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BRIEF COMMUNICATION
Year : 2016  |  Volume : 34  |  Issue : 3  |  Page : 350-352
 

Distribution of genes encoding aminoglycoside-modifying enzymes among clinical isolates of methicillin-resistant staphylococci


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

Date of Submission19-Mar-2016
Date of Acceptance28-Jun-2016
Date of Web Publication12-Aug-2016

Correspondence Address:
P Krishnan
Department of Microbiology, Dr. ALM PG Institute of Basic Medical Sciences, University of Madras, Chennai
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.188339

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 ~ Abstract 

The objective of this study was to determine the distribution of genes encoding aminoglycoside-modifying enzymes (AMEs) and staphylococcal cassette chromosome mec (SCCmec) elements among clinical isolates of methicillin-resistant staphylococci (MRS). Antibiotic susceptibility test was done using Kirby-Bauer disk diffusion method. The presence of SCCmec types and AME genes, namely, aac (6')-Ie-aph (2''), aph (3')-IIIa and ant (4')-Ia was determined using two different multiplex polymerase chain reaction. The most encountered AME genes were aac (6′)-Ie-aph (2'') (55.4%) followed by aph (3')-IIIa (32.3%) and ant (4')-Ia gene (9%). SCCmec type I (34%) was predominant in this study. In conclusion, the aac (6')-Ie-aph (2'') was the most common AME gene and SCCmec type I was most predominant among the MRS isolates.


Keywords: Aminoglycoside modifying enzyme genes, aminoglycosides, staphylococcal cassette chromosome mec, staphylococci


How to cite this article:
Perumal N, Murugesan S, Krishnan P. Distribution of genes encoding aminoglycoside-modifying enzymes among clinical isolates of methicillin-resistant staphylococci. Indian J Med Microbiol 2016;34:350-2

How to cite this URL:
Perumal N, Murugesan S, Krishnan P. Distribution of genes encoding aminoglycoside-modifying enzymes among clinical isolates of methicillin-resistant staphylococci. Indian J Med Microbiol [serial online] 2016 [cited 2020 Feb 20];34:350-2. Available from: http://www.ijmm.org/text.asp?2016/34/3/350/188339



 ~ Introduction Top


The treatment of the staphylococci infections has become problematic because of the emergence of resistance to methicillin and other antibiotics. [1] Methicillin-resistant staphylococcal (MRS) strains have acquired and integrated into their genome the staphylococcal cassette chromosome mec (SCCmec), which carries the methicillin resistance (mecA) gene, and other antibiotic resistance determinants. [2]

Aminoglycosides are often used in combination with either beta-lactam or glycopeptides, especially for the treatment of complicated staphylococcal infections, as these drugs act synergistically. [3] The main mechanism of aminoglycoside resistance in staphylococci is attributed to drug inactivation caused by aminoglycoside-modifying enzymes (AMEs) encoded within mobile genetic elements. The most frequent AMEs are the bifunctional enzyme AAC (6')/APH (2') encoded by the gene aac (6')-Ie-aph (2')-Ia, APH (3')-III enzyme encoded by aph (3')-IIIa gene and ANT (4')-I enzyme encoded by ant (4')-Ia gene. [4]

Currently, there is no report on the distribution of genes encoding AMEs among clinical isolates of MRS in India. Thus, this study was aimed to detect the diversity of SCCmec elements and AME genes among clinical isolates of MRS.


 ~ Materials and Methods Top


In this study, clinical isolates of MRS (n = 164) collected from two different tertiary care hospitals were included. Clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin resistant coagulase negative staphylococci (MR-CoNS) were obtained from various clinical specimens of hospitalised patients. Antibiotic susceptibility testing was done for aminoglycoside antibiotics by Kirby-Bauer disc diffusion method and interpreted according to the Clinical and Laboratory Standards Institute guidelines. [5] Bacterial DNA was obtained by the boiling lysis method. DNA concentration and quality were determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc.). Multiplex polymerase chain reaction (M-PCR) was performed for the simultaneous detection and differentiation of MRSA from MR-CoNS. [6] S. aureus ATCC 43300 and S. epidermidis RP62A were used as positive control strains. Speciation of CoNS isolates was done by the standard biochemical tests. [7] The diversity of SCCmec types (types I-V) was determined using M-PCR by detecting the target sequences of ccrA2-B (937 bp), ccrC (518 bp), IS1272 (415 bp) and mecA-IS431 (359 bp) elements. Amplification was done using Eppendorf Mastercycler Gradient with the following cycling conditions: Initial denaturation: 94°C for 5 min, 30 cycles of denaturation 94°C for 1 min; annealing 54°C for 30 s; extension 72°C for 1 min and final extension of 72°C for 7 min. [8] The presence of aac (6')-Ie-aph (2''), aph (3')-IIIa and ant (4')-Ia genes responsible for aminoglycoside resistance was detected by M-PCR. [9]


 ~ Results Top


Out of the 164 isolates, 74 (45%) were found to be MRSA and the remaining 90 (55%) were MR-CoNS [Table 1]. Among the aminoglycoside antibiotics tested, highest resistance was seen for gentamicin (n = 113, 69%) followed by kanamycin (n = 104, 63%), tobramycin (n = 97, 59%), amikacin (n = 86, 52%) and netilmicin (n = 61, 37%). SCCmec typing revealed type I (n = 56, 34%) to be predominant followed by type V (n = 39, 24%), type III (n = 24, 16%), type IV (n = 24, 13%), type I and V (n = 4, 2%) and type II (n = 2, 1%). 15 (9%) isolates were nontypeable (NT) [Table 2]. Among the AME genes tested, the most prevalent-resistance gene was aac (6')-Ie-aph (2'') detected singly in 59 isolates (36%) and combination with aph (3)-IIIa in 37 (22%) and ant (4)-Ia in 3 (2%) isolates. The aph (3)-IIIa was detected in 28 (31%) isolates and the ant (4)-Ia in four (4%) isolates. The aph (3)-IIIa was detected in 53 (32%) isolates and the ant (4)-Ia in 15 (9%) isolates. AAC (6')-APH (2'') displaying AAC (6') and APH (2'') activity encoded by the aac (6')-Ie-aph (2'') gene was most commonly observed in tobramycin-resistant phenotypes, while APH (3')-III encoding aph (3')-IIIa gene in kanamycin resistant phenotypes and ANT (4')-I encoding ant (4')-Ia gene in gentamicin, amikacin, tobramycin and netilmicin-resistant phenotypes.
Table 1: Species distribution of methicillin - resistant coagulase-negative staphylococci in this study


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Table 2: Diversity of staphylococcal cassette chromosome mec types and distribution of aminoglycoside modifying enzymes genes among study isolates


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 ~ Discussion Top


The study revealed the diversity of the SCCmec types along with the most clinically important aminoglycoside resistance genes among MRS isolates. Aminoglycosides play an important role in serious staphylococcal infections despite reports of increased resistance to these drugs. Drug inactivation by AME is the main mechanism of aminoglycoside resistance.

The most prevalent AME gene in our methicillin-resistant isolates were aac (6)-Ie-aph (2'') (55.4%) followed by aph (3')-IIIa (32.3%). The ant (4')-Ia gene was the least frequent AME gene among MRS isolates (9%). These results were in disagreement with the previous studies around the globe where the most encountered gene was ant (4')-Ia.[9],[10],[11],[12] This discrepancy may be due to the usage of particular aminoglycoside antibiotics in their respective countries. The bifunctional enzyme, AAC (6')/APH (2''), encoded by the aac (6')-Ie-aph (2'') gene mediates resistance mainly to gentamicin.

Several reports have stated that aminoglycoside resistance is closely related to methicillin-resistance. [10],[13] This study also shows a significant correlation between aminoglycoside and methicillin-resistance. The SCCmec is a mobile genetic element widely distributed among methicillin-resistant strains of Staphylococci. In this study, five different SCCmec types (type I-V) and the combination of SCCmec types (I + V) were detected. Fifteen isolates were found to be NT. A strong association was observed between multidrug resistance and the presence of SCCmec type I and type III. The majority of the MRSA isolates belonged to SCCmec type V, while SCCmec type I in MR-CoNS and aac (6')-Ie-aph (2'') was the predominant gene in both MRSA or MR-CoNS. Coexistence of all the three AME genes was detected in 7.3% of isolates, all of which belonged to SCCmec type III.


 ~ Conclusion Top


In conclusion, this study shows the emergence of aminoglycoside resistance among clinical isolates of MRS and high prevalence of aac (6')-Ie-aph (2'') gene.

Financial support and sponsorship

Nil

Conflicts of interest

There are no conflicts of interest.

 
 ~ References Top

1.
Smith TL, Jarvis WR. Antimicrobial resistance in Staphylococcus aureus. Microbes Infect 1999;1:795-805.  Back to cited text no. 1
    
2.
International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC). Classification of staphylococcal cassette chromosome mec (SCCmec): Guidelines for reporting novel SCCmec elements. Antimicrob Agents Chemother 2009;53:4961-7.  Back to cited text no. 2
[PUBMED]    
3.
Davies JE. Aminoglycosides: Ancient and modern. J Antibiot (Tokyo) 2006;59:529-32.  Back to cited text no. 3
    
4.
Ramirez MS, Tolmasky ME. Aminoglycoside modifying enzymes. Drug Resist Updat 2010;13:151-71.  Back to cited text no. 4
    
5.
Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk Tests; Approved Standards. Doucement M2-A9. 9 th ed., Vol. 26. Wayne, PA: Clinical and Laboratory Standards Institute; 2006.  Back to cited text no. 5
    
6.
Abimanyu N, Krishnan A, Murugesan S, Subramanian GK, Gurumurthy S, Krishnan P. Use of triplex PCR for rapid detection of PVL and differentiation of MRSA from methicillin resistant coagulase negative staphylococci. J Clin Diagn Res 2013;7:215-8.  Back to cited text no. 6
    
7.
Koneman EW. Color Atlas and Textbook of Diagnostic Microbiology. Netherlands: Lippincott-Raven Publishers; 1997.  Back to cited text no. 7
    
8.
Boye K, Bartels MD, Andersen IS, Møller JA, Westh H. A new multiplex PCR for easy screening of methicillin-resistant Staphylococcus aureus SCCmec types I-V. Clin Microbiol Infect 2007;13:725-7.  Back to cited text no. 8
    
9.
Ida T, Okamoto R, Shimauchi C, Okubo T, Kuga A, Inoue M. Identification of aminoglycoside-modifying enzymes by susceptibility testing: Epidemiology of methicillin-resistant Staphylococcus aureus in Japan. J Clin Microbiol 2001;39:3115-21.  Back to cited text no. 9
    
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. 10
    
11.
Yadegar A, Sattari M, Mozafari NA, Goudarzi GR. Prevalence of the genes encoding aminoglycoside-modifying enzymes and methicillin resistance among clinical isolates of Staphylococcus aureus in Tehran, Iran. Microb Drug Resist 2009;15:109-13.  Back to cited text no. 11
    
12.
Choi SM, Kim SH, Kim HJ, Lee DG, Choi JH, Yoo JH, et al. Multiplex PCR for the detection of genes encoding aminoglycoside modifying enzymes and methicillin resistance among Staphylococcus species. J Korean Med Sci 2003;18:631-6.  Back to cited text no. 12
    
13.
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. 13
    



 
 
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