BEL-2, an Extended-Spectrum β-Lactamase with Increased Activity toward Expanded-Spectrum Cephalosporins in Pseudomonas aeruginosa

Abstract

A Pseudomonas aeruginosa isolate recovered in Belgium produced a novel extended-spectrum ß-lactamase, BEL-2, differing from BEL-1 by a single Leu162Phe substitution. That modification significantly altered the kinetic properties of the enzyme, increasing its affinity for expanded-spectrum cephalosporins. The bla_BEL-2 gene was identified from a P. aeruginosa isolate clonally related to another bla_BEL-1-positive isolate.

Extended-spectrum ß-lactamases (ESBLs), such as TEM, SHV, PER, VEB, GES, and more recently, CTX-M variants, are reported increasingly to be found in Pseudomonas aeruginosa in various areas (1, 2, 7, 8, 10-12, 15, 17, 21, 23, 27, 28, 30). The BEL-1 ß-lactamase, distantly related to other ESBLs, was identified from a P. aeruginosa isolate from Roeselare, Belgium, which interestingly shows resistance to ticarcillin and ceftazidime but only reduced susceptibility to piperacillin, cefepime, cefpirome, and aztreonam (24). The bla_BEL-1 determinant was found as a gene cassette in the chromosome-borne class 1 integron, In120, that includes other resistance genes (aacA4, aadA5, and smr2) and that was part of a Tn1404-type transposon structure (24). Very recently, Bogaerts et al. (5) reported on the diffusion of BEL-1-producing isolates in various hospital centers of Belgium and also found that BEL-1 could be associated with other relevant β-lactamases, such as the VIM-1 metallo-β-lactamase (5).

P. aeruginosa isolate 531 (this study) was recovered from a urine sample of a patient hospitalized in Roeselare, Belgium, in February 2007 for pneumonia and was resistant to all β-lactams but imipenem (Table 1). A synergy between aztreonam or ceftazidime and clavulanic acid-containing disks suggested the synthesis of an ESBL (19). PCR followed by sequencing using ESBL gene-specific primers (24) identified a novel gene encoding BEL-2, which differs from BEL-1 by a single amino acid substitution (Leu to Phe at Ambler position 162) (3). Transfer of a ß-lactam resistance marker from P. aeruginosa 531 to Escherichia coli or to P. aeruginosa reference strains was unsuccessful by either conjugation or transformation (25). Plasmid extraction performed as described previously (14) did not identify any plasmid, suggesting a chromosomal location of the bla_BEL-2-like gene in P. aeruginosa 531. A pulsed-field gel electrophoresis (PFGE) analysis (4) showed that isolates 531 (BEL-2 positive) and 51170 (BEL-1 positive), recovered from the same geographical area, were clonally related. A PCR mapping approach confirmed the presence of a class 1 integron whose structure was identical to that of In120 of P. aeruginosa 51170 (24) and identified an identical structure in P. aeruginosa 531 (data not shown). Overall, these data suggest that the bla_BEL-2 sequence likely resulted from a mutational event that had occurred in In120-carrying P. aeruginosa strains.

TABLE 1.

MICs of β-lactams^a

β-Lactam(s)b	MIC (μg/ml)
	P. aeruginosa 531	P. aeruginosa 51170	E. coli TOP10 (pSB-2) (BEL-2)	E. coli TOP10 (pSB-1) (BEL-1)	E. coli TOP10
Amoxicillin	>512	>512	>512	>512	4
Amoxicillin and CLA	>512	>512	64	64	4
Ticarcillin	>512	>512	>512	>512	4
Ticarcillin and CLA	>512	128	64	64	4
Piperacillin	16	16	32	128	1
Piperacillin and TZB	8	8	8	32	1
Cephalothin	>512	>512	256	256	2
Cefuroxime	>512	>512	32	128	2
Cefoxitin	512	512	2	2	2
Ceftazidime	256	32	128	16	0.06
Cefotaxime	256	32	32	4	0.12
Cefepime	16	4	1	0.25	0.06
Cefpirome	64	16	0.5	0.25	0.06
Aztreonam	64	32	16	16	0.12
Imipenem	1	1	0.12	0.12	0.12

^aMICs of β-lactams for P. aeruginosa 531 and 51170 clinical isolates, producing ESBLs BEL-2 and BEL-1, respectively, E. coli TOP10 harboring recombinant plasmid pSB-2 expressing BEL-2, E. coli TOP10 harboring recombinant plasmid pSB-1 expressing BEL-1, and the E. coli TOP10 reference strain.

^bCLA, clavulanic acid at a fixed concentration of 4 μg/ml; TZB, tazobactam at a fixed concentration of 4 μg/ml.

In order to compare the contributions of BEL-1 and BEL-2 to ß-lactam resistance, the corresponding genes (amplified using primers PreBEL-A [5′-AGACGTAAGCCTATAATCTC] and PreBEL-B [5′-GCGAATTGTTAGACGTATG]) were cloned in the pCR-BluntII-TOPO vector (Invitrogen, Cergy-Pontoise, France) and subsequently introduced into E. coli TOP10, giving rise to recombinant strains E. coli TOP10(pSB-1) and E. coli TOP10(pSB-2), producing BEL-1 and BEL-2, respectively. MICs of ß-lactams were determined by solid agar dilutions following the guidelines of the CLSI (9). E. coli TOP10(pSB-2) had MICs of piperacillin, cephalothin, and cefuroxime that were lower than those of E. coli TOP10(pSB-1), but its cefotaxime, ceftazidime, ceftriaxone, and cefepime MICs were higher than those of TOP10(pSB-1), while MICs of carbapenems were the same (Table 1).

E. coli TOP10(pSB-2) produced a ß-lactamase with a pI value of 7.1 (identical to that of BEL-1) (18). Approximately 1.5 mg of BEL-2 was purified (>95% as estimated by SDS-PAGE analysis; data not shown) from an E. coli MCT236(pET-BEL-2) crude extract by using a two-step chromatography process (an anion exchange at pH 7.5 using a Q Sepharose Fast Flow column followed by a cation exchange at pH 6.2 using a 1-ml Resource S column). (The specific activity was 8,800 nmol/min·mg of protein with 100 μM of cephalothin as the substrate, purified 95-fold.) BEL-2 had a broad-spectrum hydrolysis profile, including penicillins and expanded-spectrum cephalosporins but not cephamycins and carbapenems (Table 2). BEL-2 overall showed higher catalytic efficiencies (k_cat/K_m) than BEL-1 for aztreonam and most oxyiminocephalosporins (cefotaxime, ceftazidime, ceftriaxone, and cefepime but not cefuroxime). This was due to a significant alteration of the K_m values for these substrates with BEL-2, which were decreased relative to those of BEL-1 by 300-fold (ceftriaxone) to up to three orders of magnitude (ceftazidime) (Table 2). Interestingly, a decrease of the K_m value was also observed with all the other substrates (though the variation was less important), likely reflecting a modification of the active site structure and thus substrate recognition. Overall, BEL-2 k_cat values were also lower but to a lesser extent (Table 2). The values of catalytic efficiency toward expanded-spectrum cephalosporins for BEL-2 may explain the higher MICs observed for the BEL-2-producing recombinant E. coli strains and P. aeruginosa clinical isolate. Position 162 is located at the beginning of the Ω loop, which bears the functionally important Glu166 residue, which is conserved in class A enzymes, and where mutations conferring extended-spectrum properties have been extensively reported in natural TEM and SHV variants (13). The presence of a bulky Phe residue in BEL-2 might modify the orientation of the Ω loop and the overall geometry of the active site. The further extension of the substrate profile as a consequence of a single substitution in the Ω loop observed with the BEL-2 variant may parallel that of other enzymes, e.g., CTX-M-19 (CTX-M-14 Pro167Ser variant) (26) or GES-2 (GES-1 Gly170Asn variant) (28). Inhibition studies showed that BEL-2 and BEL-1 are similarly inhibited by clavulanic acid, tazobactam, and sulbactam (50% inhibitory concentrations of 0.1, 2, and 3 μM, respectively).

TABLE 2.

Kinetic parameters of purified ß-lactamase BEL-2, in comparison with previously reported values of BEL-1 (22)^a

ß-Lactam	BEL-2			BEL-1
	kcat (s−1)	Km (μM)	kcat/Km (mM−1· s−1)	kcat (s−1)	Km (μM)	kcat/Km (mM−1· s−1)
Benzylpenicillin	1.2	4	300	3	20	150
Piperacillin	1.1	0.3	3,700	2	15	130
Cephalothin	4.3	2	2,200	150	280	540
Cephaloridine	1.2	8	150	30	130	230
Cefoxitin	<0.01	ND	ND	<0.01	ND	ND
Cefuroxime	0.88	12	73	10	40	250
Ceftriaxone	0.2	0.1	2,000	25	30	830
Cefotaxime	0.13	0.45	290	30	250	120
Ceftazidime	0.03	0.64	47	>1.5	>700	ND
Cefepime	0.003	0.3	10	1	150	7
Aztreonam	0.1	0.36	280	10	100	100
Imipenem	<0.01	ND	ND	<0.01	ND	ND

^aStandard deviations were below 15%. ND, not determinable, due to the initial rate of hydrolysis being too low.

Conclusion.

This study emphasizes the spread and evolution of BEL-type ESBLs in P. aeruginosa in Belgium. A novel integron-encoded BEL-type ß-lactamase with enhanced hydrolytic properties was identified. This evolution of an ESBL determinant expressed in a P. aeruginosa strain is similar to that reported for GES-1 in South Africa, where P. aeruginosa isolates expressing ESBL GES-2 or GES-5 with expanded-spectrum activities toward carbapenems have been reported (16, 29), and also in Brazil, where clonally related P. aeruginosa isolates expressing either GES-1 or GES-5 coexist (22).

It is tempting to hypothesize that a BEL-2 producer has been selected under antibiotic selective pressure and especially by expanded-spectrum cephalosporins. This work shows that ESBL BEL-1 has the potential to evolve with significantly altered biochemical properties. It remains to evaluate whether BEL-type ß-lactamases could further evolve to gain activity on cephamycins and/or carbapenems (as observed for several GES-type ESBLs [20]) or resistance to ß-lactamase inhibitors (as observed for several TEM and SHV variants [6]).

Nucleotide sequence accession number.

The nucleotide and protein sequences corresponding to the BEL-2 enzyme have been registered in GenBank under accession number FJ666063.