New Integron-Associated Gene Cassette Encoding a 3-N-Aminoglycoside Acetyltransferase

Abstract

A fifth gene cassette containing an aacC gene, aacCA5, was found in an aacCA5-aadA7 cassette array in a class 1 integron isolated from a multiply drug resistant Salmonella enterica serovar Kentucky strain. The AacC-A5 or AAC(3)-Ie acetyltransferase encoded by aacCA5 is related to other AAC(3)-I enzymes and confers resistance to gentamicin.

Acetyltransferases that modify the 3-amino group of aminoglycosides represent one type of enzyme that confers resistance to this important group of antibiotics. The known 3-N-aminoglycoside acetyltransferases [AAC(3) enzymes] are classified into several groups based on phenotypic differences in the specific spectra of aminoglycosides they are able to modify (22). However, they fall into only two clearly distinct groups based on the relationships between the proteins. The four aacC genes in family A, aacC1 [here designated aacCA1 and also sometimes referred to as aac(3)-Ia] (GenBank accession no. X15852) (25), a variant of aacCA1 (97.6% identical) (9, 15) previously named aacC4 (15) and here designated aacCA4, aac(3)-Ib (here designated aacCA2) (L06157) (21), and aac(3)Ic (here designated aacCA3) (AJ511268) (18) belong to the aac(3)-I phenotypic group and are found in gene cassettes. The products of these genes are all small proteins, of 154 to 156 amino acids, that are related to one another (Table 1) and confer resistance to gentamicin, sisomicin, and fortimicin but not to tobramycin, amikacin, or kanamycin. The remaining aacC genes are not found in gene cassettes and encode longer proteins, of 261 to 300 amino acids, that do not appear to be significantly related (generally less than 25% identical) to members of the AAC(3)-I group. This type B protein family currently includes at least 14 distinct members and variants (<2% difference) of some of the members.

TABLE 1.

Relationships between members of the AacC-A or AAC(3)-I protein family^a

Protein	AacC-A1	AacC-A2	AacC-A3	AacC-A4	AacC-A5
AacC-A1 AAC(3)-Ia	—	71.6	59.4	95.5	51.0
AacC-A2 AAC(3)-Ib	87.1	—	60.1	72.1	49.0
AacC-A3 AAC(3)-Ic	74.2	77.1	—	60.4	55.6
AacC-A4 AAC(3)-Id	98.7	86.4	74.0	—	51.6
AacC-A5 AAC(3)-Ie	64.1	61.4	71.9	64.1	—

^aValues represent the percent amino acid identities (top right) and similarities (bottom left) between the different AacC-A proteins. Dashes represent the 100% line.

Here, we report the identification of a further aacCA gene cassette that was found in a class 1 integron in a multiply drug resistant Salmonella enterica serovar Kentucky strain.

The isolate.

Salmonella serovar Kentucky SRC73 was isolated in 2001 from spice imported into Australia from India. The strain was serotyped by using standard procedures according to the Kauffman and White scheme (16). Salmonella serovar Kentucky SRC73 was scored as resistant to ampicillin (at 32 μg/ml), gentamicin (2.5 μg/ml), streptomycin (25 μg/ml), spectinomycin (50 μg/ml), sulfathiazole (550 μg/ml), tetracycline (20 μg/ml), and nalidixic acid (50 μg/ml) but susceptible to chloramphenicol (10 μg/ml), trimethoprim (50 μg/ml), kanamycin (10 μg/ml), and ciprofloxacin (2 μg/ml) by using the plate-replicator method as previously described (2, 3). Briefly, antibiotics at the concentrations indicated were in lysed blood Iso Sensitest agar plates (Oxoid, Hampshire, England), and the inoculum was 10⁵ CFU per spot. Plates were incubated overnight at 37°C.

Resistance genes in Salmonella serovar Kentucky SRC73.

Whole cell DNA isolated from the Salmonella serovar Kentucky strain by using standard methods (20) was screened by PCR for several known antibiotic resistance genes with primers pairs internal to the genes (Table 2). PCR amplification reactions were carried out with PCR buffer (Roche Molecular Biochemicals, Mannheim, Germany) containing 160 μM of each deoxynucleoside triphosphate, 20 pmol of each primer, approximately 10 to 50 ng of template, and 1 U of Taq DNA polymerase (Roche). Reaction conditions were generally 94 to 96°C for 3 to 5 min; 30 to 40 cycles of 94 to 96°C for 30 s, 53 to 62°C for 30 to 60 s, and 72°C for 30 s to 2 min; and a final incubation at 72°C for 10 to 15 min. A product of the appropriate size for each gene of the strAB gene pair that confers resistance to streptomycin and for the bla_TEM ampicillin resistance gene was detected. The tetracycline resistance determinant was tet(A), but spectinomycin resistance was not due to the aadA2 gene. The sul1 gene, which confers resistance to sulfonamides and is found only in association with class 1 integrons, and the sul2 gene were both present.

TABLE 2.

PCR primer pairs

Primer name	5′-to-3′ sequence	Location	Product size	Accession no.	Referencea
L1	GGCATCCAAGCAGCAAGC	5′-CS	Variable	M95287.4	11
R1	AAGCAGACTTGACCTGAT	3′-CS		U12338.2	11
L2	GACGATGCGTGGAGACC	5′-CS	297	M95287.4	19
L3	CTTGCTGCTTGGATGCC	5′-CS		M95287.4	12
QS-1	ATGAAAGGCTGGCTTTTTCTTG	3′-CS	722	U12338.2	5
QS-2	TGAGTGCATAACCACCAGCC	3′-CS		U12338.2	5
sulI-F	GTGACGGTGTTCGGCATTCT	sul1	668	U12338.2	10
sulI-R	TTTACAGGAAGGCCAACGGT	sul1		U12338.2	10
sulII-F	GGCAGATGTGATCGACCTCG	sul2	405	M28829	10
sulII-R	ATGCCGGGATCAAGGACAAG	sul2		M28829	10
aadA2-L	TGTTGGTTACTGTGGCCG	aadA2	538	X68227	14
aadA2-R2	TGCTTAGCTTCAAGTAAGACG	aadA2		X68227	4
strA-F	CTTGGTGATAACGGCAATTC	strA	548	M95402	8
strA-R	CCAATCGCAGATAGAAGGC	strA		M95402	8
strB-F	ATCGTCAAGGGATTGAAACC	strB	509	M95402	8
strB-R	GGATCGTAGAACATATTGGC	strB		M95402	8
tem-F	TTCTTGAAGACGAAAGGGC	blaTEM	1,208	L27758	6
tem-R	ACGCTCAGTGGAACGAAAAC	blaTEM		L27768	6
tet(A)-L	GCTACATCCTGCTTGCCTTC	tetA(A)	210	X61367	14
tet(A)-R	CATAGATCGCCGTGAAGAGG	tetA(A)		X61367	14
tet(B)-L	TTGGTTAGGGGCAAGTTTTG	tetA(B)	659	AP000342	14
tet(B)-R	GTAATGGGCCAATAACACCG	tetA(B)		AP000342	14
tet(G)-L	CAGCTTTCGGATTCTTACGG	tetA(G)	844	S52437	14
tet(G)-R	GATTGGTGAGGCTCGTTAGC	tetA(G)		S52437	14

^aReferences for primers are shown.

Gene cassettes in a class 1 integron.

The presence of a class 1 integron was confirmed using primers within the intI1 gene (L2 and L3) and in the 3′-conserved segment (3′-CS) (QS-1 and QS-2) (Table 2). Amplification of the gene cassettes with standard primers in the 5′-CS and 3′-CS (L1 and R1) yielded a product of 1.6 kb, indicating the presence of gene cassettes totaling 1.45 kb. The sequence of this amplicon (GenBank accession no. AY463797) revealed two gene cassettes. The first is 564 bp long and contains an open reading frame with a GTG start codon at positions 22 to 24 relative to the beginning of the cassette that is preceded by a GAGG ribosome binding site at positions 11 to 14. Translation from this GTG gives a protein of 158 amino acids that is related to the known AacC-A [AAC(3)-I] proteins (Table 1), and alignment of the sequences (Fig. 1) revealed 65 completely conserved amino acids. Using the next available number, the gene and cassette were named aacCA5 and the protein AacC-A5 or AAC(3)-Ie.

FIG. 1.

Alignment of AacC-A proteins in the AAC(3)-I family. Amino acids completely conserved in all sequences are shown as white letters on a black background and are indicated by uppercase letters below the sequence. The sequences of AacC-A1 [AAC(3)-Ia], AacC-A2 [AAC(3)-Ib], AacC-A3 [AAC(3)-Ic], and AacC-A4 were obtained from GenBank and have accession nos. U12338, L06157, AJ511268, and AF318077, respectively. AacC-A5 [AAC(3)-Ie] is from this study.

The aacCA5 cassette has a 59-be (59-base element) of 78 bp made up of two simple sites (Fig. 2) and a central region, as is characteristic for 59-be (23). This 59-be is not closely related to those of other cassettes in the aacCA group nor to those of any other known cassettes. The 59-be of the aacCA1 and aacCA4 cassettes, which are closely related, differ at only three positions, as expected if these two cassettes had diverged by accumulation of mutations over their full lengths (17). However, the more distantly related aacCA2 [aac(3)-Ib] cassette (73% identical to aacCA1), for which a complete sequence is not available, appears to have the same 59-be (Fig. 2), as the 34 bp of the aacCA2 59-be sequence present in GenBank accession no. L06157 is identical to the corresponding part of the aacCA1 59-be. In this case, the 59-be may have been acquired recently from the aacCA1 cassette. The 59-be in the aacCA3 [aac(3)-Ic] cassette is most closely related to that of the bla_GES/IBC cassette, as noted previously (18). Thus, the aacCA5 and aacCA3 cassettes appear to have separate origins.

FIG. 2.

Alignment of the 59-be of aacCA family gene cassettes. Bases in lowercase letters are derived from the beginning of the cassette. The core sites are in boldface type and are designated 1L, 1R, 2L, and 2R according to reference 23. The extents of the simple sites (LH, left-hand; RH, right-hand) and the 59-be are indicated by bars (bottom). Sequences are from sources given in Fig. 1.

The second cassette in the integron is identical to the aadA7 cassette (13) (GenBank accession no. AF224733), found in an Escherichia coli strain from the ECOR collection that was isolated from a leopard in Washington Zoo in 1973 and in a Shiga toxin-producing E. coli O157:H7 strain (AF234167) (26). In both of these strains, aadA7 is the only cassette in a class 1 integron. The aadA7 gene confers resistance to streptomycin and spectinomycin and accounts for the spectinomycin resistance of SRC73. Since our work was completed, the aacCA5-aadA7 cassette array (AB114632) has been found in Vibrio fluvialis (1), but the aacCA5 gene was incorrectly designated aac(3)-Id. The same cassette array is also found in Vibrio cholerae (AY605683) and Salmonella enterica serovar Newport (AY458224) (7). The aacCA5 cassettes are all identical, but there are minor differences in the aadA7 cassettes (T65G C837Δ in AB114631 and AY605683; T669C A786G in AY458224).

The aacCA5 cassette confers resistance to aminoglycosides.

The aacCA5-aadA7 cassette array was amplified by PCR and eluted from a 1% (wt/vol) agarose gel using an Amicon Bioseparations Ultrafree-DNA kit (Millipore Corp., Bedford, Massachusetts), and ligated into pCR-Script (PCR-Script Amp cloning kit; Stratagene, La Jolla, California) using the manufacturer's protocols. The cloned fragment was recovered by transformation with selection on LB agar containing ampicillin (50 μg/ml) and gentamicin (8 μg/ml). Susceptibilities to gentamicin, tobramycin, amikacin, netilmicin, and kanamycin for E. coli strain 294 (24) containing either pCR-Script or pCR-Script with the cassette array were determined by using the CDS method for antibiotic disks (Oxoid, Basingstoke, Hampshire, United Kingdom). The cloned fragment conferred resistance to gentamicin (5- versus 11-mm zone size), but not to tobramycin, amikacin, netilmicin, or kanamycin, consistent with the presence of an AAC(3)-I-type aminoglycoside acetyltransferase. Resistance to sisomicin has also been demonstrated (1). The cassette array also conferred resistance to streptomycin and spectinomycin, consistent with the presence of the aadA7 cassette. SRC73 was resistant to the same set of aminoglycosides, indicating that the aacCA5 and aadA7 cassettes are sufficient to account for the observed aminoglycoside resistance.

Nucleotide sequence accession number.

The nucleotide sequence reported in this paper has been submitted to GenBank under accession no. AY463797.