Recent Emergence of Aztreonam-Avibactam Resistance in NDM and OXA-48 Carbapenemase-Producing Escherichia coli in Germany

LETTER

The continuing surge of carbapenem-resistant Enterobacterales (CRE) worldwide is a challenging problem of antimicrobial resistance today. The introduction of ceftazidime-avibactam (CZA) was an important step in the treatment of infections caused by CPE. Avibactam restores ceftazidime activity against carbapenemase-producing Enterobacterales of Ambler class A (KPC) and of Ambler class D (OXA-48 and 48-like), which are all serine β-lactamases. However, avibactam as well as the other β-lactam inhibitors currently in clinical use do not inhibit metallo-β-lactamases (MBLs) such as the carbapenemases of Ambler class B. Aztreonam is stable to hydrolysis by MBLs, a unique feature compared with other β-lactams but is generally cleaved by other clinically relevant serine β-lactamases (1). Therefore, aztreonam/avibactam (ATM-AVI) is a drug combination currently undergoing clinical trials to assess its efficacy in treating infections with Gram-negative bacteria, including those that produce MBLs (2). As many Enterobacterales that produce an MBL often coproduce a serine-β-lactamase, a combination of aztreonam with avibactam offers an effective alternative to treating those CRE infections (3).

ATM has potent selective, specific activity toward the highly conserved penicillin-binding protein 3 (PBP3). Isolates carrying PBP3 variants with a specific insertion (₃₃₃YRIN/K₃₃₄) have reduced susceptibilities toward ATM-AVI. This resistance increased in isolates harboring the plasmid-borne bla_CMY AmpC-β-lactamase, particularly when it coexisted in a combination with both NDMs and variant PBP3 (4).

We selected isolates exhibiting resistance to >4 mg/ml ATM and found ATM-AVI resistance in 15 of 110 carbapenem-resistant NDM- and OXA- producing Escherichia coli collected from 2017 to 2019 as part of a regional surveillance initiative (Table 1). The highest numbers were seen in 2019, suggesting recent emergence. For 11 isolates, we detected previously described combinations of PBP3 modifications either with or without bla_NDM alleles and/or bla_CMY-42. One isolate with bla_NDM-5 and bla_CMY-42 with a point mutation in its PBP3 sequence had a MIC of 8 mg/liter toward ATM-AVI. Three other isolates, being either OXA-181 or OXA-48producers harbored bla_CMY-42 and a PBP3 insertion and exhibited resistance levels of 8 to 16 mg/liter. The bla_CMY-42 alleles are present at an identical location on IncIγ plasmids of 27 to 53 kb. Many plasmids carry deletions of transfer genes (traB-Y) and the conjugative pilus (pilI-V) (Fig. 1A) (5, 6), as previously also reported for two IncIγ plasmids harboring bla_CMY-42 (7).

TABLE 1.

Genetic characteristics and MIC^a

Isolate	Sampling yr	Country	Gender	Age (yrs)	MLST type	Serotype	Carbapenemase gene(s)	PBP3		CMY allele	blaCMY-42–encoding plasmid		MIC (mg/liter)
								Insertion	Mutation		Inc type	Size (bp)	ATM	ATM-AVI	ETP	IMI
Survcare058	2017	DE	Male	83	405	O102:H6	NDM-5	YRIK	A413V	bla CMY-42	IncIγ	48,544	64	16	>32	>32
Survcare321	2019	DE	Male	81	405	O102:H6	NDM-5	None	A413V	bla CMY-42	IncIγ	48,543b	>128	8	128	32
Survcare385	2019	DE	Male	42	361	O9:H30	NDM-5	YRIN	Q227H, E349K, I532K	bla CMY-42	IncIγ	53,219b	32	8	128	32
Survcare420	2019	DE	Male	75	361	O9:H30	NDM-5	YRIN	Q227H, E349K, I532K	bla CMY-42	IncIγ	53,209	>128	8	128	64
N590	2019	CH	Male	69	167	O89:H9	NDM-5	YRIN	Q227H, E349K, I532K	bla CMY-42	IncIγ	48,546	64	8	64	32
N640	2019	CH	Female	68	167	O89:H9	NDM-5	YRIN	Q227H, E349K, I532K	bla CMY-42	IncIγ	48,546	64	4	128	32
N1013	2019	CH	Female	31	361	O9:H30	NDM-5	YRIN	Q227H, E349K, I532K	bla CMY-42	IncIγ	45,854	128	8	>128	64
Survcare052	2017	DE	Female	59	617	O89:H9	NDEM-5	YRIK	A413V	None	NA	NA	>128	2	>32	>32
Survcare278	2019	DE	Male	64	354	O45:H6	NDEM-1	YRIP	A413V	None	NA	NA	>128	1	>32	>32
Survcare289	2019	DE	Female	15	1284	O9	NDEM-5	YRIN	Q227H, E349K, I532K	None	NA	NA	>128	8	32	16
Survcare292	2019	DE	Male	78	46	O9:H10	NDEM-5	YRIK	A413V	None	NA	NA	>128	2	64	16
Survcare293	2019	DE	Female	<1	1284	O9	NDEM-5	YRIK	A413V	None	NA	NA	>128	8	32	16
Survcare309	2019	DE	Male	<1	1284	O9	NDEM-5	YRIK	A413V	None	NA	NA	>128	8	32	16
Survcare408	2019	DE	Male	38	167	O89:H5	NDEM-5	YRIN	Q227H, E349K, I532K	None	NA	NA	>128	2	128	64
Survcare253	2019	DE	Male	76	2851	O154:H18	NDEM-5, OXA-181	YRIN	Q227H, E349K, I532K	bla CMY-42	IncIγ	26,256b	>128	16	128	32
Survcare043	2018	DE	Female	67	167	H9	OXA-181	YRIN	Q227H, E349K, I532K	bla CMY-42	IncIγ	45,585	>128	8	8	1
Survcare045	2018	DE	Female	65	167	O89:H9	OXA-181	YRIN	Q227H, E349K, I532K	bla CMY-42	IncIγ::IncF [36:A4:B-]	167,285b	>128	4	1	0.25
Survcare453	2019	DE	Female	64	410	O8:H21	OXA-48	YRIK	A413V	bla CMY-42	IncIγ	45,009	>128	16	4	2

^aATM, aztreonam; AVI, avibactam; CH, Switzerland; DE, Germany; ETP, ertapenem; IMI, imipenem; NA, not applicable; MLST, multilocus sequence type.

^bClosed genomes.

FIG 1.

(A) Comparison of the bla_CMY-42-encoding IncIγ plasmids included pCMY-42_005008 with pR621a as the reference. The GenBank accession numbers of pCMY-42_005008 and pR621a are CP058662 and AP011954, respectively. Note the absence of the conjugal transfer and/or pilus formation regions on most plasmids. (B) Genetic map of pS045CMY-42, a conjugative chimeric IncIγ::IncFII plasmid, compared with the NDM-5-encoding IncF plasmids pS321-NDM5, pES3-NDM5, and pN525NDM-5 and the CMY-42-encoding IncIγ plasmids pS321CMY-42 and pS385CMY-42. The IncIγ plasmid is between positions 35000 and 105300. Many of the NDM-5-encoding IncF plasmids carry genes for conjugative transfer as well as a region with highly homology in IncIγ plasmids. The regions between positions 35043 and 38524 and 103905 and 1078381 share a DNA identity of ∼94%.

It is unlikely that these truncated plasmids can directly promote transfer of bla_CMY-42 (horizontal transfer) (Fig. 1A). Nevertheless, they all carry the type I partition machinery (parAB) and a toxin-antitoxin pndBA postsegregational killing system, which can ensure stable presence and inheritance (vertical transfer) even without antibiotic selection (Fig. 1B) (8). Indeed, plasmids of isolates were highly stable across a 7-day serial passage in the absence of antibiotics.

The presence of these IncIγ plasmids in differing E. coli sequence types (STs), however, suggests inherent mobility (5, 9, 10). We detected a conjugative chimeric IncIγ::IncFII plasmid (pS045CMY-42) in the isolate Survcare045 and sequenced it to completion. This chimera of 167,286 bp was created through homologous recombination with subsequent duplication of a region in both plasmids containing highly related genes (Fig. 1B). This region of homology is found in all the IncFII-carrying NDM alleles and IncIγ plasmids examined here. However, resistance transfer, at a rate of 10⁻⁶, was only detected for the chimeric plasmid.

Here, we show that emergent aztreonam-avibactam resistance may be identified with MBL- producing strains but also with carbapenem-hydrolyzing class D β-lactamases, even if this combination is not yet in clinical use. This is of concern because OXA-48 and OXA-181 are commonly occurring carbapenemases found worldwide, often together with high-risk clones, and in Europe are by far the most prevalent type of carbapenemases (11, 12). Their occurrence in carbapenem-resistant E. coli, which is also associated with community spread, could limit use of this drug combination as a first-line therapy and exacerbate a situation of antibiotic resistance selection where, already, only a limited number of antibiotic options remains.

Data availability.

The sequences reported in this study have been deposited in NCBI GenBank under the BioProject accession number PRJNA732846.