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JAC Advance Access originally published online on April 2, 2007
Journal of Antimicrobial Chemotherapy 2007 59(5):996-1000; doi:10.1093/jac/dkm070
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Molecular characterization of the gene encoding a new AmpC ß-lactamase in Acinetobacter baylyi

Alejandro Beceiro, Francisco J. Pérez-Llarena, Astrid Pérez, Ma del Mar Tomás, Ana Fernández, Susana Mallo, Rosa Villanueva and Germán Bou*

Servicio de Microbiología-Unidad de Investigación, Complejo Hospitalario Universitario Juan Canalejo, Xubias de Arriba 84, 15006 La Coruña, Spain

* Corresponding author. Tel: +34-981-178359; Fax: +34-981-178216; E-mail: germanbou{at}canalejo.org

Received 27 October 2006; returned 16 December 2006; revised 29 January 2007; accepted 12 February 2007


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Objectives: The main objective of the present study was to demonstrate the presence of a ß-lactamase ampC gene in the chromosome of the non-pathogenic bacterium Acinetobacter baylyi ADP1.

Methods: ß-Lactam MICs were determined by Etest. The ampC gene was amplified by PCR, with specific oligonucleotides, then cloned into pBGS18 and pAT-RA plasmids and transformed into Escherichia coli TG1 and parental A. baylyi as hosts. The gene was sequenced and analysed. The AmpC protein was expressed, purified by affinity chromatography and the kinetic parameters determined.

Results: An ampC gene was amplified from the ADP1 genome. Sequencing of the gene showed typical SVSK and KTG domains and the typical YXN Class C motif. The amplified gene showed significant identity (48.5% to 49.3%) with the AmpC enzymes of Acinetobacter baumannii and AG3 strains, which have recently been renamed ADC-1 to ADC-7. MIC analysis revealed a cephalosporinase profile for the E. coli TG1 clone as well as for the parental A. baylyi strain that overexpressed the ampC gene cloned under the control of an external promoter. Analysis of kinetic parameters of the purified enzyme showed higher catalytic efficiency for cefalotin than for ampicillin.

Conclusions: This study represents the first report of an AmpC ß-lactamase in A. baylyi, which was shown by biochemical and microbiological experiments to have a typical cephalosporinase profile. The presence of the respective gene in the chromosome of A. baylyi ADP1 suggested that this ampC gene is the naturally occurring cephalosporinase in this species, as previously reported for other Acinetobacter spp. We tentatively named the enzyme ADC-8.

Keywords: A. baylyi , cephalosporinase , ADC-type


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The bacterial genus Acinetobacter consists of strictly aerobic Gram-negative coccobacilli, which are oxidase-negative, non-motile, nitrate-negative and non-fermentative. In this genus, at least 21 DNA homology groups or genomic species have been described on the basis of the results of DNA–DNA hybridization studies.1 A new genomic species, Acinetobacter baylyi, which has recently been separated from Acinetobacter calcoaceticus, has been reported.2 The species is frequently used in experimental research because of its high capability for natural transformation. The genome of A. baylyi (ADP1) has recently been published.3 Because of the good potential of this microorganism as a model for genetic studies in Acinetobacter spp., we decided to investigate putative genes involved in ß-lactam resistance.

The most common mechanism of resistance of Acinetobacter baumannii to ß-lactam antibiotics is attributed to the presence of a chromosomal cephalosporinase.4 Several allelic variants of the A. baumannii AmpC enzyme have also been reported.5 Recently, a uniform designation for this family of cephalosporinases has been reported5 [Acinetobacter-derived cephalosporinases (ADC)], with AmpCs of A. baumannii designated as ADC-1, -3, -4, -6 and -7, the Acinetobacter G3 AmpC as ADC-5 and one closely related AmpC found in Oligella urethralis as ADC-2.

In the present study, we investigated A. baylyi strain ADP1 for the presence of an ampC gene that may be involved in ß-lactam resistance.


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Susceptibility testing, cloning and expression of the ampC gene in Escherichia coli

The susceptibility testing of isolates used in this study was performed by Etest (AB Biodisk, Solna, Sweden).

We used the sequence of the ampC gene of the A. baumannii RyC52763/97 strain4 for comparison with the whole genome of A. baylyi ADP1 to detect putative homologues to the A. baumannii ampC gene. This search yielded the presence of a putative ampC gene named ACIAD3597. To clone this gene, chromosomal DNA from strain ADP1 was purified by use of standard protocols (MasterPure DNA Purification Kit, Epicentre, Madison, WI, USA). Five hundred nanograms of the ADP1 chromosomal DNA was used as a template for amplification by PCR with the oligonucleotides (Sigma-Genosys Ltd, UK) P1 (5'-aaggatccATGATGAAAGACATATTAGGTAA-3') and P2 (5'-aaagaattcTCGTGGACGTAAACGTTCG-3'), which hybridize in the ADP1 ampC gene (including the ATG of the coding sequence and part of the 3'-untranslated region). The PCR was performed under standard conditions. Then, the amplified fragment was cloned into the BamHI and EcoRI restriction sites of pBGS18 plasmid.6 The plasmid was electroporated into E. coli TG1 and transformants selected on Luria– Bertani (LB) plates with 40 mg/L kanamycin. This plasmid construct was named pAB-1. Another two different genetic constructs were achieved by use of the pBGS18 plasmid as vector: (i) ampC open reading frame (ORF) from ADP1 with an extensive part of its own promoter (pAB-2 plasmid), amplified with primers P3 (5'-aaaggatccAACTCTGGGTGGTTGAGAT-3'), which includes 442 bp of promoter region upstream of the translation initiation codon of the ampC gene, and P2 and (ii) ampC ORF amplified with the oligonucleotides P1/P2 and cloned under the strong promoter from the ß-lactamase CTX-M-14 gene (positions 1501–1740 of the accession number AF252622 [GenBank] ) and yielding the plasmid pAB-3.

Cloning and expression of the ampC gene in A. baylyi

As A. baylyi ADP1 was found to be susceptible to ß-lactam antibiotics, we aimed to restore the ß-lactam resistance phenotype by plasmid transformation with the microorganism's own ampC gene. For this, the ampC gene from A. baylyi strain ADP1 was cloned into the shuttle pAT-RA plasmid (made of part of pUC18 and pWH1266)7 with two different constructs: (i) with its own promoter (442 bp upstream of ATG) amplified with P3/P2 oligonucleotides and (ii) ampC ORF (amplified with P1/P2 primers) cloned with the previously reported recombinant plasmid which harbours the ß-lactamase CXT-M-14 gene promoter8 to yield the pAT-RA-ampC1 and pAT-RA-ampC2 plasmids, respectively. Recombinant plasmids were then transformed by electroporation in A. baylyi ADP1. Transformants were selected with LB agar plates supplemented with rifampicin at 20 mg/L.

IEF analysis, purification and biochemical properties

ß-Lactamases were analysed by isoelectric focusing (IEF) as described previously.4

To purify the AmpC enzyme, the blaampC gene was cloned in the pGEX-6P-1 (Amersham Pharmacia Biotech Europe GmbH, Barcelona, Spain) vector (EcoRI/SmaI) to generate a fusion protein from glutathione S-transferase and the AmpC enzyme. The oligonucleotides used for ampC amplification (lacking the signal peptide) were P4 (5'-aaagaattcCAATCGACAGTCCAACAAT-3') and P5 (5'-aaacccgggAATCATTTCTGAATATCTGC-3'). After cleavage with PreScission protease, the purified proteins appeared on SDS–PAGE as a band of 40 kDa (≥95% purity). Kinetic experiments were performed and IC50 (ß-lactamase inhibitor concentration that reduces 50% of the enzymatic activity) values determined as described previously.4

RT–PCR ampC gene expression

To confirm increased expression of the ampC gene, the original ADP1 as well as ADP1 with different plasmids were used in a real-time RT–PCR to determine the expression of ampC ß-lactamase gene relative to the house-keeping gene for the isoleucyl-tRNA synthetase gene. Total RNA was isolated using a TRIzol Max Bacterial RNA Isolation Kit (Invitrogen, Carlsbad, CA, USA), and its integrity was checked by electrophoresis on 1% (w/v) agarose gel. One microgram of RNA was reverse transcribed into single-stranded cDNA by use of a high-capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA). The cDNAs were subsequently quantified by real-time PCR amplification (annealing temperature 50ºC) with primers P6 (5'-ATGATGAAAGACATATTAGG-3')/P7 (5'-GTTGCCCATTTATGCTG-3') and P8 (5'-GTCGGTTTTGGCGTGG-3')/P9(5'-GATTTGTGGGTTGGCTT-3') for the ampC gene and the house-keeping gene, respectively; this yielded products of 202 and 535 bp. The results obtained are shown as the mean values of three independent experiments.

Induction experiments

Induction experiments with cefoxitin (at 8 mg/L) were performed with the ADP1 strain. The activity of the AmpC ß-lactamase was measured as the specific hydrolysis of 25 µM nitrocefin.


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Amplification of the ampC gene of A. baylyi ADP1 yielded a product of ~1.2 kbp, which corresponded to the theoretical size of the ampC gene. The entire sequence of the fragment contained one ORF of 1194 bp (397 amino acids long), which showed 100% identity to that described in the genome of A. baylyi ADP1 (the GenBank accession number for the AmpC ß-lactamase of ADP1 is AM293332 [GenBank] ). GenBank database searches with this protein revealed similarities to several Class C chromosomal and plasmid-mediated ß-lactamases. The highest similarity was obtained with the AmpC proteins from Mycobacterium smegmatis and Pseudomonas fluorescens (51%) and Ralstonia metallidurans (50%). A significant similarity to AmpC enzymes of A. baumannii and AG3 (48.5% to 49.3%) was also detected [Figure S1, available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)], which have recently been renamed ADC-1 to ADC-75 and with plasmid-mediated AmpC enzymes such as CMY-1 and MOX-2 (49%). The ampC gene of A. baylyi ADP1 was not associated with integron or transposon structures. Also, no ampR-like genes were found upstream of this ampC gene.

The A. baylyi ADP1 proved to be unusually susceptible to most of the antibiotics tested. The MICs (mg/L) of the non-ß-lactam antibiotics were as follows: ciprofloxacin, 0.047; gentamicin, 0.094; co-trimoxazole, 0.19; amikacin, 1; tetracycline, 1.5 and tobramycin, 0.19. Those of the ß-lactam antibiotics are given in Table 1. As the capacity of its own promoter to allow ampC expression was not known, we decided to clone the ADP1 ampC gene in plasmid constructs pAB-2 and pAB-3. The MIC values of all bacterial isolates included in this study are shown in Table 1. The ß-lactam MICs for E. coli TG1 with pAB-1 and pAB-2 did not increase, and only the pAB-3 plasmid conferred resistance to ß-lactams. A similar effect was observed when A. baylyi ADP1 was transformed with the different pAT-RA plasmids (Table 1). Only pAT-RA-ampC2 increased amoxicillin, cefalotin and cefotaxime MICs and also caused a moderate increase in piperacillin, cefuroxime and ceftazidime MIC values.


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  		Table 1.. ß-Lactam MICs (mg/L) for the A. baylyi ADP1 isolate, and E. coli TG1 and parental A. baylyi ADP1 expressing the ampC gene with the indicated constructs

 
These results were consistent with those of the IEF gel experiments, as only a band of ß-lactamase activity (pI 9.0) was detected in the sonicated extracts from E. coli TG1 (pAB-3) as well as in A. baylyi (pAT-RA-ampC2). These results were also confirmed by RT–PCR analysis. In this experiment and considering a relative value of 1 for the ampC gene expression in ADP1, the A. baylyi ADP1 expressing the ampC gene (pAT-RA-ampC2) yielded 802 ± 157 times higher expression. Almost identical levels of the ampC gene expression were obtained with the A. baylyi harbouring pAT-RA and pAT-RA-ampC1, as were obtained with ADP1.

Kinetic experiments with the AmpC from A. baylyi ADP1 were performed with purified enzyme (specific activity of 0.0216 µmol of nitrocefin/min/µg of protein). The relative enzymatic efficiency (Kcat/Km values) indicated that cefalotin was hydrolysed with higher hydrolytic efficiency than ampicillin (1.45 and 0.019 s–1µM–1, respectively), as expected for a Class C ß-lactamase. The values of Km (µM) and Kcat (s–1) were 8.95 and 0.17 for ampicillin and 4.22 and 6.13 for cefalotin, respectively. Measurement of the hydrolysis rate, at fixed antibiotic concentration of 100 µM and considering the rate for ampicillin to be 100%, yielded values of 5409, 2.1, 1.1, 1 and 0.45 for cefalotin, cefotaxime, cefoxitin, imipenem and cefuroxime, respectively. The IC50 values were >250 and 13.9 µM for clavulanic acid and sulbactam, respectively.

Induction experiments with ADP1 isolates in the presence of cefoxitin did not show any increase in the activity of the AmpC ß-lactamase when the inducer was added (data not shown), which was in agreement with the lack of ß-lactamase regulatory genes in A. baylyi ADP1 isolate.


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In this study, we report the molecular and biochemical characterization of a novel AmpC ß-lactamase from A. baylyi ADP1, which revealed typical features of a cephalosporinase. The physiological role of this enzyme in this strain is unclear since A. baylyi ADP1 is susceptible to ß-lactam antibiotics. However, when this ampC gene was cloned and expressed in an E. coli and in A. baylyi ADP1 host under the direction of a strong promoter, a pattern of resistance to ß-lactam antibiotics was observed in these isolates. The reason for low levels of the ampC gene expression in specific microorganisms remains unclear, although it is probably related to the lack of antibiotic selective pressure in this species, which usually inhabits soil.

We searched for putative insertion sequences surrounding the ampC gene sequence that may alter the gene expression by breaking promoter or regulatory sequences. The previously reported insertion sequence ISAba19 was not found in the chromosome of A. baylyi ADP1, although five copies of a 1237 bp insertion sequence IS1236 (belonging to the IS3 family) were found in the ADP1 genome.10 However, they are probably unrelated with the ampC gene regulation (activation or repression).

One interesting aspect of the ADP1 AmpC enzyme is its amino acid composition compared with those of the remaining Acinetobacter AmpC enzymes or ADC-type ß-lactamases. Whereas ADC-type enzymes (ADC-1 to -7) showed high similarity of amino acid sequences (from 97.7% to 99%), thus defining a unique family of Class C enzymes, the AmpC from ADP1 showed amino acid homology ranging from 48.5% to 49.3% (ADC-7 to ADC-5), respectively (Figure S1). Nonetheless, its phylogenetic proximity appears clear, as all ADC-type enzymes evolved from a common ancestor (Figure 1). The new AmpC enzyme from A. baylyi ADP1 was tentatively designated ADC-8 and might constitute a new phylogenetic branch among the ADC-type enzymes.


Figure 1
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  		Figure 1.. Phylogenetic analysis of the AmpC enzyme from A. baylyi with respect to the 23 ß-lactamases showing higher similarity, including ADC-1 to -7. Branch lengths indicate the numbers of amino acids exchanged (as indicated in the figure). The sizes of the branches are represented to scale. The cladogram was constructed by the Neighbour Joining method. Accession numbers are also indicated.

 

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None to declare.


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Figure S1 is available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).


   Acknowledgements
 
We thank Valerie Barbe and Patrice Nordmann for the gift of A. baylyi strain ADP1 and pAT-RA plasmid, respectively. We thank Ignacio Rego for technical assistance in RT–PCR analysis. A. B. is in receipt of a scholarship from SEIMC. This work was financially supported by the Consellería de Innovación, Industria y Comercio, Xunta de Galicia (PGIDIT04BTF916028PR) and by Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III (PI040514, PI061368) and Spanish Network for the Research in Infectious Diseases (REIPI RD06/0008).


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1 Ibrahim A, Gerner-Smidt P, Liesack W. (1997) Phylogenetic relationship of the twenty-one DNA groups of the genus Acinetobacter as revealed by 16S ribosomal DNA sequence analysis. Int J Syst Bacteriol 47:837–41.[Abstract/Free Full Text]

2 Vaneechoutte M, Young DM, Ornston LN, et al. (2006) Naturally transformable Acinetobacter sp. strain ADP1 belongs to the newly described species Acinetobacter baylyi. Appl Environ Microbiol 72:932–6.[Abstract/Free Full Text]

3 Barbe V, Vallenet D, Fonknechten N, et al. (2004) Unique features revealed by the genome sequence of Acinetobacter sp. ADP1, a versatile and naturally transformation competent bacterium. Nucleic Acids Res 32:5766–79.[Abstract/Free Full Text]

4 Bou G and Martinez-Beltran J. (2000) Cloning, nucleotide sequencing, and analysis of the gene encoding an AmpC ß-lactamase in Acinetobacter baumannii. Antimicrob Agents Chemother 44:428–32.[Abstract/Free Full Text]

5 Hujer KM, Hamza NS, Hujer AM, et al. (2005) Identification of a new allelic variant of the Acinetobacter baumannii cephalosporinase, ADC-7 ß-lactamase: defining a unique family of class C enzymes. Antimicrob Agents Chemother 49:2941–8.[Abstract/Free Full Text]

6 Spratt B, Hedge PJ, Heesen TS, et al. (1986) Kanamycin-resistant vectors that are analogues of plasmids pUC8, pUC9, pEMBL8, and pEMBL9. Gene 41:337–42.[CrossRef][Web of Science][Medline]

7 Hunger M, Schmucker R, Kishan V, et al. (1990) Analysis and nucleotide sequence of an origin of DNA replication in Acinetobacter calcoaceticus and its use for Escherichia coli shuttle plasmids. Gene 87:45–51.[CrossRef][Web of Science][Medline]

8 Tomas M, Beceiro A, Perez A, et al. (2005) Cloning and functional analysis of the gene encoding the 33- to 36-kilodalton outer membrane protein associated with carbapenem resistance in Acinetobacter baumannii. Antimicrob Agents Chemother 49:5172–5.[Abstract/Free Full Text]

9 Nordmann P and Poirel L. (2006) Carbapenem resistance in Acinetobacter baumannii: mechanisms and epidemiology. Clin Microbiol Infect 12:1138–41.[CrossRef][Web of Science][Medline]

10 Gerischer U, D'Argenio DA, Ornston LN. (1996) IS1236, a newly discovered member of the IS3 family, exhibits varied patterns of insertion into the Acinetobacter calcoaceticus chromosome. Microbiology 142:1825–1831.[Abstract/Free Full Text]


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