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Antimicrobial Agents and Chemotherapy, July 1999, p. 1813-1814, Vol. 43, No. 7
Characterization of a New Enterococcal Gene, satG,
Encoding a Putative Acetyltransferase Conferring Resistance to
Streptogramin A Compounds
Streptogramin antibiotics are mixtures of two chemically unrelated
A and B compounds that act synergistically in vivo against gram-positive pathogens, such as staphylococci, streptococci, and
enterococci (8, 11). Resistance against B compounds is very
widespread among enterococci and is mediated via the ermB gene cluster (e.g., on Tn917) that confers
macrolide-lincosamide-streptogramin B resistance (7). The
synergistic mixture of streptogramins A and B overcomes resistance to B
compounds but is inactive in resistance to A compounds.
The only known resistance mechanism against streptogramin A compounds
in enterococci is mediated by the streptogramin acetyltransferase SatA
(9). Enterococcus faecium isolates with
satA-mediated resistance have been found in samples of human
and animal origins, indicating a possible spread of resistance genes or
resistant bacteria among different ecosystems (10).
We isolated a quinupristin-dalfopristin-resistant E. faecium
UW1965 from a sewage treatment plant in Germany. The resistance determinant was transferred to a susceptible recipient, producing the
transconjugant UW1965K1. UW1965K1 is resistant to
quinupristin-dalfopristin (MIC In staphylococci, resistance to streptogramin A compounds is mediated
by two mechanisms: (i) acetylation of the streptogramin A via
acetyltransferases (Vat, VatB, and VatC [1-3]) and
(ii) efflux due to an ABC transporter (Vga and VgaB [4,
5]). PCR amplification for the vat,
vatB, vatC, and vga genes failed to produce
any product. The putative protein sequences of the known streptogramin
acetyltransferases in staphylococci and enterococci contain three
conserved motifs (2). Corresponding primers, satI and satJ,
have been made, producing a 144- to 147-bp fragment for
vat, satA, and vatB (2).
PCR performed with these primers resulted in a ca. 150-bp fragment for
UW1965K1. A digoxigenin-labelled probe of the amplified fragment was
prepared, hybridizing with a 5.5-kbp fragment of
EcoRI-digested plasmid DNA from the transconjugant. The
corresponding plasmid fragment was cloned into pUC18 and sequenced.
The resulting DNA sequence (Fig. 1) did not show significant identity
with other gene sequences from GenBank on the DNA level (6).
One suitable open reading frame (ORF) was found, giving rise to a
putative 214-amino-acid (214-aa) protein. A comparison of amino acid
similarities indicated rather significant homology between
streptogramin acetyltransferases and the new putative acetyltransferase, designated SatG (Fig.
2). Based on the sequence for
satG, two primers, satG1 and satG2, have been designed.
Preliminary results of a search for streptogramin-resistant enterococci
(E. faecium, E. hirae, and E. durans)
revealed the existence of the satG gene in 9 of 23 isolates
from sewage, 6 of 24 isolates from broiler samples, and all 17 isolates
from poultry manure. Of 62 quinupristin-dalfopristin-resistant E. faecium (QDREF) isolates from hospitals in Germany, 9 were
positive for satG. The high number of satG QDREF
isolates from poultry meat and manure may be due to selection of these
bacteria by use of virginiamycin as a feed additive, and spread of the
resistance via the food chain to humans is very likely. This hypothesis
is being investigated.
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FIG. 1.
A 904-bp sequence located on the 5.5-kbp cloned fragment
in pUC18 (GenBank accession no. AF139725). The ORF begins at nucleotide
63 with an ATG start codon preceding a putative ribosomal binding site
(RBS) (double-underlined) at positions 50 to 57. The predicted gene
sequence encodes a protein of 214 aa which shows significant homology
with other streptogramin acetyltransferases (aa motifs I, II, III; see
also Fig. 2). The locations of the primers satG1 and satG2, specific
only for the satG sequence, are underlined (plus strand).
16 µg/ml) and virginiamycin M
(A compound; MIC, 16 µg/ml), whereas the MIC of each antibiotic for
the recipient was 1 µg/ml. PCR amplification for the satA
gene was negative.
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FIG. 2.
Alignment of amino acid sequences of acetyltransferases
from staphylococci and enterococci (1-3, 9) conferring
resistance to streptogramin A antibiotics. Identical residues are
indicated by asterisks. Highly conserved regions in different
streptogramin A acetyltransferases 
motifs I, II, and IIIare
boldfaced. Primers satI and satJ have been designed on the basis of the
corresponding nucleotide sequences in motifs II and III
(2).
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G. Werner W. Witte Robert Koch Institute Wernigerode Branch Burgstrasse 37 D-38855 Wernigerode Germany |
Antimicrobial Agents and Chemotherapy, July 1999, p. 1813-1814, Vol. 43, No. 7
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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