INTRODUCTION

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A major mechanism of resistance of Pseudomonas aeruginosa to cephalosporins used in clinical practice, such as cefsulodin or ceftazidime, is the overproduction of its cephalosporinase, resulting from depression of the ampC transcription inhibition pathway (14). Alternatively, resistance to the broad-spectrum cephalosporins, including ceftazidime and cefepime, may result from the production of an extended-spectrum -lactamase (ESBL). In P. aeruginosa, as opposed to members of the family Enterobacteriaceae, production of TEM- and SHV-type ESBLs (3) seems to be rare (22), and other extended-spectrum enzymes, such as PER-1 (23), OXA-2-, or OXA-10-derived ESBLs (8-10), and a clavulanic acid-susceptible class D enzyme, OXA-18 (24), have been described for clinical strains of this species. The enzymes of molecular class D (13) comprise those of group 2d of the functional classification scheme described by Bush et al. (3) and are characterized by high relative rates of oxacillin hydrolysis and generally low susceptibility to inhibition by clavulanic acid (3, 18). Like the other active-site serine -lactamases, the class D enzymes have at least three conserved amino acid motifs, which are shown in Fig. 1 for the OXA-10 derivatives.

View larger version (11K): [in this window] [in a new window]   FIG. 1.   Amino acid differences between the -lactamases of the OXA-10 group. Amino acid numbering is according to Huovinen et al. (13); the conserved motifs typical for class D enzymes are boxed. The amino acids shown in boldface contribute to the substrate profile, with asparagine in position 157 leading to extended-spectrum variants. The amino acids of OXA-10 that are not numbered are G20, S27, S50, D55, V89, T107, Y174, A197, E229, T230, S245, and E259; , absence of an amino acid.

High-resolution crystallography studies of several class A penicillinases (34) have generated a basis for understanding the molecular mechanisms underlying the substrate profile extensions secondary to a large array of point mutations in TEM- or SHV-type ESBLs (16, 26). For the class D enzymes, no such studies have been reported, and only two amino acid changes have been implicated in the extension of the substrate profiles to ceftazidime, i.e., Gly157 to Asp in the OXA-10-derived OXA-11 (10) and OXA-14 (8) and Asp150 to Gly in the OXA-2-derived OXA-15 (9). These positions are, respectively, 14 and 2 amino acids downstream from the YGN triad in the OXA-10 and OXA-2 derivatives (6, 8-10, 13) (Fig. 1). Here, we describe a novel ESBL, OXA-19, from a clinical P. aeruginosa isolate, containing nine altered amino acids with respect to the non-ESBL OXA-10 (13) and two altered amino acids with respect to the non-ESBL OXA-13 (20), both of which appear to contribute to the level of resistance to anti-Pseudomonas penicillins and cephalosporins in Escherichia coli and P. aeruginosa.

	MATERIALS AND METHODS

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Strains and plasmids. The strains and plasmids used are summarized in Table 1. P. aeruginosa PAe191 was isolated in 1991 at the Saint-Louis Hospital, Paris, France. This strain was highly resistant to ceftazidime (MIC, 512 µg/ml). Strain PAO38(pAZ310) was selected on ceftazidime (16 µg/ml) from strain PAO38(pAZ309), which was described previously (20). Strains were grown in Mueller-Hinton (MH) medium at 37°C.

MIC determinations and antibiotics. MICs on MH agar containing serially twofold-diluted antibiotics were determined. Plates inoculated with a Steers-type inoculator and ca. 10⁴ CFU per spot were incubated at 37°C for 18 h. The MICs of -lactams were determined alone or in combination with imipenem (0.25 µg/ml), clavulanic acid (2 µg/ml), or tazobactam (4 µg/ml). MIC determinations were repeated twice, with identical results. Antibiotics were provided from the following suppliers: ampicillin, aztreonam, and amikacin, Bristol Myers Squibb; ceftazidime, Glaxo Group Research, Ltd.; cefotaxime, Hoechst Roussel Pharmaceuticals Ltd.; piperacillin and tazobactam, Lederle Laboratories; imipenem, Merck Sharp and Dohme-Chibret; gentamicin, Schering Plough; clavulanic acid and ticarcillin, SmithKline Beecham; and cefsulodin, Takeda Laboratories.

-Lactamase preparation. Crude enzyme (S100) extracts were prepared as previously described (20). The supernatants were used immediately for the determination of kinetic parameters and isoelectric points. The protein concentrations were measured by the technique described by Bradford (2), and the hydrolysis rates in phosphate buffer (50 mM, pH 7.0) were determined spectrophotometrically at 30°C with a model 550S double-beam spectrophotometer (Perkin-Elmer), with ampicillin and ceftazidime as the substrates. One unit of -lactamase was defined as the amount hydrolyzing 1 µmol of substrate per min.

IEF. Isoelectric focusing (IEF) of S100 extracts was for 2 h, with a mini-IEF cell 111 (Bio-Rad) and a gradient made up of two-thirds polyampholytes with a pH range of 3 to 9 and one-third of polyampholytes with a pH range of 2 to 11 (Serva). Extracts from OXA-10-, SHV-1-, and SHV-5-producing strains were used as standards for pIs of 6.1, 7.6, and 8.2, respectively. -Lactamases were revealed by overlay with nitrocefin (1 mg/ml) in phosphate buffer (50 mM, pH 7.0).

Cloning experiments and DNA sequencing. Standard DNA methodology (1) was used to clone two ca. 4.0-kb HindIII DNA fragments carrying either the in vitro-selected ESBL gene oxa13-1 or the in vivo-selected gene oxa19 (Table 1) (20) into the pNJR3-2 shuttle vector, allowing the expression of both genes in P. aeruginosa PAO38. These fragments were also cloned into M13mp18 and M13mp19 phage vectors and were partially sequenced as described previously (20). The sequenced stretches which have been determined for oxa19 and oxa13-1 and their adjacent integron regions are represented schematically in Fig. 1. The 35 sequences of the promoter of the aac(6')-Ib, oxa13-1 cistron were taken to be identical with that of the original oxa13-containing integron (20). For expression of the two ESBL genes in an isogenic E. coli background, both genes were cloned on a NarI-HindIII fragment into the vector pK18.

Nucleotide sequence accession numbers. The nucleotide sequences for oxa19 [including aac(6')-Ib] and oxa13-1 have been deposited in GenBank under accession numbers AF043381 and AF043558, respectively.

	RESULTS AND DISCUSSION

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Resistance phenotypes of PAe191 and PAO38(pAZ316). PAe191 was resistant to all anti-Pseudomonas -lactam antibiotics tested, except imipenem, and also to aminoglycosides, sulfonamides, and mercuric ions. Ticarcillin, piperacillin, and ceftazidime were not protected when associated with a -lactamase inhibitor, such as clavulananic acid or tazobactam. However, the high-level resistance of PAe191 to ceftazidime was reversed by imipenem (Table 2). The protective effect of imipenem on otherwise hydrolyzed -lactam compounds in P. aeruginosa has been previously observed in the presence of OXA-10-related enzymes (20). In order to characterize the -lactamase responsible for this phenotype and the nucleotide sequences controlling its production, a ca. 4.0-kb HindIII fragment from PAe191 DNA was cloned into pNJR3-2, yielding pAZ316 (Table 1). PAO38(pAZ316) showed the same -lactam resistance pattern as that of PAe191, and a MIC of gentamicin greater than that of amikacin was observed. Both PAe191 (see Fig. 3, lane 3) and PAO38(pAZ316) (data not shown) produced only one -lactamase, with an apparent pI (pI_app) of 7.6, which was then likely to be an ESBL. No plasmid was found in the clinical strain PAe191 despite the use of techniques (15, 35) allowing the extraction of large plasmids, such as the OXA-1-encoding plasmid RGN238 (12).

View this table: [in this window] [in a new window]   TABLE 2.   MICs of different -lactams for PAe191 and OXA13-1, OXA-19- and OXA-13-producing transformants

Nucleotide sequence of the gene cluster encoding aminoglycoside and -lactam resistance in PAe191. Sequence analysis of a ca. 2-kb fragment from pAZ316 revealed the presence of two resistance genes, each flanked by a recombinational element at its 3' extremity, which is typical of those found in the resistance gene cassettes in integrons (11, 30, 33). Only four mutations (Fig. 1) were observed in comparison with the sequence of a very similar integron-borne cluster described previously (20). The first was in the 35 sequence of the promoter P1, located within the integrase gene intI1, which controls the expression of the resistance genes (33). The three other mutations were in the resistance genes themselves. The gene immediately downstream from intI1 encoded an AAC(6')-Ib variant, called AAC(6')-Ib₉, with a Leu-to-Ser change at position 119 with respect to the otherwise identical AAC(6')-Ib protein encoded by pAZ301 (20) and to other typically amikacin resistance-conferring variants (36). This amino acid change has been associated previously with a shift from amikacin to gentamicin resistance in vitro after site-directed mutagenesis (27) and in clinical isolates of Pseudomonas fluorescens (17) and Enterobacteriaceae (4). The second resistance gene had two nucleotide differences accounting for two amino acid differences in comparison with OXA-13 (Fig. 1 and 2) (20), with Asn73 instead of Ser and Asp157 instead of Gly. This novel variant was named OXA-19. As already mentioned, Asp157 has been associated previously with the extension of the -lactam resistance spectrum to ceftazidime (8, 10, 21).

View larger version (9K): [in this window] [in a new window]   FIG. 2.   Structure of the integron encoding the -lactamases OXA-19 and OXA-13-1. Nucleotide sequences are identical in both clusters, except for three differences in the 35 sequences of the promoter and in the aac(6')-Ib and the oxa genes. Amino acid numbering is from Tran Van Nhieu and Collatz (36) and Huovinen et al. (13), respectively. Arrows represent the following open reading frames: intI1, for integrase; aac(6')-Ib, for the aminoglycoside acetyltransferases genes aac(6')-Ib10 on pAZ310 (20) and aac(6')-Ib9 on pAZ316; oxa, for the OXA variants indicated on the left; and qacE1, for the typically truncated quaternary ammonium resistance-conferring gene associated with the 3' conserved segment of integrons. The intI1 and qacE1 genes have been only partially sequenced here (indicated in boldface). Data for pAZ309 are from reference 20, and data for pAZ310 and pAZ316 are from the present study. Boldface, differences with respect to the OXA-13-encoding plasmid pAZ309; dashed line, the oxa13-1 region that has been sequenced.

Selection in vitro and characterization of an ESBL variant of OXA-13. To determine whether a derivative of OXA-13 conferring the resistance phenotype of OXA-19 in P. aeruginosa could be generated by a point mutation, a spontaneous mutant of PAO38(pAZ309), which was called PAO38(pAZ310), was selected on ceftazidime (Table 1). An OXA variant of pI_app 7.8 was obtained (Fig. 3). It differed from OXA-13 by only a Gly157-to-Asp change and was named OXA-13-1. Although the production of this enzyme in PAO38(pAZ310) led to a resistance pattern similar to that of OXA-19 in PAO38(pAZ316) (Table 2), the level of resistance of PAO38(pAZ316) to some of the -lactams tested, especially ticarcillin and piperacillin, appeared to be higher than that of PAO38(pAZ310). In repeated assays, the MIC of ampicillin for a pAZ316-containing E. coli strain was also found to be four times as high as that for the same strain containing pAZ310 (data not shown). These observations could be related to a ca. 30-times-higher specific activity of OXA-19 against ampicillin, in comparison with OXA-13-1 (Table 3), while the specific activities of both enzymes against ceftazidime were similar.

View larger version (94K): [in this window] [in a new window]   FIG. 3.   Isoelectric focusing of extended-spectrum OXA-10-related -lactamases. Enzymes SHV-1 (pIapp, 7.6), SHV-5 (pIapp, 8.2), OXA-13 (pIapp, 8 [18]), and OXA-10 (pIapp, 6.1) were used as pI standards. The two novel -lactamases, OXA-13-1 and OXA-19, had estimated pIapps of 7.8 and 7.6, respectively. -Lactamase activity was revealed by overlaying the gel with nitrocefin.

Effect of Asn or Ser in position 73 on the level of resistance to penicillins in OXA-19 and OXA-13-1 producers. Two of the three nucleotide differences that distinguish the aac(6')-Ib, oxa clusters of pAZ316 and pAZ310 (Fig. 2) could be involved in modulating the levels of penicillin resistance, the difference in the 35 promoter sequence and that concerning amino acid position 73. Cloning of the NarI-HindIII fragments from both plasmids carrying oxa13-1 or oxa19 into pK18 yielded pAZ326 and pAZ327, respectively, leaving only the difference in position 73 (Fig. 2). It eliminated the likely difference in promoter strength (the 35 sequence TTGACA [Fig. 2] being known to occur in promoters stronger than TGGACA in E. coli [19]) and the possible influence of secondary-structure variations in the so-called 59-bp element (5) on oxa expression. Table 2 shows the -lactam resistance patterns of E. coli DH5 harboring pAZ326 or pAZ327. In these constructs, the higher MICs of penicillins for the OXA-19-producing strain correlated with a higher specific activity of OXA-19 against ampicillin in comparison with that of OXA-13-1 (Table 3). Thus, it is conceivable that Asn in position 73 conditions the level of resistance to -lactams, and to penicillins in particular, also for P. aeruginosa PAO38(pAZ316) and, hence, the clinical strain PAe191.