To the Editor—The emergence of carbapenemase-producing Enterobacteriaceae (CPE) isolates is an important public health problem; the treatment of carbapenem-resistant isolates is extremely difficult because few options remain available for clinical use.Reference Johnson and Woodford ¹ Usually, CPE harbors only 1 carbapenemase gene, although other resistance mechanisms (ESBL, porin loss, eflux pumps) may also be present. However, relatively few studies have reported Enterobacteriaceae isolates producing more than 1 carbapenemase.Reference Meletis, Chatzidimitriou and Malisiovas ² In the present study, we describe the characteristics of 10 Enterobacteriaceae coharboring carbapenemase genes.

The isolates were selected from an epidemiologic study evaluating Enterobacteriaceae with reduced susceptibility to carbapenems in several hospitals in the southernmost state of Brazil. The methods of this epidemiologic study are detailed elsewhere.Reference Magagnin, Rozales and Antochevis ³ Briefly, those isolates harboring more than 1 of the following genes were selected for further evaluation in this report: bla _KPC, bla _VIM, bla _GES, bla _NDM, bla _OXA-48, and bla _IMP (detected by a multiplex real-time polymerase chain reaction [PCR]).

These isolates were initially identified in the original institution by the VITEK2 system (bioMeriéux, France), and isolates coharboring more than 1 carbapenemase gene were confirmed by 16S rRNA sequencing. The presence of the carbapenemase genes was confirmed by conventional PCR, and the amplicons were purified and sequenced using a BigDie Terminator kit (version 3.1) and an ABI 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA). GenBank was used to access the sequences deposited to date, and the BioEdit program was used to compare similarities among sequences.

Plasmids were extracted by alkaline lysis and were transformed into cells of an Escherichia coli TOP10 eletrocompetent by electroporation.Reference Birnboim and Doly ⁴ Transformants were selected on Luria-Bertani agar containing 2 mg/L ceftazidime. Estimation of plasmid size was performed after 0.7% agarose gel electrophoresis, using a curve obtained by plotting the distance (mm), compared to E. coli 39R861.Reference Macrina, Kopecko, Jones, Ayers and McCowen ⁵

Minimum inhibitory concentrations (MIC) of carbapenems were evaluated using the broth microdilution method and were interpreted according to the Clinical and Laboratory Standards Institute (CLSI).

Overall, 10 isolates coproducing carbapenemases were identified: 5 Enterobacter cloacae complexes with bla _NDM-1 and bla _OXA-370 genes, 3 Klebsiella pneumoniae, 1 E. cloacae complex with bla _NDM-1 and bla _KPC-2 genes, and 1 K. pneumoniae with bla _KPC-2 and bla _OXA-370 genes. We detected multiple plasmids in 8 clinical isolates (Table 1); 2 isolates presented only one plasmid.

TABLE 1 Phenotypic Characteristics of Coproducing Enterobacteriaceae Isolates

NOTE. IMP, imipenem; MEM, meropenem; ERT, ertapenem; CAZ, ceftazidime; AZT, aztreonam; CIP, ciprofloxacin; AMK, amikacin; TGC, tigeciclyne; POL, polymyxin.

^a Transformant.

Bold: Transferred gene and size of the transferred plasmid.

One Providencia rettgeri presented bla _GES and bla _IMP genes in the multiplex PCR but the sequencing of the amplicon did not yield the specific variant of these genes. Notably, the P. rettgeri presented a peculiar result: it was positive for both bla _IMP and bla _GES, which are supposed to be in a plasmid, but the plasmid was not identified in either the clinical isolate or the transformant (data not show).

The MICs of transformants were much higher than that of E. coli TOP10, which indicates that the plasmids are enough to confer resistance to antibiotics. The analysis of the antimicrobial susceptibility profile of the transformants compared to the wild-type isolates showed that most isolates present lower MICs for carbapenems. The transformants that received only the OXA-370 gene present very low MICs to both meropenem and imipenem, which may indicate that this OXA-48 variant lacks carbapenemase activity.

Plasmid analysis demonstrated a heterogeneous pattern of plasmid sizes: 92, 110, 128, 130, and 154 kbp. Moreover, we observed that carbapenemases were inserted in different plasmids, which was also observed in other studies. Balm et alReference Balm, La, Krishnan, Jureen, Lin and Teo ⁶ reported a K. pneumoniae isolate coharboring bla _NDM and bla _OXA-181 genes on ~160 kb and ~280 kb plasmids, respectively. Another study demonstrated coproduction of bla _NDM-1 and bla _OXA-232 in E. coli: the bla _NDM-1 gene was located on a plasmid of 129,085 bp and the bla _OXA-232 gene was located on a small plasmid of 6,141 bp.Reference Both, Huang and Kaase ⁷ A study of K. pneumoniae coharboring bla _VIM and bla _KPC revealed 2 plasmids of 70 and 150 kb, while the bla _VIM transconjugants had a single plasmid of 150 kb and the bla _KPC-bearing transconjugant had a single plasmid of 70 kb.Reference Pournaras, Poulou, Voulgari, Vrioni, Kristo and Tsakris ⁸ We were able to transfer at least 1 carbapenemase gene to the E. coli TOP10 receptor, with the exception of 1 (4517F) E. cloacae that transferred both carbapenemase genes (bla _NDM-1 and bla _KPC-2).

The most frequently reported Enterobacteriaceae species carrying 2 or more carbapenemases is Klebsiella pneumoniae, although other species with this property have also been reported sporadically.Reference Meletis, Chatzidimitriou and Malisiovas ² Moreover, a few studies have indicated that the number of bla _NDM-1 and bla _OXA-48 is constantly increasing, and this combination has been the most frequently described.Reference Meletis, Chatzidimitriou and Malisiovas ^{2 ,} Reference Seiffert, Marschall, Perreten, Carattoli, Furrer and Endimiani ^{9 ,} Reference Dortet, Cuzon, Ponties and Nordmann ¹⁰ Relatively few reports of Enterobacteriaceae coproducing carbapenemases are available; here, we describe 2 additional species harboring 2 carbapenemases, and we observed other combinations such as a New Delhi metallo (NDM)-codifying gene with an OXA-48 variant carbapenemase.

In the present study, we observed the occurrence of 10 clinical isolates coproducing different carbapenemases located in a variety of plasmids, demonstrating the plasticity of these mobile genetic elements. The dissemination of double-carbapenemase–producing Enterobacteriaceae is worrisome because it potentially further narrows the therapeutic options. Furthermore, isolates producing more than 1 carbapenemase may impair the detection of carbapenemase production using some phenotypic methods, which reinforces the need for further investigation of these isolates.