Nosocomial Outbreak of Extended-Spectrum β-Lactamase SHV-5-Producing Isolates of Pseudomonas aeruginosa in Athens, Greece

Abstract

Seven nonrepetitive Pseudomonas aeruginosa isolates producing the clavulanic acid-inhibited extended-spectrum β-lactamase SHV-5 were isolated in the same hospital in Athens, Greece, from 1998 to 2002. All isolates except one were clonally related, and the bla_SHV-5 gene was chromosomally located. This study underlined that this gene, which is widespread in Enterobacteriaceae in Greece, may disseminate also in P. aeruginosa.

Pseudomonas aeruginosa is a common nosocomial pathogen, especially for immunocompromised patients. It exhibits intrinsic resistance to several β-lactams and may acquire additional resistance mechanisms. Resistance to ceftazidime is due mostly to overexpression of the AmpC-type cephalosporinase (1). In addition, several acquired clavulanic acid-inhibited extended-spectrum β-lactamases (ESBLs) have been identified in P. aeruginosa (18). The ESBL PER-1 is mostly from Turkey, whereas VEB-1 is from Southeast Asia (5, 16, 18). Another group of ESBLs, the GES/IBC-like enzymes, have been found in P. aeruginosa in France, South Africa, and Greece (18). In addition, rare TEM-type ESBLs have been reported in P. aeruginosa (TEM-4, -21, -24, and -42), and only two SHV-type ESBLs have been reported in that species, SHV-2a in France and SHV-12 in Thailand (18).

The ESBL SHV-5 was reported first in a Klebsiella pneumoniae isolate from Chile in 1989 (6). Subsequently, the bla_SHV-5 gene was identified in several enterobacterial species scattered throughout the world and especially in Greece (3, 7, 17). The β-lactamase SHV-5 confers a high level of resistance to ceftazidime and to monobactams.

The aim of our study was to analyze the spread of P. aeruginosa isolates expressing an ESBL phenotype and recovered in different units of a pediatric hospital in Athens, Greece, from 1998 to 2002. Between July 1998 and February 2002, 50 consecutive ceftazidime-resistant P. aeruginosa strains identified by using the API32GN system (bioMérieux, Marcy-l'Etoile, France) were isolated from different patients in the clinical microbiology laboratory of P. and A. Kyriakou Children's Hospital in Athens, Greece. Among these strains, seven isolates had an ESBL phenotype according to results of a double-disk synergy test performed with cefepime, ceftazidime, and ticarcillin-clavulanic acid disks on Mueller-Hinton agar plates, as described previously (10). P. aeruginosa isolates 1 to 7 were from hospitalized patients, and their clinical backgrounds are detailed in Table 1. Four out of the seven patients were colonized by the ESBL-positive P. aeruginosa isolates, whereas three of the patients had infections and received antibiotic regimens consisting of piperacillin-tazobactam (two patients) or meropenem (one patient) (Table 1). Isolates 1 to 6 were resistant to ceftazidime, cefotaxime, cefpirome, cefepime, and aztreonam. They were susceptible to ticarcillin-clavulanate, piperacillin-tazobactam, imipenem, and meropenem (Table 2). Isolate 7 exhibited a higher level of resistance for most β-lactams. For all the strains, MICs of ceftazidime and cefotaxime were lowered by addition of clavulanate, which was consistent with ESBL expression (Table 2). In addition, all isolates were resistant to fluoroquinolones, gentamicin, amikacin, and tobramycin except for isolate 7, which remained susceptible to fosfomycin and ciprofloxacin. All isolates were susceptible to colistin, according to the guidelines of Gales et al. (2) using an agar dilution method.

TABLE 1.

Clinical features of bla_SHV-5-positive P. aeruginosa isolates

Isolate	Date of isolation (mo-day-yr)	Dates of hospitalization (mo-day-yr)	Hospitalization unit(s)	Source	Underlying disease, predisposing factor(s)	Age	Treatment	Outcome
1	07-25-98	05-25 to 10-15-98	Nephrology	Urine	Chronic renal failure	10 yr	No treatment
2	10-05-98	06-27 to 07-14-98, then 10-05 to 10-21-98	Neonatal unit, then internal medicine ward	Otic discharge	Prematurity	3 mo	Piperacillin-TZBa	Cured
3	12-23-98	12-22 to 12-24-98	Surgical ward	Urine	Neurogenic bladder, catheterizations	14 yr	No treatment
4	04-08-99	04-07 to 05-02-99	Internal medicine ward	Urine	Vesico-ureteric reflux, cystostomy	1 mo	Piperacillin-TZB	Cured
5	11-01-00	08-18-00b	Oncology	Feces	Leukemia	6 yr	No treatment
6	02-25-02	07-01-99b	Oncology	Blood	Leukemia	7 yr	Meropenem	Cured
7	06-23-99	03-18 to 06-17-99, 06-17 to 07-02-99	PICUc, then surgical ward	Skin	Burns	2 yr	No treatment

^aTZB, tazobactam.

^bAfter the first admission, the patient had continuous admissions in order to complete the treatment or because of relapse.

^cPICU, pediatric intensive care unit.

TABLE 2.

MICs of β-lactams for P. aeruginosa isolates 1 to 6, isolate 7, and reference strain PU21

β-Lactam(s)a	MIC (μg/ml)
	P. aeruginosa 1 to 6	P. aeruginosa 7	PU21
Ticarcillin	>512	>512	8
Ticarcillin + CLA	16	16	4
Piperacillin	16	64	8
Piperacillin + TZB	2	4	16
Cefotaxime	256	256	8
Cefotaxime + CLA	32	32	16
Ceftazidime	64	256	1
Ceftazidime + CLA	4	4	4
Cefepime	64	256	1
Cefpirome	128	512	4
Aztreonam	128	512	4
Moxalactam	32	32	8
Imipenem	2	32	1
Meropenem	1	4	1

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

PCR experiments using primers specific for bla_TEM, bla_SHV, bla_VEB-1, bla_GES-1, and bla_PER-1 (4) gave a positive result only for the bla_SHV gene. Sequence analysis of the entire gene revealed its perfect identity with the bla_SHV-5 gene.

Isoelectric focusing analysis performed as described elsewhere (14) showed that P. aeruginosa isolates expressed two β-lactamases with pI values of 8.2 and 8.4 that likely corresponded to SHV-5 and the naturally occurring AmpC-type enzyme, respectively.

Transfer of the ceftazidime resistance marker of P. aeruginosa isolate 7 by conjugation or electroporation as described previously (13) using ciprofloxacin-resistant P. aeruginosa PU21 as the recipient strain failed. The location of the bla_SHV-5 gene was determined precisely by using the endonuclease I-CeuI technique (9). Pulsed-field gel electrophoresis (PFGE) gave four DNA fragments from P. aeruginosa isolates 1 (0.5, 1.5, 1.7, and 2.2 Mb) and 7 (0.7, 1.3, 1.7, and 2.2 Mb) and also from the P. aeruginosa PU21 reference strain (0.5, 1.1, 1.9, and 2.2 Mb) (Fig. 1). The DNA probe for rRNA consisting of a 1,504-bp PCR fragment for 16S and 23S rRNA genes (4) hybridized with all the fragments of all P. aeruginosa DNAs except those of 2.2 Mb (Fig. 1). Hybridization with DNA probe internal to bla_SHV-5 consisting of a 300-bp PCR fragment generated from whole-cell DNA of P. aeruginosa gave a single signal for isolate 1, suggesting that bla_SHV-5 was chromosomally located (Fig. 1). Hybridization with a DNA probe for bla_SHV-5 gave three signals for isolate 7, indicating that at least three copies of the bla_SHV-5 gene were scattered on the chromosome of that strain (Fig. 1).

FIG. 1.

(A) PFGE profiles of I-CeuI-digested whole-cell DNA of three P. aeruginosa isolates. Lane 1, P. aeruginosa isolate 1; lane 2, P. aeruginosa isolate 7; lane 3, P. aeruginosa PU21 reference strain. (B and C) Southern hybridization was performed with a specific probe for the 16S-23S rRNA gene (B) and an internal probe for the bla_SHV-5 gene (C).

PCRs using primers annealing to bla_SHV-5 and the IS26 transposase gene did not provide positive results, whereas IS26 was found to be associated with bla_SHV-2a (11), and detection of the bla_SHV-5 gene as part of a class 1 integron by PCR also failed (8).

The SHV-5-producing P. aeruginosa isolates were recovered in different units of the P. and A. Kyriakou Children's Hospital during a 5-year period. Thus, it was hypothesized that one clone may have persisted in that hospital. Consequently, genotypic comparison was performed in order to evaluate the clonality of the isolates. PFGE analysis performed with the restriction enzyme SpeI as described previously (5) showed that six out of the seven isolates likely corresponded to a single clone, whereas isolate 7 was not clonally related (Fig. 2) (15).

FIG. 2.

PFGE patterns of SpeI-digested whole-cell DNA of eight P. aeruginosa isolates. Lane M, bacteriophage lambda DNA ladder; lanes 1 to 7, P. aeruginosa isolates 1 to 7; lane 8, P. aeruginosa PU21 reference strain.

The present work described ESBL-producing P. aeruginosa isolates in several hospitalization units of a pediatric hospital. ESBL production is frequently reported in Enterobacteriaceae but rarely in P. aeruginosa (18). As previously observed (13), the infected patients were at risk of acquiring P. aeruginosa infections. Two distinct clonal isolates were found, demonstrating coexistence of distinct ESBL-positive clones in the same unit. Another threatening aspect was that the same P. aeruginosa clone had been involved in nosocomial infections during a 4-year period of time, suggesting probable persistence of the isolate in the hospital environment.

Interestingly, the presence in the chromosome of a single isolate of several copies of the same β-lactamase gene may explain in part the higher level of resistance to β-lactams of that strain compared to others. It may indicate a probable location of the β-lactamase gene bla_SHV-5 on a transposable element, although this has not been identified.

This study represents the first description of the bla_SHV-5 gene in the Pseudomonas species and it adds to the list of ESBLs identified in P. aeruginosa. While this work was in progress, an outbreak of 11 P. aeruginosa isolates producing SHV-5 was reported in Heraklion, Crete (12), which is in the vicinity of Athens.

In our study, the bla_SHV-5 gene was chromosomally located and might have resulted from transfer of a bla_SHV-5-positive plasmid from Enterobacteriaceae followed by its chromosomal integration (7). Finally, this study suggested that when ESBLs become very much prevalent in Enterobacteriaceae, they may become the source of acquired resistance in P. aeruginosa that may remain hidden if not systematically investigated.