Detection of 16S rRNA Methylase Genes in Gram-Negative Bacilli Isolated from Hospitals in Changchun, China

Methylation of 16S rRNA is an important mechanism of aminoglycoside resistance among gram-negative pathogens. In this report, 16S rRNA methylase genes were amplified using PCR among gram-negative bacillus isolates from hospitals in the Changchun area of China and 16S rRNA methylase genotypes (armA, rmtB, rmtA, rmtC, rmtD, and npmA) were identified by direct sequencing. Fifty of the isolates (43.1%) harbored 16S rRNA methylase genes. The common 16S rRNA methylase genes were armA and rmtB (12.1% and 31.0%, respectively), whereas the rmtA, rmtC, rmtD, and npmA genes were absent from the sample. It suggests that the predominant 16S rRNA methylase genes among gramnegative bacilli in the Changchun area are armA and rmtB.


Introduction
Aminoglycosides have strong antibacterial activity against gram-negative bacilli and gram-positive bacilli.Aminoglycosides are well received by clinicians because of their broad antimicrobial spectrum and efficacy.The irrational use of antibiotics is causing increasingly acute problems associated with antimicrobial resistance, however.The methylation of 16S rRNAs in gram-negative bacilli is one of the mechanisms underlying strong resistance to aminoglycosides.Recent studies [1][2] showed that 16S rRNA methylase could methylate the 30S ribosomal subunit in gram-negative bacilli.16S rRNA methylase can protect the target sites of the 30S ribosomal subunit, preventing the aminoglycosides from combining with the 30S ribosomal subunit.
Following the first discovery of 16S rRNA methylase gene armA in France [3], other methylases such as rmtB, rmtA, and rmtC were found among gram-negative pathogens [4,5].The predominant methylase genes in southern China are armA and armB [6]; however, the prevalence of 16S rRNA methylases among clinical isolates of gramnegative bacilli in Changchun, Northeast of China, has not been previously assessed.16S rRNA methylase me-diates high-level resistance to aminoglycosides in gramnegative isolates, and some spices in gram-negative bacilli are major causes of nosocomial infections [7].The aim of this study was to investigate the prevalence of 16S rRNA methylases in aminoglycoside-resistant isolating from three hospitals in Changchun and to characterize the host bacteria.

Isolates and Drugs
One hundred and sixteen strains of gram-negative bacilli were isolated from China-Japan Union Hospital of Jilin University (Changchun, China), the Affiliated Hospital to Changchun University of Chinese Medicine (Changchun, China), and the Clinical Laboratory of Jilin Province People's Hospital (Changchun, China).The clinical isolates consisted of 33 Escherichia coli strains, 25 Klebsiella pneumoniae strains, 14 Enterobacter cloacae strains, 15 Acinetobacter baumannii strains, 19 Pseudomonas aeruginosa strains, and 10 Serratia marcescens strains.Identification of these isolates was done using the VITEK-32 system (Mérieux, France).Amikacin was provided by the National Institute for the Control of Pharmaceutical and Biological Products (batch number 130335-200204) (Bei-jing, China).Gentamicin was supplied by Beijing Dingguo Changsheng Biotech Co., Ltd.(batch number 1B310330) (Beijing, China).

Testing for Antibiotic Susceptibility by Agar Dilution
The susceptibilities of the 116 strains to amikacin and gentamicin were determined by agar dilution.Isolates that were able to grow at antibiotic concentrations above 16 mg/L were regarded as being resistant to the antibiotics; and those that were not able to grow at concentrations above 2 mg/L were regarded as sensitive.

Extraction of Bacteria DNA
One milliliter of bacterial culture was placed in a 1.5 mL centrifuge tube and centrifuged at 10,000 rpm for 1 min.The supernatant was removed, and 100 μL TE Buffer (1 mol/L Tris-HCl, pH 8.0; 500 mmol/L EDTA, pH8.0) was added.Then, an equal volume of mixed phenol, chloroform, and isoamyl alcohol (25:24:1) was added.Vortex oscillation was then performed for 30 s, and the mixture was subsequently centrifuged at 10,000 rpm for 5 min.
The resulting supernatant was then stored at −20˚C until later use as the template for genetic testing.

Primer Design and Detection of 16S rRNA Methylase Genes
Primers were self-designed for six different 16S rRNA methylase gene sequences available from GenBank.The primer sequences, target genes, and primer lengths are shown in Table 1.The conditions for producing PCR products with lengths greater than 500 bp were: 2 min at 93˚C; 35 cycles of 1 min at 93˚C, 1 min at 55˚C, and 1 min at 72˚C; followed by 5 min at 72˚C.The conditions for producing PCR products with lengths less than 500 bp were: 5 min at 93˚C; 35 cycles of 30 s at 93˚C, 30 s at 55˚C, and 1 min at 72˚C; followed by 5 min at 72˚C.After 2% agarose gel electrophoresis, the PCR products were observed under a gel imager and photos were taken.

PCR Product Sequencing
The PCR products were sent to Beijing Genomics Institute, (Beijing, China) for sequencing.The sequences were detected using the Chromas software and compared with those released by GenBank.

Results of 16S rRNA Methylase Genetic Testing
Fifty (43.1%) of the 116 isolates harbored 16S rRNA methylase genes.The common 16S rRNA methylase genes were armA (12.1%, 14/116) and rmtB (31.0%, 36/ 116).All of the isolates were negative for the rmtA, rmtC, rmtD, and npmA genotypes (Table 4).Additionally, three of the S. marcescens strains harbored both armA and rmtB and were highly resistant to both amikacin and gentamicin.An electrophoregram of the products of PCR amplification of armA and rmtB is shown in Figures 1  and 2.

Sequencing of 16S rRNA Methylase
The results of the direct sequencing of the armA and rmtB genes isolated from our sample were compared with the corresponding sequences in the GenBank database, and the sequences in our sample were found to be identical to those in the database (arm A:HQ204573.1; rmt B:FJ539137.1).

Discussion
Aminoglycosides exert their antibacterial action by binding to the highly conserved A site of the 16S rRNA of the bacterial 30S ribosomal subunits, interfering with protein synthesis with subsequent bacterial death.Furthermore, aminoglycosides have a broad antimicrobial Open Access AID   spectrum and produce synergistic effects with other kinds of antibiotics.Because of massive use of antibiotics including aminoglycosides, problems related to bacterial resistance to aminoglycosides are becoming very serious.Such resistance is achieved by enzymatic modification, changes in cellular membrane permeability, efflux pump activity, the phoP-phoQ system, or 16S rRNA methylase activity [10][11][12].16S rRNA methylase is usually encoded   by plasmids, and its transfer via plasmids has led to the rapid spread of 16S rRNA methylase genes among bacilli [13,15], causing great difficulties for clinical treatment.
Our study showed that 43.1% and 75% of a sample of 116 gram-negative bacillus isolates were resistant to amikacin and gentamicin, respectively.Except for the low resistance rates among the K. pneumoniae and E. cloacae strains, the resistance rates among the other strains of gram-negative bacilli were all greater than 45%, indicating high rates of aminoglycoside resistance among gramnegative bacilli in the Changchun area.In terms of the 16S rRNA methylase genetic testing, the frequency of rmtB (31.0%) in our sample was higher than that of armA (12.1%).Furthermore, rmtB is mainly distributed among E. coli (54.5%) and P. aeruginosa (73.7%) strains; whereas armA is mainly distributed among A. baumannii (33.3%) and S. marcescens (70%) strains.We did not detect any 16S rRNA methylase genes among 25 K. pneumoniae strains; and we only found 1 rmtA gene among 14 strains of E. cloacae (7.1%).The three S. marcescens strains that carried both armA and rmtB showed strong resistance to both amikacin and gentamicin.In our study, the frequency of aminoglycoside resistance among the 116 strains of gram-negative bacilli was higher than the frequency of 16S rRNA methylase genes, suggesting that the drug-resistant phenotypes were not totally consistent.Therefore, some of the aminoglycoside resistance in our sample was likely caused by other antibiotic resistance mechanisms such as the production of AME genes, changes in cellular membrane permeability, efflux pump activity, or the phoP-phoQ system.
In terms of the distribution of 16S rRNA methylase ge-nes, only the armA and rmtB genes were detected in our study, suggesting that armA and rmtB are the two main 16S rRNA methylase genes in the Changchun area, which is consistent with relevant domestic studies [16,17].The armA and rmtB genes are also the most common genotypes in other Asian countries such as South Korea [18] and Japan [19].The most commonly detected 16S rRNA methylase genes in European countries such as Belgium [20] and Bulgaria [21] is armA, whereas in Brazil it is rmtB [22].According to our data, high-level aminoglycoside resistance in clinical isolates conferred by 16S rRNA methylase is of great concern in Changchun area.Hence, clinicians must pay more attention to the rational use of such drugs to reduce the frequencies of drug-resistant bacteria under the selective pressure by antibiotics.

Conclusion
In conclusion, our experimental results suggest that the predominant 16S rRNA methylase genes among gramnegative bacillus isolates from Changchun area, Northeast of China, are armA and rmtB.Aminoglycoside-resistant isolates producing armA or rmtB may become a major therapeutic threat in the future.