AbaR4-Type Resistance Island Including the bla_OXA-23 Gene in Acinetobacter nosocomialis Isolates

Abstract

This study reports for the first time the AbaR4-type resistance island with the bla_OXA-23 gene in two carbapenem-resistant A. nosocomialis isolates from South Korea and Thailand.

TEXT

OXA-23, an acquired class D β-lactamase with carbapenemase activity, confers carbapenem resistance in Acinetobacter baumannii. The presence of the bla_OXA-23 gene has increasingly been reported worldwide and has emerged as a serious threat (5). Although several transposons, such as Tn2006, Tn2007, and Tn2008, have been identified as genetic structures harboring the bla_OXA-23 gene, Tn2006 is part of the ISAba1-linked AbaR4-type resistance island of A. baumannii related to clonal dissemination of the carbapenem-resistant A. baumannii European clone II, ST92, in many countries (5). Acinetobacter radioresistens was identified as a source of the bla_OXA-23-like genes (8), but an AbaR4-type resistance island including the bla_OXA-23-like gene has not been found in species other than A. baumannii. In this article, we report the AbaR4-type resistance island with the bla_OXA-23 gene in two Acinetobacter nosocomialis (formerly Acinetobacter genomospecies 13TU) isolates.

In a molecular epidemiology study of Acinetobacter sp. isolates from Asian countries, we found two A. nosocomialis isolates harboring the bla_OXA-23-like gene, which was identified by multiplex PCR (11). This species was identified using sequences of 16S rRNA and partial rpoB genes (2). The sequences displayed 100% similarity to the reference strains of A. nosocomialis LMG 10619^T and RUH503 in the 16S rRNA and rpoB gene analyses, respectively. Two bla_OXA-23-positive A. nosocomialis isolates, K08-39 and Th01-06, were isolated from South Korea and Thailand, respectively. Both were isolated from patients who developed hospital-acquired pneumonia: K08-39 from the endotracheal aspirate and Th01-06 from the sputum. In vitro susceptibility testing was performed by measuring the MIC using the broth microdilution method according to Clinical and Laboratory Standards Institute (CLSI) guidelines (1). K08-39 from South Korea and Th01-06 from Thailand were resistant to both imipenem and meropenem (Table 1). K08-39 showed very high MICs for imipenem and meropenem (>64 and 64 mg/liter, respectively), while Th01-06 did not. K08-39 was susceptible to antimicrobial agents other than carbapenem, but Th01-06 was resistant to piperacillin-tazobactam and intermediate in resistance to cefepime.

Table 1.

Characteristics of two A. nosocomialis isolates with bla_OXA-23

Characteristic	Value or description for isolatea
	K08-39	Th01-06
Species	A. nosocomialis	A. nosocomialis
Locality	South Korea	Thailand
Source	Endotrachial aspirate	Sputum
MIC of antimicrobial agent, mg/liter (susceptibility)
Gentamicin	2 (S)	0.25 (S)
Ceftazidime	4 (S)	2 (S)
Cefotaxime	4 (S)	8 (S)
Cefepime	2 (S)	16 (I)
Ciprofloxacin	0.12 (S)	0.12 (S)
Imipenem	>64 (R)	16 (R)
Meropenem	64 (R)	16 (R)
Trimethoprim-sulfamethoxazole	0.12, 2.37 (S)	2, 38 (S)
Piperacillin-tazobactam	≤0.25, 4 (S)	256, 4 (R)
Colistin	0.5 (S)	1 (S)
Polymyxin B	0.5 (S)	1 (S)

^aR, resistant; I, intermediate; S, susceptible.

The comM gene was evaluated for the presence of a transposon containing the bla_OXA-23 gene. Both the Th01-06 and K08-39 isolates failed to produce a comM amplicon, indicating the interruption of the comM gene. They were PCR positive for the AbaR-comM junction, confirming the presence of an AbaR-type resistance island. The structure of the AbaR-type resistance islands was determined by several PCR procedures and sequencing using previously published primers (9, 10). Both showed the AbaR4 type, although their subtypes were also identified (see Fig. S1 in the supplemental material). While a primer set spanning tniB-tniE produced a 388-bp amplicon in the K08-39 isolate (referred to as the AB210 type), a 3,238-bp amplicon was produced by the same primer set in the Th01-06 isolate (referred to as the D36 type). In K08-39, the AB210 type, the tniB gene was 500 bp in size, while the tniD gene was absent. In Th01-06, the D36 type, however, the tniB gene was 921 bp in size, while the tniD gene was present. These AbaR4-type resistance islands have been identified nearly exclusively in the global clone II (GC II; formerly European clone II) of A. baumannii (9, 10), which is the most frequently identified clone in Asian countries, including South Korea (6). Thus, we hypothesize that AbaR4-type resistance islands, including the bla_OXA-23 gene in A. nosocomialis, have been transferred from A. baumannii GC II clones. Although the resistance islands of both isolates belonged to the AbaR4 type, they might have been transferred independently because they are of different subtypes and different localities.

The expression of OXA-23 in the two A. nosocomialis isolates was evaluated by quantitative reverse transcription-PCR (qRT-PCR) and normalized against the housekeeping gene rpoB. To compare the expression of OXA-23, two bla_OXA-23-positive carbapenem-resistant, one bla_OXA-23-negative carbapenem-resistant, and one bla_OXA-23-negative carbapenem-susceptible A. baumannii isolate were also included in the qRT-PCR. Both bla_OXA-23-positive A. nosocomialis isolates showed high mRNA levels of OXA-23 (see Fig. S2 in the supplemental material). Since we know that the bla_OXA-23 gene confers resistance to carbapenems in A. baumannii isolates, the high expression of bla_OXA-23 genes in the two A. nosocomialis isolates likely contributes to the resistance to carbapenems as well.

A. nosocomialis, which was long referred to as Acinetobacter genomospecies 13TU, belongs to the A. calcoaceticus-A. baumannii (Acb) complex or A. baumannii group along with Acinetobacter pittii (formerly Acinetobacter genomospecies 3) because they could not be differentiated easily by phenotypic or biochemical methods. A. nosocomialis is the second-most-frequent species isolated from blood among the genus Acinetobacter in South Korea and displays a low carbapenem resistance rate but a high polymyxin resistance rate, unlike A. baumannii (6). In our recent study, two carbapenem-resistant A. nosocomialis isolates from South Korea contained the bla_SIM-1 gene, which has been identified in other Acinetobacter species, including A. baumannii (3, 4, 7, 12). Although bla_OXA-23 has been identified in A. radioresistens and Acinetobacter baylyi (9, 12), it was first identified in A. nosocomialis. Thus, our findings in this study suggest that interspecies transfer of the resistance island may occur, which could contribute to the dissemination of the antimicrobial-resistant isolates of Acinetobacter species.