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  Table of Contents  
Year : 2019  |  Volume : 37  |  Issue : 3  |  Page : 418-422

Diverse aminoglycoside phosphotransferase types conferring aminoglycoside resistance in Enterobacteriaceae: A single-centre study from Northeast India

1 Department of Microbiology, Assam University, Silchar, Assam, India
2 Department of Microbiology, Silchar Medical College and Hospital, Silchar, Assam, India

Date of Submission15-Jul-2019
Date of Decision14-Sep-2019
Date of Acceptance29-Nov-2019
Date of Web Publication29-Jan-2020

Correspondence Address:
Dr. Amitabha Bhattacharjee
Department of Microbiology, Assam University, Silchar, Assam
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmm.IJMM_19_262

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 ~ Abstract 

The present study investigates the molecular basis of aph-mediated aminoglycoside resistance and their transmission dynamics in a tertiary care hospital of Northeast India. Two hundred forty one isolates (230 Escherichia coli and 11 Klebsiella pneumoniae) were collected and screened for aminoglycoside resistance genes. Various aph types were amplified using polymerase chain reaction (PCR) assay. Plasmid incompatibilty, horizontal transferability and ERIC-PCR based typing were carried out for all the positive isolates. Among them, 67 isolates showed the presence of aph gene. Aph (3“)-IIIa and aph (3')-Via were predominant and horizontally transferable. All the plasmids were of incompatibility I1 group. Twenty-eight different haplotypes of E. coli were found harbouring aph gene types. This study was able to identify diverse aph types in a single centre and their corresponding phenotypic trait.

Keywords: Aminoglycoside resistance, antibiotic resistance, aminoglycoside phosphotransferase, Escherichia coli

How to cite this article:
Wangkheimayum J, Bhattacharjee M, Das BJ, Singha K M, Chanda DD, Chanda DD, Bhowmik D, Bhattacharjee A. Diverse aminoglycoside phosphotransferase types conferring aminoglycoside resistance in Enterobacteriaceae: A single-centre study from Northeast India. Indian J Med Microbiol 2019;37:418-22

How to cite this URL:
Wangkheimayum J, Bhattacharjee M, Das BJ, Singha K M, Chanda DD, Chanda DD, Bhowmik D, Bhattacharjee A. Diverse aminoglycoside phosphotransferase types conferring aminoglycoside resistance in Enterobacteriaceae: A single-centre study from Northeast India. Indian J Med Microbiol [serial online] 2019 [cited 2020 Oct 25];37:418-22. Available from:

 ~ Introduction Top

Aminoglycoside antibiotics were among the frontline drugs used for therapeutic options in hospital settings and constitute as one of the potent agents for life-threatening infections caused by Gram-negative bacteria.[1] However, the efficacy of this antibiotic has been compromised by the emergence of acquired resistance and enzymatic modifications. The enzymes responsible are methyltransferase, acetyltransferase, nucleotidyltransferase and phosphotransferase. Aminoglycoside O-phosphotransferases (APHs) catalyse the transfer of a phosphate group to aminoglycoside molecule.[2] In recent studies, Aph-mediated aminoglycoside resistance is reported in  Escherichia More Details coli and Klebsiella pneumoniae.[3],[4] In India, no previous study has been carried out to characterize aph gene with their corresponding resistance profile. Hence, the current study aimed to characterize aph variants and their transmission dynamics in a tertiary care hospital of Northeast India.

 ~ Materials and Methodology Top

Bacterial strains

A total of 241 consecutive nonduplicate clinical isolates of E. coli and K. pneumoniae were collected from Silchar Medical College and Hospital, Silchar, between June 2018 and January 2019 from the patients who were admitted or attended the OPD of the tertiary referral hospital. Clinical specimens used were urine, pus, aspirates and catheter tip. Isolates were identified based on cultural characteristics on CHROMagar (HiMedia, India) and standard biochemical reactions.[5]

Antimicrobial susceptibility testing

Minimum inhibitory concentration (MIC) of isolates against aminoglycoside antibiotics, namely kanamycin, tobramycin, gentamicin, netilmicin, amikacin and streptomycin (HiMedia, India) was determined by agar dilution method. Disc-diffusion method was also used for the detection of susceptibility pattern towards imipenem (10 μg), cefepime (30 μg), aztreonam (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), ceftriaxone (30 μg) and ciprofloxacin (5 μg). The results were interpreted in accordance with the Clinical and Laboratory Standards Institute guidelines 2017.[6]

Molecular detection of aminoglycoside O-phosphptransferases gene

Any organism that was resistant to at least one of the aminoglycosides was selected further for the molecular analysis. Two multiplex polymerase chain reaction (PCR) assays were performed targetting various aph genes, namely aph (2“

;)-Ib, aph (2“)-Ic, aph (2')-Id, aph (3')-IIb, aph (3')-I, aph (3“)-IIIa, aph (3')-Via and aph (4)-Ia.[7] The PCR mixture was composed of 12.5 μl Go Taq green Master mix (Promega, Madison, USA); 10 pmol of each primer ~100 ng DNA template was prepared by boiling centrifugation method (82°C for 20 min). The PCR assay was performed in T100™ Thermal cycler (Bio-Rad, USA) with the condition as follows: initial denaturation at 94°C for 3 min, denaturation at 95°C for 20 s, annealing at 52°C for 45 s, extension at 72°C for 1 min and final extension at 72°C for 5 min.

Horizontal transferability assay

A horizontal transferability assay was done for all the 67 aph carrying isolates. Plasmid was extracted by QIAprep Spin Miniprep Kit (Qiagen, Germany) and isolated plasmids were subjected to transformation by heat shock method using E. coli DH5α as recipient.[8] Transformants were selected onto the Luria Bertani (LB) agar (HiMedia, Mumbai, Maharashtra, India) containing 2 μg/ml of kanamycin. Conjugation assay was performed using E. coli, harbouring aph as donor and azide-resistant E. coli J53 as recipient. Cells were mixed at a ratio of 1:5 donor-to-recipient and transconjugant was selected on LB medium (HiMedia, Mumbai, Maharashtra, India) containing 2 μg/ml of kanamycin and 100 μg/ml of sodium azide. Transformants and transconjugant were also confirmed by aph PCR assay.

Plasmid incompatibility typing

Plasmids harbouring aph genes were typed by PCR-based replicon typing to identify the different incompatibility (Inc) types.[9]

DNA fingerprinting by enterobacterial repetitive intergenic consensus-polymerase chain reaction

Enterobacterial repetitive intergenic consensus (ERIC)-PCR was performed to determine the clonal relatedness of all the isolates using primers ERIC-F (5'-ATGTAAGCTCCTGGGGATTCAC-3') and ERIC-R (5'-AAGTAAGTGACTGGGGTGAGCG-3'), and the banding patterns were determined by agarose gel electrophoresis.

 ~ Results Top

Of the 241 isolates, 230 were E. coli isolates and 11 of them were K. pneumoniae. Among them, 111 E. coli and 5 K. pneumoniae were found to be resistant to at least one of the aminoglycosides tested and were further selected for the molecular analysis. Among the study isolates, imipenem came up with the highest efficacy as E. coli and K. pneumoniae showed 89% and 73% susceptibility, respectively. However, against other antibiotics, susceptibility was very low [Supplementary Table 1]. While testing MIC, majority of the isolates were within susceptible range against tobramycin (143/241) followed by kanamycin (141/241), netilmicin (141/241), amikacin (136/241), gentamicin (131/241) and streptomycin (125/241). A total of 67 isolates (64 E. coli and 3 K. pneumoniae) were found to harbour single and multiple aph genes. Among them, 30 isolates were harbouring single aph gene types and 37 were found to harbour more than one aph types [Table 1]. All the aph gene types were conjugatively transferable, and Inc typing of the plasmids that harboured these genes showed that the isolates harbouring multiple aph genes were originated from a single IncI1 group [Figure 1] when transconjugants and transformants were confirmed by PCR assay for replicon typing and aph genes. DNA fingerprinting by ERIC-PCR suggested that 28 different haplotypes of E. coli were detected within this centre and were found to be responsible for the spread of this aph genes [Figure 2]. However, ERIC-PCR result showed that only a single haplotype of K. pneumoniae was responsible for the carriage of aph gene.

Table 1: Genotypic phenotypic correlation of isolates based on the minimum inhibitory concentration results

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Figure 1: Inc I1 plasmid type (Lane 1–8)

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Figure 2: Enterobacterial repetitive intergenic consensus polymerase chain reaction results showing 28 haplotypes. Lane 1 and 18; DNA Ladder, Lane 2; type 1, Lane 3; type 2; Lane 4; type 3; Lane 5–7; type 4, Lane 8; type 5, Lane 9; type 6, Lane 10; type 7; Lane 11; type 8, Lane 12; type 9, Lane 13; type 10, Lane 14; type 11, Lane 15; type 12, Lane 16; type 13, Lane 19; type 14, Lane 20; type 15, Lane 21; type 16, Lane 22; type 17, Lane 23; type 18, Lane 24; type 19, Lane 25; type 20; Lane 26; type 21, Lane 27-28; type 22, Lane 29; type 23, Lane 30; type 24, Lane 31; 25, Lane 32; type 26, Lane 33; type 27, Lane 34; type 28

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 ~ Discussion Top

Till the late 1990s, aph (3“) family was more predominant among clinical isolates of Gram-negative pathogen.[2] In the current study, we found that most of our isolates were resistant to kanamycin and gentamicin. Another highlight of our study is the presence of diverse aph types within a single centre. We observed that aph (3“) and aph (2“) were more common than other types. The presence and maintenance of different aph genes in isolate from a single centre underscores their diverse origin. In other studies, aph (3“)-IIIa was found to be responsible for kanamycin resistance.[10],[11] In agreement with their studies, we too could select transformants and transconjugant receiving aph genes successfully on a screening media containing kanamycin, whereas the same was not successful with other aminoglycosides. Similar to our study, aph (2) was found conferring resistance to kanamycin, tobramycin, dibekacin and gentamicin.[12] In a more recent study, it reported for the first time that aph (2)-If gene was highly predominant and chromosomally encoded in Camphylobacter and it is also increasingly resistant to gentamicin.[13]

The present study was able to underscore that the IncI1 plasmid type was responsible for the expansion of aph genes in this clinical setting. However, no previous Indian study is on record to compare the local epidemiology of this resistance determinant. Thus, the present study was able to highlight a resistance mechanism which is not commonly investigated.

 ~ Conclusion Top

The finding of the current study underscores the role of aph genes in resistance within hospital settings which warrants further investigation to decipher its origin and source of acquisition to implement proper infection control strategy against antimicrobial resistance problem.


The authors would like to acknowledge the Jawaharlal Nehru Memorial Trust for providing a scholarship to Jayalaxmi Wangkheimayum.

Financial support and sponsorship

JNMF scholarship was awarded to Jayalaxmi Wangkheimayum Vide letter no; SU-1/068/2018-19/89 dated 4th December 2017.

Conflicts of interest

There are no conflicts of interest.

 ~ References Top

Shokravi Z, Mehrad L, Ramazani A. Detecting the frequency of aminoglycoside modifying enzyme encoding genes among clinical isolates of methicillin-resistant Staphylococcus aureus. Bioimpacts 2015;5:87-91.  Back to cited text no. 1
Wright GD, Thompson PR. Aminoglycoside phosphotransferases: Proteins, structure, and mechanism. Front Biosci 1999;4:D9-21.  Back to cited text no. 2
Abo-State MA, Saleh YE, Ghareeb HM. Prevalence and sequence of aminoglycosides modifying enzymes genes among E. coli and Klebsiella species isolated from Egyptian hospitals. J Radiat Res Appl Sci 2018;11:408-15.  Back to cited text no. 3
Nasiri G, Peymani A, Farivar TN, Hosseini P. Molecular epidemiology of aminoglycoside resistance in clinical isolates of Klebsiella pneumoniae collected from Qazvin and Tehran provinces, Iran. Infect Genet Evol 2018;64:219-24.  Back to cited text no. 4
Colee JG, Diguid JP, Mackie Fraser AG, Microbiology McCartney Practical Medical. 14th ed. Edinburg: Churchill, Livingstone; 1996.  Back to cited text no. 5
Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-First Informational Supplement. M100-S27. Wayne, PA: Clinical and Laboratory Standards Institute; 2017.  Back to cited text no. 6
Nie L, Lv Y, Yuan M, Hu X, Nie T, Yang X, et al. Genetic basis of high level aminoglycoside resistance in Acinetobacter baumannii from Beijing, China. Acta Pharm Sin B 2014;4:295-300.  Back to cited text no. 7
Paul D, Bhattacharjee A, Bhattacharjee D, Dhar D, Maurya AP, Chakravarty A. Transcriptional analysis of and blaNDM-1 and copy number alteration under carbapenem stress. Antimicrob Resist Infect Control 2017;6:26.  Back to cited text no. 8
Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods 2005;63:219-28.  Back to cited text no. 9
Kobayashi N, Alam M, Nishimoto Y, Urasawa S, Uehara N, Watanabe N. Distribution of aminoglycoside resistance genes in recent clinical isolates of Enterococcus faecalis, Enterococcus faecium and Enterococcus avium. Epidemiol Infect 2001;126:197-204.  Back to cited text no. 10
Schmitz FJ, Fluit AC, Gondolf M, Beyrau R, Lindenlauf E, Verhoef J, et al. The prevalence of aminoglycoside resistance and corresponding resistance genes in clinical isolates of staphylococci from 19 European hospitals. J Antimicrob Chemother 1999;43:253-9.  Back to cited text no. 11
Toth M, Frase H, Antunes NT, Vakulenko SB. Novel aminoglycoside 2''-phosphotransferase identified in a gram-negative pathogen. Antimicrob Agents Chemother 2013;57:452-7.  Back to cited text no. 12
Ho PL, Leung LM, Chow KH, Lai EL, Lo WU, Ng TK. Prevalence of aminoglycoside modifying enzyme and 16S ribosomal RNA methylase genes among aminoglycoside-resistant Escherichia coli isolates. J Microbiol Immunol Infect 2016;49:123-6.  Back to cited text no. 13


  [Figure 1], [Figure 2]

  [Table 1]


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