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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, January 1999, p. 157-160, Vol. 43, No. 1
Hospital Infections Program, National Center
for Infectious Diseases, Centers for Disease Control and Prevention,
Atlanta, Georgia 303331;
Department of
Medical Microbiology, School of Medicine, University of Tromsø,
Tromsø, Norway2; and
Department of
Pathology and Laboratory Medicine, University of Wisconsin Hospital
and Clinics, Madison, Wisconsin 537923
Received 16 April 1998/Returned for modification 15 July
1998/Accepted 27 October 1998
Genes encoding streptomycin/spectinomycin adenylyltransferases
[ANT(3")(9)] have been reported to exist in gram-negative organisms and Staphylococcus aureus. During a study of high-level
aminoglycoside resistance in enterococci, we encountered an isolate of
Enterococcus faecalis that was streptomycin resistant but
did not appear to contain the 6'-adenylyltransferase gene
(aadE) when examined by PCR with specific primers.
Phosphocellulose paper binding assays indicated the presence of an
ANT(3")(9) enzyme. Streptomycin and spectinomycin MICs of 4,000 and
8,000 µg/ml, respectively, were observed for the isolate. PCR primers
corresponding to a highly conserved region of the aadA gene
were used to amplify a specific 284-bp product. The product hybridized
with a digoxigenin-labeled PCR product from E. coli
C600(pHP45 Streptomycin continues to be an
important drug for synergistic therapy of serious enterococcal
infections (10). Resistance to streptomycin can be mediated
by ribosomal mutation or by enzymatic modification of the drug.
Ribosomal resistance is usually associated with streptomycin MICs of
>32,000 µg/ml (11), while enzyme-mediated resistance
produces lower MICs. The aadA gene, which encodes resistance to streptomycin and spectinomycin via an adenylyltransferase
[ANT(3")(9)] enzyme, has been reported to exist in gram-negative
organisms (3, 6, 15, 16, 33) and Staphylococcus
aureus (7). Although there have been reports of
streptomycin adenylyltransferases in enterococci (6, 8, 18),
none of the isolates reported were resistant to spectinomycin,
suggesting a mechanism other than one involving the ANT(3")(9) enzyme.
In large surveys of clinical isolates, neither Leclercq et al. nor
Ounissi et al. reported the identification of an ANT(3")(9) in
enterococci (20, 25). However, a clinical isolate of
Enterococcus faecalis exhibiting high-level resistance to
streptomycin and spectinomycin (MICs of The goal of this study was to characterize the mechanism of
streptomycin resistance in this isolate.
Bacterial isolates.
The E. faecalis strain
used in this study (W4770) was originally isolated from a patient at
the University of Wisconsin Hospital, Madison, Wis., and was sent to
the Centers for Disease Control and Prevention, where it was confirmed
to be E. faecalis by the performance of biochemical
tests described by Facklam and Collins (12). Strains were
grown on Trypticase soy agar with 5% sheep blood (Becton Dickinson
Microbiology Systems, Cockeysville, Md.) at 35°C. The control strains
used throughout the study were as follows: rifampin-resistant
(Rifr) and fusidic acid-resistant (Fusr)
E. faecalis JH2-2 (plasmid free) (17);
streptomycin-resistant (Strr), spectinomycin-resistant
(Spr), and ampicillin-resistant Escherichia coli
C600(pHP45 Antimicrobial susceptibility testing.
All isolates were
tested by the broth microdilution method as described by the
National Committee for Clinical Laboratory Standards, using
cation-adjusted Mueller-Hinton broth (Difco Laboratories, Detroit,
Mich.) (23). The antimicrobials tested were ampicillin, chloramphenicol, ciprofloxacin, gentamicin, imipenem, penicillin, quinupristin/dalfopristin, rifampin, spectinomycin, streptomycin, teicoplanin, tetracycline, and vancomycin.
AME assays.
Lysates of bacterial strains were screened for
the presence of aminoglycoside-modifying enzymes (AMEs) by the
phosphocellulose binding assay as described by Cooksey et al.
(4). The only modification of their procedure was that the
wash buffer (10 mM Tris, 1 mM EDTA; pH 7.4) and the suspension buffer
(10 mM Tris, 10 mM MgCl2, 10 mM dithiothreitol; pH 6.8)
used for the enterococci were those described by Chen and Williams
(2).
PCR amplification of resistance genes.
The oligonucleotide
primers chosen for amplification of the 6'-streptomycin
adenylyltransferase (aadE) and the 3"-adenylyltransferase (aadA) genes (Table 1) were
selected from published sequences (13, 16) with the
assistance of OLIGO software, version 4.0 (National Biosciences, Hamel,
Maine). The PCR mixtures and conditions for amplification of the
aac(6')-aph(2") and the aadE genes
have been described previously (32).
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Detection of a Streptomycin/Spectinomycin
Adenylyltransferase Gene (aadA) in Enterococcus
faecalis
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
) known to contain the aadA gene. The
aadA gene was transferred via filter matings from the E. faecalis donor to E. faecalis
JH2-2. PCR primers designed for analysis of integrons were used to
amplify a 1-kb product containing the aadA gene, which was
cloned into the vector pCRII and transformed into Escherichia
coli DH5-
competent cells. D-Rhodamine dye
terminator cycle sequencing was used to determine the gene sequence,
which was compared to previously reported sequences of aadA
genes. We found the aadA gene in E. faecalis to be identical to the aadA genes reported
by Sundström et al. for E. coli plasmid R6-5 (L. Sundström, P. Rådström, G. Swedberg, and O. Sköld,
Mol. Gen. Genet. 213:191-201, 1988), by Fling et al. for the
aadA within transposon Tn7 (M. E. Fling, J. Kopf, and C. Richards, Nucleic Acids Res. 13:7095-7106,
1985), and by Hollingshead and Vapnek for E. coli
R538-1 (S. Hollingshead and D. Vapnek, Plasmid 13:17-30, 1985).
Previous reports of the presence of the aadA gene in
enterococci appear to be erroneous and probably describe an
aadE gene, since the isolates were reported to be
susceptible to spectinomycin.
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
2,000 µg/ml) was recently
recognized in a multilaboratory study of screening methods for
detection of high-level aminoglycoside resistance in enterococci
(32). The isolate did not exhibit ribosomal resistance since
the streptomycin MIC was <32,000 µg/ml, failed to produce an
aadE (also referred to as the ant6'
gene)-specific product when tested by PCR, and did not hybridize with
an aadE-specific gene probe, suggesting an alternate
mechanism of streptomycin resistance.
MATERIALS AND METHODS
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Abstract
Introduction
Materials and methods
Results
Discussion
References
) (aadA) (26); E. faecalis ATCC 49532 [aac(6')-aph(2")]
(32), ATCC 49533 (aadE) (32), and
Strr (ribosomal) E1-R (11); Spr
S. aureus 8325-4 [ant(9')] (24);
E. coli C600(pFCT3103) [ant(2")] (34); and Bacillus subtilis pUBH2
[ant(4')] (9).
TABLE 1.
PCR amplification of aminoglycoside resistance genes
Resistance gene probes.
The probe used for detection of the
aadE gene was that described by Swenson et al.
(32) and consisted of a digoxigenin-labeled 597-bp PCR
product from E. faecalis JH2-2(pJH-1) (17).
For detection of the aadA gene, a 284-bp PCR product from
E. coli plasmid pHP45 (26) was labeled
with digoxigenin by using a Genius kit (Boehringer Mannheim), and
hybridization was performed under stringent conditions at 68°C
according to the manufacturer's recommendations (1).
Transfer of resistance genes. Filter matings were performed by using E. faecalis W4770 as the donor and E. faecalis JH2-2 (17) as the recipient, as previously described (35). Overnight Trypticase soy broth (TSB) cultures of Strr, Spr E. faecalis W4770 (donor) and Rifr, Fusr E. faecalis JH2-2 (recipient) were mixed in a 20:1 ratio and filtered through a 0.22-µm-pore-size Nalgene filter (Nalgene Nunc International, Rochester, N.Y.). The membrane was placed on a brain heart infusion agar (BBL) plate and incubated for 24 h at 37°C. The growth was resuspended in 5 ml of TSB, and transconjugants were selected on brain heart infusion agar containing 50 µg of rifampin, 20 µg of fusidic acid, 50 µg of streptomycin, and 50 µg of spectinomycin/ml.
The transconjugants were examined for the presence of the resistance gene by PCR with specific aadA gene primers. Expression of the gene was demonstrated by determination of streptomycin, spectinomycin, and tetracycline MICs by the broth microdilution method (23).Sequencing of the resistance gene. Using the oligonucleotide primers designed by Lévesque et al. for PCR analysis of integrons (21, 22), we generated a PCR product of approximately 1 kb, using cesium chloride-purified plasmid DNA as the template (27). The 100-µl reaction mixture included 1× PCR buffer II; 2 mM MgCl2; 0.2 mM (each) dATP, dCTP, dGTP, and dTTP; 10 µl of each primer (2.5 pmol/ml stock); and 2.5 U of AmpliTaq Gold DNA polymerase (Perkin-Elmer Applied Biosystems, Foster City, Calif.). The thermocycler was programmed as follows: an initial denaturation for 12 min at 95°C; 30 cycles consisting of a 30-s denaturation step at 94°C, a 30-s annealing at 55°C, and a 30-s extension step at 72°C; a 10-min extension step at 72°C; and incubation at 4°C until the analysis was performed. The products were purified on QIAquick PCR spin columns (Qiagen, Inc., Chatsworth, Calif.), and the DNA was eluted from the columns with 10 mM Tris, pH 9.0.
The integron PCR DNA was ligated into the pCRII vector (TA Cloning Kit; Invitrogen, Carlsbad, Calif.) and transformed into E. coli Library Efficiency DH5- competent cells (Gibco/BRL Life Technologies, Inc., Gaithersburg, Md.). The DH5- competent cells are
Strs, allowing streptomycin to be used for selection of the
transformants; the competent cells normally supplied with the TA
Cloning Kit are resistant. The transformants were inoculated into 5 ml
of TSB containing 100 µg of ampicillin/ml and incubated at 37°C
with shaking for 6 h. The plasmid DNA was purified by using a
Qiagen plasmid minikit and then digested with EcoRI
(Gibco/BRL Life Technologies, Inc.) to check for insertions into the
vector DNA. The purified transformant plasmid DNA from multiple clones
with an insert of approximately 1 kb was used as the template for
the sequencing reaction.
The samples for sequencing were prepared in a 10-µl volume by using
an ABI PRISM dRhodamine terminator cycle sequencing kit with AmpliTaq
DNA polymerase, FS (Perkin-Elmer Applied Biosystems), and 3.2 pmol of
stocks of both the forward and reverse integron and aadA
gene PCR primers. The thermocycler was preheated to 96°C, the program
was aborted, and the samples were added; then the thermocycler was
programmed for 25 cycles of a 10-s denaturing step at 96°C, a 5-s
annealing step at 50°C, a 4-min extension step at 60°C, and a
holding step at 4°C. The dye terminators were removed from the
samples by passing the samples through CentriSep columns (Princeton
Separations, Adelphia, N.J.), and the samples were dried in a SpeedVac
vacuum centrifuge (Savant Instruments, Inc., Holbrook, N.J.) without
heat. The samples were electrophoresed in a 5% Gene Page-Plus
acrylamide gel with 6 M urea in 1× Tris-borate-EDTA buffer (Ultrapure
grade TBE; AMRESCO, Solon, Ohio) on an ABI PRISM model 377 DNA
sequencer (Perkin-Elmer Applied Biosystems).
Nucleotide sequence accession number. The E. faecalis aadA gene sequence has been deposited in the GenBank database under accession no. AF052459.
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RESULTS |
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Abstract
Introduction
Materials and methods
Results
Discussion
References
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Antimicrobial susceptibility testing. E. faecalis W4770 was resistant to streptomycin and spectinomycin (MICs, 4,000 and 8,000 µg/ml, respectively), as well as to tetracycline (MIC, >16 µg/ml). The isolate was susceptible to ampicillin, chloramphenicol, ciprofloxacin, gentamicin, imipenem, penicillin, quinupristin/dalfopristin, rifampin, teicoplanin, and vancomycin.
AME assays.
The isolate and controls were lysed and tested for
AME activity. Only the assays for adenylyltransferase and
phosphotransferase were performed, since streptomycin and spectinomycin
are modified only by those classes of enzymes. W4770 gave a
modification pattern consistent with the production of the ANT(3")(9)
enzyme by modifying streptomycin and spectinomycin but not amikacin,
butirosin, gentamicin, kanamycin, neomycin, paromomycin, or tobramycin,
as did E. coli C600(pPHP45), which is known to
produce ANT(3")(9) enzyme. No phosphotransferase activity was
demonstrated except in the control isolate E. faecalis
ATCC 49532.
PCR amplification of resistance genes and specific gene
probing.
The specific 284-bp aadA gene product was
amplified from both plasmid and total DNA obtained from W4770, and the
specificity of the PCR product was confirmed with the
digoxigenin-labeled product from E. coli
C600(pHP45). Control strains containing the aadA
gene were positive by PCR for the 284-bp product; no product was
demonstrated with strains containing other AME genes [aadE, ant(2"), ant(4"),
ant(9'), aac(6'), or aph(2")]. No
gene amplification was seen in the isolate with the aadE-
and aph(2")-aac(6')-specific primers.
Transfer of resistance genes.
A resistance plasmid
(pNCC801) of approximately 80 kb was transferred by conjugation
from strain W4770 to E. faecalis JH2-2 at a frequency
of ~3 × 10
10. The MICs of streptomycin and
spectinomycin for the transconjugants were 2,000 and 4,000 µg/ml,
respectively; the transconjugants were also resistant to
tetracycline. The 284-bp aadA gene product was
amplified from the transconjugants. The BamHI,
EcoRI, and HindIII restriction profiles for
pNCC801 and the plasmids present in the JH2-2 transconjugants
were identical (data not shown).
Cloning and sequencing of the resistance gene. The 1,009-bp PCR product amplified by integron primers 1 and 2 from E. faecalis W4770 was cloned into pCRII. The sequence from the integron containing the aadA gene is presented in Fig. 1. Our 1,009-bp sequence is identical to that reported by Sundström et al. for E. coli plasmid R6-5 (GenBank accession no. X12870) from bases 1192 to 2201 (30), as well as to bases 5298 to 6306 of the recently assembled sequence of transposon Tn21 (GenBank accession no. AF071413). The 791-bp enterococcal aadA structural gene sequence (bases 107 to 898) was identical to that in plasmid R538-1 in E. coli K-12 (bases 399 to 1190 of GenBank accession no. M10241) and was 99.4% homologous to the sequence reported by Fling et al. (15) for Tn7 (GenBank accession no. X03043, bases 82 to 873), except for the substitutions of an A for a G at position 790 and a C for a T at position 856, neither of which resulted in an amino acid change. However, an AGA insertion in the enterococcal sequence (bases 814 to 816) did result in an additional glutamic acid (E) residue.
|
) containing the aadA gene from
R100.1 (26) is identical to that from W4770.
The 59-bp element sequences containing the hs1 hot spot
(28), located downstream of the aadA gene, were
identical in W4770, Tn7, and pHP45 (Fig. 1). However, the
Tn7 integron sequence upstream of the 5' insertion site
(GTTA) also contains an hs1 hot spot (28). Thus, it differs
from the W4770, pHP45, and R538-1 sequences, which are identical to
each other and contain hs2 hot spots (28) (Fig. 1).
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DISCUSSION |
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Abstract
Introduction
Materials and methods
Results
Discussion
References
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In 1981, Kono et al. reported the presence of ANT(3") activity in an isolate of E. faecalis (18). Similar results were reported by Courvalin et al. in 1978 for the E. faecalis transconjugants of three clinical isolates (8), including strains JH2-14 (derived from Enterococcus faecium JH-7), JH2-15 (derived from E. faecalis JH-1 containing pJH-1), and JH2-12 (derived from E. faecalis JH-6). All of the isolates had high streptomycin MICs of >2,000 µg/ml but were susceptible to spectinomycin (MIC, 64 µg/ml). Courvalin et al. (8) attributed the adenylylation of streptomycin to the ANT(6') (aadE) activity in their strains, since they were susceptible to spectinomycin as is typical for S. aureus strains carrying this determinant (31).
In 1992, Leclercq et al. noted that the AME ANT(3")(9) was produced in
staphylococci but not in enterococci (20). Our report of the
ANT(3")(9) enzyme in enterococci was not surprising since many other
enterococcal genes, e.g., 
-lactamase, ant(4'), and aac(6')-(aph(2"), were originally recognized in
staphylococci (5, 6, 20, 25, 36). The ANT(9) enzyme,
which mediates resistance to spectinomycin only, also appears to
have its origins in staphylococci (19, 20).
Integrons are found either as part of transposons or independently on broad-host-range plasmids, but to our knowledge their presence has only been reported in gram-negative organisms (21, 29). Integrons typically have two conserved segments, 5' and 3', separated by a variable region which includes integrated antimicrobial resistance genes or cassettes. The 5' conserved segment contains the integrase gene and, on the opposite strand, a common promoter region, P1-P2, which is directed toward the site of integration. Most genes inserted into integrons lack their own promoters and are expressed by the use of the common promoter region. The 3' conserved segment contains the genes that determine resistance to quaternary ammonium compounds and sulfonamide, in addition to an open reading frame (21, 29). At the 3' end of each resistance gene cassette is a short inverted-repeat element called the 59-bp element. The 59-bp element, which can vary to a certain degree in sequence and length, is important in the site-specific recombination event that results in the insertion of individual genes.
The integron sequence containing the aadA gene
from E. faecalis W4770 was located on a plasmid
of approximately 80 kb (pNCC801) which was transferable at a low
frequency to E. faecalis JH2-2. The DNA sequences of
the 1,009-bp amplification product from W4770 and the E. coli plasmids R6-5 (30) and pHP45 (26)
were identical. The differences between our sequences and those of
Hollingshead and Vapnek (16) for R538-1 were probably due to
technical factors relating to earlier sequencing methods, as previously
noted by Fling et al. (15). Our aadA sequence was
preceded by an hs2 hot spot and followed by an hs1 hot spot, which
included the characteristic 59-bp element described by Schmidt et al.
(28). Our results suggested that the aadA gene
was the only resistance gene inserted within the integron, since the
PCR product was not of sufficient size to include other cassettes. It
is unclear whether this integron is part of a larger transposon, but
based on the sequence data, the integron does appear to have originated
from an enteric organism.
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ACKNOWLEDGMENTS |
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We thank Kamile Rasheed and Linda Weigel for technical assistance and helpful discussions and members of the Biotechnology Core Facility Branch at the Centers for Disease Control and Prevention for oligonucleotide primer preparation and supportive expertise.
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FOOTNOTES |
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* Corresponding author. Mailing address: Nosocomial Pathogens Laboratory Branch, Centers for Disease Control and Prevention, 1600 Clifton Rd., N.E., Mailstop G08, Atlanta, GA 30333. Phone: (404) 639-0195. Fax: (404) 639-1381. E-mail: ncc1{at}cdc.gov.
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Abstract
Introduction
Materials and methods
Results
Discussion
References
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