Multidrug-resistant pseudomonas aeruginosa producing PER-1 extended-spectrum serine-[beta]-lactamase and VIM-2 metallo-[beta]-lactamase. (Letters).

To the Editor: In Pseudomonas aeruginosa, secondary beta-lactamases with extended substrate specificity can be responsible for acquired resistance to the most powerful antipseudomonal beta-lactams, such as expanded-spectrum cephalosporins and carbapenems (1). A number of these enzymes have been described, including extended-spectrum serine-betadactamases (ESBLs) of groups 2be and 2d (e.g., PER-1 and various OXA-type enzymes) (2,3) and metallobeta-lactamases of group 3 (e.g., IMP-1 and the recently described VIM-1 and VIM-2 enzymes) (2,4,5). The secondary ESBLs can degrade penicillins, expanded-spectrum cephalosporins, and monobactams (but not carbapenems) and are often susceptible to serine-beta-lactamase inhibitors (1-3). The secondary metallo-beta-lactamases, on the other hand, are notable for their carbapenemase activity and can degrade virtually all beta-lactams except monobactams, while being resistant to the currently available inhibitors (1,2,5,6).

On March 2000, a multidrug-resistant P. aeruginosa (isolate VA-182/00) was isolated in pure culture from a bronchial washing of a 58-year-old patient with multiple myeloma. The patient had been admitted 15 days earlier to the Varese University Hospital with a diagnosis of pneumonia and had been treated with ciprofloxacin (0.5 g twice a day) plus piperacillin (2 g three times a day) for 12 days, and then with imipenem/cilastatin (0.5 g three times a day). No cultures of respiratory tract specimens were done earlier in hospitalization. Multiple myeloma had been diagnosed in 1997, and the patient had been treated with multiple cycles of antiproliferative chemotherapy and had received autologous peripheral blood stem cell transplantation. According to clinical records, P. aeruginosa had not been isolated previously during this patient's protracted illness. In vitro susceptibility testing showed that the P. aeruginosa isolate was resistant to mezlocillin, ceftazidime, cefepime, aztreonam, imipenem, meropenem, gentamicin, tobramycin, netilmicyn (MICs, > 128 [micro]g/mL), amikacin (MIC, 64 [micro]g/mL), ciprofloxacin and levofloxacin (MICs, >32 [micro]g/mL). Only piperacillin and piperacillin/tazobactam had MIC values slightly lower than the breakpoints for resistance (64 [micro]g/mL and 48/4 [micro]g/ mL, respectively), although-considering the normal MICs of piperacillin for susceptible P. aeruginosa (2-8 [micro]g/mL)--it was evident that the isolate also had considerable biological resistance to these drugs. A double disk-diffusion test, carried out with standard disks placed 20 mm apart (center-to-center), showed synergy between clavulanate and aztreonam. The treatment was changed to piperacillin/tazobactam (4 g four times a day), and a slow recovery ensued over a 30-day period. The patient died 3 months later following a relapse of the underlying malignancy.

The unusually high carbapenem MICs exhibited by VA182/00 suggested production of a secondary metallo-beta-lactamase, while the synergy between clavulanate and aztreonam suggested production of a secondary serine ESBL. A crude extract of that isolate, assayed spectrophotometrically (7), exhibited imipenem-hydrolyzing activity (94 nmol/min/ mg protein, inhibited by EDTA) as well as aztreonam-hydrolyzing activity (11 nmol/min/mg protein, resistant to EDTA). Analytic isoelectric focusing (IEF) of the extract, followed by development with the nitrocefin chromogenic substrate (7), showed three bands of beta-lactamase activity of pIs 5.4, 5.6, and 6.3, suggesting the presence of at least three different secondary enzymes. A colony-blot hybridization with probes for the [bla.sub.IMP], [bla.sub.VIM], and [bla.sub.PER] resistance genes (all of which have been previously detected in P. aeruginosa clinical isolates from the same hospital [8,9; Luzzaro F, unpub. data]) yielded positive results with both the [bla.sub.VIM] and the [bla.sub.PER] probes. Amplification of the resistance genes by polymerase chain reaction (PCR) with primers VIM/DIA-f (5'-CAgATTgCCgATggTgTTTgg) and VIM/DIA-r (5'-AggTgggC-CATTCAgCCAgA) for [bla.sub.VIM] genes (4,5) and BLAPER-f (5'-gggACA(g/A)TC(g/C)(g/T)ATgAATgTCA) and BLAPER-r (5'-ggg(C/T)(g/C)gCTTAgATAgTgCTgAT) for blapER genes (9), yielded amplicons of the expected sizes (522 and 966 bp, respectively). Direct amplicon sequencing identified the two beta-lactamase determinants as [bla.sub.VIM-2] (5) and [bla.sub.PER-1] (10), respectively, a finding consistent with the pIs 5.6 and 5.4 beta-lactamase bands detected in IEF (3,5). Conjugative transfer of the resistance determinants to Escherichia coli proved unsuccessful. In a Southern blot analysis of total undigested DNA from VA-182/00, both the [bla.sub.VIM] and [bla.sub.PER] probes apparently hybridized to the chromosomal DNA band; no plasmid bands recognized by either probe were detected. A PCR experiment with primers OXA10-f (5'-ggAA-CAAAgAgTTCTCTgCC) and OXA105-r (5'-TTAgCCAC-CAATgATgCC(C/T)TC), suitable for amplification of [bla.sub.OXA] genes of the OXA-10 group, did not yield an amplicon of the expected size (719 bp), suggesting that the pI 6.3 beta-lactamase band detected by IEF did not correspond to an enzyme of this group.

This is the first observation of a P. aeruginosa clinical isolate simultaneously producing a secondary PER-1 ESBL and a secondary metallo-betadactamase. The finding, observed in a hospital where both the resistance genes ([bla.sub.PER-1] and [bla.sub.VIM-2]) had been detected separately among clinical isolates, underscores the possibility of the emergence of new threatening combinations of resistance determinants among nosocomial pathogens. In fact, the recruitment of similar resistance determinants within a single P. aeruginosa strain can determine a resistance phenotype to virtually all the available antipseudomonal beta-lactams, an occurrence that can be particularly dramatic when, as in the present case, resistance to beta-lactams is associated with resistance against aminoglycosides and fluoroquinolones. In this case, only piperacillin (which appears to be a relatively poor substrate for both enzymes [3,5]) retained moderate activity in vitro and, administered at high dosage in combination with tazobactam, was apparently effective in vivo. Should a similar resistance phenotype disseminate, it might have strategic implications for the development of new beta-lactamase inhibitors and for selection of beta-lactam compounds to associate wth inhibitors.

Acknowledgments

This work was supported in part by grant no. FMRX-CT98-0232 from the European Training and Mobility of Researchers Network on metallo-beta-lactamases.

Jean-Denis Docquier, * Francesco Luzzaro, ([dagger]) Gianfranco Amicosante, ([double dagger]) Antonio Toniolo, ([dagger]) and Gian Maria Rossolini *

* Dipartimento di Biologia Molecolare, Sezione di Microbiologia, Universita di Siena, Siena, Italy; ([dagger]) Laboratorio di Microbiologia, Ospedale di Circolo e Universita dell'Insubria, Varese, Italy; and ([double dagger]) Dipartimento di Scienze e Tecnologie Biomediche, Universita di L'Aquila, L'Aquila, Italy

References

(1.) Livermore DM. Beta-Lactamases in laboratory and clinical resistance. Clin Microbiol Rev 1995;8:557-84.

(2.) Bush K, Jacoby GA, Medeiros AA. A functional classification scheme for beta-lactamases and its correlation with molecular structure. Antimicrob Agents Chemother 1995;39:1211-33.

(3.) Nordmann P, Ronco E, Naas T, Duport C, Michel-Briand Y, Labia R. Characterization of a novel beta-lactamase from Pseudomonas aeruginosa. Antimicrob Agents Chemother 1993;37:962-9.

(4.) Lauretti L, Riccio ML, Mazzariol A, Cornaglia G, Amicosante G, Fontana R, et al. Cloning and characterization of [bla.sub.VIM], a new integron-borne metallo-beta-lactamase gene from a Pseudomonas aeruginosa clinical isolate. Antimicrob Agents Chemother 1999;43:1584-90.

(5.) Poirel L, Naas T, Nicholas D, Collet L, Bellais S, Cavallo JD, et al. Characterization of VIM-2, a carbapenem-hydrolyzing metallo-beta-lactamase and its plasmid- and integron-borne gene from a Pseudomonas aeruginosa clinical isolate in France. Anti-microb Agents Chemother 2000;44:891-97.

(6.) Franceschini N, Caravelli B, Docquier JD, Galleni M, Frere JM, Amicosante G, et al. Purification and biochemical characterization of the VIM-1 metallo-beta-lactamase. Antimicrob Agents Chemother 2000;44:3003-7.

(7.) Livermore DM, Williams JD. Beta-lactams: mode of action and mechanisms of bacterial resistance. In: Lorian V, editor. Antibiotics in Laboratory Medicine. 4th ed. Baltimore: William & Wilkins; 1996. p. 502-78.

(8.) Luzzaro F, Mantengoli E, Perilli M, Lombardi G, Orlandi V, Orsatti A, et al. Dynamics of a nosocomial outbreak of multi-drug-resistant Pseudomonas aeruginosa producing the PER-1 extended-spectrum beta-lactamase. J Clin Microbiol 2001;39:1865-70.

(9.) Pereira M, Perilli M, Mantengoli E, Luzzaro F, Toniolo A, Rossolini GM, et al. PER-1 extended-spectrum beta-lactamase production in an Alcaligenes faecalis clinical isolate resistant to expanded-spectrum cephalosporins and monobactams from a hospital in Northern Italy. Microb Drug Resist 2000;6:85-90.

(10.) Nordmann P, Naas T. Sequence analysis of PER-1 extended-spectrum beta-lactamase from Pseudomonas aeruginosa and comparison with class A beta-lactamases. Antimicrob Agents Chemother 1994;38:104-14.