Clinical Carbapenem-Resistant Acinetobacter baylyi Strain Coharboring bla_SIM-1 and bla_OXA-23 from China

Abstract

bla_SIM-1 and bla_OXA-23 were codetected in clinical carbapenem-resistant Acinetobacter baylyi strain NB09A30. Both of carbapenemase genes were located on a large plasmid (ca. 360 kb). bla_SIM-1 was found as a gene cassette inserted into a class 1 integron identical to that determined in Acinetobacter sp. isolates from South Korea. The genetic structure of bla_OXA-23 in NB09A30 was different from that in the prevalent Acinetobacter baumannii of clonal complex 92 (CC92) from the same hospital.

TEXT

Carbapenem-resistant Acinetobacter spp. are one of the most frightening threats in the current antibiotic era (14). The mechanisms of Acinetobacter sp. resistance to carbapenem are production of carbapenemases (the main mechanism), overexpression of efflux pumps, and reduced expression or loss of outer membrane proteins (8). The metallo-β-lactamase (MBL) gene, bla_SIM-1, was first identified in Acinetobacter spp. from South Korea in 2005 (11) and then in Acinetobacter genomic species 10 from two hospitals of South Korea in 2010 (10) but, as far as we know, has not yet been detected in other countries. Here, we report the emergence of Acinetobacter baylyi coharboring bla_SIM-1 and bla_OXA-23 in China.

A 59-year-old male was admitted for cerebral hemorrhage at the Affiliated Hospital of Ningbo University in June 2009. After surgery to remove the intracerebral hematoma, he was transferred to the intensive care unit and received antibiotic therapy with cefminox, which was later replaced with cefodizime plus amikacin. Imipenem-resistant Acinetobacter sp. NB09A30 was isolated from the cloudy secretion of the surgical incision 10 days after the operation. MICs were determined by Etest and interpreted according to the CLSI 2011 recommendation (7). The strain was resistant to almost all β-lactams, including imipenem (MIC ≥ 32 μg/ml) and meropenem (MIC ≥ 32 μg/ml), but remained susceptible to minocycline, tigecycline, ciprofloxacin, levofloxacin, and colistin. The MICs of gentamicin and amikacin were ≥256 μg/ml and 16 μg/ml, respectively. Etest MBL strip testing against NB09A30 indicated the presence of metallo-β-lactamase (imipenem/imipenem plus EDTA MIC of >16 μg/ml). Surgical site infection was suspected, and the antibiotic regimen was changed to levofloxacin. The man was cured finally, and NB09A30 was no longer recovered.

PCR detection of the bla_OXA-51-like gene in NB09A30 was negative, implying that NB09A30 is not an Acinetobacter baumannii strain (15). The 16S-23S rRNA intergenic spacer region (ISR) of NB09A30 was amplified and cloned into the pGEM-T Easy vector for sequencing (5). This showed that NB09A30 possessed ISRs with different lengths (long ISR, 650 bp; short ISR, 611 bp), a unique organization of the 16S-23S rRNA ISRs of A. baylyi (12). The ISR sequences were 94% identical to those of A. baylyi strain 93A2 (GenBank accession no. EU042163) and A. baylyi ADP1 (GenBank accession no. CR543861); they were less than 85% identical to those of the other genomic species, which indicated that NB09A30 is an A. baylyi strain (12).

PCR was performed to check for the presence of carbapenem-hydrolyzing class D β-lactamase (CHDL) genes (bla_OXA-23, bla_OXA-24, and bla_OXA-58), MBL genes (bla_IMP-type and bla_VIM-type genes, bla_SIM-1, bla_SPM-1, bla_GIM-1, and bla_NDM-1), and the bla_KPC-type gene, as previous reported (13, 16). bla_SIM-1 and bla_OXA-23 were detected and confirmed by sequencing.

Repeated mating-out assays and electrotransformation to transfer bla_SIM-1 and bla_OXA-23 failed. Therefore, we used an S1 nuclease assay and an I-CeuI assay to identify the locations of the two carbapenemase genes as previously described (2). Two 23S rRNA gene probes were designed to target the sequences upstream and downstream of the I-CeuI cut site, respectively, according to the genomic sequence of A. baylyi strain ADP1 (GenBank accession no. CR543861). All seven I-CeuI-generated DNA fragments can be hybridized with 23S rRNA gene probe 1 plus 23S rRNA gene probe 2 but did not cohybridize with bla_SIM-1 or bla_OXA-23 probes, excluding chromosomal locations for the two carbapenemase genes (Fig. 1). In contrast, hybridization signals of bla_SIM-1 and bla_OXA-23 probes were codetected in a ca. 360-kb large plasmid after S1 nuclease treatment, suggesting plasmid locations for the two carbapenemase genes (Fig. 1). bla_SIM-1 was confirmed to have a chromosomal location in Acinetobacter spp. of South Korea (10, 11), but it has a large-plasmid location in A. baylyi strain NB09A30. Using the A. baumannii plasmid replicon typing scheme constructed by Bertini et al. (4), we detected four replicase (rep) gene groups (GRs), GR2, GR3, GR7, and GR13, in A. baylyi strain NB09A30. Further hybridization with corresponding probes is needed to define the large plasmid's rep group.

Fig. 1.

Analysis of the locations of bla_SIM-1 and blaOXA-23 in A. baylyi strain NB09A30 using the S1 nuclease assay and I-CeuI assay. Shown are pulsed-field gel electrophoresis (PFGE) profiles of total DNA after digestion (A) and Southern blotting and hybridization results with bla_SIM-1 (B), bla_OXA-23 (C), 23S rRNA gene probe 1 (D), and 23S rRNA gene probe 2 (E). The largest chromosomal fragment of the I-CeuI profile was not hybridized using 23S rRNA gene probe 1 (D). The smallest chromosomal fragment was not hybridized using 23S rRNA gene probe 2 (E). Arrows indicate the hybridization signals of bla_SIM-1 and bla_OXA-23. Lane 1, Salmonella enterica serotype Braenderup strain H9812 DNA digested by XbaI, used as a molecular marker (kb); lanes 2, A. baylyi strain NB09A30 digested by S1 nuclease; lanes 3, A. baylyi strain NB09A30 DNA digested by I-CeuI.

Primers targeting 5′ and 3′ conserved segments of the class I integron were used for PCR amplification (11). bla_SIM-1 of A. baylyi strain NB09A30 was found to be class I integron borne. It is interesting that the sequence of the entire integron was 100% identical to that in Acinetobacter sp. isolates from South Korea (GenBank accession no. EF125010) (11). The genetic structure of bla_OXA-23 was determined by a cloning experiment as previously reported (1). bla_OXA-23 in A. baylyi strain NB09A30 was part of transposon Tn2008 (1), which was inserted into a noncoding region and flanked by two copies of an 11-bp direct repeat (ATATTCTGTTT).

In order to trace the source of bla_SIM-1 and bla_OXA-23, we further screened for the presence of the two carbapenemases in clinical A. baumannii isolates. From October 2008 to June 2009, we collected 29 carbapenem-resistant A. baumannii isolates from the same hospital. Multilocus sequence typing (MLST) identified an identical sequence type, ST138 (http://pubmlst.org/abaumannii/), in all isolates (3); this sequence type was clustered into clonal complex 92 (CC92), the dominant clonal complex of carbapenem-resistant A. baumannii in China (9). bla_OXA-23 was identified in all isolates, but bla_SIM-1 was absent. The patient in this case had no history of visiting South Korea. Moreover, we failed to detect bla_SIM-1 in A. baumannii isolates prevalent in the hospital at the time. Therefore, the source of bla_SIM-1 in A. baylyi NB09A30 is still unknown. The genetic structure of bla_OXA-23 in the prevalent A. baumannii isolates with ST138 included Tn2009, a novel bla_OXA-23-containing transposon also identified in CC92 A. baumannii strain MDR-ZJ06 from China (17). Distinct from Tn2008, which was flanked on one side by ISAba1, Tn2009 was a composite transposon flanked by two ISAba1 sequences with identical orientations (17), implying different origins of bla_OXA-23 in A. baylyi NB09A30 and A. baumannii ST138.

In conclusion, our study identified bla_SIM-1 for the first time in China. Since the potential source and details of the epidemiological situation are unclear, more study should be carried out to survey the presence of bla_SIM-1 in Acinetobacter spp. A. baylyi used to be considered an environment species, existing in soil and activated sludge, and nonpathogenic (8). Though nosocomial infections caused by A. baylyi have been reported (6), carbapenem resistance is still rare. More attention should be paid to the emergence of carbapenem resistance in this potential pathogen.

Nucleotide sequence accession numbers.

The nucleotide sequences of the 16S-23S rRNA intergenic spacer region, the bla_SIM-1-containing class I integron, Tn2008 of A. baylyi strain NB09A30, and Tn2009 of the prevalent A. baumannii isolates of ST138 are deposited in the GenBank database under accession no. JF731031 to JF731040, JF731030, JF731029, and JF731028, respectively.