Emergence of Carbapenem-Hydrolyzing Enzymes in Acinetobacter baumannii Clinical Isolates

In recent years, the number of nosocomial infections caused by Acinetobacter baumannii has increased significantly (4). Many outbreaks have been reported, especially among patients confined to hospital intensive care units, where the widespread use of antibiotics may select multidrug-resistant strains. The difficulty of treating A. baumannii nosocomial infection is associated with the high resistance to a wide range of antimicrobial agents frequently observed in this species (8). Often, imipenem remains one of the few therapeutic alternatives. Fortunately, imipenem resistance is relatively rare among Acinetobacter clinical isolates. Carbapenem resistance can arise by a decrease in expression of an outer membrane protein (3) or by alteration in penicillin-binding proteins (5). In general, the emergence of carbapenem-hydrolyzing enzymes has been limited compared to the prevalence of other β-lactamases (1). However, in 1985 in Scotland, an A. baumannii strain that produced a plasmid-mediated carbapenemase, ARI-I, was isolated (7), and recently, imipenem-hydrolyzing enzymes have been noted in some Acinetobacter isolates in a United Kingdom burns unit (10).

This communication reports the production of imipenem-hydrolyzing enzymes in two A. baumannii isolates obtained from urine cultures in 1998 in a Portuguese teaching hospital.

MICs were determined by the E-test method according to the manufacturer’s instructions, and resistance was defined according to National Committee for Clinical Laboratory Standards guidelines (6). Crude sonicates of cell suspensions were assayed by spectrophotometry (UV/Vis Perkin-Elmer Lambda 6 spectrophotometer) with 0.1 mM imipenem and 0.1 mM nitrocefin solutions. One unit of β-lactamase activity was defined as the amount of enzyme hydrolyzing 1 nmol of the substrate per min per mg of protein at 30°C in 0.1 M phosphate buffer, pH 7.0. Isoelectric focusing (IEF) was performed in precast polyacrylamide gels, pI 3 to 9 (Pharmacia), by using a PhastSystem apparatus according to the manufacturer’s instructions. Prior to staining with nitrocefin, one of the gels was overlaid with 0.1 mM cloxacillin solution, which is known to inhibit Bush group 1 β-lactamases (2), enzymes that are usually produced by Acinetobacter spp. (9). Two control strains were used in the experiments: an A. baumannii clinical isolate susceptible to imipenem and A. baumannii ATCC 19606. In an attempt to detect transfer of imipenem resistance, filter mating experiments were performed at 37°C by using Escherichia coli K802N as a recipient cell.

The results are shown in Table 1. The isolates (strains 122FFC and 65FFC) presented similar resistance patterns, with high-level resistance to imipenem and meropenem (MICs of both compounds, >32 mg/liter) and cephalosporins, including a new cephalosporin, cefepime (MICs, >256 mg/liter). They were susceptible to ciprofloxacin (MICs of 1.5 and 0.5 mg/liter, respectively) and to aminoglycosides (data not shown).

TABLE 1.

Characteristics of the isolates of A. baumannii studied

Characteristic	Value for isolate:
	122FFC	65FFC	3625a	ATCC 19606
Date isolated (mo/yr)	4/98	9/98
MIC (mg/liter)
Amoxicillin	>256	>256	>256	>256
Amoxicillin-clavulanate	8	16	16	4
Piperacillin	48	48	>256	16
Ceftazidime	>256	>256	48	8
Ceftriaxone	>256	>256	>256	32
Cefepime	>256	>256	24	16
Aztreonam	64	64	96	16
Imipenem	>32	>32	2	0.38
Meropenem	>32	>32	NDb	ND
Ciprofloxacin	1.5	0.5	ND	ND
Hydrolysis by crude extracts (U/mg of protein)c
Imipenem	247.79	205.5	0.95	0.32
ZnCl2d	273.8	229.96	ND	ND
EDTAe	37.34	45.32	ND	ND
Nitrocefin	2,087.47	1,871.75	13,452	6.15

^aClinical strain from laboratory collection. 

^bND, not determined. 

^cNanomoles of substrate hydrolyzed per minute per milligram of protein. 

^dEnzyme activity in the presence of 1 mM ZnCl₂ solution. 

^eEnzyme activity in the presence of 1 mM EDTA solution after 10 min of preincubation with extract at 30°C. 

IEF revealed a large β-lactamase band with a pI of >8, presumably a chromosomal Bush group 1 enzyme. A sharp β-lactamase band with a pI of >8 was observed after the treatment with cloxacillin, suggesting the production of another β-lactamase not inhibited by cloxacillin.

Imipenem was readily hydrolyzed by crude extracts of imipenem-resistant isolates, but not by extracts of the controls, confirming the presence of an imipenem-hydrolyzing enzyme. The observed carbapenem resistance correlated with hydrolytic activity. A slight improvement of the β-lactamase activity was observed during the measurement of imipenem hydrolysis in the presence of 1 mM ZnCl₂ solution. Preincubation of the extract with 1 mM EDTA solution for 10 min at 30°C resulted in a decrease in β-lactamase activity (between 70 and 80% inhibition). All attempts to transfer imipenem resistance to E. coli K802N were unsuccessful.

The results obtained suggest the possibility of a metalloenzyme. Neither of the A. baumannii carbapenemases reported in the literature are zinc-dependent enzymes (7, 10). This is the first report of an imipenem-hydrolyzing enzyme in this species found in Portugal. The increase in carbapenem therapy might be associated with the emergence of A. baumannii strains that produce imipenem-hydrolyzing enzymes, which is a serious concern due to the large spectrum of these enzymes. Therefore, it is crucial to rationalize the use of this class of compounds in an attempt to minimize the selection of these new β-lactamases.