Dissemination of clonal complex 2 Acinetobacter baumannii strains co-producing carbapenemases and 16S rRNA methylase ArmA in Vietnam

Background Acinetobacter baumannii strains co-producing carbapenemase and 16S rRNA methylase are highly resistant to carbapenems and aminoglycosides. Methods Ninety-three isolates of multidrug-resistant A. baumannii were obtained from an intensive care unit in a hospital in Vietnam. Antimicrobial susceptibility tests and whole genome sequencing were performed. Multilocus sequence typing and the presence of drug resistant genes were determined and a maximum-likelihood phylogenetic tree was constructed by SNP alignment of whole genome sequencing data. Results The majority of isolates belonged to clonal complex 2 (ST2, ST570 and ST571), and carried carbapenemase encoding genes blaOXA-23 and blaOXA-66. Two isolates encoded carbapenemase genes blaNDM-1 and blaOXA-58 and the 16S rRNA methylase encoding gene armA and did not belong to clonal complex 2 (ST16). Conclusion A. baumannii isolates producing 16S rRNA methylase ArmA and belonging to clonal complex 2 are widespread, and isolates co-producing NDM-1 and ArmA are emerging, in medical settings in Vietnam. Electronic supplementary material The online version of this article (doi:10.1186/s12879-015-1171-x) contains supplementary material, which is available to authorized users.

Aminoglycosides are effective antibiotics for the treatment of infectious diseases caused by Gram-negative bacteria. These agents block bacterial protein synthesis by binding to the 30S ribosomal subunit [31]. Methylation of 16S rRNA by 16S rRNA methylases, however, makes Gram-negative bacteria highly resistant to all clinically important aminoglycosides [32]. In 2003, clinical isolates of highly aminoglycoside-resistant Gram-negative bacteria producing 16S rRNA methylases were identified in France [33] and Japan [34]. Since then, 16S rRNA methylaseproducing Gram-negative bacteria have been isolated in other parts of the world, including Asian countries, such as Afghanistan, Bangladesh, China, Hong Kong, India, Japan, Korea, Oman, and Pakistan [35].

Bacterial samples and drug susceptibility tests
From 2011 to 2013, 93 clinical isolates of A. baumannii were obtained from respiratory tract samples taken from patients hospitalized in an intensive care unit (ICU) in Cho Ray Hospital in Ho Chi Minh City, Vietnam.

Whole genome sequences
Genomic DNA from the 93 multidrug-resistant isolates were extracted using DNeasy Blood & Tissue kits (QIAGEN, Tokyo, Japan) and sequenced by MiSeq (Illumina, San Diego, CA). MiSeq data, including total length, number of contig, N50, average contig length and % GC content, were shown in Additional file 1: Table S1. To identify SNPs among these genomes, all reads of each isolate were aligned against the A. baumannii TYTH-1 sequence (Accession no. CP003856) using CLC genomics workbench, version 5.5 (CLC bio, Tokyo, Japan). SNP concatenated sequences were aligned by MAFFT (http://mafft.cbrc.jp/alignment/server/). A maximumlikelihood phylogenetic tree was constructed from the SNP alignment with PhyML 3.0 [37]. The probability of node branching was evaluated with 100 bootstrappings. Raw reads of all isolates were assembled into more than 500 bp contigs by CLC genomics workbench. Contigs around drug-resistant genes were annotated using the BLAST database (http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHome). Multilocus sequence typing (MLST) based on contig data was deduced using CLC genomics workbench, and matched against the Institut Pasteur MLST (http://pubmlst.org/abaumannii/) databases. The result of STs according to PubMLST (http://pubmlst.org/abaumannii/) scheme was shown in Additional file 2: Table S2. Annotations using the RAST server (http://rast.nmpdr.org/) were performed to compare numbers of prophages and resistance factors. All raw read data of the 93 isolates have been deposited at GenBank as accession numbers DRX032164 to DRX032256.

Ethical approval
The study protocol was carefully reviewed and approved by the ethics committee of Cho Ray Hospital (approval number: 1644/QD-BVCR), the ethics committee of the National Center for Global Health and Medicine (No. 1268), and the Biosafety Committee of the National Center for Global Health and Medicine (approval number: 27-M-52), respectively. Individual informed consent was waived by the ethics committee listed above because this study used currently existing sample collected during the course of routine medical care and did not pose any additional risks to the patients.
Genomic environments surrounding armA, bla OXA-23 bla NDM-1 , bla OXA-51-like , and bla OXA-58 The genetic environment surrounding armA in NCGM346 belonging to Clade ST571 (Accession no. LC030435) is shown in Fig. 2a. This genetic environment, from nt 1 to nt 17,473, was more than 99.99 % homologous to the analogous region of A. baumannii strains MDR-TJ isolated in China [40] and NCGM253 isolated in Japan [41]. The sequence surrounding armA from nt 5838 to nt 9879 was identical to the transposon Tn1548 (Accession no. EU014811) detected in an A. baumannii isolate from North America [42] and included the ISCR1 insertion sequence. Putative transposase genes were located both upstream (tnpU) and downstream (tnpD) of armA (Fig. 2a). Four additional isolates, NCGM165, NCGM169, NCGM175, and NCGM194, belonging to Clades ST570, ST215, ST23, and ST2, respectively, had the same genetic organization surrounding armA as the NCGM346 isolate. None of these five isolates contained plasmids, indicating that armA is chromosomally encoded in each.

Structures of the genomic resistance islands of CC2 isolates
The resistance island (RI) of the isolate NCGM196 belonging to Clade ST2 contained two Tn6021-like copies and one Tn5393-like copy. The resistance genes in the RI included sul1, which encodes sulfonamide resistance protein, and tetB and tetR, which regulate tetracycline resistance, as well as the streptomycin resistance genes strA and strB. The RI structure of the other ST2 isolate (NCGM194) was identical to that of A. baumannii MDT-TJ [40] and TYTH-1 [43]. RIs of the isolates belonging to Clades ST570 (NCGM165) and ST571 (NCGM346) were identical to those of AbaR4 [44], a compound transposon containing a Tn6022 backbone.

Discussion
To our knowledge, this is the first report of A. baumannii isolates co-producing NDM-1 and ArmA emerging in a medical setting in Vietnam. Enterobacteriaceae producing only NDM-1 had been reported in Vietnam [18,45,46], including NDM-1-producing K. pneumoniae isolated from environmental samples [45] and NDM-1-producing Enterobacteriaceae isolated from samples in a Vietnamese surgical hospital [47]. There have been no reports of A. baumannii co-producing NDM-1 and ArmA and belonging to international clone 2, although NDM-1 producers belonging to international clone2 were reported in East Africa in 2013 [48]. It is important to continue the surveillance of NDM-1-producing pathogens, including A. baumannii, in medical settings in Vietnam.
The high prevalence of Gram-negative bacteria producing ArmA in Vietnam may result from the inadequate use of aminoglycosides in that country. An analysis of patients hospitalized in Vietnam showed that 67.4 % received antibiotics, with 18.9 % receiving aminoglycosides, although  NCGM321 (b). orf1, gene encoding a hypothetical protein; orf2, gene encoding a DNA-binding protein; orf3, gene encoding a DNA replication protein; orf4 and orf5, genes encoding hypothetical proteins 30.8 % of the prescribed antibiotics were considered inappropriate [49]. This latter rate was higher than the rates of inappropriately prescribed antibiotics in Malaysia (4.0 %) [50], Turkey (14.0 %) [51], Hong Kong (20.0 %) [52] and European countries (17.8-32.0 %) [53,54].
A similar genetic environment surrounding bla NDM-1 has been reported in A. baumannii stains isolated in China [55], Colombia (accession no. CP010399), France [22], Germany [56] and the United States (accession no. CP010370); in A. lwoffii isolated in China [57]; in E. coli isolated in Colombia (accession no. CP010373); in K. pneumoniae isolated in Colombia (CP010391) and the United States [58]; and in Providencia rettgeri isolated in Canada [59]. A similar environment surrounding armA was reported in A. baumannii strains isolated in China [40], Japan [41], and Nepal (accession no. AP014649). The genetic organization of bla NDM-1 has spread worldwide, whereas that of armA has spread in Asian countries.
A. baumannii isolates belonging to international clone 2 must have been disseminated throughout medical settings in Vietnam, since 69.9 % of all isolates tested belonged to this clone (ST2, ST570, and ST571). Epidemiological studies of A. baumannii isolates obtained from a hospital in Hanoi are currently ongoing to clarify whether A. baumannii isolates belonging to international clone 2 are disseminating throughout Vietnam. The isolates belonging to Clades ST16, ST23, and ST215 were not identified as belonging to any previously described international clones [38]. To date, one A. baumannii isolate belonging to Clade ST16 was isolated in 2001 in the Netherlands, 3 isolates belonging to ST23 were isolated in the Netherlands (in 1964) and Sweden (in 2006 and 2007), and 6 isolates belonging to Clade ST215 were isolated in 2008 in China. Clones ST570, ST571, and ST575 were novel STs. Of the isolates belonging to CC2, those in Clades ST570 and ST571 may have evolved in a unique manner in Vietnam because the structures of resistant islands in ST570 and ST571 isolates were different from those in ST2 isolates.

Conclusions
This study showed that 16S rRNA methylase ArmAproducing A. baumannii isolates belonging to clonal complex 2 have spread, and that NDM-1-and ArmA-coproducers not belonging to clonal complex 2 are emerging, in medical settings in Vietnam.

Additional files
Additional file 1: Table S1. Assembly summary report of 93 A. baumannii isolates using CLC Genomics Workbench version 5.