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Year : 2019  |  Volume : 149  |  Issue : 6  |  Page : 795-798

Linezolid-resistant Staphylococcus haemolyticus: Emergence of G2447U & C2534U mutations at the domain V of 23S ribosomal RNA gene in a tertiary care hospital in India

1 Department of Microbiology, All India Institute of Medical Sciences, New Delhi 110 029, India
2 Department of Anaesthesiology, All India Institute of Medical Sciences, New Delhi 110 029, India
3 Department of Biostatistics, All India Institute of Medical Sciences, New Delhi 110 029, India

Date of Submission08-Feb-2018
Date of Web Publication3-Sep-2019

Correspondence Address:
Benu Dhawan
Department of Microbiology, All India Institute of Medical Sciences, New Delhi 110 029
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmr.IJMR_283_18

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How to cite this article:
Kumari S, Rawre J, Trikha A, Sreenivas V, Sood S, Kapil A, Dhawan B. Linezolid-resistant Staphylococcus haemolyticus: Emergence of G2447U & C2534U mutations at the domain V of 23S ribosomal RNA gene in a tertiary care hospital in India. Indian J Med Res 2019;149:795-8

How to cite this URL:
Kumari S, Rawre J, Trikha A, Sreenivas V, Sood S, Kapil A, Dhawan B. Linezolid-resistant Staphylococcus haemolyticus: Emergence of G2447U & C2534U mutations at the domain V of 23S ribosomal RNA gene in a tertiary care hospital in India. Indian J Med Res [serial online] 2019 [cited 2021 Sep 23];149:795-8. Available from:


Linezolid (an oxazolidinone drug available in both parenteral and oral formulations) has emerged as a novel alternative to vancomycin and other second-generation drugs for the treatment of infections from Gram-positive cocci. The first clinical isolates of linezolid-resistant staphylococci and enterococci were reported in 2001[1]. Since then, linezolid-resistant strains have become an increasing problem worldwide. The most frequently reported mechanisms of linezolid resistance include the mutation in 23S ribosomal RNA (23S rRNA) and presence of cfr gene.

At our hospital, a tertiary care hospital in north India, methicillin-resistant coagulase-negative staphylococci (CoNS) and vancomycin-resistant enterococci have become a worrisome clinical problem[2],[3]. This situation brings about new challenges for the treatment of these infections and patient safety.

This study was aimed to determine the distribution of linezolid-resistant isolates in an inpatient setting of the All India Institute of Medical Sciences (AIIMS), New Delhi, India, and to evaluate the resistance mechanisms among these isolates. In addition, the clonal diversity of the isolates was determined by pulsed-field gel electrophoresis (PFGE). The study included linezolid resistance Staphylococcus haemolyticus (LR-SH) isolates [linezolid resistance screening was assessed by linezolid (30 μg) discs] recovered from pus specimens of patients with chronic osteomyelitis and pemphigus vulgaris hospitalized in the departments of Orthopaedics and Dermatology & Venereology of the AIIMS, New Delhi, respectively, from June 2015 to December 2016. The study was approved by the Institutional Ethics Committee.

Bacterial identification was performed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)[4]. Antimicrobial susceptibility testing was performed by disc diffusion method according to Clinical and Laboratory Standards Guidelines (2015)[5] and minimum inhibitory concentration (MICs) of linezolid, vancomycin and teicoplanin by E-test method (bioMérieux, USA).

Isolates were screened for the presence of cfr (chloramphenicol - florfenicol resistance) gene and mutations in the 23S rRNA gene by PCR and DNA sequencing as described previously[6],7. Amplicons were sequenced on both strands and were compared with S. aureus ATCC 29213 (bioMérieux).

The clonal relatedness of the LR-SH isolates was examined by PFGE of Sma-I-digested genomic DNA according to the protocol described by Goering and Winters[8], with some modifications. Genomic DNA was prepared in agarose blocks and digested with Sma I (Promega, USA). The DNA fragments were separated on one per cent agarose gel using CHEF Mapper System III (Bio-Rad, USA) for 20 h at 6 V/cm at 14°C, with a pulse angle of 120° and a ramped pulse time of 1-40 sec. S. aureus NCTC 8325 was used as a reference marker. Comparison and grouping of PFGE patterns were performed with InfoQuest FP Software v.5.4 (Bio-Rad).

A total of 13 LR-SH isolates were recovered from 16 pus specimens. The rate of linezolid resistance among S. haemolyticus isolates was 81.3 per cent. All patients had received multiple antibiotics before referral. Three patients had received linezolid, the duration of which varied from 10 days to two weeks. The characteristics of the patients and their isolates are presented in the [Table 1].
Table 1: Clinical characteristics of patients with linezolid-resistant Staphylococcus haemolyticus (n=13)

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MIC testing by E-test confirmed linezolid MIC of ≥256 μg/ml in all the isolates of S. haemolyticus including susceptibility to teicoplanin and vancomycin. All the isolates were cefoxitin resistant and showed similar multidrug-resistant phenotype, exhibiting uniform resistance to chloramphenicol, clindamycin, ciprofloxacin and rifampicin. However, variable susceptibility to erythromycin (84.6%) and amikacin (92.3%) was observed in all the isolates irrespective of prior linezolid exposure.

Sequencing results revealed G2576T mutations in eight, G2447U in four and C2534U in one isolate of S. haemolyticus. All three isolates of S. haemolyticus from patients with prior linezolid exposure showed G2447U mutation. One isolate of S. haemolyticus showed two simultaneous mutations (G2576T and G2447U) in the domain V region of 23S rRNA gene. Sequences were submitted to GenBank with accession numbers- KT277663, KT277664, KT277666, KT277667, KT277668, KT277669, KT277670, KT277671, KT277672, KT277673, KT277674, KT277665 and KU379673. All the 13 isolates carried the cfr gene.

Eleven clones (I-XI) were identified on PFGE [Figure 1]. Of these, clones I and II had two isolates each. Isolates of clone I exhibited identical band pattern with the previous isolates of LR-SH isolated from department of Orthopaedics. Similarly, isolates of clone II also shared same band pattern with the previous LR-SH isolates from department of Dermatology & Venereology of our centre[9].
Figure 1: Dendrogram based on the similarities using InfoQuest FP software v5.4 (Bio-Rad). Pulsed-field gel electrophoresis patterns of Sma-I macrorestriction fragments of linezolid-resistant S. haemolyticus isolates are shown.

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In a hospital setting, knowledge of clonal spread and resistance patterns of LR isolates are important in patient management and formulation of infection control measures. Linezolid resistance was observed only in S. haemolyticus. Neither LR- S. aureus nor LR- enterococci were found during this study. Worldwide, the incidence of LR-CoNS is 28 times that of LR- S. aureus[10]. All the isolates exhibited high-level resistance to linezolid. Our results were similar to previous studies from China where high-level resistance (MIC values ≥256 μg/ml) was described in most strains of LR-CoNS[11]. On the contrary, reports from other parts of the world demonstrated a predominance of low to medium level LR-CoNS with a complete absence of high-level LR-CoNS strains[12],[13]. The LR-SH isolates had the cfr-associated PhLOPS (phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin) pattern, thereby further reducing treatment options available.

Similar to our previous report[9], all the isolates demonstrated a dual mechanism of resistance with a mutation at domain V of 23S rRNA gene and presence of cfr gene. However, contrary to our previous findings[9], in addition to G2576T mutation, several previously described mutations including G2447U and C2534U were identified[9],[14]. The presence of mutations highlights excessive or inadequate exposure to linezolid, but their chromosomal location does not threaten rampant spread of such infections. In contrast to our previous report[9] where we had documented clonal dissemination, the present study documented the emergence of multiple clones of LR-SH. Linezolid resistance is known to be associated with prolonged linezolid treatment or inappropriate linezolid dosage. In our study, most of the patients had not received linezolid.

In conclusion, this study highlights the importance of continuous monitoring of linezolid resistance in staphylococci. Rationalizing the use of linezolid and implementing methods to control the spread of hospital clones are of paramount importance to prevent further dissemination of these strains.

Financial support & sponsorship: None.

Conflicts of Interest: None.

   References Top

Gonzales RD, Schreckenberger PC, Graham MB, Kelkar S, DenBesten K, Quinn JP. Infections due to vancomycin-resistant Enterococcus faecium resistant to linezolid. Lancet 2001; 357 : 1179.  Back to cited text no. 1
Ghosh A, Singh Y, Kapil A, Dhawan B. Staphylococcal cassette chromosome mec (SCCmec) typing of clinical isolates of coagulase-negative staphylocci (CoNS) from a tertiary care hospital in New Delhi, India. Indian J Med Res 2016; 143 : 365-70.  Back to cited text no. 2
Meena S, Mohapatra S, Sood S, Dhawan B, Das BK, Kapil A. Revisiting nitrofurantoin for vancomycin resistant enterococci. J Clin Diagn Res 2017; 11 : DC19-22.  Back to cited text no. 3
Dubois D, Grare M, Prere MF, Segonds C, Marty N, Oswald E. Performances of the Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry system for rapid identification of bacteria in routine clinical microbiology. J Clin Microbiol 2012; 50 : 2568-76.  Back to cited text no. 4
Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; 25th informational supplement. CLSI Document M100-S25. Wayne, PA: CLSI; 2015.  Back to cited text no. 5
Kehrenberg C, Schwarz S, Jacobsen L, Hansen LH, Vester B. A new mechanism for chloramphenicol, florfenicol and clindamycin resistance: Methylation of 23S ribosomal RNA at A2503. Mol Microbiol 2005; 57 : 1064-73.  Back to cited text no. 6
Bonora MG, Solbiati M, Stepan E, Zorzi A, Luzzani A, Catania MR, et al. Emergence of linezolid resistance in the vancomycin-resistant Enterococcus faecium multilocus sequence typing C1 epidemic lineage. J Clin Microbiol 2006; 44 : 1153-5.  Back to cited text no. 7
Goering RV, Winters MA. Rapid method for epidemiological evaluation of Gram-positive cocci by field inversion gel electrophoresis. J Clin Microbiol 1992; 30 : 577-80.  Back to cited text no. 8
Brijwal M, Dhawan B, Rawre J, Sebastian S, Kapil A. Clonal dissemination of linezolid-resistant Staphylococcus haemolyticus harbouring a G2576T mutation and the cfr gene in an Indian hospital. J Med Microbiol 2016; 65 : 698-700.  Back to cited text no. 9
Gu B, Kelesidis T, Tsiodras S, Hindler J, Humphries RM. The emerging problem of linezolid-resistant Staphylococcus. J Antimicrob Chemother 2013; 68 : 4-11.  Back to cited text no. 10
Yang XJ, Chen Y, Yang Q, Qu TT, Liu LL, Wang HP, et al. Emergence of cfr-harbouring coagulase-negative staphylococci among patients receiving linezolid therapy in two hospitals in China. J Med Microbiol 2013; 62 (Pt 6) : 845-50.  Back to cited text no. 11
Mendes RE, Hogan PA, Streit JM, Jones RN, Flamm RK. Zyvox® Annual Appraisal of Potency and Spectrum (ZAAPS) program: report of linezolid activity over 9 years (2004-12). J Antimicrob Chemother 2014; 69 : 1582-8.  Back to cited text no. 12
Flamm RK, Mendes RE, Ross JE, Sader HS, Jones RN. An international activity and spectrum analysis of linezolid: ZAAPS Program results for 2011. Diagn Microbiol Infect Dis 2013; 76 : 206-13.  Back to cited text no. 13
Tewhey R, Gu B, Kelesidis T, Charlton C, Bobenchik A, Hindler J, et al. Mechanisms of linezolid resistance among coagulase-negative staphylococci determined by whole-genome sequencing. mBio 2014; 5 : e00894-14.  Back to cited text no. 14


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