Detection of PER-2-Producing Enterobacter cloacae in a Brazilian Liver Transplantation Unit

LETTER

High rates of extended-spectrum-β-lactamase (ESBL)-producing Klebsiella pneumoniae isolates have been documented in many Brazilian hospitals, with CTX-M-2 being the most frequent ESBL reported (1). Resistance to broad-spectrum cephalosporins among Enterobacter cloacae isolates is usually due to hyperproduction of AmpC; however, production of ESBL represents an important cause of resistance to these antimicrobial agents among E. cloacae isolates from Brazilian hospitals (2). PER-2 was first identified in 1996 in a Salmonella enterica serovar Typhimurium strain isolated from a patient in Argentina who had previously been hospitalized in 1990 (3). Since then, this enzyme has also been described in Enterobacteriaceae isolated from Uruguay, Bolivia, and Argentina and in Acinetobacter spp. and P. aeruginosa isolates from Argentina (4–7). Here, we report the first occurrence of bla_PER-2 in Brazil.

In 2006, two cefepime-resistant E. cloacae strains were isolated from blood cultures of two distinct patients hospitalized in the Liver Transplant Unit of a tertiary care hospital located in Curitiba, a southern Brazilian city. Antimicrobial susceptibility testing was interpreted using the CLSI broth microdilution (8) method, except for polymyxin B, for which the EUCAST criteria were applied (9). The genetic relatedness of the isolates was evaluated by pulsed-field gel electrophoresis (PFGE) using XbaI (10). Isoelectric focusing analysis was performed with polyacrylamide gel containing ampholines (Amersham Pharmacia Biotech, Sweden; pH range, 3.5 to 9.5). The presence of bla_CTX-M-1, _-2, _-8, _-9, and _-25, bla_OXA-23, _-24, _-48, _-51, and _-58, bla_BES, bla_TEM, bla_SHV, bla_PER, and bla_GES was determined by PCR using specific primers (1, 2). Plasmid DNA (11) and chromosomal DNA (QIAamp DNA minikit; Qiagen, Germany) was extracted, and conjugation and transformation were carried out with Escherichia coli J53, E. coli DH5∞, and E. coli TOP10 recipient strains. Southern blotting and hybridization were performed using the digoxigenin (DIG) DNA labeling and detection kit (Roche Diagnostics, GmbH, Germany).

Furthermore, both isolates showed susceptibility to polymyxin B, imipenem, and meropenem and resistance to broad-spectrum cephalosporins, cefepime, and ertapenem (Table 1). Both isolates showed the ESBL phenotype by disk approximation test (12). bla_PER-2 and bla_TEM-1 were identified in both clinical isolates, which possessed unique PFGE profiles. Isoelectric focusing analysis showed a pI of 5.4, which may correspond to the PER-2 and/or TEM-1 pI. Multiple attempts to transfer the bla_PER-2 gene by electroporation and conjugation failed, and hybridization with bla_PER-2- and bla_TEM-1-specific probes showed that the bla_TEM-1 gene was located on an ∼140-kb plasmid, while bla_PER-2 was located on the chromosome in both isolates.

TABLE 1.

Antimicrobial susceptibility profiles of two E. cloacae clinical isolates carrying bla_PER-2 and bla_TEM-1 in Brazil

Antimicrobial	MIC(s) (mg/liter)a
	ECL532	ECL635
Polymyxin B	0.125	0.125
Imipenem	0.25	0.25
Meropenem	0.5	1
Ertapenem	2	2
Amikacin	8	8
Gentamicin	>64	>64
Cefotaxime	64	128
Ceftazidime	>64	>64
Cefepime	64	128
Ciprofloxacin	>32	>32
Levofloxacin	32	32
Aztreonam	>32	>32
Ticarcillin-clavulanic acid	>256, >2	>256, >2
Piperacillin-tazobactan	>128, >4	>128, >4
Trimethoprim-sulfamethoxazole	>64, >1,216	>64, >1,216

^aMICs were determined by the CLSI broth microdilution method.

Our study constitutes the first report of PER-2 in Brazil. bla_PER-2 and other PER variant-encoding genes are usually found on a conjugative plasmid. In contrast, in these E. cloacae isolates, bla_PER-2 was located on the chromosome. The clonal relatedness demonstrated by the two E. cloacae clinical isolates suggests that the patients may have acquired these strains from a common source. Although chromosomal AmpC production may have masked the identification of the ESBL phenotype, both isolates were phenotypically identified as ESBL producers by the disk approximation test. It probably occurred because the chromosomal bla_AmpC gene was not derepressed, as shown by the isoelectric focusing results. Although more studies are needed to evaluate the prevalence of PER-2 among Brazilian isolates, a previous study showed that this enzyme was infrequent. It was detected only in these 2 strains out of 205 Enterobacter species isolates from bloodstream samples over a 5-year period (2). The reason why PER-2 has not been as frequently detected in Brazil as it is in Argentina, a neighbor country, remains to be further investigated.

The project was approved by the Research Ethics Committee of the Hospital de Clínicas da Universidade Federal do Paraná (protocol no. 2288.182/2010-07).

(This report was presented in part at the 52th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, USA.)