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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, May 1998, p. 1229-1232, Vol. 42, No. 5
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
A New High-Level Gentamicin Resistance Gene,
aph(2")-Id, in Enterococcus spp.
Shane F.
Tsai,1
Marcus J.
Zervos,2
Don B.
Clewell,3
Susan M.
Donabedian,2
Daniel F.
Sahm,4,
and
Joseph W.
Chow1,5,*
Research and Medical Service, Department of
Veterans' Affairs Medical Center,1 and
Wayne State University School of
Medicine,5 Detroit, Michigan 48201;
William Beaumont Hospital, Royal Oak, Michigan
480732;
Departments of Biologic and
Materials Sciences and Microbiology and Immunology, University of
Michigan, Ann Arbor, Michigan 481093; and
Department of Pathology, Jewish Hospital at Washington
University Medical Center, St. Louis, Missouri 631104
Received 5 August 1997/Returned for modification 3 December
1997/Accepted 18 February 1998
 |
ABSTRACT |
Enterococcus casseliflavus UC73 is a clinical blood
isolate with high-level resistance to gentamicin. DNA
preparations from UC73 failed to hybridize with intragenic probes
for aac(6')-Ie-aph(2")-Ia and aph(2")-Ic. A
4-kb fragment from UC73 was cloned and found to confer resistance to
gentamicin in Escherichia coli DH5
transformants. Nucleotide sequence analysis revealed the presence
of a 906-bp open reading frame whose deduced amino acid sequence had a
region with homology to the aminoglycoside-modifying enzyme APH(2")-Ic and to the C-terminal domain of the bifunctional enzyme
AAC(6')-APH(2"). The gene is designated aph(2")-Id, and its
observed phosphotransferase activity is designated APH(2")-Id. A
PCR-generated intragenic probe hybridized to the genomic DNA from 17 of
118 enterococcal clinical isolates (108 with high-level gentamicin
resistance) from five hospitals. All 17 were vancomycin-resistant
Enterococcus faecium isolates, and pulsed-field typing
revealed three distinct clones. The combination of
ampicillin plus either amikacin or neomycin exhibited synergistic
killing against E. casseliflavus UC73. Screening and
interpretation of high-level aminoglycoside resistance in enterococci
may need to be modified to include detection of APH(2")-Id.
| |
INTRODUCTION |
High-level gentamicin resistance
(MIC 
2,000 µg/ml) in enterococci is known to be associated
with the aac(6')-Ie-aph(2")-Ia gene, which encodes
the bifunctional aminoglycoside-modifying enzyme AAC(6')-APH(2")
(14). The presence of this gene eliminates the
synergism between a cell wall-active agent, such as ampicillin or
vancomycin, and virtually all commercially available
aminoglycosides
including gentamicin, tobramycin, netilmicin,
kanamycin, and amikacinexcept streptomycin (17).
aph(2")-Ic is a midlevel gentamicin resistance gene
(MIC = 256 µg/ml), found less commonly than
aac(6')-Ie-aph(2")-Ia in enterococci, that eliminates the
synergism between ampicillin and gentamicin (6). We describe
a new high-level gentamicin resistance gene initially found in
Enterococcus casseliflavus that is distinct from
aac(6')-Ie-aph(2")-Ia and aph(2")-Ic.
(This work was presented in part at the Infectious Diseases Society of
America 34th Annual Meeting, New Orleans, La., 18 to 20 September 1996 [25], and the 97th General Meeting of the American
Society for Microbiology, Miami Beach, Fla., 4 to 8 May 1997 [26].)
| |
MATERIALS AND METHODS |
Bacterial strains, media, and antimicrobial
susceptibilities.
Enterococci were identified by conventional
biochemical criteria (13). UC73 is an E. casseliflavus blood isolate from a patient in Chicago, Ill.
(20). Escherichia coli DH5 was used as the
recipient for electroporation and the host to maintain recombinant
plasmids. Enterococcus faecium GE1 (12) and
Enterococcus faecalis FA2-2 (7) were the
recipient strains in mating experiments. Gentamicin was obtained from
Fluka (Buchs, Switzerland). Netilmicin, 6'-N-ethylnetilmicin, and 5-episisomicin were a gift from
Karen J. Shaw (Schering-Plough Research Institute, Kenilworth, N.J.). All other antimicrobial agents were obtained from Sigma Chemical Company (St. Louis, Mo.). Transformants from electroporation were selected on Luria-Bertani plates containing gentamicin (20 µg/ml). Filter matings were performed as previously described (23). Antimicrobial susceptibilities were determined by a standardized broth
microdilution method (28). Tests of synergistic killing were
performed at least in triplicate to ensure reproducibility of results
and were done by previously described methods (19). Synergism was defined as a 2-log10 decrease in the number
of CFU per milliliter between the combination (ampicillin plus
aminoglycoside) and its most active constituent (ampicillin) after
24 h (the aminoglycoside had no effect on the growth curve); the
number of surviving organisms in the presence of the combination was
2-log10 CFU/ml below that in the starting inoculum. One
hundred eighteen enterococcal clinical isolates (108 with high-level
gentamicin resistance) from five Detroit, Mich., area hospitals were
obtained to screen for the presence of the new gene.
DNA preparation and cloning.
Plasmid DNA minipreparations
and total genomic DNA were obtained by a modified alkaline lysis method
(27). Restriction endonuclease digestion, agarose gel
electrophoresis of DNA, contour-clamped homogeneous electric field
(CHEF) electrophoresis of genomic DNA, and electroporation were
performed as previously described (3, 10). DNA typing of
isolates was done by visual inspection of gel bands by using published
criteria (22). For detection of DNA-DNA homology,
biotin-labeled probes were prepared as instructed by the manufacturer
(GIBCO BRL Life Technologies, Gaithersburg, Md.). The probe for
aac(6')-Ie-aph(2")-Ia was a 1.5-kb AluI fragment from E. faecalis plasmid pSF815A (14). The
probe for aph(2")-Ic was generated by PCR as previously
reported (6). DNA was transferred to MagnaGraph nylon
membranes (Micron Separation, Inc., Westboro, Mass.) by the method of
Southern and exposed to probes for hybridization (11). DNA
to be sequenced was obtained as described in the Qiagen plasmid
handbook (Qiagen, Inc., Chatsworth, Calif.). The vector pBluescript II
KS+ (Stratagene Cloning Systems, La Jolla, Calif.) was used in standard
cloning experiments (3).
DNA sequencing and PCR.
Nested deletions of cloned DNA were
made by using the Erase-a-Base System from Promega (Madison, Wis.). The
nucleotide sequences of both strands were determined by a modification
of the dideoxynucleotide chain termination method
with a Sequenase kit (version 1.0; United States Biochemical,
Cleveland, Ohio) and [-32P]dATP (Amersham Life
Science, Arlington Heights, Ill.) (15, 21). PCR was
performed with a GeneAmp PCR Reagent kit with AmpliTaq DNA polymerase
(Perkin-Elmer, Norwalk, Conn.) (15). Computer analysis was
performed by using MacVector software, version 6.0, and AssemblyLIGN,
version 1.0 (Oxford Molecular Group, Oxford, United Kingdom). The
GenBank database was searched by using the BLAST program from the
National Center for Biotechnology Information (1). Amino
acid sequences were compared by using the Gap Analysis Program from the
University of Wisconsin Genetics Computer Group, version 8.1 (9).
Enzyme assays.
Aminoglycoside phosphotransferase activity
was confirmed through a modified phosphocellulose paper binding assay
as previously described (6, 18). A substrate was considered
to be modified if its radioactive counts were greater than five times
those of negative controls (8).
Nucleotide sequence accession number.
The nucleotide
sequence data for the new high-level gentamicin resistance gene,
aph(2")-Id, are available from GenBank under accession no.
AF016483.
| |
RESULTS |
Microbiological characterization.
Aminoglycoside MICs for
E. casseliflavus UC73 are shown in Table
1. The ampicillin MIC was 1.0 µg/ml,
and the streptomycin MIC was 32 µg/ml. Ampicillin at 0.5 µg/ml was
used in synergy studies of UC73 because ampicillin alone at 1.0 µg/ml
exhibited too much killing at 24 h to easily detect
synergistic killing. Synergistic killing was not seen
for UC73 in time-kill studies using ampicillin (0.5 µg/ml) and
either gentamicin (16 µg/ml) or netilmicin (16 µg/ml). Synergistic
killing was exhibited against UC73, however, when ampicillin (0.5 µg/ml) was combined with amikacin (8 and 16 µg/ml) or with neomycin
(8 and 16 µg/ml).
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|
TABLE 1.
Susceptibilities of E. casseliflavus
UC73, E. coli NC95, and E. coli
DH5(pBluescript II KS+) to aminoglycosides
|
|
The probes for aac(6')-Ie-aph(2")-Ia and
aph(2")-Ic did not hybridize to Southern blots of total
cellular DNA from E. casseliflavus UC73. Filter matings
with UC73 as the donor and E. faecium GE1 or
E. faecalis FA2-2 as the recipient did not result in
transfer of gentamicin resistance (frequency, <10
9 per
recipient CFU). Electroporation of a plasmid preparation from UC73 into
competent E. faecalis FA2-2 cells also did not result
in the selection of gentamicin-resistant transformants.
Cloning and expression of the gentamicin resistance gene.
Partial Sau3AI digestions of total genomic DNA from UC73
were ligated to pBluescript II KS+ digested with BamHI.
After electroporation of the ligated products, selection for
gentamicin-resistant transformants yielded an E. coli
DH5 derivative that contained a 7.5-kb cloned fragment. Further
subcloning resulted in a gentamicin-resistant transformant, named NC95,
which contained a 4-kb cloned fragment. Aminoglycoside MICs for NC95
and DH5(pBluescript II KS+) are shown in Table 1.
Nucleotide sequencing of the gentamicin resistance
gene.
Nucleotide sequencing revealed the presence
of only one open reading frame (ORF) whose predicted amino acid
sequence showed homology with aminoglycoside-modifying enzymes
(Fig. 1). Only recombinants that
contained the entire ORF (906 bp), and not recombinants that contained
a part of the ORF, were gentamicin resistant. Gap analysis revealed
54.4% similarity and 31.1% identity of APH(2")-Id with
AAC(6')-APH(2") and 48.6% similarity and 28% identity with APH(2")-Ic. This gentamicin resistance gene was designated
aph(2")-Id.
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FIG. 1.
Nucleotide sequence of aph(2")-Id from
E. casseliflavus UC73. The potential  35 and 10
sites and the potential ribosome binding site (RBS) are underlined. The
stop codon of the ORF is designated by an asterisk. Inverted repeats
(IR) (two mismatched nucleotides) are designated by arrows.
|
|
Determination of phosphotransferase activity.
The crude
extract prepared from negative-control E. coli
DH5(pBluescript II KS+) showed no phosphorylation of
gentamicin over time (86, 95, and 95 cpm at 1, 5, and 15 min,
respectively), whereas the reaction mixture containing extract from
NC95 showed a significant increase in radioactivity observed over time
(2,795, 6,215, and 6,415 cpm at 1, 5, and 15 min, respectively), thus showing that gentamicin phosphotransferase activity, designated APH(2")-Id, is associated with the presence of aph(2")-Id.
Identification of the new gentamicin resistance gene in
E. faecium.
An 849-bp intragenic probe for
aph(2")-Id was generated by PCR with synthetic
oligonucleotide primers (5'-GACCAGGTAGAAAAGGCAATAGAGCAG-3' and 5'-ATACCAATCCATATAACCATATTCCTT-3'). The probe
hybridized to the Southern blots of total cellular DNA from 17 of 118 enterococcal clinical isolates. These 118 isolates were composed of 108 with high-level resistance to gentamicin (78 E. faecalis isolates, 28 E. faecium isolates, 1 E. raffinosus isolate, and 1 E. gallinarum isolate) and 10 that were gentamicin sensitive (1 E. faecalis isolate and 9 E. faecium isolates). Forty
of the 118 isolates were vancomycin resistant (6 E. faecalis isolates, 33 E. faecium isolates, and 1 E. gallinarum isolate). All 17 isolates positive for
the aph(2")-Id probe were vancomycin-resistant E. faecium and came from 16 patients in four of the five hospitals.
The DNA from these 17 E. faecium isolates did not
hybridize to the aac(6')-Ie-aph(2")-Ia probe. CHEF gel
electrophoresis showed three distinct strain types (data not shown).
The probe also hybridized to the DNA on the CHEF gel from all 17 isolates. The 17 E. faecium isolates were ampicillin resistant (MIC range, 64 to 256 µg/ml). The amikacin MICs
ranged from 256 to 512 µg/ml, and the netilmicin MICs ranged from 512 to 2,000 µg/ml. In contrast to results for E. casseliflavus UC73, tests of synergism performed on the two
isolates (NC103 and SF13485) for which the ampicillin MICs were the
lowest (64 µg/ml), in which ampicillin was used at 64 µg/ml and
amikacin was used at 32 µg/ml, showed less than a 2-log10
difference in killing with the combination compared to the most active
agent alone (average 1.34- and 0.65-log10 difference,
respectively).
| |
DISCUSSION |
To date, high-level gentamicin resistance in enterococci has
been associated only with the presence of the
aac(6')-Ie-aph(2")-Ia gene. Therefore, clinical
laboratories test enterococcal isolates for high-level
aminoglycoside resistance by using only gentamicin and
streptomycin, since enterococci resistant to gentamicin are assumed to
be resistant to the other clinically available aminoglycosides, except
streptomycin. Our small survey showed that 16% (17 of 108) of
enterococci with high-level gentamicin resistance possess
aph(2")-Id and not aac(6')-Ie-aph(2")-Ia. If
these results are confirmed in more-extensive surveys, clinical
laboratories may need to add amikacin to their screening protocols,
since aph(2")-Id does not confer high-level resistance to
amikacin and ampicillin-amikacin synergistic killing of E. casseliflavus UC73, which contains aph(2")-Id, has been
shown. E. faecium NC103 and E. faecium
SF13485 both contain aph(2")-Id, but the ampicillin and
amikacin MICs for these strains were high, which may explain why they
were not killed as effectively by ampicillin-amikacin as E. casseliflavus UC73 was. The high amikacin MICs (256 to 512 µg/ml) for the 17 E. faecium isolates containing
aph(2")-Id may be due to the presence of another
aminoglycoside resistance gene(s). Although an enterococcus for which
the amikacin MIC is <256 µg/ml might contain aph(2")-Id
and be susceptible to killing by ampicillin-amikacin, a low amikacin
MIC does not always imply susceptibility to synergism. Enterococci that
possess the aminoglycoside resistance gene aph(3')-IIIa,
aac(6')-Ie-aph(2")-Ia, or ant(4')-Ia
are resistant to ampicillin-amikacin synergism but may not have
high-level resistance to amikacin (MICs as low as 64 to 256 µg/ml)
(2, 4, 5, 16, 17, 24). Tests for synergistic killing may
thus prove to be a useful adjunct to confirm the utility of amikacin in
combination therapy for isolates thought to contain
aph(2")-Id. Alternatives might include the use of probes or
PCR to test for the presence of aph(2")-Id and for the
absence of the other three aminoglycoside resistance genes.
| |
ACKNOWLEDGMENTS |
This study was supported in part by the Department of Veterans'
Affairs; in part by the William Beaumont Hospital Research Institute;
and in part by the General Clinical Research Center at the University
of Michigan, funded by a grant (MO1RR00042) from the National Center
for Research Resources, NIH, USPHS.
We thank Karen J. Shaw and Stephen A. Lerner for helpful discussions
and Deborah D. Jaworski for assistance with computer analysis.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Infectious Diseases, Wayne State University School of Medicine, 4160 John R, Suite 2140, Detroit, MI 48201-2021. Phone: (313) 745-9649. Fax:
(313) 763-9905. E-mail: waichung{at}umich.edu.
Present address: MRL Pharmaceutical Services, Inc., Reston, VA
20190.
| |
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Antimicrobial Agents and Chemotherapy, May 1998, p. 1229-1232, Vol. 42, No. 5
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
