Characterization of Aminoglycoside-Modifying Enzymes in Enterobacteriaceae Clinical Strains and Characterization of the Plasmids Implicated in Their Diffusion

Abstract

A total of 788 clinical Enterobacteriaceae were collected to describe the aminoglycoside-modifying genes (AME genes) and to characterize the plasmids that carry these genes. Among the 788 strains collected, 330 (41.8%) were aminoglycoside-resistant: 264 Escherichia coli (80%), 33 Proteus mirabilis (10%), 10 Klebsiella pneumoniae (3%), six K. oxytoca (1.8%), five Enterobacter cloacae (1.5%), three Morganella morganii (0.9%), three Providencia stuartii (0.9%), two Salmonella enterica (0.6%), and one each Citrobacter freundii, C. koseri, Proteus vulgaris, and Shigella sonnei. The most affected aminoglycoside was streptomycin (92.7%), followed by kanamycin (26.3%), gentamicin (18%), tobramycin (16.9%), netilmicin (3.6%), and amikacin (1.5%). The AME genes found were aph(3″)-Ib (65.4%), ant(3″)-Ia (37.5%), aph(3′)-Ia (13.9%), aac(3)-IIa (12.4%), aac(6′)-Ib (4.2%), ant(2″)-Ia (3.6%), and aph(3′)-IIa (1.2%). Thirty-four percent of the strains showed more than one enzyme. The most frequent association was ant(3″)-Ia plus aph(3″)-Ib (35 strains). From 66 selected AME genes, 24 were plasmid located: 12 aac(3)-IIa, six aph(3′)-Ia, three ant(3″)-Ia, two ant(2″)-Ia, and one aac(6′)-Ib. These genes were located in plasmids belonging to incompatibility groups F, FIA, FIB, or HI2. In conclusion, the AME genes involved in aminoglycoside-clinical resistance were aac(3)-IIa, aac(6′)-Ib, and ant(2″)-Ia, genes that confer resistance to tobramycin, gentamicin, and amikacin.

Introduction

Aminoglycosides are a large family of drugs that act by inhibiting one or more of the biochemical steps involved in translation of the ribosome. They are extensively used in the treatment of serious bacterial infections, particularly in combination with β-lactams or glycopeptides.^3,10

Susceptibility of common gram-negative bacteria to clinical aminoglycosides has changed little over time. The SENTRY surveillance program, conducted in the United States, Canada, South America, and Europe in 1997 found that the most common gram-negative bacteria were susceptible to nearly all the aminoglycosides tested. Among the various species evaluated, they found percentages of susceptibility ranging from 96% to 100% for amikacin, from 90% to 96% for gentamicin, and from 94% to 97% for tobramycin.¹¹ We also found high percentages of susceptibility in 2010 in the routine of our laboratory. We observed 97% susceptibility for amikacin, 81.7% for gentamicin, and 84% for tobramycin.

Acquired aminoglycoside resistance mainly occurs through the presence of aminoglycoside-modifying enzymes (AMEs). Despite the existence of more than 50 AME, only ANT(2″)-I, AAC(6′)-I, and, to a lesser extent, AAC(3)-I to AAC(3)-VI, have been identified in gram-negative bacteria.^10,11,13 This enzymatic mechanism of resistance has frequently been found in genetically transferable vectors, such as plasmids or transposable elements. The presence of these transferable elements could explain the rapid acquisition of aminoglycoside resistance among a large variety of bacterial species around the world.¹³ Other mechanisms of aminoglycoside resistance have been described, such as methylation of 16S rRNA, ribosomal alterations, or loss of permeability.^9,13,14,16

Information is lacking regarding the prevalence of aminoglycoside resistance, the type of AME genes involved, and the mobile genetic elements responsible for their diffusion. The purpose of the present study is to describe the enzymatic mechanisms responsible for aminoglycoside resistance in clinical isolates of Enterobacteriaceae and to characterize the plasmids that carry them.

Materials and Methods

Isolates

From January to March 2006, a total of 788 Enterobacteriaceae clinical isolates were collected at Hospital de la Santa Creu i Sant Pau. Isolates showing resistance or decreased susceptibility to any of the aminoglycosides mentioned below were selected for further studies. Only one clinically significant isolate per patient was studied.

Antibiotic susceptibility testing

The susceptibility test was performed by disc diffusion method according to Clinical Laboratory Standards Institute (CLSI) guidelines,² except in the case of spectinomycin and neomycin for which guidelines are not available.

The drugs tested were: ampicillin (10 μg), piperacillin (100 μg), amoxicillin-clavulanic acid (20/10 μg), cefazolin (30 μg), cefepime (30 μg), cefotaxime (30 μg), cefoxitin (30 μg), ceftazidime (30 μg), cefuroxime (30 μg), aztreonam (30 μg), imipenem (10 μg), amikacin (30 μg; A), gentamicin (10 μg; G), kanamycin (30 μg; K), neomycin (30 μg; Nm), netilmicin (30 μg; N), spectinomycin (100 μg; Sp), streptomycin (10 μg; S), tobramycin (10 μg; T), ciprofloxacin (5 μg), nalidixic acid (30 μg), co-trimoxazole (1.25/23.75 μg), fosfomycin (200 μg), and nitrofurantoin (300 μg) (NEO-SENSITABS^™ ROSCO).

Enterobacterial Repetitive Intragenic Consensus-polymerase chain reaction

The clonal relationships between Escherichia coli isolates were analyzed by ERIC-polymerase chain reaction (PCR) using the oligonucleotide ERIC2 (5′-AAGTAAGTGACTGGGGTGAGCG-3′), as described by Versalovic et al.¹⁵

Pulsed-field gel electrophoresis

The clonal relationships between isolates other than E. coli were analyzed by comparing pulsed-field gel electrophoresis (PFGE) profiles of genomic DNA digested with XbaI (GE Healthcare Bioscience). Isolates were typed by PFGE of XbaI-digested genomic DNA using the Chef-DR^® III System (Bio-Rad). The electrophoresis were run at 14°C at 6 V/cm on a 120° angle in 0.5×Tris/borate/EDTA buffer (TBE). Different linear pulse times for the different members of the Enterobacteriaceae were used: 5–20 sec for 6 hr, followed by 25–40 sec for 18 hr for Enterobacter cloacae; 1–20 sec for 21 hr for Morganella morganii; 4–25 s for 4 hr, followed by 25–45 sec for 20 hr for Klebsiella pneumoniae and K. oxytoca; 5–25 sec for 4 hr, followed by 26–50 sec for 20 hr for Proteus mirabilis; and 5–50 sec for 24 hr for Providencia stuartii.

Enzymes detected

Nomenclature used was that defined by Shaw et al.¹³ The initial screening for AME was performed according to the phenotype observed in each isolate. The expected enzyme for each phenotype was APH(3″)-Ia (resistant to S), APH(3″)-Ib (S), ANT(3″)-Ia (S, Sp), APH(3′)-Ia (KNm), ANT(2″)-Ia (KGT), ANT(4″)-IIa (KTA), AAC(2′)-Ia (GTN), AAC(3)-Ia (G), AAC(3)-Ib (G), AAC(3)-IIa (KGTN), AAC(6′)-Ia (KTAN), AAC(6′)-Ib (KTAN), and AAC(6′)-Ic (KTAN).^3,10 [A, amikacin, G, gentamicin, K, kanamycin, Nm, neomycin, N, netimicin, S, streptomycin, Sp, spectinomycin, T, tobramycin]

Characterization of antimicrobial resistance genes

Primer pairs used in this study are summarized in Table 1 and references 4 and 8. PCRs were performed using the following conditions: denaturing for 5 min at 94°C, 30 cycles for 30 sec at 94°C, annealing for 30 sec at 55°C and elongation for 1 min at 72°C, followed by a final elongation step at 72°C for 10 min. As mentioned, PCR was performed for one or more of the genes mentioned in Table 1 according to the phenotype.

Table 1.

Primers Used in Characterization of Aminoglycoside-Modifying Genes

Genes	Primer name	Primer sequence	GenBank number		Weight (pb)
aac(2′)-Ia	AAC-2′-Ia FW	5′-AGAAGCGCTTTACGATTTATTA-3′	L06156	317–338	406
	AAC-2′-Ia R	5′-GACTCCGCCTTCTTCTTCAA-3′		722–703
aac(3)-Ia	AAC-3-Ia FW	5′- GCAGTCGCCCTAAAACAAA-3′	X15852	1284–1302	441
	AAC-3-Ia R	5′-CACTTCTTCCCGTATGCCCAACTT-3′		1724–1747
aac(3)-Ib	AAC-3-Ib FW	5′-GCAGTCGCCCTAAAACAAA-3′	L06157	589–607	417
	AAC-3-Ib R	5′-GGATCGTCACCGTAGTCTGC-3′		1006–987
aac(3)-IIa	AAC-3-IIa FW	5′-GGCAATAACGGAGGCGCTTCAAAA-3′	X13543	200–223	563
	AAC-3-IIa R	5′-TTCCAGGCATCGGCATCTCATACG-3′		762–739
aac(6′)-Ia	AAC-6′-Ia FW	5′-ATGAATTATCAAATTGTG-3′	M18967	757–774	558
	AAC-6′-Ia R	5′-TTACTCTTTGATTAAACT-3′		1314–1297
aac(6′)-Ib	AAC-6′-Ib FW	5′-CAAAGTTAGGCATCACA-3′	M21682	402–418	540
	AAC-6′-Ib R	5′-ACCTGTACAGGATGGAC-3′		941–924
aac(6′)-Ic	AAC-6′-Ic FW	5′-CTACGATTACGTCAACGGCTGC-3′	M94066	1742–1763	130
	AAC-6′-Ic R	5′-TTGCTTCGCCCACTCCTGCACC-3′		1871–1850
ant(2″)-Ia	ANT-2″Ia FW	5′-ACGCCGTGGGTCGATGTTTGATGT-3′	X04555	1213–1236	572
	ANT-2″Ia R	5′-CTTTTCCGCCCCGAGTGAGGTG-3′		1784–1763
ant(3″)-Ia	ANT-3″-Ia FW	5′-TCGACTCAACTATCAGAGG-3′	X02340	430–448	245
	ANT-3″-Ia R	5′-ACAATCGTGACTTCTACAGCG-3′		674–654
ant(4″)-IIa	ANT-4″-IIa FW	5′-CCGGGGCGAGGCGAGTGC-3′	M98270	231–248	423
	ANT-4″-IIa R	5′-TACGTGGGCGGATTGATGGGAACC-3′		653–630
aph(3′)-Ia	APH-3′-Ia FW	5′-CGAGCATCAAATGAAACTGC-3′	AY260546	865–885	625
	APH-3′-Ia R	5′-GCGTTGCCAATGATGTTACAG-3′		1489–1468
aph(3″)-Ia	STRa-FW	5′-CGGCGTGGGCGGCGACTG-3′	X53527	45–62	557
	STRa-R	5′-CCGGATGGAGGACGATGTTGG-3′		601–581
aph(3″)-Ib	STRb-FW	5′-GTGGCTTGCCCCGAGGTCATCA-3′	M28829	189–210	612
	STRb-R	5′-CCAAGTCAGAGGGTCCAATC-3′		800–781

Fifty-two amplicons, representative of all AME genes obtained, were purified with the Wizard^® SV Gel and PCR clean-up system kit (Promega Corporations) for sequencing. DNA sequencing of purified PCR products was performed by Macrogen (Macrogen Inc.). Nucleotide and amino-acid sequences were analyzed using Mega-BLAST and PSI-BLAST, respectively (www.ncbi.nlm.nih.gov).

PCR-based replicon typing

PCR-based Inc/rep typing was performed to identify the major incompatibility groups of the plasmids present.¹ Template DNA was prepared by extraction of total DNA using the GenElute^™ Bacterial Genomic DNA commercial kit (Sigma).

Plasmid profiles and Southern blot analysis

Plasmid profiles were visualized after DNA linearization with S1 enzyme followed by PFGE as previously described.^1,4 Plasmid sizes were estimated using Fingerprinting II Informatix^™ software. S1-PFGE was then transferred onto a nylon-membrane by Southern blotting. Purified DNA products were obtained from the PCR of aminoglycoside-modifying genes and the replicons were used as probes for hybridization of the S1-PFGE blots. These probes were labeled with the commercial kit Amersham ECL Direct Nucleic Acid Labelling and Detection Systems, as recommended by the manufacturer (GE Healthcare).

Results

A total of 344 aminoglycoside-resistant Enterobacteriaceae strains were collected from among 788 clinical isolates (44%). All strains that had identical PFGE or ERIC-PCR pattern were excluded. Therefore, only 330 out of the 344 resistant strains were selected for enzyme characterization: 264 Escherichia coli (80%), 33 P. mirabilis (10%), 10 K. pneumoniae (3%), six K. oxytoca (1.8%), five E. cloacae (1.5%), three M. morganii (0.9%), three P. stuartii (0.9%), two Salmonella enterica (0.6%), and one each Citrobacter freundii, C. koseri, Proteus vulgaris, and Shigella sonnei.

Only six of the eight aminoglycosides tested (gentamicin, tobramycin, amikacin, kanamycin, netilmicin, and streptomycin) have interpretative criteria.² Using these criteria, the most affected drug was streptomycin (306/330; 92.7%), followed by kanamycin (87/330; 26.3%), gentamicin (61/330; 18.4%), tobramycin (56/330; 16.9%), netilmicin (12/330; 3.6%), and amikacin (5/330; 1.5%).

The most frequent gene found was aph(3″)-Ib (216/330, 65.4%) and ant(3″)-Ia (124/330, 37.5%). All these genes encoded for enzymes responsible for streptomycin and spectinomycin resistance. Respect to aminoglycosides of clinical use, the most frequent gene found was aph(3′)-Ia (46/330, 13.9%) and aac(3)-IIa (41/330, 12.4%), followed by aac(6′)-Ib (14/330, 4.2%), ant(2″)-Ia (12/330, 3.6%), and aph(3′)-IIa (4/330, 1.2%). In 16 of 330 aminoglycoside-resistant isolates, no AME genes were found. Fourteen out of these unresolved strains showed streptomycin resistance and had halos between 9 and 11 mm (13 E. coli and one P. mirabilis). One was a gentamicin-resistant E. coli (9 mm) and the other was a gentamicin (9 mm), tobramycin (14 mm), and streptomycin (13 mm)-resistant C. koseri strain.

We sequenced 52 amplicons, representative of all AME genes obtained, and found 100% concordance with the GenBank sequences stated in Table 1. Among the 14 aac(6′)-Ib amplicons, eight showed the–cr variant (57.1%). Four out of these eight strains carrying aac(6′)-Ib-cr were also ESBL producers.

Resistance to streptomycin by aph(3″)-Ib gene was predominantly found in E. coli isolates (188/264, 71.2%), whereas the ant(3″)-Ia, which also confers resistance to spectinomycin, was the enzyme most frequently found in P. mirabilis (24/33, 72.7%) and Klebsiella species (11/16, 68.7%). Resistance to the other aminoglycosides was due to the presence of the aph(3′)-Ia in E. coli (31/264, 11.7%) and in P. mirabilis (12/33, 36.3%), to the presence of aac(3)-IIa in E. coli (37/264, 14%) and to the presence of aac(6′)-Ib (5/16, 31.2%) in Klebsiella species (Table 2).

Table 2.

Aminoglycosides-Modifying Genes Present in the Enterobacteriaceae Isolates Studied

		Bacterial species												Resistance to
AME gene	Number of isolates	E. coli	P. mirabilis	K. pneumoniae	K. oxytoca	E. cloacae	M. morganii	P. stuartii^a	S. enterica	S. sonnei	P. vulgaris	C. freundii	C. koseri	AMP	AMC	PIP	CEF	FOX	CXM	CAZ	CTX	CPM	ATM	NAL	CIP	SXT	FOS	NIT	ESBLs/pAMPC
aph(3″)-Ib	122	112	3	2	1	3						1		106	12	95	56	10	13	1	5			60	49	89	4	10	CTX-M-14 (4); CMY-2 (1)
ant(3″)-Ia	56	33	10	3	2	1		3	2	1	1			39	8	32	20	4	8	5	6	3	6	25	14	33	2	20	CTX-M-1 (3)
ant(3″)-Ia+aph(3″)-Ib	35	27	7	1										31	2	23	13	4	7	1	3	1	1	18	12	26		8	CTX-M-14 (2); CTX-M-1 (1)
aph(3′)-Ia+aph(3″)-Ib	16	13	3											15	3	10	5	2	2					12	7	16	2	4
aac(3)-IIa	13	13												12	1	10	9	2	3					12	11	2
aac(3)-IIa+aph(3″)-Ib	13	13												13	2	11	8	1	1					13	13	11		1
ant(3″)-Ia+aph(3′)-Ia+aph(3″)-Ib	7	6	1											7	3	6	4	2	3		2	1	2	6	6	7	1	2	CTX-M-14 (1); CTX-M-9 (1)
aph(3′)-Ia	5	4					1							5	2	4	2		1					3	3	3	1	2
aac(6′)-Ib+ant(3″)-Ia	3				3									3	3	3	3	2	1	3	3	3	3	3	3	3	2	1
ant(3″)-Ia+aph(3′)-Ia	4	2	1				1							2	1	1	2		1					3	2	3	1	2
aac(3)-IIa+ant(3″)-Ia+aph(3″)-Ib	4	4												4		4	4		1		1		1	3	2	4			CTX-M-32 (1)
aac(3)-IIa+aph(3′)-Ia+aph(3″)-Ib	4	4												4	1	4	2	1	1	1	1		1	2	2	4		1	CMY-4 (1)
aac(6′)-Ib	3	1		2										3	3	3	3		3	2	3		3	3	3	2	1	2	CTX-M-15 (3)
ant(2″)-Ia+ant(3″)-Ia	3	3												3		2	3		2	1	2		1	3	1	1			CTX-M-9 (1)
ant(2″)-Ia+ant(3″)-Ia+aph(3″)-Ib	3	2	1											3	2	3	2	2	2	2	2		2	3	3	3	1	2	CMY-2 (1)
Others^b	23	13	6	2		1	1							23	9	19	12	3	7	6	7	3	5	16	11	12	3	8	CTX-M-3 (1); CTX-M-9 (1);CTX-M-15 (1); VEB-4 (1)
Unresolved	16	14	1										1	14	2	13	9	0	2	1	1	0	1	10	3	8	0	2	SHV-12 (1)
Total strains	330	264	33	10	6	5	3	3	2	1	1	1	1	287	54	243	157	33	58	23	36	11	26	195	145	227	18	65
%		80	10	3	1.8	1.5	0.9	0.9	0.6	0.3	0.3	0.3	0.3	86.9	16.3	73.6	47.6	10	17.6	7	11	3.3	7.9	59	44	68.8	5.4	19.7

These strains showed its natural aac(2′)-Ia gene.

Other combinations found were aac(3)-IVa+aph(3″)-Ib; ant(2″)-Ia+ant(3″)-Ia+aph(3′)-Ia+aph(3″)-Ib; aph(3″)-Ib+aph(3′)-IIa;

aph(3′)-Ia+streptomycin resistance; aph(3′)-IIa; aac(3)-IIa+aac(6′)-Ib; aac(3)-IIa+aac(6′)-Ib+ant(3″)-Ia+aph(3′)-Ia+aph(3″)-Ib; aac(3)-IIa+aac(6′)-Ib+aph(3″)-Ib; aac(3)-IIa+ant(2″)-Ia+aph(3″)-Ib; aac(3)-IIa+ant(3″)-Ia; aac(3)-IIa+ant(3″)-Ia+aph(3′)-Ia+aph(3″)-Ib;

aac(3)-IIa+aph(3′)-Ia; aac(6′)-Ib+ant(2″)-Ia+ant(3″)-Ia; aac(6′)-Ib+ant(2″)-Ia+aph(3′)-Ia+aph(3″)-Ib; aac(6′)-Ib+aph(3″)-Ib; aac(6′)-Ib+streptomycin resistance; ant(2″)-Ia+ant(3″)-Ia+aph(3′)-Ia; ant(2″)-Ia+aph(3′)-Ia+aph(3″)-Ib.

E. coli, Escherichia coli; P. mirabilis, Proteus mirabilis; K. pneumoniae, Klebsiella pneumonia; E. cloacae, Enterobacter cloacae; M. morganii, Morganella morganii; P. stuartii, Providencia stuartii; S. enterica, Salmonella enterica; S. sonnei, Shigella sonnei; P. vulgaris, Proteus vulgaris; C. freundii, Citrobacter freundii; C. koseri, Citrobacter koseri.

Following AME genes detection, we analyzed the correlation between the breakpoints proposed by CLSI and the halos that we had obtained for the aminoglycosides tested. We observed that in more than 80% of cases, the strains carrying aac(3)-IIa and aac(6′) -Ib genes did not express the complete expected phenotype (KGTN and KTAN, respectively) because the diameters of the inhibition (φ) found for tobramycin, netilmicin, or amikacin, were higher than the CLSI cut-off. Only gentamicin was clearly resistant in all cases (Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/mdr).

Breakpoints have not yet been established for spectinomycin and neomycin. Our results showed that inhibition diameters for spectinomycin ranged from 9 to 29 mm (φ₅₀ 24 mm and φ₉₀ 27 mm) in 56 strains carrying ant(3″)-Ia, and from 22 to 48 mm in 143 strains without ant(3″)-Ia (Supplementary Table S1). In the case of neomycin, the five strains carrying aph(3′)-Ia showed diameters of 13 mm or less (Supplementary Table S1).

The numbers underlined in Supplementary Table S1 indicate reduced susceptibility to other aminoglycosides not justified by the presence of an AME gene. This reduced susceptibility could be explained by the presence of other nonenzymatic mechanisms such as alterations in permeability.

Thirty-four percent of the strains presented more than one gene (Supplementary Table 2). The most frequent association was ant(3″)-Ia plus aph(3″)-Ib (35 strains). This association can also be found alone or together with aac(3)-IIa (four strains) or aph(3′)-Ia (seven strains). ant(2″)-Ia was always associated with other AME genes. Also of note was the presence of four or five AME genes in five P. mirabilis strains (Table 2).

Table 2 also shows resistance to β-lactams, quinolones, and other antimicrobial drugs associated with aminoglycoside resistance. Twenty-two of the 330 AME-producing strains also produced an extended-spectrum-β-lactamase (ESBL) (seven CTX-M-14, four CTX-M-15, four CTX-M-1, three CTX-M-9, one CTX-M-3, one CTX-M-32, one SHV-12, and one VEB-4) and three produced plasmid-mediated AmpC β-lactamases (two CMY-2 and one CMY-4). More than 50% of the strains were also resistant to quinolones or co-trimoxazole.

To characterize the plasmids involved in AME gene diffusion, we selected 48 strains carrying 66 different AME genes. Out of these genes, 24 were located in a plasmid: 12 aac(3)-IIa, six aph(3′)-Ia, three ant(3″)-Ia, two ant(2″)-Ia, and one aac(6′)-Ib (Supplementary Table S2). These plasmids belonged to incompatibility groups F, FIA, FIB, or HI2. Different AME genes were found in the same plasmid in only four strains (Supplementary Table S2). In the other 24 genes, either no bands were observed in the S1-PFGE gel, or the bands that were present did not hybridize with the different probes tested.

Discussion

The level of aminoglycoside resistance in Enterobacteriaceae has changed little over the past few decades. We found approximately half the isolates were resistant to aminoglycosides, but about 90% of these were resistant to streptomycin and spectinomycin, drugs not used in clinical practice. AME genes responsible for streptomycin resistance were aph(3″)-Ib and ant(3″)-Ia, with a frequency of 65.4% and 37.5%, respectively. These percentages are higher than those found in the literature.^13,14 It should be noted that the selection criteria may be different in each study and that hybridization methods were used rather than PCR.

Regarding aminoglycosides used in clinical practice (amikacin, gentamicin, or tobramycin), we found percentages of resistance similar to those described in an European SENTRY surveillance study in Enterobacteriaceae, that showed percentages of resistance between 0.4% to 3% for amikacin, 2% to 13% for gentamicin, and 2.5% to 15.3% for tobramycin.¹¹ We found that the mechanisms responsible for this resistance were aac(3′)-IIa, followed by aac(6′)-Ib and ant(2″)-Ia.

aph(3′)-I was identified on plasmids and transposons in many gram-negative bacteria.¹² The high frequency of occurrence described for this gene (up to 46% in 1993) resulted in the clinical obsolescence of kanamycins.¹⁴ The subsequent lack of use of kanamycin as a therapeutic option has resulted in a corresponding decline in the importance of these enzymes in clinical isolates as we can confirm, because in our study in 2006 the prevalence detected was 13.9%.

Another enzyme whose frequency has decreased is AAC(3)- II. In 1993, Shaw et al.¹³ described a prevalence of AAC(3)- II of 60.3% in 2445 strains of gram-negative bacteria, whereas we found a prevalence of only 14%.

Among the AAC(6′), the most prevalent enzyme was AAC(6′)-Ib.^13,14 These enzymes are important because they are among the few that confer resistance to amikacin, and they can also confer resistance to quinolones. In our case, 1.5% of studied strains were amikacin resistant and all of them produced the AAC(6’)-Ib enzyme, emphasizing that most of them produced the–cr variant that also confers resistance to quinolones.

In a recent study⁷ conducted in 116 strains of E. coli and K. pneumoniae isolated from blood cultures from 2000 to 2009, 49 strains were aminoglycoside resistant, the authors found a prevalence of 79.3% for aac(3)-IIa, 37.9% for aac(6′)-Ib, and 28.4% harbored both genes.

The ANT(2″)-Ia enzyme is widespread among gram-negative bacteria and some authors relate its prevalence to the use of gentamicin. Vakulenko et al.¹⁴ stated that this enzyme is more frequent in countries that use gentamicin more frequently than amikacin. Galimand et al.⁶ found a prevalence of ANT(2″) of about 11%, whereas in our hospital, this prevalence was lower (3.6%), and curiously, the enzyme was always associated with other AME.

Several surveys have shown that most of the resistant strains carry combinations of several aminoglycoside resistance mechanisms.^10,12,14 Thirty-four percent of our strains expressed more than one enzyme, and in one P. mirabilis strain we found as many as five AME. The most frequent association we found was ANT(3″)-Ia plus APH(3″)-Ib. It can also be found alone or together with AAC(3)-IIa or with APH(3′)-Ia.

Most of the 16 strains resistant to aminoglycosides without AME genes were resistant to streptomycin. One mechanism of aminoglycoside resistance is reduced drug uptake, due, for example, to membrane alterations. This reduced uptake is clinically relevant since it affects all aminoglycosides and results in a moderate level of resistance (intermediate susceptibility). Another mechanism of resistance that could affect aminoglycosides is the active efflux of these drugs. This mechanism has been related to neomycin and kanamycin resistance but its clinical significance is still uncertain.¹³

A third enzymatic resistant mechanism is the methylases, enzymes that confer a high level of resistance to all aminoglycosides.⁵ As we did not detect any strain with this resistant pattern we did not test these enzymes in the present study.

The interpretative antibiogram for β-lactams has been reasonably established in the literature but the interpretative antibiogram for aminoglycosides is not so clear. The aminoglycosides used in the susceptibility test of gram-negative bacilli are usually gentamicin, tobramycin, and amikacin, and these three aminoglycosides proved to be sufficient to suspect the genes that we found most frequently [aac(3)-II, aac(6′)-I, and ant(2″)-Ia]. Nevertheless, in most cases, the inhibition zones found for tobramycin, amikacin, and netilmicin were outside the cut-off values established by CLSI breakpoints. Therefore, other values perhaps need to be established for these drugs.

Another important open question is whether it is convenient to modify the antibiogram for each aminoglycoside when a specific enzyme is suspected. Additional clinical studies should be performed to clarify this point, and as these antimicrobial agents are used in combined therapies in most cases, conclusions will be difficult to obtain.

There are no specific associations between the genes involved in aminoglycoside resistance and other antibiotic resistance genes.³ But among the 330 aminoglycoside-modifying-producing strains characterized in this study, 6.7% were ESBLs-producing, 0.9% produced plasmid-mediated AmpC β-lactamases, and more than 50% were also resistant to quinolones or co-trimoxazole.

Many of the AME genes are located in transposable genetic elements or integron sequences.^10,13,14 However, few data in the literature have described the kind of plasmids that carry these genes. Witchitz et al.¹⁶ found that the plasmids mediating gentamicin resistance belonged to the incompatibility group 6-C or 7-M. Our results showed that most plasmids implicated in aminoglycoside resistance belong to the F, FIA, or FIB incompatibility groups and are not related to the plasmids harboring ESBLs or plasmid mediated AmpC- β-lactamases. IncF plasmids are low copy-number plasmids that often carry more than one replicon and are common in naturally occurring fecal flora of humans and animals, regardless of resistance genes.¹

In conclusion, the prevalence of,AME has not significantly changed over the years, with the exception of AAC(6′)-Ib, the enzyme responsible for amikacin resistance, the frequency of which has doubled. Finally, we detected aminoglycoside-modifying genes in multireplicon IncF plasmids, seen to be widely distributed in clinical and environment settings.

Footnotes

Acknowledgments

We thank C. Newey for revising the English. This study was partially supported by the Ministry of Health and Consumer Affairs, Instituto de Salud Carlos III—FEDER, Spanish Network for the Research in Infectious Diseases (REIPI RD06/0008), and by the grant from the “Fondo de Investigación Sanitaria” (PS05/1751).

Disclosure Statement

The authors report no conflicts of interest relevant to this article.

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