OXA-23 Is a Prevalent Mechanism Contributing to Sulbactam Resistance in Diverse Acinetobacter baumannii Clinical Strains

LETTER

Acinetobacter baumannii is one of the most important and threatening pathogens for health care-associated infections (HAI), and the treatments for multidrug-resistant A. baumannii (MDRAB) are limited (1, 2). Sulbactam serves as an alternative and effective option to combat these infections because of its intrinsic antimicrobial activity against the Acinetobacter genus (2). Unfortunately, increasing resistance to sulbactam compound in A. baumannii has been reported during the last decade (3, 4). Previous studies have demonstrated that β-lactamases such as TEM-1 and ADC confer sulbactam resistance in A. baumannii; however, they did not fully explain the sulbactam resistance phenomenon in each clinical strain (5–7). Therefore, we screened all β-lactamase genes in clinical isolates by whole-genome sequencing and aimed to assess the role of other β-lactamases apart from TEM-1 and ADC in sulbactam resistance of A. baumannii.

Eighty-eight clinical isolates, including 57 sulbactam-nonsusceptible isolates and 31 sulbactam-susceptible isolates, were selected from a broad geographic distribution across China (7). The presence of the β-lactamase genes bla_TEM-1, ISAba1-bla_ADC, bla_OXA-23, bla_OXA-58, bla_PER-1, and bla_CARB-2 were screened by whole-genome sequencing (Fig. 1a). Of note, in the sulbactam-nonsusceptible group, 54 (94.74%) strains possessed the bla_OXA-23 gene, while in the sulbactam-susceptible group, only 4 (12.90%) isolates were positive for the bla_OXA-23 gene (P < 0.001). These results indicated that bla_OXA-23 might facilitate sulbactam resistance in A. baumannii.

FIG 1.

(a) Heatmap of the presence of the β-lactamase genes of all selected clinical A. baumannii strains. Due to the lack of a breakpoint for sulbactam alone, we chose ≤4 mg/liter as the temporary susceptibility breakpoint on the basis of the CLSI susceptibility breakpoint for ampicillin-sulbactam (≤8/4 mg/liter) in A. baumannii. On the basis of the breakpoint, all clinical isolates were divided into sulbactam-nonsusceptible or sulbactam-susceptible groups. Sul-S group, sulbactam-susceptible group; Sul-NS group, sulbactam-nonsusceptible group; A, bla_TEM-1; B, ISAba1-bla_ADC; C, bla_OXA-23; D, bla_OXA-58; E, bla_PER-1; F, bla_CARB-2. Red color represents positive, and the blue color represents negative. (b) Correlation of the characteristics of the bla_OXA-23 gene associated with sulbactam MIC. All the bla_OXA-23-positive strains were divided into two groups according to the transposase types carrying bla_OXA-23 or copy number of bla_OXA-23: Tn2009 and Tn2006, as well as single-copy group (copy number <1.5) and multicopy group (copy number >1.5).

To further elucidate the correlation between bla_OXA-23 and sulbactam resistance, deletion and complementation of this gene were performed. See Table S1 in the supplemental material for a list of the strains and plasmids used in this study. The bla_OXA-23 gene was deleted in a clinical A. baumannii isolate, A2265, which carried one copy of bla_OXA-23 without bla_TEM-1 or ISAba1-bla_ADC. The strain A2265 Δbla_OXA-23 was complemented with the recombinant plasmid pYMAb2-Hyg^r::ISAba1-bla_OXA-23. The changes in the MICs were determined (Table 1). Notably, A2265 Δbla_OXA-23 exhibited increased susceptibility to sulbactam (8-fold) compared to that of A2265, and a similar trend was observed in the MICs of cefoperazone-sulbactam, imipenem, and meropenem, but no obvious change in MIC values was found in other agents. The MICs of A2265 Δbla_OXA-23(pYMAb2-Hyg^r::ISAba1-bla_OXA-23) for sulbactam, cefoperazone-sulbactam, imipenem, and meropenem returned to levels at or above those of strain A2265.

TABLE 1.

MICs of the knockouts and the transformants

Isolatea	MIC (mg/liter)b
	SUL	CPS	IPM	MEM	CTX	CAZ	CIP	MIN	GEN	CST	TGC	ATM
A2265	8	16	32	32	16	2	>32	4	>256	0.25	1	12
A2265 ΔblaOXA-23	1	1.5	1	1	8	4	>32	2	>256	0.25	1	12
A2265 ΔblaOXA-23(pYMAb2-Hygr)	1	0.75	1	1	4	6	>32	8	>256	0.25	1	6
A2265 ΔblaOXA-23(pYMAb2-Hygr::ISAba1-blaOXA-23)	64	64	128	128	>32	3	>32	4	>256	0.25	1	24

^aA2265 Δbla_OXA-23, the clinical strain A2265 without the bla_OXA-23 gene; A2265 Δbla_OXA-23(pYMAb2-Hyg^r), the strain A2265 was transformed with a plasmid pYMAb2-Hyg^r; A2265 Δbla_OXA-23(pYMAb2-Hyg^r::ISAba1-bla_OXA-23), the strain A2265 Δbla_OXA-23 was complemented with the recombinant plasmid pYMAb2-Hyg^r::ISAba1-bla_OXA-23.

^bSUL, sulbactam; CPS, cefoperazone-sulbactam; IPM, imipenem; MEM, meropenem; CTX, cefotaxime; CAZ, ceftazidime; CIP, ciprofloxacin; MIN, minocycline; GEN, gentamicin; CST, colistin; TGC, tigecycline; ATM, aztreonam.

Among the 58 bla_OXA-23-positive strains, 47 isolates were correlated with Tn2009, 8 isolates were associated with Tn2006, and the remaining 3 isolates in the sulbactam-susceptible group were found to carry only a partial sequence of bla_OXA-23. Tn2007 and Tn2008 were not detected. No significant difference in the sulbactam MICs (log2) of the Tn2009-group and Tn2006-group was found (P > 0.05) (Fig. 1b). The copy numbers of all bla_OXA-23-positive strains were measured by quantitative real-time PCR. A total of 25 isolates were found to carry a single copy of bla_OXA-23, while 30 isolates had at least two copies of bla_OXA-23. The sulbactam MICs of the multicopy group were similar to that of the single-copy group (P > 0.05) (Fig. 1b). Furthermore, the MIC of sulbactam did not correlate with the copy number of bla_OXA-23 (r = 0.263) (Fig. 1b).

OXA-23 is notoriously not inhibited by sulbactam, but sulbactam is also a well-known agent used directly against the Acinetobacter genus as an inhibitor. Although OXA-23 manifests considerable hydrolytic activity on a variety of β-lactam antibiotics, its effect on sulbactam has not been fully established (8, 9). Our research described the effect of OXA-23 on the sulbactam MIC in a clinical A. baumannii strain by gene deletion and complementation experiments. Nevertheless, we found that the sulbactam MIC did not relate to the transposon type or copy number of bla_OXA-23. Similar results were found in studies concerning bla_OXA-23 associated with carbapenem resistance (10, 11). The unstable copy number due to the plasmid location of bla_OXA-23 mentioned in prior research may be one of the factors (11). Another possible explanation may be the coexistence of other mechanisms, such as bla_TEM-1, ISAba1-bla_ADC, mutations of penicillin-binding proteins, and overexpression of the efflux pump, which can affect the sulbactam MIC simultaneously (12).

Altogether, the above-described findings suggest that sulbactam resistance in A. baumannii is multifactorial and can indeed be affected via OXA-23.