Emergence of Klebsiella pneumoniae Harboring the aac(6ʹ)-Ian Amikacin Resistance Gene

LETTER

Carbapenemase-producing Enterobacteriaceae (CPE) have become a major public health concern worldwide (1). Isolates producing NDM and KPC carbapenemases often carry 16S rRNA methyltransferases (16S-RMTases) that confer resistance to several aminoglycosides (2), while OXA-48- and VIM-producing Enterobacteriaceae most often produce aminoglycoside-modifying enzymes that confer resistance to gentamicin and tobramycin but spare amikacin. In the Mediterranean region, OXA-48 and VIM are predominant, and susceptibility to amikacin remains high (>90%) (3–5).

We have recently reported pandrug resistance in a VIM-1-producing Klebsiella pneumoniae sequence type 54 (ST54) isolate obtained from a pediatric patient (6). The isolate harbored a plasmid-carried aac(6ʹ)-Ian gene coding for an aminoglycoside 6ʹ-N-acetyltransferase that conferred resistance to amikacin. The aim of this study was to investigate the presence of this gene in clinical enterobacterial isolates obtained from pediatric patients in our hospital. The study was approved by the local review board. A retrospective search for amikacin-resistant Enterobacteriaceae was done using the microbiology department database. We recovered 26 amikacin-resistant (MIC, >16 µg/ml) isolates that had been obtained between January 2010 and August 2017. The aac(6ʹ)-Ian gene was searched by PCR using primers aac(6ʹ)-Ian-F (5ʹ-GGTGCTTTATACTTCCTATA-3ʹ) and aac(6ʹ)-Ian-R (5ʹ-ATACTGATCTTCTGGAGAT-3ʹ). Thirteen isolates were positive for the gene (Table 1), and all of them were Klebsiella pneumoniae. Two ST54-specific regions were selected, and two pairs of specific primers were designed (7), as follows: St-54-c100-F (5ʹ-AACTGTTCTGTGGCTGGTCC-3ʹ) and St-54-C100-R (5ʹ-TGCCAATCCTGACGAGTCTC-3ʹ), and St-54-C40-F (5ʹ-CCGACAATGGCTGCAATCC-3ʹ) and St-54-C40-R (5ʹ-GCCTTACCGTTACGCTTCAC-3ʹ). Nine of the isolates belonged to ST54. The clonality of the four non-ST54 isolates was determined by randomly amplified polymorphic DNA (RAPD) analysis, and they were found to be different sequence types. The aac(6ʹ)-Ian gene harboring plasmid pKP1050-4 (6) was searched by PCR among these four isolates using primers targeting the IncN repE gene, Inc-N-F (5ʹ-AGCGCGTTCTCTGGTTATGT-3ʹ) and Inc-N-R (5ʹ-GGAGCGAGTAGGTGGTGAAC-3ʹ). One of the non-ST54 isolates had the same plasmid, while the other three did not.

All 13 patients were in severe condition and had long hospital stays. Eight of the patients had been admitted to transplant units and had stays longer than 3 months. Ten patients had been in the pediatric intensive care unit for at least 4 weeks, and two patients had been in the neonatal intensive care unit.

The aminoglycoside acetyltransferase aac(6ʹ)-Ian gene was described in 2015 in Serratia marcescens isolates from Japan (8). We did a database search and identified 13 additional matches, with all but one being clinical isolates of human origin (Table 2). Amikacin is the aminoglycoside in clinical use that is most resistant to aminoglycoside-modifying enzymes (9), and it is used as a last-resort drug in our environment to treat severe infections and late-onset neonatal sepsis. The spread of VIM-producing strains harboring the aac(6ʹ)-Ian gene adds to that of the NDM- and KPC-producing strains harboring 16S-RMTases and might become a serious challenge for the treatment of CPE.