Substrate Spectrum Extension of PenA in Burkholderia thailandensis with a Single Amino Acid Deletion, Glu168del

Abstract

bWe describe a deletion mutation in a class A ␤-lactamase, PenA, of Burkholderia thailandensis that extended the substrate spectrum of the enzyme to include ceftazidime. Glu168del was located in a functional domain called the omega loop causing expansion of the space in the loop, which in turn increased flexibility at the active site. This deletion mutation represents a rare but significant alternative mechanical path to substrate spectrum extension in PenA besides more common substitution mutations. Burkholderia pseudomallei is the etiological agent of septicemic melioidosis, which is endemic in Southeast Asia and northeastern Australia (4). Burkholderia mallei, the cause of glanders, is a species derived from a clone of B. pseudomallei (20). Bcc, which is a complex composed of more than 10 Burkholderia species, including Burkholderia cepacia, Burkholderia cenocepacia, and Burkholderia multivorans, is a group of nosocomial pathogens that cause respiratory and systemic infections in patients with cystic fibrosis or chronic granulomatous disease and in other immunocompromised patients (10). The antibiotic regimen used to treat infections with these bacteria generally includes ceftazidime (10,26 (15,17,18,22). Similarly, two orthologs of PenA, PenB2 and PenB3, with seven and two amino acid alterations, respectively, were shown to be associated with ceftazidime resistance in clinical isolates of B. cenocepacia (13).Here we report a new mutation that extends to ceftazidime the substrate spectrum of PenA of B. thailandensis (3) (BTH_II1450 of B. thailandensis strain E264), which is closely related to PenA enzymes of the pathogenic species B. pseudomallei, B. mallei, and Bcc (13,17,22). The resistant isolate arose against a high level of ceftazidime (5 g/ml) in LB medium. The MIC of ceftazidime for this isolate was 48 g/ml, which was significantly higher than the 1.75 g/ml of the wild type ( Fig. 1). By PCR amplifying penA from its genomic DNA using primers penA-F (5=-CGTCAATCCGATG CAGTACC-3=) and penA-R (5=-GCCGTTATCGCACCTTTAT C-3=) and sequencing the amplicon in both directions using a 3730XL DNA analyzer (Applied Biosystems, Foster City, CA), we found a three-base deletion mutation in the coding region of penA (Fig. 1). To confirm that this penA gene with the mutation was responsible for the ceftazidime resistance that developed in the mutants, we inactivated the penA gene by replacing a region spanning 196 bp in the middle of the coding region with a Tet r cassette, which was obtained from broad-host-range vector pRK415K (9). The penA-null mutant was verified by PCR using primers penA_LF (5=-AACAGATCGCCGAGATGG-3=) and penA_LR (5=-GCGAACGTTGCCCGATAC-3=), which hybridize to the genomic regions outside penA. The mutant strain with inactivated penA-Glu168del (that is, with ⌬penA-Glu168del) lost resistance to ceftazidime, whose MIC was comparable to that for the wild-type strain with ⌬penA-WT (Fig. 1). The MICs were measured by the Etest (8) in accordance with the manufacturer's instructions (AB Biodisk, Solna, Sweden...

Deletion Mutations Conferring Substrate Spectrum Extension in the Class A β-Lactamase

“…The R164-to-D179 distance might reflect an unsecured salt bridge between the two residues that clamps the omega loop structure at both ends (30), destabilizing the omega loop. Consistently, the space in the omega loop calculated as the distance between amino acid residues 163 and 174 (or 175 for P174del) and between residues 164 and 173 (or 174 for I173del) showed greater variation in PenA proteins with deletion mutations, including E168del (14), than in wild-type PenA (Fig. 3).…”

“…The mutations we isolated also conferred decreased activity on an original substrate, amoxicillin ( Table 1). The enzyme activity associated with the deletion mutations was effectively inhibited by clavulanic acid (Table 1), similar to PenA amino acid substitution mutants (7) and E168del (14). None of the deletion mutations conferred resistance to cefepime, a fourth-generation cephalosporin, or to meropenem, a carbapenem subgroup member (Table 1).…”

“…The P174del mutation conferred decreased activity against ceftriaxone, unlike the other three mutations (Table 1). That the three deletion mutations except P174del did not produce reduced activities against cefotaxime and ceftriaxone (Table 1) indicated that the mutations were distinctive from most amino acid substitution mutations (7) and E168del (14).…”

“…1A). The P174del mutation was generated in a TEM-1 derivative with E168del (11), which was previously identified and characterized in PenA (14).…”

A Structure-Based Classification of Class A β-Lactamases, a Broadly Diverse Family of Enzymes

“…Interestingly, a single-amino-acid deletion (Glu168del) may expand the in vitro spectrum of inactivation to ceftazidime in B. thailandensis (165). Additional singleamino-acid deletions have recently been reported (27). Another mechanism of acquired resistance involves the overexpression of the chromosomal ␤-lactamase of B. pseudomallei or the loss of a penicillin-binding protein or (PBP) and also porin (164,(166)(167)(168).…”