Plasmid-Encoded Carbapenem-Hydrolyzing β-Lactamase OXA-48 in an Imipenem-Susceptible Klebsiella pneumoniae Strain from Belgium

Emergence and dissemination of Enterobacteriaceae isolates harboring carbapenemases represent a significant threat to the management of nosocomial infections (6). Carbapenem-hydrolyzing β-lactamases can be metallo-β-lactamases, Ambler class A enzymes, or more rarely expanded-spectrum oxacillinases (6, 10). The Ambler class D OXA-48 β-lactamase has been found only in enterobacterial species (one Escherichia coli isolate and four Klebsiella pneumoniae isolates) from Turkey (1, 5, 9). Here we describe a K. pneumoniae isolate from Belgium with reduced susceptibility to carbapenems that produced the β-lactamase OXA-48.

On 27 November 2007, a 37-year-old man undergoing chemotherapy treatment for a lymphoma was hospitalized in the hematology ward of the hospital of St-Luc, Brussels, Belgium, for confusion due to herpetic encephalitis caused by herpes simplex virus type 2. On 29 November, a K. pneumoniae UCL-1 isolate from urine samples was found to be resistant to amoxicillin (AMX) and amoxicillin-clavulanate (AMC) but remained susceptible to expanded-spectrum cephalosporins and to meropenem (MIC of <1 μg/ml) as determined by the Phoenix Microbiology system (Becton-Dickinson, Erembodegem, Belgium) and Vitek (bioMérieux, Marcy l'Etoile, France). As routinely performed for AMX/AMC-resistant isolates, a disk diffusion antibiogram revealed a reduced susceptibility to meropenem (23 mm) and imipenem (22 mm) but intermediate susceptibility to ertapenem (18 mm) (3). In addition, the isolate was also resistant to fluoroquinolones, cotrimoxazole, kanamycin, and tetracycline according to disk diffusion susceptibility testing results. The patient was not treated for its infection and did not develop a systemic infection with K. pneumoniae UCL-1 requiring antibiotic treatment. No other K. pneumoniae isolate with a similar antibiotic resistance pattern was recovered from the hospital during this same period of time.

The MICs of β-lactams determined by the Etest method (AB BIODISK, Solna, Sweden) and interpreted according to CLSI criteria (3) for K. pneumoniae UCL-1 showed MICs of penicillins not modified after addition of clavulanic acid, of imipenem and meropenem in the susceptibility range, and of ertapenem in the intermediate range (Table 1). A crude β-lactamase extract of that isolate showed significant carbapenem-hydrolyzing activity as measured spectrophotometrically (1.6, 0.05, and 0.04 μmol of imipenem, meropenem, and ertapenem, respectively/min/mg of total protein) (4).

TABLE 1.

MICs of β-lactams for K. pneumoniae 11978, K. pneumoniae UCL-1, their E. coli transconjugants, and E. coli recipient strain J53

β-Lactam(s)a	MIC (μg/ml) for strain
	K. pneumoniae 11978	E. coli J53 Tc11978	K. pneumoniae UCL-1	E. coli J53 TcUCL-1	E. coli J53
Amoxicillin	>256	>256	>256	>256	4
Amoxicillin + CLA	>256	>256	>256	>256	4
Ticarcillin	>256	>256	>256	>256	2
Ticarcillin + CLA	>256	>256	>256	>256	2
Piperacillin	>256	128	256	128	1
Piperacillin + TZB	>256	128	256	128	1
Cephalothin	>256	8	16	8	4
Cefoxitin	128	2	2	2	2
Cefotaxime	64	0.38	0.125	0.38	0.06
Ceftazidime	>256	0.75	1.0	0.75	0.06
Aztreonam	>256	0.064	0.064	0.064	0.03
Cefepime	32	0.125	0.125	0.125	0.03
Imipenem	64	1	0.75	0.75	0.12
Meropenem	64	0.5	0.5	0.5	0.03
Ertapenem	>32	2	4	2	0.03
Tigecycline	1.5	0.5	2.0	0.5	0.5

^aCLA, clavulanic acid at a fixed concentration of 2 μg/ml; TZB, tazobactam at a fixed concentration of 4 μg/ml.

PCR experiments with primers for detection of Ambler class A, class D, and class B β-lactamase genes (4), followed by sequencing, identified the carbapenemase bla_OXA-48 gene. Using a series of PCR primers, two IS1999 elements were found surrounding the bla_OXA-48 gene in a similar manner to that found in the prototype K. pneumoniae 11978 strain from Turkey (2, 8). Plasmid analysis detected a 70-kb self-conjugative plasmid of similar size to pA-1 (OXA-48) in K. pneumoniae strain 11978 (9). This plasmid conferred a β-lactam resistance pattern for E. coli transconjugants with reduced carbapenem susceptibility (Table 1). A β-lactamase extract from a transconjugant culture subjected to analytical isoelectric focusing (7) identified one β-lactamase with an isoelectric point of 7.2.

Up to now, carbapenemases of the OXA-48 type have seemed to be limited to Turkey (1, 5, 9). This is the first evidence of the type's identification outside of Turkey for a patient with no apparent relation with this country. Moreover, the fact that reduced susceptibility to carbapenem was not detected by automated systems (Phoenix and Vitek) may suggest that this novel resistance determinant could be more prevalent than expected.

Reduced susceptibility to imipenem and meropenem but with the MIC still in the susceptible range was also observed using Etest. It is likely that resistance may occur only if additional mutations are present beyond OXA-48 or if the plasmid carrying OXA-48 is transferred to bacteria with already-reduced susceptibility to carbapenems. A similar low level of resistance to carbapenems may be observed for KPC and VIM-IMP producers, potentially leading to detection failures (4, 11). Ertapenem seems to be more affected by these enzymes in Enterobacteriaceae and therefore is a better indicator for their detection.