Genetic and Functional Variability of AmpC-Type β-Lactamases from Acinetobacter baumannii

Abstract

The incidence of naturally occurring AmpC β-lactamases with extended activities toward several cephalosporins was evaluated among 17 ceftazidime (CAZ)-resistant Acinetobacter baumannii isolates. Five AmpC β-lactamases (named ADC β-lactamases) were identified, among which those possessing the Val208Ala (inside the omega-loop) or Asn283Ser (helix H-10) substitution conferred higher levels of resistance (4- to 64-fold higher) to CAZ and to cefotaxime in Escherichia coli. This study demonstrates that peculiar AmpCs playing a role in resistance to broad-spectrum cephalosporins in A. baumannii may be identified.

Acinetobacter baumannii is commonly associated with serious nosocomial infections (4, 10, 14). A growing number of β-lactamases conferring resistance to broad-spectrum cephalosporins in Acinetobacter spp. have been identified. Even though resistance to oxyiminocephalosporins (ceftazidime and cefotaxime) may be related to production of extended-spectrum β-lactamases (ESBLs), it is usually associated with overproduction of an AmpC-type β-lactamase (1, 2, 9). Overexpression of the bla_AmpC gene of A. baumannii may occur as a result of an insertion sequence, ISAba1, providing strong promoter sequences (5, 8, 13), being located upstream.

Most AmpC-type β-lactamases naturally produced by Gram-negative bacteria hydrolyze amino- and ureidopenicillins, cephamycins (cefoxitin and cefotetan), and, at a low level, oxyiminocephalosporins, such as ceftazidime, cefotaxime, and ceftriaxone, and monobactams, such as aztreonam (3). AmpCs possessing a broad substrate activity have been reported in Enterobacteriaceae and Pseudomonas aeruginosa (11, 12, 16, 19, 20). These AmpCs (or extended-spectrum AmpCs [ESACs]) with activities against several cephalosporins may confer reduced susceptibility to those molecules (6, 11, 12, 19). They differ from “regular” cephalosporinases by amino acid substitutions or insertions/deletions in four specific regions that are all located in the vicinity of the active site: the Ω loop, the H-10 helix, the H-2 helix, and the C-terminal extremity of the protein (15).

Recently, we have identified the first ESAC (named ADC-33, according to the numbering of AmpC β-lactamases from A. baumannii [9]) from a single A. baumannii isolate (21). ADC-33, possessing a Pro210Arg substitution together with a duplication of an Ala residue at position 215 (inside the Ω loop), hydrolyzed ceftazidime, cefepime, and aztreonam at high levels (21).

The present study aimed to evaluate the extent of the variability of ADC β-lactamases in A. baumannii. Seventeen nonrepetitive A. baumannii clinical isolates that were recovered in 2007-2008 from patients hospitalized at the Bicêtre Hospital and were resistant to ceftazidime were included in this study. Genotyping was performed by using pulsed-field gel electrophoresis (PFGE) according to the instructions of the manufacturer (Bio-Rad, Marnes-la Coquette, France) as previously described (17, 22), and it identified eight distinct clones among the 17 isolates (Table 1).

TABLE 1.

Features of the A. baumannii isolates studied^d

Clone (PFGE)	AmpC overproducerb	ISAba1-blaADC colinearity	ADC variant	OXA-51-like variant	MIC (μg/ml)c
					CAZ	CTX	FEP	CPO	ATM	IPM
A (strain CIP)a	−	−	ADC-50	OXA-64	2	8	1	2	8	0.25
B (strain AYE)	+	+	ADC-11	OXA-69	>256	>256	>256	>256	>256	1
C	+	+	ADC-51	OXA-69	>256	>256	64	>256	>256	>32
D	+	+	ADC-30	OXA-66	128	>256	32	256	32	4
E	−	−	ADC-52	OXA-90	32	64	32	64	128	0.25
F	+	+	ADC-30	OXA-66	64	>256	32	128	128	2
G	+	+	ADC-51	OXA-69	>256	>256	32	>256	>256	>32
H	+	+	ADC-30	OXA-66	64	>256	32	64	128	2
I	+	+	ADC-26	OXA-69	32	>256	16	64	32	2
J	+	+	ADC-53	OXA-51	64	>256	8	32	32	>32

^aClones A and B correspond to A. baumannii CIP7010 and A. baumannii AYE, coding for regular AmpC β-lactamases.

^bAmpC overproduction evaluated by cloxacillin test (250 μg/ml).

^cCAZ, ceftazidime; CTX, cefotaxime; FEP, cefepime; CPO, cefpirome; ATM, aztreonam; IPM, imipenem.

^dThere is no ISAba1-bla_OXA-51-like colinearity among any of the clones in the table.

In addition to being resistant to ceftazidime, most of the clones were resistant to cefotaxime, ceftriaxone, aztreonam, cefepime, and cefpirome (Table 1). By use of cloxacillin-containing plates as described previously (17), the susceptibility to ceftazidime was recovered for most of the clones (excluding clones C, E, and G), indicating that overproduction of the ADC β-lactamase was responsible for the resistance to ceftazidime. Double-disk synergy tests using clavulanic acid-containing disks were performed as described previously (17) for those isolates belonging to clones C, E, and G, but no ESBL production was detected.

Insertion sequence ISAba1, providing strong promoter sequences (−35 [TTAGAA] and −10 [TTATTT]) immediately upstream of the bla_AmpC gene, was identified in almost all clones, similar to previous observations (8).

Analysis of the naturally occurring carbapenem-hydrolyzing bla_OXA-51-like sequences was also performed as described previously (Table 1) (7, 18). No colinearity between ISAba1 and the bla_OXA-51-like genes was observed in any of these isolates.

The bla_AmpC genes of the A. baumannii isolates were sequenced and cloned with or without the P_ISAba1 promoter as described previously (21). The inserts containing the P_ISAba1 promoter were further named with “P+.” The A. baumannii AYE clinical isolate and A. baumannii CIP7010 reference strain were used to clone wild-type bla_ADC genes expressing ADC-11 and ADC-50, respectively. Five ADC variants (ADC-26, ADC-30, ADC-51, ADC-52, and ADC-53) were identified (Fig. 1), with ADC-51, ADC-52, and ADC-53 corresponding to newly identified enzymes. Amino acid changes were not identified either in the SVSK, YSN, and KTG conserved motifs or in helix H-2, previously associated with the extended activity of ESACs identified in other Gram-negative species (11, 15). However, substitutions Val208Ala located in the omega loop of ADC-53 and Asn283Ser located in helix H-10 of ADC-51 were identified.

FIG. 1.

Amino acid sequence alignment, including ADC-7 taken as a reference sequence as previously published (9) and ADC-11 and ADC-50 as wild-type ADCs, together with the newly characterized ADC-26, ADC-30, ADC-51, ADC-52, and ADC-53. Identical amino acids are indicated by dashes. The typical ADC β-lactamase domains (SVSK, YSN, and KTG) are underlined. Helices H-2 and H-10 are boxed in gray. The Ω loop is boxed in gray and double underlined. Differences observed inside the Ω loop and helix H-10 are in bold. The vertical arrow indicates the position of the +1 amino acid (cleavage site for signal peptide). Numbering is according to the sequence of the mature protein. Asterisks indicate ADC β-lactamases possessing ESAC properties.

Subsequent cloning showed that MICs of broad-spectrum cephalosporins differed significantly among the recombinant Escherichia coli strains that did not include the strong P_ISAba1 promoter upstream of the bla_ADC genes (Table 2). Higher MICs of ceftazidime were observed for E. coli TOP10(pADC-30), E. coli TOP10(pADC-51), and E. coli TOP10(pADC-53), producing β-lactamases ADC-30, ADC-51, and ADC-53, respectively.

TABLE 2.

MICs of β-lactams for E. coli TOP10 strains harboring recombinant plasmids and a TOP10 reference strain

β-Lactam(s)a	MIC(μg/ml) for E. coli TOP10 with:
	pADC-26	pADC-30	pADC-51	pADC-52	pADC-53	pADC-11	No plasmid
Amoxicillin	>256	>256	32	256	>256	>256	2
Amoxicillin + CLA	>256	>256	32	256	>256	256	2
Ticarcillin	8	8	256	8	16	8	2
Ticarcillin + CLA	8	8	256	8	16	8	2
Piperacillin	8	8	4	8	16	8	1
Piperacillin + TZB	8	8	4	8	16	8	1
Cefuroxime	64	>256	64	64	32	128	2
Ceftazidime	0.5	2	32	0.5	4	0.5	0.125
Cefotaxime	0.25	1	4	0.5	2	0.5	0.06
Cefepime	0.03	0.03	0.06	0.03	0.03	0.032	0.03
Cefpirome	0.03	0.03	0.06	0.03	0.03	0.032	0.03
Aztreonam	0.125	0.25	0.25	0.125	0.5	0.094	0.03
Imipenem	0.25	0.25	0.25	0.25	0.5	0.19	0.125
Meropenem	0.023	0.023	0.047	0.023	0.023	0.023	0.03

^aSusceptibility testing performed by using Etest strips (AB bioMérieux, Solna, Sweden). CLA, clavulanic acid (4 μg/ml); TZB, tazobactam (4 μg/ml).

MICs of ceftazidime, cefotaxime, and aztreonam were higher for the clones expressing ADC-30, ADC-51, and ADC-53, suggesting that they might correspond to ESACs (Table 3).

TABLE 3.

MICs of β-lactams for E. coli TOP10 strains harboring recombinant plasmids and a TOP10 reference strain

β-Lactam(s)a	MIC(μg/ml) for E. coli TOP10 with:
	pADC-26-P+	pADC-30-P+	pADC-51-P+	pADC-53-P+	pADC-11-P+	No plasmid
Amoxicillin	>256	>256	>256	>256	>256	2
Amoxicillin + CLA	>256	>256	256	>256	>256	2
Ticarcillin	128	128	>256	>256	256	2
Ticarcillin + CLA	128	128	>256	>256	256	2
Piperacillin	>256	256	>256	>256	>256	1
Piperacillin + TZB	256	128	128	256	256	1
Cefuroxime	>256	>256	>256	>256	>256	2
Ceftazidime	8	64	>256	256	8	0.125
Cefotaxime	8	64	>256	128	8	0.06
Cefepime	0.125	0.25	0.5	0.25	0.125	0.03
Cefpirome	0.125	0.25	2	0.5	0.125	0.03
Aztreonam	2	8	8	16	2	0.06
Imipenem	0.25	0.5	0.25	0.25	0.25	0.125
Meropenem	0.03	0.03	0.03	0.03	0.03	0.03

^aSusceptibility testing performed by using Etest strips (AB bioMérieux, Solna, Sweden). CLA, clavulanic acid (4 μg/ml); TZB, tazobactam (4 μg/ml).

In order to better evaluate the impact of the ADC variants with extended activities (ADC-30, ADC-51, and ADC-53), recombinant plasmids containing the ISAba1-bla_ADC fragments were electrotransformed into E. coli HB4 with permeability defects associated with a lack of OmpC and OmpF porins (12). Besides that of ceftazidime, the MICs of cefepime, cefpirome, and aztreonam were higher for the ADC-51 producer (16, 64, and 32 μg/ml, respectively) than for the wild-type ADC-26 producer (2, 4, and 8 μg/ml, respectively).

Several amino acid substitutions were identified here among ADC β-lactamases (Fig. 1). These residues were located at positions 188, 242, 255, 274, 275, and 313 (Fig. 1), but none of those was located in domains supposed to be crucial for β-lactamase activity, such as the Ω loop, helix H-2, or helix-H10. A steric view of these substitutions in the overall fold structure of the modeled wild-type ADC-26 variant seems to indicate that these amino acid substitutions are located far from the active site of the enzyme (data not shown). In an analysis of sequences of ADC variants possessing ESAC properties, compared to those of wild-type β-lactamases ADC-11 and ADC-26, it is interesting that both ADC-51 and ADC-53 variants possess a Val208Ala substitution located in the omega loop and an Asn283Ser substitution located in helix H-10, respectively. These substitutions may explain their extended-spectrum activity. However, ADC-30 possesses two specific residues relative to the other ADC sequences (Lys122 and Ser139) and a Thr at position 313 that was identified in the ADC-53 ESAC. Whether those unusual amino acid substitutions (alone or combined) might play a significant role in the broadened activity of ADC-30 remains to be determined.

We report here the identification and characterization of five bla_ADC genes from ceftazidime-resistant A. baumannii isolates. Three of them encode novel ADC variants and three of them possess significant extended activity toward ceftazidime and cefotaxime. After the identification of ESACs in Enterobacteriaceae, the demonstration of their wide diffusion among P. aeruginosa, and the recent and initial finding of such an enzyme in A. baumannii, further investigations are required to evaluate whether ADC β-lactamases with extended activities might be widely distributed in A. baumannii.