Dissemination of blaOXA-23 in Acinetobacter spp. in China: main roles of conjugative plasmid pAZJ221 and transposon Tn2009

Abstract

Production of the OXA-23 carbapenemase is the most common reason for the increasing carbapenem resistance in Acinetobacter spp. This study was conducted to reveal the genetic basis of blaOXA-23 dissemination in Acinetobacter spp. in China. A total of 63 carbapenem-resistant OXA-23-producing Acinetobacter sp. isolates, representing different backgrounds, were selected from 28 hospitals in 18 provinces for this study. Generally, two patterns of plasmids carrying blaOXA-23 were detected according to S1-nuclease pulsed-field gel electrophoresis and Southern blot hybridization. A ca. 78-kb plasmid, designated pAZJ221, was found in 23 Acinetobacter baumannii and three Acinetobacter nosocomialis isolates, while a novel ca. 50-kb plasmid was carried by only two other A. baumannii isolates. Three of these isolates had an additional copy of blaOXA-23 on the chromosome. Transformation of the two plasmids succeeded, but only pAZJ221 was conjugative. Plasmid pAZJ221 was sequenced completely and found to carry no previously known resistance genes except blaOXA-23. The blaOXA-23 gene of the remaining 35 isolates was chromosome borne. The blaOXA-23 genetic environments were correlated with Tn2009 in 57 isolates, Tn2008 in 5 isolates, and Tn2006 in 1 isolate. The MIC values for the carbapenems with these isolates were not significantly associated with the genomic locations or the copy numbers of blaOXA-23. Overall, these observations suggest that the plasmid pAZJ221 and Tn2009 have effectively contributed to the wide dissemination of blaOXA-23 in Acinetobacter spp. in China and that horizontal gene transfer may play an important role in dissemination of the blaOXA-23 gene.

FIG 1

Analysis of the localization of bla_OXA-23 using the S1 nuclease-PFGE method. Shown are PFGE profiles after S1 nuclease digestion (A) and Southern blot hybridization with a bla_OXA-23 probe (B). The 10 isolates displayed were clustered into one of four groups: Acinetobacter nosocomialis isolates (298, 2295, and 2464), A. baumannii isolates carrying the 78-kb plasmid (126, 489, and 221), A. baumannii isolates carrying the 50-kb plasmid (557 and 2964), and A. baumannii isolates of the same clone with chromosomal bla_OXA-23 (2008 and 2154). Salmonella enterica serotype Braenderup strain H9812 DNA digested by XbaI was used as a molecular marker (in kb).

FIG 2

PFGE files of BamHI-digested genomic DNA of isolates with plasmid-borne bla_OXA-23 (A) and Southern blot hybridization with a bla_OXA-23 probe (B). T1 and T2 indicate the transformants associated with the 78-kb and 50-kb plasmids, respectively. The two isolates carrying the 50-kb plasmid have an additional copy of bla_OXA-23 on the chromosome; thus, the donors have one more hybridization signal than the transformants. Detailed information of other isolates is given in the Fig. 1 legend. Salmonella enterica serotype Braenderup strain H9812 DNA digested by XbaI was used as a molecular marker (in kb).

FIG 3

Analysis of the localization of bla_OXA-23 using the ApaI-PFGE method. Shown are PFGE profiles after ApaI digestion (A) and Southern blot hybridization with a bla_OXA-23 probe (B). The 10 isolates displayed are in the same order as in Fig. 1. Salmonella enterica serotype Braenderup strain H9812 DNA digested by XbaI was used as a molecular marker (in kb).

FIG 4

Circular map of plasmid pAZJ221. The two inner circles indicate the G+C content plotted against the average G+C content of 34.03% (black circle) and GC skew information (green and purple circles). The outer circles display the open reading frames (ORFs) in opposite orientations. Regions related to conjugation, replication, and Tn2009 are marked in blue.