INTRODUCTION

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The aminoglycoside resistance gene, aac(6')-Ie-aph(2")-Ia, encodes a bifunctional enzyme, AAC(6')-APH(2"), that confers resistance to a broad spectrum of aminoglycosides and has to date been detected only in gram-positive bacteria, including Enterococcus spp., Staphylococcus aureus, Streptococcus agalactiae (group B), Streptococcus mitis, and group G Streptococcus (3, 6, 8, 13, 19). Several amikacin-resistant gram-negative bacterial clinical isolates from Slovakia and Germany express an aminoglycoside resistance profile similar to that conferred by aac(6')-Ie-aph(2")-Ia (11). In order to determine whether an aminoglycoside resistance gene similar to aac(6')-Ie-aph(2")-Ia was responsible for the resistance profile in these gram-negative bacteria, we chose one of these resistant isolates, Escherichia coli SCH92111602, for further study. We describe here the characterization of the aac(6')-Im and aph(2")-Ib genes from this E. coli isolate, and the detection of this pair of genes also in clinical isolates of Enterococcus faecium.

	MATERIALS AND METHODS

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E. coli SCH92111602 is a clinical isolate from University Hospital, Bratislava, Slovakia. Aminoglycoside MICs were determined by a standard broth microdilution method (16). Gentamicin was obtained from Fluka (Buchs, Switzerland). Netilmicin, dibekacin, and arbekacin were donated by Meiji Seika Kaisha (Tokyo, Japan). All other antibiotics were purchased from Sigma Chemical Company (St. Louis, Mo.). Plasmid DNA from clinical strains was isolated using a Qiagen (Chatsworth, Calif.) plasmid column or a modified alkaline lysis method (26). Plasmid DNA from transformants was prepared with either a Wizard miniprep (Promega, Madison, Wis.) or a Qiagen column. DNA was digested with restriction enzymes and ligated with T4 ligase from New England Biolabs (Beverly, Mass.) or Life Technologies (Rockville, Md.) according to the manufacturers' recommendations, and products were analyzed in an agarose gel system of Tris-borate-EDTA. Cloned Pfu DNA polymerase enzyme (Stratagene) or recombinant Taq DNA polymerase (Life Technologies) was used for PCRs. Oligonucleotide primers were synthesized by Research Genetics (Huntsville, Ala.) or by Life Technologies. The vectors pBluescript II KS(+) (Stratagene Cloning Systems, La Jolla, Calif.) and pACYC184 (New England Biolabs) were used in standard cloning experiments (2). Competent E. coli DH5 cells were used as the recipients in transformation experiments. The phosphocellulose paper-binding assay was performed as previously described (18). Nucleotide sequencing was performed by Lark Technologies, Inc. (Houston, Tex.), and by the DNA Sequencing Core, University of Michigan. Computer analysis was performed with Mac Vector software, version 6.0, and OMIGA software, version 2.0 (Genetics Computer Group, Madison, Wis.). 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 (5).

Conditions for dot blot hybridizations were performed at 42°C and have been described previously (21). DNA hybridization was performed with a collection of 4,625 aminoglycoside-resistant bacterial clinical isolates acquired by one of the coauthors (G. H. Miller) within the past decade. Some of these isolates had been included in previous studies to detect the presence of other aminoglycoside resistance genes (15). These 4,625 isolates were from Belgium (n = 455), the Czech Republic (n = 49), France (n = 907), Guatemala (n = 123), Mexico (n = 516), the Philippines (n = 41), Portugal (n = 378), Slovakia (n = 71), South Africa (n = 868), Thailand (n = 255), Turkey (n = 725), the United States (n = 81), and Venezuela (n = 156). A 303-bp internal fragment (nucleotides 1282 to 1584) of the aac(6')-Im gene was used as the probe for DNA hybridizations. This fragment was generated by PCR amplification using primers 5'-GGCTGACAGATGACCGTGTTCTTG-3' and 5'-GTAGATATTGGCATACTACTCTGC-3'. PCR conditions were as follows: DNA was initially denatured for 4 min at 94°C, followed by 30 cycles of 1 min of denaturation at 94°C, 1 min of annealing at 55°C, and 2 min of extension at 72°C. The amplified DNA fragment was removed from 1% agarose gels by repeated electroelution in dialysis tubing and purified by phenol-chloroform-isoamyl alcohol (25:24:1) extraction and ethanol precipitation. The purified DNA fragment was labeled with [-³²P]ATP using an oligonucleotide labeling kit from Amersham Pharmacia Biotech, Inc. (Piscataway, N.J.). Unincorporated nucleotides were separated from labeled product by column chromatography on Sephadex G-50 columns (5 Prime-3 Prime, Inc., Boulder, Colo.). The PCR primers used for detecting the aac(6')-Im gene in enterococcal isolates were 5'-GCGAGTTTCCTTTCGCCC-3' and 5'-CACCGCATCGGCATCC-3'. PCR conditions were as follows: an initial 5-min denaturation at 94°C, followed by 30 cycles of 30 s of denaturation at 94°C, 20 s of annealing at 58°C, and 1 min of extension at 72°C.

Nucleotide sequence accession number. The nucleotide sequence for aac(6')-Im has been deposited in GenBank under accession number AF337947.

	RESULTS AND DISCUSSION

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SCH92111602 is an E. coli clinical isolate resistant to a number of aminoglycoside antibiotics, including gentamicin, tobramycin, and amikacin (Table 1), and contains an approximately 50-kb plasmid. Plasmid DNA isolated from this strain was introduced into E. coli DH5 by transformation, and colonies were selected on Luria-Bertani agar plates containing 10 µg of tobramycin (Eli Lilly & Co., Indianapolis, Ind.) per ml. Analysis of restriction digests on agarose gels of DNA from a tobramycin-resistant transformant confirmed the presence of the same 50-kb plasmid that was isolated from E. coli SCH92111602. The 50-kb plasmid was digested with XmnI, and the fragments were ligated into the EcoRV site of the vector pACYC184. After electroporation, selection for tobramycin-resistant transformants yielded an E. coli DH5 derivative that contained a 3.7-kb cloned fragment. Further subcloning experiments using the vector pBluescript II KS(+) yielded a tobramycin-resistant E. coli DH5 transformant that contained a 2.5-kb AvaI fragment ligated to pBluescript (designated pSCH075). Nucleotide sequencing revealed the presence of an 897-bp open reading frame (ORF), whose predicted amino acid sequence was identical to that of the APH(2")-Ib aminoglycoside-modifying enzyme reported in E. faecium, except for three amino acid changes that resulted from four nucleotide differences (12). A putative promoter sequence composed of TTGAAA and TATAAT (35 and 10) was noted 36 bases upstream of the ATG start codon, which was identical to that noted in E. faecium. APH(2")-Ib from E. faecium has 33% identity and 51% similarity with the deduced APH(2")-Ia domain of the bifunctional enzyme AAC(6')-APH(2") (12).

View this table: [in this window] [in a new window]   TABLE 1.   Susceptibility profiles conferred by aph(2")-Ib and/or aac(6')-Im cloned in the vector pBluescript in E. coli DH5

Beginning 44 nucleotides downstream from the aph(2")-Ib gene in the DNA cloned from the E. coli plasmid, a second ORF of 534 nucleotides was detected (G+C content of 40%), which exhibited 65% nucleotide identity with the aac(6')-Ie portion of aac(6')-Ie-aph(2")-Ia. A putative ribosome binding site (GAGG) was located 7 nucleotides upstream from the start codon of this ORF, but no nucleotide sequences consistent with a 35 and 10 promoter sequence were detected. The predicted 178-amino acid sequence of this ORF showed similarity to sequences of aminoglycoside acetyltransferases in the GenBank database. The highest homology was seen with the deduced protein of the aac(6')-Ie-aph(2")-Ia gene from Enterococcus [56% identity and 80% similarity to the AAC(6')-Ie domain] (6). Other acetyltransferases with lower degrees of homology include AAC(6')-IIa from Pseudomonas aeruginosa (20) and AAC(6')-Ib, initially detected in Serratia marcescens (24) and subsequently in Klebsiella pneumoniae, Salmonella enterica Serovar Typhimurium, P. aeruginosa, and Pseudomonas fluorescens (7, 9, 14, 17). Our newly observed ORF has been designated aac(6')-Im (23). The aac(6')-Im designation has been used by others to describe another aminoglycoside acetyltransferase, which was initially named aac(6')-Il (10), then had its name changed to aac(6')-Im (25), and subsequently was renamed aac(6')-Ip (4).

Since the aac(6')-Im gene was found in such close proximity downstream from the aph(2")-Ib gene in E. coli SCH92111602, we attempted to determine if aac(6')-Im was also present in the E. faecium SF11770 isolate in which we had initially detected aph(2")-Ib (12). A 3.3-kb HindIII fragment from E. faecium SF11770 cloned in the vector pWM119 in previous experiments was known to contain the aph(2")-Ib gene (S. J. Kao, I. You, and J. W. Chow, unpublished data). Nucleotide sequencing of this fragment downstream from aph(2")-Ib determined that the aac(6')-Im gene was also present, as in E. coli, 44 bases from aph(2")-Ib. The nucleotide sequence of aac(6')-Im from E. faecium was identical to that from E. coli except for two nucleotides, whose presence did not alter the predicted amino acid sequence. The sequence of the 200 nucleotides upstream of aph(2")-Ib and that of the 44 nucleotides between the aph(2")-Ib and aac(6')-Im ORFs were identical in the E. faecium and E. coli strains. The 200 nucleotides downstream from aac(6')-Im were identical in the two strains except for one base. PCR results showed that all of the nine other E. faecium clinical isolates in our collection known to possess the aph(2")-Ib gene were also positive for the aac(6')-Im gene.

The aac(6')-Im gene was subcloned by digesting with HincII the 3.3-kb HindIII fragment [derived from E. faecium SF11770 and containing both aph(2")-Ib and aac(6')-Im]. A 1.5-kb HincII fragment [which contained aac(6')-Im but not aph(2")-Ib] was ligated to pBluescript digested with HincII, and the ligation products were transformed into E. coli DH5. The aminoglycoside MICs for the E. coli DH5 transformant containing aac(6')-Im are listed in Table 1. As seen in Table 1, the presence of both the aph(2")-Ib and aac(6')-Im genes in E. coli confers a higher level of resistance to some aminoglycosides than the presence of either gene alone. The aminoglycoside acetyltransferase activity, designated AAC(6')-Im, was confirmed by the phosphocellulose paper-binding assay (data not shown). Crude extracts from E. coli DH5 containing only aac(6')-Im exhibited acetyltransferase activity with tobramycin, amikacin, dibekacin, netilmicin, and kanamycin A, but not with gentamicin, neomycin, or arbekacin. The aminoglycoside phosphotransferase activity of APH(2")-Ib from E. faecium has been reported previously (12).

A 303-bp internal fragment of the aac(6')-Im gene from E. coli SCH92111602 was amplified by PCR and used as a probe. DNA hybridization results showed that 46 of 4,625 (1%) bacterial isolates hybridized to the aac(6')-Im probe. The isolates that hybridized with this probe were from Slovakia (n = 38), the Czech Republic (n = 6), and Belgium (n = 2). Several genera were represented among the isolates that hybridized to the aac(6')-Im probe, including Aeromonas (1 of 6), Citrobacter (1 of 132), Enterobacter (9 of 616), Escherichia (5 of 590), Klebsiella (16 of 1,117), Pseudomonas (4 of 765), and Serratia (10 of 267).

Although the aph(2")-Ib and aac(6')-Im genes show a high degree of homology with the aac(6')-Ie-aph(2")-Ia gene from Enterococcus, there are significant differences. aac(6')-Ie-aph(2")-Ia appears to be a fusion of two genes and has one start (ATG) and one stop (TAA) codon, whereas aph(2")-Ib and aac(6')-Im are separate ORFs, each with its own putative ribosome binding site, although we have some preliminary (unpublished) data suggesting that transcription of aph(2")-Ib and aac(6')-Im may come from a single promoter. In addition, the order of the AAC and the APH domains of aac(6')-Ie-aph(2")-Ia is reversed compared to the set of aph(2")-Ib and aac(6')-Im genes. Furthermore, the AAC(6')-Ie domain of aac(6')-Ie-aph(2")-Ia confers resistance to fortimicin (22), whereas AAC(6')-Im does not (J. Petrin, Kuvelkar, M. Kettner, R. S. Hare, G. H. Miller, and K. J. Shaw, Abstr. Cold Spring Harbor Bacteria Phage Meet., abstr. 197, 1995). Finally, whereas aac(6')-Ie-aph(2")-Ia has been detected only in gram-positive cocci, aph(2")-Ib and aac(6')-Im have now been detected in both enterococci and gram-negative bacilli. Our results show that a member of the aph(2") family of genes is present in gram-negative bacteria and provide evidence for the horizontal transfer of aph(2")-Ib and aac(6')-Im as a unit between gram-positive and gram-negative bacteria. The G+C content of the aph(2")-Ib (32%) and aac(6')-Im (40%) genes suggests that they may have originated in enterococci (G+C content, approximately 35%) or another bacterial genus and were subsequently transferred to E. coli (G+C content, approximately 50%). The presence of these two linked aminoglycoside resistance genes that appear to have been transferred together between such diverse bacterial species is another example of how bacteria evolve to become more resistant to a broader spectrum of antimicrobial agents.