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Codetection of blaOXA-23-Like Gene (blaOXA-133) and blaOXA-58 in Acinetobacter radioresistens: Report from the SENTRY Antimicrobial Surveillance Program

We read with great interest the report from Poirel et al. (7) describing Acinetobacter radioresistens as a source of blaOXA-23-like genes. During the 2006 SENTRY Antimicrobial Surveillance Program, the occurrence of acquired class D carbapenemases and metallo-β-lactamases in Acinetobacter spp. from the Asia-Pacific region was evaluated (4). In this study, one A. radioresistens strain (251-39C; identified by 16S rRNA gene sequencing) showing decreased susceptibility to penicillins and imipenem was observed (Manipal, India). However, since blaOXA-23-like genes from all A. radioresistens strains described by Poirel et al. (7) were silent and chromosome borne and did not confer a resistance phenotype, we were intrigued by the elevated MICs displayed by the isolate 251-39C. Therefore, further screening for class D carbapenemase (6, 10) detected blaOXA-58 in addition to the intrinsic blaOXA-23-like gene. 
 
Flanking sequences of both blaOXA genes were characterized by PCR using primers targeting ISAba1, -2, or -3 or a degenerate primer approach (5, 9). The upstream region of the blaOXA-23-like gene showed the highest identity with a putative O-sialoglycoprotein endopeptidase gene detected in the Acinetobacter baumannii strain AYE ({"type":"entrez-nucleotide","attrs":{"text":"CU459141","term_id":"169147133","term_text":"CU459141"}}CU459141); no putative promoter was located in this region. Sequencing analysis of the blaOXA-23-like gene revealed a new gene, named blaOXA-133. The putative amino acid sequence displayed the most identity with OXA-102 (99.6%; one amino acid substitution difference [Leu-5 Phe]). Other close variants were OXA-103 (98.5%), OXA-23 (97.4%), OXA-73 (97.1%), OXA-27 and -49 (96.7%), and OXA-105 (96.3% [data not shown]) (7). A truncated ATPase located downstream of blaOXA-133 was detected, consisting of the same genetic context as that reported previously (7). Sequencing confirmed the presence of blaOXA-58 and ISAba3 upstream, thus providing a promoter region. 
 
Plasmid analysis demonstrated nine plasmid bands (ca. 54, 35, 14, 5.6, 4.0, 3.0, 2.8, 2.5, and 2.4 kb) and a blaOXA-58-specific probe hybridized with the 54-, 35-, and 5.6-kb bands (5). It is worthwhile to mention that the 4.0-kb and smaller plasmid bands may represent different forms of at least two distinct plasmids. However, blaOXA-58 was considered to be carried by three different plasmids, since they showed greater size differences. Further experiments should be performed to determine the exact number of plasmid DNAs carried by isolate 251-39C. Curing experiments were performed to investigate whether β-lactam MICs would decrease after removing blaOXA-58-carrying plasmids (3). Curing was confirmed by a negative PCR result for blaOXA-58. Index and cured strains were tested for susceptibility by the broth microdilution method (2) and Etest (AB BioDisk, Solna, Sweden). The cured A. radioresistens isolate became susceptible and showed MICs for penicillins and carbapenems between 128- and 16-fold and 4- to 32-fold lower than the index strain, respectively (Table ​(Table11). 
 
 
 
TABLE 1. 
 
MICs for A. radioresistens index and cured strains tested against several antimicrobial agents 
 
 
 
In addition, we evaluated the transcriptional levels of blaOXA-133 and blaOXA-58 by using quantitative real-time PCR. RNA was extracted using the RNeasy mini kit (Qiagen GmbH, Hilden, Germany). Relative quantification of target gene expression (blaOXA-133 and blaOXA-58) was performed in triplicate by normalization to an endogenous reference (16S rRNA). Quantitative real-time PCR demonstrated that blaOXA-133 was transcribed at very low levels (mean threshold cycle = 34.74), ca. 1,500-fold lower than those for blaOXA-58 (mean threshold cycle = 24.00). Although the index strain harbored the chromosomal blaOXA-133, the loss of β-lactam resistance displayed by the cured strain suggests OXA-58 as the main β-lactam resistance mechanism (8), possibly enhanced by multiple gene copies and increased production of enzyme (1). Detection of blaOXA-58 in the index strain, highly disseminated in A. baumannii (4), indicates the occurrence of DNA exchange between these two species (7). Furthermore, our findings emphasize the ability of blaOXA-58 mobilization.