, macB-120 |
| DH5α (pTZ3519) | mphA-143, mphA-144, macA-114, macB-119, macB-120 | |
| HB101 (pVA891) | ermB-17, ermB-123, macA-114, macB-119, macB-120 | |
| DB10 (pAT421) | vatD-46, vatD-47, mphA-143, mphA-144, macA-114, macB-119, macB-120 | |
| BM2506 (pTZ3721 and pTZ3723) | mphB-63, mphB-64, ermB-17, ermB-123, macA-114, macB-119, macB-120 | |
| BM2570 (pIP1527) | ereB-22, ermB-17, ermB-123, macA-114, macB-119, macB-120 | |
| Enterococcus faecalis | JH2-2 (pAMβ1) | ermB-17, ermB-123, lsa-130 |
| JH2-2 (pAM401) | ermB-17, ermB-123, lsa-130 | |
| Enterococcus faecium | A41 | vatE3-42, vatE-45, ermB-17, ermB-123 |
| UW1965 | vatE-44, vetE-45, ermB-17, ermB-123 | |
| Staphylococcus aureus | BM12235 (pIP1633) | vgaB-36, vgaB-37, vatB-50, vatB-51, vgaAv-39* |
| BM12392 (pIP1714) | vgbB-34, vgbB-35, vatC-48, vatC-49 | |
| BM3093 (pIP680) | vgaA-40, vgaA-41, vatA-52, vatA-53, vgbA-127, vgbA-128 | |
| N315 | ermA-13, ermA-14 | |
| BM4611 | lnuA-69, ermC-19*, ermC-20* | |
| Staphylococcus haemolyticus | BM4610 (pIP855) | lnuA-72 |
| Streptococcus pyogenes | A200 | ermTR-15, ermTR-16 |
| Streptococcus pneumoniae | MF4 | mefA-8, mefA-10, mefE-1, msrD-27 |
| PN150 | mefE-12, mefE-1, mel-28, msrD-27 |
Identification of additional MLS genes in control strains.
Microarray data indicating the presence of unexpected MLS genes in control strains were confirmed by DNA sequencing of the entire open reading frame, using templates obtained by PCR, as previously described (23). In B. fragilis strain V503, carrying the methylase gene erm(FU), sequence data indicated the concomitant presence of an efflux gene identical to msr(SA) (100% identity at the DNA level) of S. aureus (GenBank accession no. AB013298). The msr(SA) gene is considered typical of Staphylococcus spp. and has never been found in gram-negative bacteria. In S. aureus strain BM12235, carrying the major facilitator streptogramin efflux gene vga(B) and the streptogramin acetyltransferase gene vat(B), it was possible to identify also the presence of vga(A)v, an ATP-binding transporter gene which is commonly associated with vga(B) and vat(B) (11, 12). DNA sequence analysis showed that vga(A)v of BM12235 was essentially identical (99% identity at the DNA level) to vga(A)v of S. aureus BM3327 (GenBank accession no. AF186237). In S. aureus strain BM4611, carrying the lincomycin nucleotidyltranferase gene lnu(A), an associated methylase gene of the erm(C) class was found, with up to 90% identity at the nucleotide level with several erm(C) genes present in GenBank.
Conclusions.
This work provides detailed information for construction of a simple and powerful tool to investigate the genetic basis of macrolide resistance in bacterial isolates. Careful analysis of DNA sequences deposited in public databases allowed compilation of a list of 65 bacterial genes encoding resistance to macrolides and related drugs. Oligonucleotide DNA microarrays designed to detect these 65 genes in bacterial genomes were produced and used to test a collection of strains carrying well-characterized MLS genes. Results provided both (i) validation of the microarray chip and (ii) proof of concept that the microarray approach is effective in detecting associations of MLS genes not necessarily inferred by the resistance phenotype. Unlike other DNA microarrays developed to detect the most common resistance genes (8, 21), this one, by its comprehensive approach, is well-suited for surveillance studies specific for MLS resistance, where characterization of the resistance genotype is sought. This DNA microarray could significantly contribute to molecular epidemiology studies by allowing simultaneous testing for the presence of known MLS genes and in particular could help to define and understand the clustering of different MLS genes in genetic elements and genomes.
Acknowledgments
We thank Annalisa Pantosti for advice and all researchers listed in Table 1 for kindly providing control strains.
The work was funded in part by grants from Istituto Superiore di Sanità, from the University of Siena (PAR), and from MIUR (FIRB, RBAU01X9TB).
REFERENCES
- 1.Allignet, J., and N. El Solh. 1997. Characterization of a new staphylococcal gene, vgaB, encoding a putative ABC transporter conferring resistance to streptogramin A and related compounds. Gene 202:133-138. [DOI] [PubMed] [Google Scholar]
- 2.Allignet, J., N. Liassine, and N. El Solh. 1998. Characterization of a staphylococcal plasmid related to pUB110 and carrying two novel genes, vatC and vgbB, encoding resistance to streptogramins A and B and similar antibiotics. Antimicrob. Agents Chemother. 42:1794-1798. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Arthur, M., D. Autissier, and P. Courvalin. 1986. Analysis of the nucleotide sequence of the ereB gene encoding the erythromycin esterase type II. Nucleic Acids Res. 14:4987-4999. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1989. Current protocols in molecular biology, p. 12.0.1-12.2.10. Wiley Interscience, Hoboken, N.J.
- 5.Brisson-Noel, A., and P. Courvalin. 1986. Nucleotide sequence of gene linA encoding resistance to lincosamides in Staphylococcus haemolyticus. Gene 43:247-253. [DOI] [PubMed] [Google Scholar]
- 6.Brisson-Noel, A., P. Delrieu, D. Samain, and P. Courvalin. 1988. Inactivation of lincosaminide antibiotics in Staphylococcus. Identification of lincosaminide O-nucleotidyltransferases and comparison of the corresponding resistance genes. J. Biol. Chem. 263:15880-15887. [PubMed] [Google Scholar]
- 7.Call, D. R., M. K. Bakko, M. J. Krug, and M. C. Roberts. 2003. Identifying antimicrobial resistance genes with DNA microarrays. Antimicrob. Agents Chemother. 47:3290-3295. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Call, D. R., M. K. Borucki, and F. J. Loge. 2003. Detection of bacterial pathogens in environmental samples using DNA microarrays. J. Microbiol. Methods 53:235-243. [DOI] [PubMed] [Google Scholar]
- 9.Del Grosso, M., F. Iannelli, C. Messina, M. Santagati, N. Petrosillo, S. Stefani, G. Pozzi, and A. Pantosti. 2002. Macrolide efflux genes mef(A) and mef(E) are carried by different genetic elements in Streptococcus pneumoniae. J. Clin. Microbiol. 40:774-778. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Halula, M. C., S. Manning, and F. L. Macrina. 1991. Nucleotide sequence of ermFU, a macrolide-lincosamide-streptogramin (MLS) resistance gene encoding an RNA methylase from the conjugal element of Bacteroides fragilis V503. Nucleic Acids Res. 19:3453. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Haroche, J., J. Allignet, and N. El Solh. 2002. Tn5406, a new staphylococcal transposon conferring resistance to streptogramin A and related compounds including dalfopristin. Antimicrob. Agents Chemother. 46:2337-2343. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Haroche, J., A. Morvan, M. Davi, J. Allignet, F. Bimet, and N. El Solh. 2003. Clonal diversity among streptogramin A-resistant Staphylococcus aureus isolates collected in French hospitals. J. Clin. Microbiol. 41:586-591. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Katayama, J., H. Okada, K. O'Hara, and N. Noguchi. 1998. Isolation and characterization of two plasmids that mediate macrolide resistance in Escherichia coli: transferability and molecular properties. Biol. Pharm. Bull. 21:326-329. [DOI] [PubMed] [Google Scholar]
- 14.Kuroda, M., T. Ohta, I. Uchiyama, T. Baba, H. Yusawa, I. Kobayashi, L. Cui, A. Oguchi, K. Aoki, Y. Nagai, J. Lian, T. Ito, M. Kanamori, H. Matsumaru, A. Maruyama, H. Murakami, A. Hosoyama, Y. Mizutani-Ui, N. K. Takahashi, T. Sawano, R. Inoue, C. Kaito, K. Sekimizu, H. Hirakawa, S. Kuhara, S. Goto, J. Yabuzaki, M. Kanehisa, A. Yamashita, K. Oshima, K. Turuya, C. Yoshino, T. Shiba, M. Hattori, N. Ogasawara, H. Hayashi, and K. Hiramatsu. 2001. Whole genome sequencing of methicillin-resistant Staphylococcus aureus. Lancet 357:1225-1240. [DOI] [PubMed] [Google Scholar]
- 15.Lonks, J. R., J. Garau, and A. A. Medeiros. 2002. Implications of antimicrobial resistance in the empirical treatment of community-acquired respiratory tract infections: the case of macrolides. J. Antimicrob. Chemother. 50:87-92. [DOI] [PubMed] [Google Scholar]
- 16.Lynch, J. P., and R. Martinez. 2003. Clinical relevance of macrolide-resistant Streptococcus pneumoniae for community-acquired pneumonia. Clin. Infect. Dis. 34:S27-S46. [DOI] [PubMed] [Google Scholar]
- 17.Macrina, F. L., R. P. Evans, J. A. Tobian, D. L. Hartley, D. B. Clewell, and K. R. Jones. 1983. Novel shuttle plasmid vehicles for Escherichia-Streptococcus transgenic cloning. Gene 25:145-150. [DOI] [PubMed] [Google Scholar]
- 18.Martin, B., G. Alloing, V. Mejean, and J. P. Claverys. 1987. Constitutive expression of erythromycin resistance mediated by the ermAM determinant of plasmid pAMβ1 results from deletion of 5′ leader peptide sequences. Plasmid 18:250-253. [DOI] [PubMed] [Google Scholar]
- 19.Moellering, R. C., Jr., W. Craig, M. Edmond, D. J. Farrell, M. J. Ferraro, T. M. File, Jr., J. Klein, J. Lonks, J. P. Metlay, D. Sahm, and G. H. Talbot. 2002. Clinical and public health implications of macrolide-resistant Streptococcus pneumoniae. J. Chemother. 14:42-56. [DOI] [PubMed] [Google Scholar]
- 20.Noguchi, N., A. Emura, H. Matsuyama, K. O'Hara, M. Sasatsu, and M. Kono. 1995. Nucleotide sequence and characterization of erythromycin resistance determinant that encodes macrolide 2′-phosphotransferase I in Escherichia coli. Antimicrob. Agents Chemother. 39:2359-2363. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Perreten, V., L. Vorlet-Fawer, P. Slickers, R. Ehricht, P. Kuhnert, and J. Frey. 2005. Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria. J. Clin. Microbiol. 43:2291-2302. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.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]
- 23.Santagati, M., F. Iannelli, M. R. Oggioni, S. Stefani, and G. Pozzi. 2000. Characterization of a genetic element carrying the macrolide efflux gene mef(A) in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 44:2585-2587. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Seoane, A., and J. M. Garcia Lobo. 2000. Identification of a streptogramin A acetyltransferase gene in the chromosome of Yersinia enterocolitica. Antimicrob. Agents Chemother. 44:905-909. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Seppälä, H., M. Skurnik, H. Soini, M. C. Roberts, and P. Huovinen. 1998. A novel erythromycin resistance methylase gene (ermTR) in Streptococcus pyogenes. Antimicrob. Agents Chemother. 42:257-262. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Soltani, M., D. Beighton, J. Philpott-Howard, and N. Woodford. 2001. Identification of vat(E-3), a novel gene encoding resistance to quinupristin-dalfopristin in a strain of Enterococcus faecium from a hospital patient in the United Kingdom. Antimicrob. Agents Chemother. 45:645-646. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Werner, G., and W. Witte. 1999. Characterization of a new enterococcal gene, satG, encoding a putative acetyltransferase conferring resistance to streptogramin A compounds. Antimicrob. Agents Chemother. 43:1913-1914. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Wirth, R., F. Y. An, and D. B. Clewell. 1986. Highly efficient protoplast transformation system for Streptococcus faecalis and a new Escherichia coli-S. faecalis shuttle vector. J. Bacteriol. 165:831-836. [DOI] [PMC free article] [PubMed] [Google Scholar]