Identification of Novel VEB β-Lactamase Enzymes and Their Impact on Avibactam Inhibition

Abstract

Ceftazidime-avibactam has activity against Pseudomonas aeruginosa and Enterobacteriaceae expressing numerous class A and class C β-lactamases, although the ability to inhibit many minor enzyme variants has not been established. Novel VEB class A β-lactamases were identified during characterization of surveillance isolates. The cloned novel VEB β-lactamases possessed an extended-spectrum β-lactamase phenotype and were inhibited by avibactam in a concentration-dependent manner. The residues that comprised the avibactam binding pocket were either identical or functionally conserved. These data demonstrate that avibactam can inhibit VEB β-lactamases.

TEXT

The acquisition and spread of β-lactamase enzymes among Gram-negative bacterial species has led to widespread resistance to many β-lactam agents, which is becoming an increasingly significant burden on human health. The β-lactamases are an extremely diverse group of enzymes, and it has been shown that small amino acid sequence differences can make significant functional impacts, either in the substrate spectrum, the rate of hydrolysis, or the ability to be inhibited by clinically used β-lactamase inhibitors (1–7). The class A group of β-lactamases can be divided into multiple families, and with only 12 confirmed variants, the VEB group represents one of the smaller subgroups of class A β-lactamases. The VEB enzymes appear to be frequently observed in the nonfermenter species like Pseudomonas aeruginosa and Acinetobacter baumannii as well as in other Enterobacteriaceae spp., and the prevalence of them is increasing (8–10). Surveillance programs are an important way to broadly monitor changing trends in susceptibility as well as identify potential emerging resistance mechanisms. During the ceftazidime-avibactam surveillance program, isolates with reduced susceptibility were routinely characterized. A common feature among several isolates was the presence of class A VEB β-lactamases (Table 1). Genomic DNA from P. aeruginosa and Escherichia coli isolates that had reduced susceptibility to ceftazidime-avibactam analysis was sequenced on a MiSeq sequencer (Illumina, San Diego, CA, USA) and identified three previously described and three novel VEB enzymes (VEB-13, -14 and -15) (Table 1). Whereas the ceftazidime-avibactam MIC value of >512 μg/ml in P. aeruginosa ARC4865 can be explained by the presence of a VIM metallo-β-lactamase, the β-lactamase content of the other isolates raised the question of the ability of avibactam to effectively inhibit the VEB subgroup of class A β-lactamases. The entire genes encoding VEB-9, VEB-5, VEB-13, VEB-14, VEB-15, VEB-16, and the Shine-Dalgarno sequence were amplified from the respective strains, ligated into pSU19 downstream of the lac promoter (11), sequence verified, and expressed in E. coli DH5α. This ensured that any contributions to the reduced ceftazidime-avibactam susceptibility from other genetic determinants in the original isolate were eliminated. The MIC against each isolate was determined using the broth microdilution method following the guidelines of the Clinical and Laboratory Standards Institute (12) in the absence of isopropyl-β-D-thiogalactopyranoside induction. The recombinant VEB enzymes, including VEB-5, VEB-9 (formerly known as VEB-1a), and VEB-16 (formerly known as VEB-1b), all exhibited an extended-spectrum β-lactamase phenotype, with ceftazidime MIC values for the recombinant strains of 16 to 256 μg/ml (Table 2). The susceptibility spectrum of the three novel VEB enzymes was similar to that of VEB-5, VEB-9, and VEB-16, although the microbiological susceptibility values suggest a lower hydrolytic capacity of VEB-13 and VEB-14, especially against the second-generation cephalosporins (cefuroxime and cefpodoxime) and the monobactam (aztreonam). Of note, the susceptibility of E. coli carrying any of the VEB isoforms to ceftaroline was significantly less impacted (MIC values of 4 to 32 μg/ml) than CTX-M-15 (MIC of >512 μg/ml). The ability of avibactam to inhibit these enzymes was tested in combination with amoxicillin, aztreonam, and ceftazidime (Table 2). The amoxicillin-avibactam combination was used to minimize any variability in substrate hydrolysis between the enzymes and to maximize the understanding of β-lactamase inhibition by avibactam. Testing with a combination of 4 μg/ml of avibactam demonstrated good inhibition of all of the novel VEB isoforms. However, it should be noted that, although the isolate carrying VEB-5 had a ceftazidime-avibactam MIC value of 2 μg/ml, it also exhibited the highest MIC against ceftazidime (256 μg/ml), such that the fold restoration of ceftazidime activity was equal to or greater than with the other VEB enzymes. Susceptibility testing was also performed with amoxicillin and ceftazidime in the presence of decreasing amounts of avibactam, and the dose-dependent effect was particularly noticeable against VEB-5 and VEB-16 with both the amoxicillin and ceftazidime substrates (Table 2).

TABLE 1.

Susceptibility profiles of the clinical isolates carrying VEB β-lactamases

Strain	Species	Country	Year	β-Lactamase content	MIC (μg/ml) ofa:
					CAZ	CAZ-AVIb	ATM	ATM-AVIb	MER	CPT	CFX	POD	AMX	AVI
TRN6372	P. aeruginosa	Thailand	2012	VEB-9, AZPC-35c	>512	128	>512	64	16	>512	>512	>512	>1,024	>64
ARC3531	E. coli	France	2007	VEB-5, TEM-1, AmpC	>512	64	>512	8	0.03	256	>512	512	>1,024	8
ARC3859	P. aeruginosa	India	2010	VEB-13. OXA-21, SHV-11, AZPC-35	>512	64	512	32	8	>512	128	>512	1,024	>64
ARC4865	P. aeruginosa	India	2007	VEB-14. OXA-10, VIM-5, AZPC-11	>512	>512	256	128	>64	>512	>512	>512	>1,024	>64
ARC3554	E. coli	Norway	2003	VEB-15, OXA-1, TEM-1, AmpC	>512	16	>512	4	0.125	256	>512	512	>1,024	16
TRN6384	P. aeruginosa	Thailand	2012	VEB-16, AZPC-35	512	256	>512	128	16	>512	>512	>512	>1,024	>64

^aATM, aztreonam; AMX, amoxicillin; AVI, avaibactam; CAZ, ceftazidime; CFX, cefuroxime; CPT, ceftaroline; MER, meropenem; POD, cefpodoxime.

^bAvibactam held at a constant concentration of 4 μg/ml.

^cAZPC is the AmpC naming nomenclature in P. aeruginosa consistent with that defined by Lahiri et al. (20).

TABLE 2.

Susceptibility profiles of E. coli carrying isogenically expressed VEB β-lactamases

Compounda (β-lactamase inhibitor concn, μg/ml)	MIC (μg/ml) of E. coli alone or carrying different class A β-lactamases:
	No β-lactamase	CTX-M-15	VEB-5	VEB-9	VEB-13	VEB-14	VEB-15	VEB-16
AMX	8	>1,024	512	256	128	128	128	512
AMX-AVI (0.5)	2	8	64	8	16	16	8	32
AMX-AVI (1)	2	4	32	8	8	8	4	8
AMX-AVI (2)	2	4	16	4	4	4	4	4
AMX-AVI (4)	1	2	8	2	2	2	4	4
AMX-CLAV (1)	8	16	16	8	8	8	8	8
AMX-CLAV (2)	8	16	8	8	8	8	8	8
AMX-CLAV (4)	8	8	8	8	8	8	8	4
AMX-CLAV (8)	4	8	4	4	8	8	4	4
CEF	8	>512	512	128	8	16	64	256
POD	2	512	128	32	8	8	32	64
AVI	16	16	16	16	16	16	16	16
CLAV	32	32	32	32	32	32	32	32
ATM	0.125	32	256	64	8	8	32	64
ATM-AVI (4)	0.125	0.06	0.5	0.125	0.06	0.125	0.125	0.06
CPT	0.125	>512	32	4	1	4	4	8
MER	0.03	0.03	0.015	0.015	0.015	0.015	0.03	0.015
CAZ	0.25	4	256	128	16	64	128	128
CAZ-AVI (0.5)	0.25	0.25	32	1	1	16	4	2
CAZ-AVI (1)	0.25	0.25	8	1	0.5	8	2	1
CAZ-AVI (2)	0.25	0.25	4	0.5	0.25	2	1	0.5
CAZ-AVI (4)	0.25	0.25	2	0.5	0.25	1	0.5	0.5

^aAMX, amoxicillin; ATM, aztreonam; AVI, avibactam; CAZ, ceftazidime; CEF, cefuroxime; CLAV, clavulanic acid; CPT, ceftaroline; MER, meropenem; POD, cefpodoxime.

The sequences of the VEB β-lactamases have been deposited in GenBank (accession numbers KR021283 for VEB-13; KR021284 for VEB-14; KR021285 for VEB-15; and KR021286 for VEB-16), and the alignment can be seen in Figure 1. They were all similar to VEB-9, which was formerly called VEB-1a (13). In particular, 7 of the 12 key residues that form the avibactam binding pocket in class A β-lactamases based on the crystal structure of avibactam in complex with CTX-M-15 (14) were fully conserved, including the catalytic residues Ser70, Lys73, Ser130, and Glu166, which are involved in acylation and deacylation of avibactam. The hydrophobic residue Tyr105 that interacts with the piperidine ring of avibactam was replaced with a Trp in these novel VEB enzymes, as is the case with all of the VEB β-lactamases characterized to date. This conservative Tyr105Trp substitution is also observed in the GES, PER, and Klebsiella pneumoniae carbapenemase subgroups of class A β-lactamases and does not impact the inhibitory properties of avibactam (15, 16). Both Asn104 and Asn170 contribute strong hydrogen bonds to either the sulfonate or the carboxamide groups of avibactam in the structure (14), and all of the VEB enzymes characterized to date, including these four novel isoforms, carry Asn104Pro and Asn170His substitutions. The H-bond with carboxamide of avibactam will be maintained by His170 but not by Pro104.

FIG 1.

Sequence alignment of VEB β-lactamases. The protein sequence of the novel VEB enzymes were aligned with VEB-9 (formerly VEB-1a, GenBank accession number AF324833) and VEB-5 (GenBank accession number EF420108). The differences compared to VEB-9 are shown. The 12 key residues that line the avibactam binding pocket based on the CTX-M-15–avibactam structure (14) are shown above the VEB-9 sequence, and those that are conserved within the VEB enzymes are highlighted with a gray box. The binding residues that differ from CTX-M-15 (although conserved among the VEB enzymes) are indicated in an open box in the VEB-9 sequence.

Although class A β-lactamase structures are well conserved, allowing interpretation of binding modes across the subgroups, it should be noted that there is no VEB structure available and that slight differences in binding mode may cause different impacts of these substitutions. The Thr216 residue has no direct interaction with the inhibitor but is close enough to the bound avibactam such that some substitutions could impair the binding efficiency. However, the Thr216Ala substitution, which is also observed in VEB-8, is to a smaller Ala residue and is unlikely to impact avibactam binding. Interestingly, the VEB-14 enzyme carries a one-residue deletion in this region where there are only two consecutive Thr residues instead of three (Fig. 1). Finally, there is a Ser237Thr substitution observed in all of the VEB isoforms; however, this is a relatively common variation among class A β-lactamases, which is seen in all KPC and some TEM. This Ser237Thr substitution is also not expected to affect avibactam inhibition based on structural modeling and on the efficient inhibition of class A β-lactamases with this difference (15, 17).

In summary, all VEB variants were inhibited by avibactam and did not represent the cause of the reduced susceptibility to ceftazidime-avibactam. Indeed, genomic analysis failed to definitively identify the underlying mechanism of decreased susceptibility in the original clinical isolates, with the exception of P. aeruginosa ARC4865 that contained a VIM-5 metallo-β-lactamase. This is not surprising, as β-lactam resistance can be a complex phenotype, with a large variety of genetic determinants, not just β-lactamase enzymes, impacting the overall susceptibility profile (18, 19). With the increasing awareness of emerging antibacterial resistance, surveillance programs that monitor the erosion of activity of clinically useful drugs are crucial. As novel β-lactamase variants are identified, it is important to understand the spectrum of activity and the ability of different β-lactamase inhibitors to be able to protect the partner β-lactam compound from degradation.

Nucleotide sequence accession numbers.

The sequences of the VEB β-lactamases have been deposited in GenBank under accession numbers KR021283 for VEB-13; KR021284 for VEB-14; KR021285 for VEB-15; and KR021286 for VEB-16.