Emergence of 16S rRNA methylase-producing Acinetobacter baumannii and Pseudomonas aeruginosa isolates in hospitals in Vietnam

Background 16S rRNA methylase-producing Gram-negative bacteria are highly resistant to all clinically important aminoglycosides. We analyzed clinical strains of 16S rRNA methylase-producing Acinetobactor baumannii and Pseudomonas aeruginosa obtained from clinical isolates in medical settings in Vietnam. Methods From 2008 to 2011, 101 clinical strains of A. baumannii and 15 of P. aeruginosa were isolated from patients in intensive care units (ICUs) in two medical settings in Vietnam. Antimicrobial susceptibilities were determined using the microdilution method and epidemiological analysis was performed by pulsed-field gel electrophoresis and MLST. Genes encoding the 16S rRNA methylases, OXAs and CTX-Ms were analyzed by PCR and sequence analysis. Results 16S rRNA methylase-producing Gram-negative pathogens were detected in two hospitals in Vietnam. Of the 101 clinical isolates of A. baumannii and the 15 of P. aeruginosa isolated from two ICUs in these hospitals, 72 (71.3%) were highly resistant to amikacin, arbekacin and gentamicin, with MICs greater than 1,024 mg/L. The 16S rRNA methylases ArmA and RmtB were produced by 61 and 9 isolates of A. baumannii, respectively, and RmtB was produced by 2 isolates of P. aeruginosa. Moreover, 52 of the A. baumannii isolates producing 16S rRNA methylases harbored both blaOXA-23-like and blaOXA-51-like genes. Most A. baumannii isolates producing 16S rRNA methylase obtained in hospital A in Hanoi were ST91 and ST231, whereas most from hospital B in Ho Chi Minh City were ST136, ST195, and ST254. The two P. aeruginosa isolates harboring rmtB showed different patterns on PFGE, one each corresponding to ST217 and ST313. Conclusions Gram-negative bacteria producing the 16S rRNA methylases ArmA and RmtB are emerging in medical settings in Vietnam. A. baumannii isolates in northern and southern regions of Vietnam may be of different lineages.


Background
Aminoglycosides widely used to treat infectious diseases caused by Gram-negative bacteria have a high affinity for the 16S rRNA of the bacterial 30S ribosome and block protein synthesis [1]. Enzymatic modification [1] and the methylation of 16S rRNA makes these bacteria highly resistant to all clinically important aminoglycosides [2]. In 2003, clinical isolates of highly aminoglycoside-resistant Gram-negative bacteria producing16S rRNA methylase were identified in France [3] and Japan [4]. Since then, 16S rRNA methylase-producing 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 [5]. To date, however, there have been no reports of these isolates from South-East Asian countries, including Vietnam.

Bacterial strains
From 2008 to 2011, 50 clinical strains of A. baumannii and 15 of P. aeruginosa were isolated from patients in an ICU in hospital A in Hanoi, Vietnam; and 51 strains of A. baumannii were isolated from patients in an ICU in hospital B in Ho Chi Minh City, Vietnam. Of the 101 A. baumannii strains isolated, 98 were from patients' respiratory tracts and 3 from blood. Of the 15 P. aeruginosa strains, 14 were from respiratory tracts and 1 from pus. Most patients were on ventilators, and the samples were mostly aspirates from ventilation tubes. All clinical isolates used in this study were obtained during standard patient care.

Multilocus sequence typing (MLST)
MLST of 16S rRNA methylase-producing pathogens was performed according to the protocols described on the A. baumannii (http://pubmlst.org/abaumannii/) and P. aeruginosa (http://pubmlst.org/paeruginosa/) MLST Database websites. Seven chromosomal genes were PCR amplified and sequenced, with their nucleotide sequences compared with the sequences submitted to the MLST database to determine allele numbers and STs.
Whole genomes of methylase-negative A. baumannii and P. aeruginosa, which had MICs 128 mg/L to amikacin, 32 mg/L to arbekacin and 128 mg/L to gentamicin, were extracted by DNeasy Blood & Tissue kit (QIAGEN, Tokyo, Japan) and sequenced by MiSeq (Illumina, San Diego, CA). The sequence data were used to confirm aminoglycosideresistant genes.

Determination of the genetic environment surrounding rmtB
A draft genome sequence of an isolate of A. baumannii, NCGM36, harboring rmtB was determined using the GS Junior System (Roche Diagnostics K.K, Tokyo).

Antimicrobial susceptibility and aminoglycoside-resistant genes
The MICs at which 50% and 90% of the 101 A. baumannii and 15 P. aeruginosa isolates were inhibited (MIC50 and MIC90, respectively) were determined ( Table 1). Seventy of the 101 A. baumannii isolates (71.3%) had MICs >1,024 mg/L to all aminoglycosides tested, including amikacin, arbekacin and gentamicin. All 70 isolates had 16S rRNA methylases, with 61 having armA and the remaining 9 having rmtB ( Figure 1). The remaining 31 isolates had MICs ≤128 mg/L to amikacin, ≤32 mg/L to arbekacin and ≤128 mg/L to gentamicin and no methylase genes. Whole genome sequencing of 2 methylase-negative isolates showing relative resistance to aminoglycosides revealed that one had aac(6')-IIb and aadB and that the other had aac(6')-IIb and aadA2.

PFGE analysis and MLST
PFGE analysis of the 101 A. baumannii isolates revealed 8 clusters (Figure 1). Isolates from Clusters I, III, IV, V, VI, VII, and VIII were obtained from either one or the other hospital, whereas isolates from Clusters II and III were obtained from both. These results indicate that A. baumannii isolates had expanded in a clonal manner in both hospitals and that some isolates may spread among hospitals in Vietnam.
The16S rRNA methylase-encoding the rmtB gene was detected in Cluster I A. baumannii isolates, whereas armA was present in isolates from Clusters I, III, IV, V, VI, VII, and VIII. Isolates harboring rmtB were obtained from one hospital and isolates harboring armA were from both hospitals.
The two isolates harboring rmtB showed different patterns on PFGE, belonging to ST217 and ST313.

Genetic environment surrounding rmtB
The rmtB gene was associated with an ISCR3 mobile element upstream and a Tn3 transposon structure blaTEM-1-tnpR-tnpA downstream (data not shown). The genetic environment of rmtB had more than 99.9% nucleotide sequence identity, from nucleotide 1 to 8,337, to plasmid pXD2 (Gen bank accession no. JN315966) in E. coli, which causes bovine milk mastitis in China [12]. NCGM36, which harbored rmtB, had the blaOXA-23 and blaOXA-68 genes, but had neither the aac(6')-Ib-cr nor the blaCTX-Ms gene.

Discussion
The high prevalence of 16S rRNA methylase producing Gram-negative bacteria in hospitals in Vietnam may have resulted from the high rate of use of aminoglycosides. It has been estimated that 67.4% of hospitalized patients in Vietnam received antibiotics, including 18.9% who received aminoglycosides, with many 30.8% of these prescriptions considered inappropriate [13]. This rate of antibiotic use was much higher than in European countries (17.8%-32.0%) [14,15]. Moreover, the rate of inappropriate indications for antibiotic prescriptions in hospitals in Vietnam was much higher than rates reported in Malaysia (4.0%) [16], Turkey (14.0%) [17], and Hong Kong (20.0%) [18].   Pseudomonas aeruginosa isolates. Eight clusters with more than 70% similarity were detected. Two clusters (I and II) with more than 70% similarity were identified. Isolates harboring rmtB (○) are shown in the column on the right.
Malaysia and 1 in China;, 5 strains showing ST231 have been isolated in Brazil and 1 strain showing ST254 has been isolated in China (http://pubmlst.org/abaumannii/). ST136 and ST195 belong to clonal complex 92, the most widely disseminated complex worldwide [19]. Two strains of P. aeruginosa producing RmtB, showing ST217 and ST313, may have originally derived from Europe or Australia, because, to date, P. aeruginosa ST217 isolates were obtained only in the United Kingdom and ST313 isolates only in Australia, France and Hungary [20] (http://pubmlst.org/paeruginosa/).
To our knowledge, this is the first report showing that A. baumannii strains harboring a 16S rRNA methylase (ArmA or RmtB) and with blaOXA-23-like and blaOXA-51-like genes are emerging in medical settings in Vietnam. ArmA and OXA-23-like producing Gram-negative pathogens have been reported in Bulgaria [21], France [22], India [23], Korea [24], Norway [25] and the United States of America [26], and ArmA and OXA-51-like producing strains have been reported in Japan [27]. Moreover, armA and rmtB have been linked to blaCTX-Ms [28,29], but almost all ArmA producing isolates in Vietnam did not harbor blaCTX-Ms.
We found that some A. baumannii clinical isolates harbored rmtB. The genetic environment of the rmtB regions was very similar to the nucleotide sequence, from nt 1 to nt 8,337, of the plasmid pXD2. However, the plasmid of NCGM36 likely differs from pXD2 (Gen bank accession no. JN315966), in that the former NCGM36 did not have aac(6')-Ib or blaCTX-Ms.
Since 16S rRNA methylase genes in A. baumannii and P. aeruginosa are located in transferable plasmids [5], the absence of methylase genes was found in the same PFGE clusters. The details of these plasmids will be reported elsewhere.

Conclusions
This is the first report describing the presence of methylase producing Gram-negative bacteria in medical settings in Southeast Asia, specifically in Vietnam. A. baumannii isolates from northern and southern regions of Vietnam may be of different lineages.