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 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
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BRIEF COMMUNICATION
Year : 2017  |  Volume : 35  |  Issue : 2  |  Page : 282-285
 

Emergence of rmtC and rmtF 16S rRNA methyltransferase in clinical isolates of Pseudomonas aeruginosa


Department of Microbiology, Dr. ALM PG Institute of Basic Medical Sciences, University of Madras, Chennai, Tamil Nadu, India

Date of Web Publication5-Jul-2017

Correspondence Address:
Thangam Menon
Department of Microbiology, Dr. ALM PG Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai - 600 113, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_16_231

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 ~ Abstract 

Occurrence of aminoglycoside (AG) resistance in clinical isolates of Pseudomonas aeruginosa is investigated in this study. Antimicrobial susceptibility test and minimum inhibitory concentration (MIC) for amikacin and gentamicin were performed followed by polymerase chain reaction amplifications of AG modifying enzyme genes (aac(6´)-I, aac(6´)-II, aac(3)-II/VI, ant(2´´)-I, aph(3´)-VI) and 16S methylases (rmtA-D, rmtF and armA). MIC50and MIC90were 64, 128 and > 256, >256 for amikacin and gentamicin, respectively. Four types of genes (aac(6´)-I, aac(3)-II/VI, ant(2´´)-I and aph(3´)-VI) were found in 53 (57.6%) isolates. ant(2´´)-I was the most predominant gene (28 isolates) followed by aac(6´)-I (23 isolates). Nineteen (20.6%) isolates were positive for 16S RMTases (rmtB, rmtC, rmtF and armA) and two isolates co-harboured rmtB + rmtC + rmtF.


Keywords: 16S rRNA methylase, aminoglycoside modifying enzymes, aminoglycosides, ISEcp1, Pseudomonas aeruginosa


How to cite this article:
Mohanam L, Menon T. Emergence of rmtC and rmtF 16S rRNA methyltransferase in clinical isolates of Pseudomonas aeruginosa. Indian J Med Microbiol 2017;35:282-5

How to cite this URL:
Mohanam L, Menon T. Emergence of rmtC and rmtF 16S rRNA methyltransferase in clinical isolates of Pseudomonas aeruginosa. Indian J Med Microbiol [serial online] 2017 [cited 2017 Sep 26];35:282-5. Available from: http://www.ijmm.org/text.asp?2017/35/2/282/209576



 ~ Introduction Top


Aminoglycosides (AGs) are often administered along with other β-lactam antibiotics for the treatment of infections caused by Pseudomonas aeruginosa. Resistance to AGs is mainly through aminoglycoside-modifying enzymes (AMEs) which is mediated by acetylation, adenylation and phosphorylation through aminoglycoside acetyltransferases, aminoglycoside nucleotidyltransferases (ANT or AAD) and aminoglycoside phosphotransferases, respectively.[1] By acquisition of these enzymes, organism does not develop resistance to all AGs because of the substrate specificity. However, methylation of 16S rRNA has emerged as a mechanism of high-level AG resistance towards all AGs in recent years. Many types of 16S rRNA enzymes have been identified so far (ArmA, RmtA-H and NpmA) and these gene determinants are found to be associated with mobile genetic determinants which confers resistance to other classes of antibiotics.[2] Transposition and expression of rmtC is mediated by insertion sequence ISEcp1. This ISEcp1 element belongs to IS1380 family which is located at 5′ end of rmtC and contains a transposase gene (tnpA) and provides a promoter activity for expression of the adjacent rmtC. This structure enables the rmtC gene to be transposed onto another plasmid.[3] Because of the spread of resistance genes, continuous monitoring is required and thus, this study was aimed to investigate the prevalence of AME enzymes and 16S rRNA methylases genes among clinical isolates of P. aeruginosa.


 ~ Materials and Methods Top


Ninety-two P. aeruginosa isolates which were collected between 2014 and 2015 from ESIC hospital, Chennai and Sri Manakula Vinayagar Medical College and Hospital, Puducherry were included in this study. These isolates were resistant to one of the tested AGs (amikacin, gentamicin, tobramycin and netilmicin) by antimicrobial susceptibility test and were recovered from clinical specimens such as pus (n = 77), urine (n = 4), tracheal wash (n = 4), sputum (n = 4), and blood (n = 3). The minimum inhibitory concentrations (MICs) of amikacin and gentamicin were determined by agar dilution method and interpreted according to the Clinical Laboratory Standards Institute guidelines 2013.[4] ATCC 27853 P. aeruginosa was used as a quality control strain.

DNA extraction was performed by boiling lysis method.[5] The detection of AME genes (aac(6´)-I, aac(6´)-II, aac(3)-II/VI, ant(2´´)-I, aph(3´)-VI) and 16S rRNA methylase genes (armA, rmtA-D and rmtF) was carried out by polymerase chain reaction (PCR) using specific primers.[6],[7],[8] The association of AMEs and 16S rRNA methylases with Class I integron was investigated by PCR.[9] ORF513 and ISEcp1 was previously shown to promote both expression and transposition of rmtC; hence to assess the association of ISEcp1 with rmtC, PCR was performed on the rmtC positive isolates with primer pairs rmtC-F and ISEcp1 with rmtC-R.[10],[11] PCR products were analysed by electrophoresis with 1.5% agarose in 1X Tris Borate EDTA buffer. The gel was stained with ethidium bromide and the PCR products were visualised using the gel documentation system (Carestream Gel Logic 21 PRO). The PCR amplicon of ISEcp1 with rmt C-R were sequenced by Sanger's technique (Xcelris, Ahmedabad). Transfer of resistant genes was evaluated through conjugation assay using 16S methylase positive strains as donors and rifampin-resistant P. aeruginosa PU21 as the recipient.[12] An overnight culture of donor (0.1 ml) and recipient cells (0.4 ml) were incubated at 37°C for 18–24 h and transconjugants were selected after 48 h of incubation at 37°C on trypticase soy agar plates containing rifampin (100 μg/ml) and gentamicin (32 μg/ml).


 ~ Results Top


Of the 92 P. aeruginosa isolates, the overall resistance rates to amikacin, gentamicin, tobramycin and netilmicin were 65.2% (n = 60), 86.9% (n = 80), 77.1% (n = 71) and 67.3% (n = 62), respectively. The rates of high-level resistance to AGs were 23.9% (n = 22) for amikacin and 47.8% (n = 44) for gentamicin [Table 1]. Four types of AME genes (aac(6´)-I, aac(3)-II/VI, ant(2´´)-I and aph(3´)-VI) were found in 53 (57.6%) isolates and ant(2´´)-I was the most predominant gene (28 isolates) followed by aac(6´)-I (23 isolates). Fifteen isolates harboured two or more AME genes and their distributions were as follows: aac(6´)-I + aac(3)-II/VI in 4 (4.3%); aac(6´)-I + ant(2´´)-I in 4 (4.3%); aac(3)-II/VI + ant(2´´)-I in 5 (5.4%); aac(6´)-I + ant(2´´)-I + aph(3´)-VI in 1 (1%); ant(2´´)-I + aph(3´)-VI in 1 (1%) isolates. Among 53 AME positive isolates, the MICs ranged from 2 μg/ml to >256 μg/ml for amikacin and gentamicin (MIC50 64 μg/ml and MIC90 > 256 μg/ml). Isolates having high MIC (128 μg/ml, 256 μg/ml and >256 μg/ml) to amikacin harboured aac(6´)-I (12 isolates), aac(3)-II/VI (6 isolates), ant(2´´)-I (10 isolates), aph(3´)-VI (1 isolate), whereas isolates with low MIC values (2 μg/ml, 4 μg/ml, 8 μg/ml and 6 μg/ml) of amikacin harboured aac(6´)-I (6 isolates), aac(3)-II/VI (1 isolate), ant(2´´)-I (12 isolates) and aph(3´)-VI (4 isolate), respectively. Isolates with high (128 μg/ml, 256 μg/ml and >256 μg/ml) MICs of gentamicin harboured aac(6´)-I (8 isolates), aac(3)-II/VI (2 isolate), ant(2´´)-I (8 isolates) and aph(3´)-VI (2 isolates). Genes such as aac(6´)-I (9 isolates), aac(3)-II/VI (2 isolate), ant(2´´)-I (15 isolates) and aph(3´)-VI (2 isolates) were observed in isolates with lower MICs of gentamicin (2 μg/ml, 4 μg/ml and 8 μg/ml).
Table 1: Aminoglycoside susceptibility profiles and 16S rRNA methyltransferase (rmtB, rmtC, rmtF and armA) and aminoglycoside modifying enzymes profiles

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Of 92 isolates 19 (20.6%) were positive for 16S RMTase; rmtB in 7 (7.6%); rmt C in 4 (4.3%); rmtF in 2 (2.1%); rmtB + rmtF in 2 (2.1%); rmtB + rmtC in 1 (1%); arm A + rmtB in 1 (1%) and rmtB + rmtC + rmtF in 2 (2.1%) isolates. All isolates were negative for rmt A and rmt D. Of 13 rmtB positive isolates, three isolates had low MIC value of 2 μg/ml, 8 μg/ml and 16 μg/ml towards amikacin. One arm A positive isolate had MIC value of 64 μg/ml to amikacin and 128 μg/ml to gentamicin. The 73 methylase-negative isolates had MICs ranging from 1 μg/ml to >256 μg/ml to amikacin (MIC50 64 μg/ml and MIC90 > 256 μg/ml) and 2 μg/ml–>256 μg/ml to gentamicin (MIC50 64 μg/ml and MIC90 > 256 μg/ml). The combinations of AME genes and 16S RMTases are listed in [Table 2]. More than one RMTase was observed in 6/19 isolates; rmtB + rmtF in combination was observed in two isolates of which one isolate showed MIC of 64 μg/ml to amikacin and >256 μg/ml to gentamicin and the other isolate showed >256 μg/ml to both amikacin and gentamicin. One rmtB with arm A positive isolate showed MIC of 64 μg/ml and 128 μg/ml to amikacin and gentamicin, respectively. In this study, we also found two strains harboring three different 16S rRNA methylase genes, that is, rmtB + rmtC + rmtF in combination and 1 strain possessing rmtB + rmtC showing high level resistance to both amikacin and gentamicin (MIC value >256 μg/ml).
Table 2: Distribution of aminoglycoside modifying enzyme and 16S rRNA methylases

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Attempts to transfer rmtB, rmtC and rmtF from the clinical isolates to P. aeruginosa PU21 through conjugation were unsuccessful. Class I integron was amplified in 77 (83.6%) isolates. Four of 19 isolates harboured ORF 513 bearing Class I integron which had rmtB, rmtF, rmtB + armA, rmtB + rmtC. rmtC-R/ISEcp1 were amplified in 4 of 7 isolates, and the amplicon size was ~1000 bp in all the four isolates. One isolate was sequenced, and it revealed the association of intact ISEcp1 with rmtC. However, the ISEcp1 element was not amplified in three isolates, suggesting that there was either a partial deletion of this element or involvement of a different ISEcp1-like element in spread of rmtC.


 ~ Discussion Top


In India, only a few studies are available on the 16S rRNA methylases in P. aeruginosa and this type of resistance mechanism in Gram-negative pathogens is increasingly recognised worldwide since the initial reports in 2003. Resistance mediated by AME and 16S rRNA methylases are predominant though other resistance mechanisms of AGs including cellular impermeability, active efflux and rarely, nucleotide substitution of the target molecule are reported. In the present study, ant(2´´)-I was predominant (30%) followed by aac(6´)-I gene (25%) and this finding is in contrast to studies conducted in the Europe, USA and Korea where aac(6´)-I and aph(3´)-VI are more frequent.[6] There is no phenotypic method available for the detection of 16S rRNA methylases in routine laboratories and resistance is suspected when a strain belonging to either Enterobacteriaceae or non-fermentative species shows phenotypic resistance to multiple AGs by disk diffusion test. The positive predictive value of the method is >90% when tested with arbekacin disk, compared with ~60% with amikacin, although arbekacin is not recommended for therapeutic use.[7] According to Wachino and Arakawa,[13] the high MIC values (≥128 μg/ml) of both amikacin and gentamicin are a good indicator of N7-G1405 16S-RMTase-producers. In the current study, isolates with MICs of 2 μg/ml, 8 μg/ml, 16 μg/ml and 64 μg/ml to amikacin and 8 μg/ml, 16 μg/ml, 32 μg/ml and 64 μg/ml to gentamicin carried 16S rRNA methylase genes, implying that their presence need not necessarily confer high resistance. In comparison, a study which included 140 isolates of Enterobacteriaceae with MIC values of >200 mg/L of amikacin and gentamicin carried armA (46%), rmtB (20%), rmtC (27%) and rmtF (24%) gene.[8] Among P. aeruginosa, rmtA and rmtD have been frequently reported in the European and American countries and other RMTases (rmtB, rmtC, armA and rmtF) are common in Enterobacteriaceae, whereas in Asian countries, rmtB and armA are the most common genotypes. In Vietnam, 2 rmtB producing isolates of P. aeruginosa had MICs of >1024 mg/L to amikacin, arbekacin and gentamicin,[14] whereas in China, rmtB was found in 14/17 isolates [15] and in another study, 26 armA and 5 rmtB positive strains were found among 35 P. aeruginosa isolates.[16] In this study, we report the occurrence of rmtB, rmtC, armA and rmtF which has not been reported from the European and American countries but has been reported from the Southeast Asian countries including India.[17]

Isolates with more than one 16S RMTase showed MIC ranging from 64 μg/ml to >256 μg/ml indicating that both the genetic context of the 16S RMTase and the presence of multiple AME genes might play a role in increased resistance to amikacin. The association of rmtC with ISEcp1 element raises a concern that further spread may occur. These findings are of concern since 16S rRNA methylase genes are already disseminated among P. aeruginosa although the overall prevalence appears to remain low. This limits the therapeutic options in patients requiring highly potent AGs such as amikacin and tobramycin. Spread of multidrug-resistant isolates that express 16S rRNA methyltransferases might be due to the selective pressure for the organisms to acquire 16S methylase genes, possibly from nonpathogenic environmental actinomycetes that intrinsically produced AGs or 16S rRNA inhibitors which disseminates through mobile genetic elements.[7] Our findings underline the emerging threat of 16S rRNA methylase in India. The presence of these enzymes compromise all parenterally formulated AGs in clinical use, and the detection of this resistance mechanism may not be possible in current routine susceptibility testing because MICs are performed only close to the breakpoints of each AG. Further studies are needed to find the association with other β-lactamases and metallo β-lactamases genes.


 ~ Conclusion Top


Resistance to aminoglycosides mediated by AME appeared to be more common than 16s RMTases; however one third of the resistant strains were negative for both AME and 16s RMTases indicating that other mechanisms are not uncommon. Presence of a single 16S RMTase gene was not associated with high MIC values; however the fact that these genes are disseminated among P.aeruginosa strains and can spread by horizontal transfer is a matter of concern.

Acknowledgements

The authors would like to thank Dr. George A. Jacoby, Infectious Disease Specialist, Lahey Clinic Medical Center, Burlington, for providing PU21 P. aeruginosa. We thank Dr. Sunil Santaji Shivekar, Sri Manakula Vinayagar Medical College and Hospital, Puducherry and Dr. Lakshmi Priya, Dr. Esther Mary, ESIC Hospital, Chennai for providing clinical isolates.

Financial support and sponsorship

The authors acknowledge the financial support from UGC, New Delhi, India.

Conflicts of interest

There are no conflicts of interest.



 
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Wachino J, Yamane K, Kimura K, Shibata N, Suzuki S, Ike Y, et al. Mode of transposition and expression of 16S rRNA methyltransferase gene rmtC accompanied by ISEcp1. Antimicrob Agents Chemother 2006;50:3212-5.  Back to cited text no. 3
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Wachino J, Arakawa Y. Exogenously acquired 16S rRNA methyltransferases found in aminoglycoside-resistant pathogenic Gram-negative bacteria: An update. Drug Resist Updat 2012;15:133-48.  Back to cited text no. 13
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Zhou Y, Yu H, Guo Q, Xu X, Ye X, Wu S, et al. Distribution of 16S rRNA methylases among different species of gram-negative bacilli with high-level resistance to aminoglycosides. Eur J Clin Microbiol Infect Dis 2010;29:1349-53.  Back to cited text no. 16
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