Detection of the High-Level Aminoglycoside Resistance Gene aph(2")-Ib in Enterococcus faecium

High-level gentamicin resistance (MIC ≥ 500 μg/ml) in enterococci is predominantly mediated byaac(6′)-Ie-aph(2")-Ia, which encodes the bifunctional aminoglycoside-modifying enzyme AAC(6′)-APH(2") (10). Found less commonly is aph(2")-Id, another gene recently reported to be associated with high-level gentamicin resistance in enterococci (19). The presence of high-level gentamicin resistance in enterococci eliminates the synergistic killing usually seen when gentamicin is combined with a cell wall-active agent, such as ampicillin or vancomycin. aph(2")-Ic is a mid-level gentamicin resistance gene isolated infrequently from enterococci that eliminates the synergism between ampicillin and gentamicin, even though the gentamicin MIC for strains containing this gene is typically only ca. 256 μg/ml (4). We describe the cloning and sequencing of another aminoglycoside resistance gene, aph(2")-Ib, that is associated with high-level gentamicin resistance inEnterococcus faecium.

(This work was presented in part at the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Ontario, Canada, 28 September to 1 October 1997.)

Enterococci were identified by conventional biochemical criteria (9). E. faecium SF11770 is a blood culture isolate from a patient in Detroit, Mich. Escherichia coli DH5α and E. coli DH10b were used as recipients for electroporation and as hosts for maintaining recombinant plasmids.E. faecium GE1 (8) and Enterococcus faecalis FA2-2 (5) were the recipient strains in mating experiments. Gentamicin was obtained from Fluka (Buchs, Switzerland). Arbekacin and netilmicin were provided by Meiji Seika Kaisha (Tokyo, Japan). All other antimicrobial agents were purchased from Sigma Chemical Company (St. Louis, Mo.). Antimicrobial susceptibilities were determined by a standard broth microdilution method (15). Tests of synergistic killing were performed using a previously described method (12), with some modifications. Synergy was defined as a ≥2-log₁₀ decrease in CFU/ml between the combination and its most active constituent after 24 h, with the number of surviving organisms in the presence of the combination being ≥2 log₁₀ CFU/ml below the number of organisms in the starting inoculum. Also, at least one of the drugs must be present in a concentration that does not affect the growth curve of the test organism when used alone. One hundred twenty-one enterococcal clinical isolates (78 E. faecalis, 41 E. faecium, 1Enterococcus raffinosus, and 1 Enterococcus gallinarum isolate) with high-level gentamicin resistance from separate patients in six Detroit, Mich., area hospitals were used to screen for the presence of the new gene. This collection included isolates that were part of clonal outbreaks in the Detroit area and had been previously screened for aph(2")-Id (19).

Plasmid DNA minipreparations and total genomic DNA were obtained by a modified alkaline lysis method (20). Methods for restriction endonuclease digestion, agarose gel electrophoresis of DNA, transformation by using calcium chloride or electroporation, and Southern blot hybridization were performed as previously described (2, 7, 19). The phosphocellulose paper binding assay was performed as previously described (4, 17). Oligonucleotide primers were synthesized by GIBCO BRL Life Technologies (Gaithersburg, Md.). The probes for aac(6′)-Ie-aph(2")-Ia,aph(2")-Ic, and aph(2")-Id were prepared as described previously (4, 10, 19). DNA to be sequenced was obtained as described in the Qiagen (Chatsworth, Calif.) plasmid handbook. The vectors pBluescript II KS(+) (Stratagene Cloning Systems, La Jolla, Calif.) and pMW119 (Nippon Gene Corporation, Toyama, Japan) were used in standard cloning experiments (2). Nucleotide sequencing was performed at the DNA Sequencing Core, University of Michigan, Ann Arbor. Computer analysis was performed with MacVector software, version 6.0, and AssemblyLIGN, version 1.0.9 (Oxford Molecular Group, Oxford, United Kingdom). The GenBank database was searched with 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 (6). The PCR primers used for detecting the aac(6′)-Ie-aph(2")-Ia gene were 5′-GAGCAATAAGGGCATACCAAAAATC-3′ and 5′-CCGTGCATTTGTCTTAAAAAACTGG-3′. The primers for detectingaph(2")-Ib were 5′-TATGGATCCATGGTTAACTTGGACGCTGAG-3′ and 5′-AT TAAGCTTCCTGCTAAAATATAAACATCTCTGCT-3′. The primers for detecting aph(2")-Ic were as previously reported (4). The primers for detecting aph(2")-Id were 5′-GGTGGTTTTTACAGGAATGCCATC-3′ and 5′-CCCTCTTCATACCAATCCATATAACC-3′.

Aminoglycoside MICs for E. faecium SF11770 are shown in Table 1. The ampicillin MIC was 128 μg/ml, the streptomycin MIC was >2,000 μg/ml, and the vancomycin MIC was >256 μg/ml. The probes for theaac(6′)-Ie-aph(2")-Ia, aph(2")-Ic, andaph(2")-Id genes did not hybridize to Southern blots of total cellular DNA from E. faecium SF11770. Filter matings, with SF11770 as the donor and E. faecium GE1 or E. faecalis FA2-2 as the recipient, did not result in the transfer of gentamicin resistance (the frequency was <1 × 10⁻⁹per recipient CFU). Electroporation of a plasmid preparation from SF11770 into competent E. faecalis OG1RF cells also did not result in the selection of gentamicin-resistant transformants. In tests of bactericidal synergistic killing against E. faeciumSF11770, ampicillin at 64 μg/ml combined with arbekacin at 8 or 16 μg/ml produced a 3- to 4-log₁₀ decrease in CFU per milliliter at 24 h compared to the most active agent (ampicillin) (Fig. 1A and B). However, since the growth curve of the organism with either ampicillin or arbekacin alone was slightly different than the growth with no antimicrobial agent, this did not fulfill the strict criteria for synergistic killing. Ampicillin at 64 μg/ml combined with arbekacin at 2 or 4 μg/ml did not produce greater killing at 24 h than either drug alone (Fig.1A and C). The combination of ampicillin (64 μg/ml) and gentamicin (16 μg/ml) also did not produce greater killing at 24 h than either drug alone (Fig. 1D).

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Fig. 1.

Tests of synergistic killing against E. faecium SF11770. ●, no antimicrobial; ○, ampicillin (64 μg/ml); ▾, arbekacin (16 μg/ml); ▿, ampicillin (64 μg/ml) plus arbekacin (16 μg/ml); ■, arbekacin (8 μg/ml); □, ampicillin (64 μg/ml) plus arbekacin (8 μg/ml); ⧫, arbekacin (4 μg/ml); ◊, ampicillin (64 μg/ml) plus arbekacin (4 μg/ml); ▴, arbekacin (2 μg/ml); ▵, ampicillin (64 μg/ml) plus arbekacin (2 μg/ml); ⬢, gentamicin (16 μg/ml); and ⬡, ampicillin (64 μg/ml) plus gentamicin (16 μg/ml).

Partial Sau3AI digestions of total genomic DNA from SF11770 were ligated to pBluescript II KS(+) digested with BamHI and dephosphorylated with calf intestine alkaline phosphatase. After electroporation of the ligated products, selection on Luria-Bertani plates containing 25 μg of gentamicin per ml yielded small numbers of gentamicin-resistant E. coli DH5α transformants. Additional subcloning experiments using HindIII andEcoRI and the low-copy-number vector pMW119 yielded a gentamicin-resistant E. coli DH10b transformant, designated KHE5-2, which contained a 1.5-kb cloned fragment. Aminoglycoside MICs for E. coli KHE5-2 and E. coli DH10b(pMW119) are shown in Table 1.

Nucleotide sequencing of the 1.5-kb fragment revealed the presence of an 897-bp open reading frame, with a G+C content of 32%, whose predicted amino acid sequence showed homology with sequences of aminoglycoside phosphotransferases deposited in GenBank. The highest homology was seen with the deduced proteins from theaph(2")-Id (32% identity and 49% similarity) andaac(6′)-Ie-aph(2")-Ia genes (33% identity and 51% similarity with the APH(2")-Ia domain). Less closely related was theaph(2")-Ic gene (25% identity and 44% similarity). The gentamicin resistance gene has been designated aph(2")-Ib. The aminoglycoside phosphotransferase activity, designated APH(2")-Ib, was confirmed by the phosphocellulose paper binding assay. Crude extracts from E. coli KHE5-2 exhibited phosphotransferase activity with all of the aminoglycoside substrates listed in Table 1, except for neomycin. aph(2")-Ib was cloned by PCR into the shuttle vector pWM401 (20), the recombinant DNA was electroporated into competent E. faecalis JH2-2 cells (gentamicin MIC = 16 μg/ml), and high-level gentamicin-resistant (MIC > 2,000 μg/ml) E. faecalis transformants were obtained.

Evaluation of the presence of gentamicin resistance genes by PCR showed that 6 of 121 isolates (5%) were negative for theaac(6′)-Ie-aph(2")-Ia, aph(2")-Ic, andaph(2")-Id genes. All six were positive for theaph(2")-Ib gene by PCR. These six isolates (five from blood and one from stool cultures) were all vancomycin-resistant E. faecium from three hospitals in Detroit, Mich., and five belonged to the same clonal group, as determined by pulsed-field gel electrophoresis. The gentamicin MICs for all six were ≥1,024 μg/ml, and the arbekacin MICs ranged from 8 to 32 μg/ml. All of the 121 isolates possessed one of the four gentamicin resistance genes tested, and none possessed more than one of the genes tested.

aph(2")-Ib is now the fourth aminoglycoside resistance gene detected in Enterococcus spp. that eliminates the bactericidal synergistic effect of ampicillin and gentamicin against enterococci. The origin of the aph(2")-I family of genes remains uncertain. aac(6′)-Ie-aph(2")-Ia is also found in staphylococci and streptococci (18; M. Galimand, G. Gerbaud, P. Courvalin, and T. Lambert, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 833, 1999), and it has been proposed that aac(6′)-Ie-aph(2")-Ia was transmitted initially from staphylococci to enterococci (14). However,aph(2")-Ib, aph(2")-Ic, and aph(2")-Idhave not yet been detected in staphylococci and thus are less likely to have been transmitted from staphylococci (21). When we compared the nucleic acid sequence of aph(2")-Ib to that of an unpublished aph(2") resistance gene from an E. coli clinical isolate from the former republic of Czechoslovakia (J. Petrin, R. Kuvelkar, M. Kettner, R. S. Hare, G. H. Miller, and K. J. Shaw, Cold Spring Harbor Bacteria Phage Meet., abstr. 197, 1995), we found that the two sequences were identical, except for four nucleotides (resulting in three amino acid changes). The 32% G+C content of aph(2")-Ib raises the possibility that this gene may have first originated in enterococci and then been transmitted to E. coli, since the G+C contents ofEnterococcus spp. and E. coli are approximately 35 and 50%, respectively.

The aminoglycoside MIC profile of E. faecium SF11770 indicates various degrees of resistance to most of the clinically available aminoglycosides, except for arbekacin (Table 1). Arbekacin is a new aminoglycoside available in Japan, where it is used to treat gentamicin- and methicillin-resistant staphylococci (11, 13, 16). Although the combination of ampicillin and arbekacin at achievable concentrations in serum did not strictly fulfill the criteria for synergism against E. faecium SF11770, the combination with higher arbekacin concentrations did exhibit significantly greater killing of the organism than either agent alone. These preliminary findings need to be confirmed by in vivo data before arbekacin can be recommended for use in combination therapy against enterococci that possess aph(2")-Ib.

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