Genetic and Biochemical Characterization of OXA-535, a Distantly Related OXA-48-Like β-Lactamase

OXA-535 is a chromosome-encoded carbapenemase of Shewanella bicestrii JAB-1 that shares only 91.3% amino acid sequence identity with OXA-48. Catalytic efficiencies are similar to those of OXA-48 for most β-lactams, except for ertapenem, where a 2,000-fold-higher efficiency was observed with OXA-535.

ABSTRACT

OXA-535 is a chromosome-encoded carbapenemase of Shewanella bicestrii JAB-1 that shares only 91.3% amino acid sequence identity with OXA-48. Catalytic efficiencies are similar to those of OXA-48 for most β-lactams, except for ertapenem, where a 2,000-fold-higher efficiency was observed with OXA-535. OXA-535 and OXA-436, a plasmid-encoded variant of OXA-535 differing by three amino acids, form a novel cluster of distantly related OXA-48-like carbapenemases. Comparison of bla_OXA-535 and bla_OXA-436 genetic environments suggests that an ISCR1 may be responsible for bla_OXA-436 gene mobilization from the chromosome of Shewanella spp. to plasmids.

TEXT

OXA-48, a carbapenemase, has become a major public health threat because of its rapid spread worldwide (1–3). Along with this rapid dispersion, several OXA-48 variants have been reported (4–6). For a complete list of variants, see the β-lactamase database (http://bldb.eu/BLDB.php?class=D#OXA) (4). Analysis of the genetic context of bla_OXA-48-like genes in Enterobacteriaceae has shown their association with insertion sequences (IS1999, ISEcp1, and IS4321) and with various plasmids (7–10). Shewanella spp. constitute the reservoir of bla_OXA-48-like genes. So far, the gene variants bla_OXA-48, bla_OXA-199, bla_OXA-204, and bla_OXA-416 genes have been reported in Shewanella xiamenensis, although some researchers have identified bla_OXA-48-like genes in other Shewanella spp. (11–17). OXA-535, the naturally occurring oxacillinase of Shewanella bicestrii JAB-1, share only 91.3% and 98.9% amino acid sequence identities with OXA-48 and the plasmid-borne OXA-436, respectively (5, 18) (Fig. 1A). The aim of this study was to characterize the biochemical properties of OXA-535 and investigate its genetic environment in S. bicestrii JAB-1.

FIG 1.

Amino acid sequence alignment of different OXA-48-like variants and molecular modeling. (A) Dashes indicate identical residues among all the amino acid sequences. Amino acid motifs that are well conserved among class D β-lactamases are indicated by gray boxes, and the single black-outlined box corresponds to the β5-β6 loop. Numbering is according to DBL (1). OXA sequences were from the β-lactamase database (http://bldb.eu/BLDB.php?class=D#OXA) (4) and ANA-3_OXA-48-like, MR-7_OXA-48-like, MR-4_OXA-48-like, and Shew256_OXA-48-like were from GenBank (accession nos. WP_011718232.1, WP_011627184.1, WP_011621491.1, and WP_088210206.1, respectively). (B) Superposition of crystal structures of OXA-535 (gray) and OXA-48 (red; PDB accession no. 4S2P). Residues Ser70 and Lys73 are shown in a Corey-Pauling-Koltun (CPK) representation and shown in yellow and blue, respectively. (C) Covalent docking pose of ertapenem (green, stick representation) in the binding site of OXA-535 (gray, surface representation). Residues Ala104, Arg107, and Tyr185 are orange, cyan, and magenta, respectively. Images were generated using Chimera (23). (D) Highlight of the favorable interaction between ertapenem (covalent docking pose) and residues Ala104, Arg107, and Tyr185 of OXA-535. (E) Highlight of the sterical hindrance between ertapenem and the Thr104 residue of OXA-48 superimposed on the OXA-535 structure.

MIC values were determined as previously described (18). Escherichia coli TOP10 (Invitrogen, Saint-Aubin, France) expressing OXA-535 from the pTOPO plasmid was resistant to penicillins, including temocillin, and reduced susceptibility to carbapenems; whereas the expanded-spectrum cephalosporins remained in the susceptibility range, in a manner similar to that of E. coli TOP10 expressing OXA-48 from a pTOPO plasmid (6, 18) (Table 1).

TABLE 1.

MIC of β-lactams for Shewanella spp. JAB-1 and E. coli TOP10 pTOPO-OXA-535, TOP10 pTOPO-OXA-48, and TOP10

Antibiotic	MIC (mg/liter)
	Shewanella sp. JAB-1	E. coli TOP10		E. coli TOP10
		pTOPO-OXA-535	pTOPO-OXA-48
Amoxicillin	>256	>256	>256	2
Amoxicillin + CLAa	8	128	>256	2
Piperacillin	>256	>256	>256	1.5
Temocillinb	0.25	>1,024	>1,024	4
Cefotaximeb	>32	0.06	0.75	0.06
Ceftazidimeb	2	0.19	0.19	0.12
Cefepime	8	0.023	0.19	0.023
Imipenemb	0.38	1	0.75	0.25
Ertapenemb	1	0.25	0.25	0.003
Meropenemb	0.38	0.19	0.25	0.016

^aCLA, clavulanic acid (2 mg/liter).

^bMIC data for these antibiotics were retested in this work but are similar to those presented by Jousset et al. (18).

A DNA fragment corresponding to the mature OXA-535, generated with the primers INF-OXA-48Fw (5′-AAGGAGATATACATATGGTAGCAAAGGAATGGCAAG-3′) and INF-OXA-48Rv (5′-GGTGGTGGTGCTCGAAGGGAATAATTTTTTCCTGTTTGAG-3′), was cloned into the expression vector pET41b (+) (Novagen, VWR International, Fontenay-sous-Bois, France), and recombinant plasmid pET41-OXA-535 was electroporated into the electrocompetent E. coli strain BL21(DE3) (Novagen). OXA-535 was overexpressed and purified, and steady-state kinetic parameters were determined and compared with those of OXA-48 as previously described (6, 19).

Initial hydrolysis tests using pure undiluted OXA-535 revealed an absence of hydrolysis of penicillins (ampicillin, oxacillin, piperacillin, amoxicillin, and benzylpenicillin), whereas the other β-lactams tested were rapidly hydrolyzed by OXA-535 in a manner similar to that of OXA-48 (Table 2). The addition of 50 mM sodium hydrogen carbonate (NaHCO₃) as a source of CO₂ did not modify the rates of penicillin hydrolysis, suggesting that the absence of penicillinase activity with concentrated OXA-535 may not be due to Lys73 decarboxylation (20). When using 100-fold-diluted OXA-535 preparations, penicillin hydrolysis was restored in a manner similar to that of OXA-48. This peculiar behavior of OXA-535 in respect to penicillin hydrolysis may be the result of two mechanisms: (i) an alteration in the affinity of the enzyme for penicillins at high enzymatic concentrations and/or (ii) a modification in the hydrolysis mechanism that may involve an alternative catalytic process specific for penicillins, active only with low concentrations of the enzyme, which may be a result of enzyme concentration-dependent conformational changes. Structural analyses should be performed to explain this peculiar OXA-535 behavior.

TABLE 2.

Comparison of the steady-state kinetic parameters of OXA-48 and OXA-535 β-lactamases

Substrate	Km (μM)		kcat (s−1)		kcat/Km (mM−1/s−1)
	OXA-48	OXA-535	OXA-48	OXA-535	OXA-48	OXA-535
Ampicillin	395	67	955	41	2,418	615
Oxacillin	95	53	130	64	1,368	1,210
Temocillin	45	357	0.30	1.8	6.6	5.1
Cefalotin	195	197	44	15	226	74
Cefoxitin	>200	268	>0.05	0.07	0.26	0.27
Ceftazidime	NHa	>1,000	NH	>0.01	NH	0.01
Cefotaxime	>900	>1,000	>9.0	>3.9	10	3.9
Cefepime	>550	>1,000	>0.60	>0.34	1.1	0.34
Imipenem	13	105	4.8	7.1	369	67
Meropenem	11	3.0	0.07	0.20	6.2	68
Ertapenem	100	0.06	0.13	0.17	1.3	2,646

^aNH, hydrolysis was not detected with concentrations of substrate and enzymes up to 1,000 and 400 nM, respectively.

Steady-state kinetic parameters for penicillins were determined by using diluted OXA-535 preparations. OXA-535 and OXA-48 presented similar catalytic efficiencies for oxacillin and temocillin (Table 2) (6). Compared with OXA-48, OXA-535 catalytic efficiency was (i) ∼4-fold lower for ampicillin due to a lower turnover number; (ii) 3-fold lower for cephalothin due to a lower k_cat; (iii) 5-fold lower for imipenem as a result of a lower affinity (8-fold increased k_m); (iv) 11-fold higher for meropenem as a result of better affinity and turnover values; and, most surprisingly, (v) ∼1,800-fold higher for ertapenem, because of a 1,660-fold-higher affinity, with similar velocity of hydrolysis to that of OXA-48 (Table 2). This reduced activity against imipenem of OXA-535 may explain the negative results observed by Jousset et al. (18), who used Carba NP tests. The fact that OXA-535-like enzymes are difficult to detect with biochemical tests, such as Carba NP, may lead to the silent spread of this type of enzyme, in a manner similar to that of OXA-244 (18, 21).

Suitable conditions for the crystallization of OXA-535 were found at facilities of the Institute for Integrative Biology of the Cell (I2BC, Gif-sur-Yvette, France), and the resulting crystals were used for acquisition of X-ray diffraction data at the SOLEIL synchrotron (Saclay, France). A preliminary crystallographic study (data not shown) showed that the structures of OXA-535 and OXA-48 are very similar, with the exception of three regions situated relatively far from the binding site, i.e., 49 to 55, 94 to 106, and 252 to 261 (class D β-lactamase [DBL] numbering system) (Fig. 1B) (1). Covalent docking of ertapenem using GOLD and the GoldScore scoring function showed a strong ionic interaction between the terminal carboxylate group of the ligand and the side chain of Arg107 and a C-H…π interaction between the side chain of Tyr185 and the terminal aromatic group of ertapenem (Fig. 1C) (22). Comparison with the OXA-48 structure showed that the residue in position 104 plays a key role in ertapenem binding, thus explaining the huge difference in affinity between OXA-48 and OXA-535 determined for this substrate. Indeed, Ala104 in OXA-535 is compatible with ertapenem binding, given the small size of the side chain and the modified conformation of loop 94 to106 (Fig. 1D), whereas Thr104 in OXA-48 is bulkier and completely blocks ertapenem binding in this region (Fig. 1E). These differences may have led to the observed 1,800-fold difference in catalytic efficiency.

Upstream of the bla_OXA-535 gene, we identified sprT and endA genes encoding an SprT-like protein of unknown function and endonuclease I, respectively. The same genetic organization was described for closely related chromosomally encoded bla_OXA-48-like genes, such as the bla_{OXA-48-like-MR-4} gene (Fig. 2). The downstream sequence of the bla_OXA-535 gene is commonly present downstream of bla_OXA-48-like genes on the chromosomes of Shewanella spp. (11, 12). The 2,394-bp upstream and 4,074-bp downstream sequences of the bla_OXA-535 gene were identical to those of bla_OXA-436, further supporting that the chromosomally encoded bla_OXA-535 gene may be the progenitor of the plasmid-encoded bla_OXA-436 gene (5). An ISCR1 element found upstream of the bla_OXA-436 gene but absent upstream of the bla_OXA-535 gene may be responsible for the bla_OXA-535-like gene mobilization from the chromosome of Shewanella spp. to plasmids (Fig. 2). ISCR1 carries a single orf that is responsible for a rolling-circle type of transposition mechanism (24). Flanking gene acquisition is thought to occur when the termination mechanism fails and rolling-circle replication extends into neighboring DNA, where it may encounter a second surrogate end (24). In the case of the bla_OXA-436 gene, the DNA sequence putatively mobilized by ISCR1-mediated rolling-circle transposition corresponded to an ∼6.5-kb fragment present also in S. bicestrii JAB-1 and surrounding the bla_OXA-535 gene. Our findings illustrate the increasing complexity of genetic vehicles at the origin of OXA-48-type carbapenemase spread (7–10). The finding of ISCR1 upstream of bla_OXA-48-like genes is worrisome, because this ISCR1plays an important role in the assembly and transmission of multiple antibiotic resistance genes (5, 18, 24).

FIG 2.

Genetic contexts of the bla_OXA-535 gene. Major genetic features of the environment of the different bla_OXA-48-like genes in Shewanella spp., i.e., MR-4 (accession no. CP000446.1) and S. bicestrii JAB-1 (accession no. NZ_CP022358.1), and of plasmid pOXA-436 (accession no. KY863418.1) isolated from Enterobacter asburiae strain AMA 497. Common features are highlighted in gray. sprT codes for a SprT-like protein of unknown function, endA encodes an endonuclease I, lysR encodes a putative lysR-type transcriptional regulator gene, and cps encodes a carbamoyl-phosphate synthase.