Genetic variability among ampC genes from acinetobacter genomic species 3

Abstract

As a part of a nationwide study in Spain, 15 clinical isolates of Acinetobacter genomic species 3 (AG3) were analyzed. The main objective of the study was to characterize the ampC genes from these isolates and to determine their involvement in beta-lactam resistance in AG3. The 15 AG3 isolates showed different profiles of resistance to ampicillin (range of MICs, 12 to >256 microg/ml). Nucleotide sequencing of the 15 ampC genes yielded 12 new AmpC enzymes (ADC-12 to ADC-23). The 12 AG3 enzymes showed 93.7 to 96.1% amino acid identity with respect to the AmpC enzyme from Acinetobacter baumannii (ADC-1 enzyme). Eight out of fifteen ampC genes were expressed in Escherichia coli cells under the control of a common promoter, and with the exception of one isolate (isolate 65, which showed lower beta-lactam MICs), significant differences in overall beta-lactam MICs for E. coli cells expressing AG3 ampC genes were not revealed. No significant differences in ampC gene expression in AG3 clinical isolates were revealed by reverse transcription-PCR analysis. A detailed analysis of the 12 AmpC protein sequences revealed that amino acid replacements (in comparison with those of ADC-1) occurred mainly in the same positions, although none were located in important functional domains such as the Omega- loop or conserved beta-lactamase motifs. Kinetic experiments performed with three representative AmpC enzymes (ADC-14, -16, and -18) in some cases revealed dramatic changes in K(m) and k(cat) values for beta-lactams. No ISAba1 was detected upstream of the ampC genes. Our results reveal 12 new ampC genes in AG3. The enzymes showed a moderate degree of variability, and they are tentatively named ADC-12 to ADC-23.

FIG. 1.

Amino acid sequence alignment among the 12 AmpC β-lactamases from AG3 (ADC-12 to -23) with respect to that of A. baumannii (ADC-1). Amino acid differences are indicated. The typical β-lactamase domains (SVSK, YSN, and KTG) and the Ω-loop are double underlined. The amino acid replacements present in at least four or more proteins (relative to that of ADC-1) with more than one residue are underlined. The vertical arrow indicates the position of the +1 amino acid (after cleavage of signal peptide). ADC-1, AmpC from A. baumannii (see reference 6).

FIG. 2.

Western blotting with anti-ADC-7 antibody and with protein extracts (10 μg) obtained from the 15 different AG3 isolates indicated above the gel. Purified ADC-7 enzyme (15 ng) (a gift from R. A. Bonomo) (C+) and protein extracts of an Acinetobacter sp. isolate overexpressing the AmpC enzyme (isolate 92) were included as positive controls.

FIG. 3.

Theoretical model of the crystallographic structure of the ADC-12 AmpC β-lactamase. Amino acids belonging to the main β-lactamase domains (SVSK, YSN, and KTG) are represented in the figure as blue circles (60-63SXXK, Y146, and K308). The SVSK domain is also shown in green. The Ω-loop is shown in turquoise. Residues at positions 1, 17, 37, 51, 75, 78, 82, 122, 139, 177, 229, 242, and 275 (Fig. 1), which were replaced in four or more ADC-type enzymes (relative to ADC-1), are represented by orange circles (starting at position +1 of the mature protein with D). The diagram was drawn with the USCF Chimera software package.