Increased copy number of 23S ribosomal RNA gene with point mutation in MRSA associated with linezolid resistance in a patient treated with long-term linezolid

Background


Background
Methicillin-resistant Staphylococcus aureus (MRSA) infections are one of the most di cult infections for us to treat.We usually treat MRSA infections with glycopeptide antibiotics such as vancomycin and teicoplanin, and oxazolidinone antibiotics such as linezolid.Among these, linezolid is one of the most promising therapeutic options in clinical use, and is effective against most Gram-positive bacteria, including MRSA and vancomycin-resistant enterococci.Linezolid binds to the domain V region of the 23S ribosomal RNA (rRNA) and exhibits antimicrobial activity by inhibiting protein synthesis in susceptible cells and by inactivating the function of the 50S ribosomal subunit [1,2].Linezolid was rst approved for clinical use in the United States in 2000.In 2001, shortly after the start of clinical use, the emergence of linezolid-resistant MRSA was reported in North America [3].
The most common mechanism of linezolid resistance is a single base substitution in chromosomal DNA encoding the domain V region of 23S rRNA.The most frequent mutation associated with linezolid resistance in clinical strains of S. aureus is the G2576T substitution (Escherichia coli 23S rRNA gene number) [3][4][5]7].In vitro, the frequncy of linezolid resistance was very low, proving to be less than 10 − 9 , and the emergence of linezolid-resistant bacteria was considered to be low.[7]. Tis mutation was con rmed not only in a single copy of the 23S rRNA gene, but also in multiple copies [8].Other mutations found in linezolid-resistant MRSA include a T2500A substitution in the domain V region of the rRNA gene [9].Besides mutations in the 23S rRNA gene, two other mechanisms of linezolid resistance have been reported.One is the expression of the chloramphenicol orfenicol resistance (cfr) gene, which encodes 23S rRNA methyltransferase [10], and the other is genetic mutation of the 50S ribosomal subunit proteins L3 and L4 (referred to as rplC and rplD, respectively) [11,12].The S. aureus chromosome encodes ve to six independent rRNA genes (rrn) or operons [13].When the G2576T mutation accumulates in different copies of the 23S rRNA gene in one cell, it can be imagined that the level of resistance to linezolid gradually increases.Indeed, in vitro studies showed that stepwise passaging of linezolid-sensitive cells into medium containing progressively higher concentrations of linezolid resulted in mutants with progressively higher minimum inhibitory concentrations (MICs) of linezolid [5].Analysis of such mutants showed that they accumulated the G2576T mutation in multiple copies of the 23S rRNA gene, and the number of mutations roughly correlated with the level of resistance [14].However, the evolution of resistance factors over time in a single strain is not clear.In the present study, we investigated MRSA isolated from a patient treated with linezolid for a long period of 75 days.
The isolates were susceptible to linezolid before the start of treatment, but became less susceptible by prolonged treatment.The evolution of resistance factors in these clinical isolates of MRSA was discussed.

Bacterial strains
A Japanese 65-year-old man with refractory pyothorax after lung cancer surgery was treated with linezolid for 75 days.A total of 16 MRSA strains isolated from sputum, airway secretions, pleural uid, and wound pus of the patient before and during the treatment with linezolid were analyzed.These 16 clinical isolates were named as KUB3961 to KUB3976.We also used the strain ATCC29213 as a control strain of S. aureus and the strain ATCC29212 as a control strain of E. faecalis.(Table 1) This patient had not been previously treated with Linezolid.

Antibiotic susceptibility test
We performed antibiotic susceptibility test of 16 clinical isolates for linezolid (LZD), oxacillin (MPIPC), vancomycin (VCM), teicoplanin (TEIC), daptomycin (DAP), rifampicin (RFP) and cefoxitin (CFX).The MIC of each antimicrobial agent was determined by the agar dilution method according to the protocols of the Clinical and Laboratory Standards Institute (CLSI) [15].We used S. aureus strain ATCC29213 and E. faecalis strain ATCC29212 as control strains for antibiotic susceptibility test as well.

23S rRNA genes sequencing analysis
The genomic DNA was extracted from 16 isolates of S. aureus by the QIAGEN DNeasy Blood & Tissue kits (Qiagen GmbH, Hilden, Germany), according to the manufactureer's instruction [18].Chromosomal genes encoding the 23S rRNA of ve independent operons were ampli ed by polymerase chain reaction (PCR) using the primers reported previously [8].Brie y, long-range PCR with product sizes of 5.6 to 6.5 kbp was performed to amplify each operon of 23S rRNA gene using primer sets (rrn1 to rrn5) [8].The puri ed 23S rRNA gene fragment was then used as a template to amplify the domain V region.Next, the PCR products were sequenced (Takara Bio, Mie, Japan or Nihon Gene Research Laboratories, Miyagi, Japan) and aligned with the corresponding nucleotide sequences obtained from linezolid-susceptible S. aureus strain NCTC8325 (GenBank accession no.X68425) [18]..

Pulsed-eld gel electrophoresis (PFGE)
Chromosomal DNA was extracted from 16 isolates of S. aureus and then digested with Sma I according to the method described by Bannerman et al. [16,17].PFGE was carried out with a CHEF DRIII electrophoresis cell (Bio-Rad) at 6 V/cm for 20 h at 14˚C, with initial and nal pulses conducted for 5.3 and 34.9 s, respectively.The gel was stained with GelRed (Biotim) according to the manufacturer's manual and visualized under a 254-nm ultraviolet light [18].

PFGE typing of 16 clinical isolates
To determine whether these linezolid-resistant MRSA strains were derived from different or similar clones, DNA typing by PFGE of Sma I restricted DNA was performed (Fig. 1).The results showed that the 16 clinical isolates were 100% identical, indicating a single clone.

23S rRNA domai V sequencing of 16 clinical isolates
We performed 23S rRNA domai V sequencing of linezolid-susceptible strain KUB3961 and linezolidresistant strains KUB3970, KUB3971, KUB3972, KUB3974 and KUB3976.The linezolid-susceptible strain KUB3961 (Linezolid MIC of 2 µg/mL) showed no mutations in the ribosomal operon at mutation sites G2447T, T2500A, T2571C, and G2576T, which are reported to be point mutations for linezolid resistance.On the other hand, three linezolid-resistant strains KUB3970, KUB3971 and KUB3972 with linezolid MIC of 8 µg/mL showed G2576T mutation in ribosomal operons 4 and 5. Two linezolid-resistant strains KUB3974 and KUB3976 with linezolid MIC of > 16 µg/mL showed G2576T mutation in ribosomal operons 1, 3, 4, and 5.These results indicated that the copy number of G2576T mutation in 23S rRNA gene of MRSA isolates increased with the use of linezolid.(Table.2)The above-mentioned gene mutation was not found in the 23S rRNA gene of E. faecalis wild-type strain ATCC2912.

Discussion
This is the rst report showing that MRSA isolated from one long-term linezolid-treated patient gradually became linezolid-resistant with an increase in the number of copies of the 23S rRNA point mutation.
Previous reports have suggested that the cause of linezolid resistance in Staphylococcus aureus is mainly a base substitution of G2576T in the domain V of 23S rRNA gene.In fact, Yurika Ikeda-Dantsuji et al. [18] and Yoshida et al. [19] reported that the clinical linezolid-resistance MRSA strains were found to have the G2576T mutation in the 23S rRNA gene domain V. S. aureus is well known to have ve to six copies of the 23S rRNA operon [8,13].Yurika Ikeda-Dantsuji et al. [18] reported that all clinical linezolid-resistant MRSA strains showed G2576T mutation in the 23S rRNA gene of at least one operon, which was found to be a possible cause of linezolid resistance.A previous in vitro study revealed that accumulation of G2576T mutation in different operons can lead to a stepwise increase in linezolid resistance levels [5], indicating that the number of mutant operons may correlate with the linezolid resistance levels of MRSA.
In this study, linezolid-resistant MRSA was isolated from pus 17 days after treatment with linezolid was started.Fifty-two days after the start of treatment, strains with increased linezolid-tolerance were isolated from pus.These linezolid-resistant strains had the same PFGE pattern as the linezolid-sensitive strains before the start of treatment, thus they were considered to be the same strains.In other words, a single strain may have become more linezolid-resistant by continued exposure to linezolid.These strains did not change to be resistant to antibiotics other than linezolid.The G2576T mutations in the 23S rRNA gene were found in all linezolid-resistant MRSA isolates.The strains with mutant operon numbers 2 and 4 showed MIC of 8 and > 16 µg/mL, respectively.T Therefore, it is suggested that the longer the exposure to linezolid in vivo, the higher the number of mutant operons of 23S rRNA of infected MRSA and the increased resistance of MRSA to linezolid.
We believe that there are three limitations of our study.First, we investigated only one patient in this study and could not con rm if the same phenomenon was always seen in any patient.Second, patients with linezolid-resistant isolates were treated for 75 days, but the relationship between the duration of linezolid treatment and the risk of G2576T mutation could not be known by this study alone.Third, linezolidresistant strains were isolated only from pus, and it was unclear why MRSA isolated from other sites did not show linezolid resistance.The resistant strains can only appear in the areas where the organisms are growing.
From this study, long-term use of linezolid carries the risk of developing linezolid-resistant MRSA.

Conclusion
Long-term use of linezolid in a patient or reuse of linezolid in a patient who has been previously treated with linezolid can lead to the emerging of linezolid-resistant MRSA in the host.Declarations

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