Resistance to linezolid in enterococci and staphylococci referred to the national reference laboratory

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1 Resistance to linezolid in enterococci and staphylococci referred to the national reference laboratory Dr Danièle Meunier, AMRHAI BSAC - Antimicrobial Susceptibility Testing User Days

2 Oxazolidinone Linezolid released by US FDA in 2000 absence of inherent cross-resistance to other ATB classes and lack of rapid in vitro resistance development in UK: for the treatment of pneumonia and skin and skin structures infections active against MRSA, MSSA, VRE and high-level penicillin resistant S. pneumoniae Tedizolid licensed in Europe in 2015 in UK: for the treatment of acute (short-term) bacterial infections of the skin and of skin structures such as cellulitis, skin abscesses and wound infections. both linezolid and tedizolid are bacteriostatic against enterococci, staphylococci, and streptococci in vitro. 2 BSAC - Antimicrobial Susceptibility Testing User Days

3 3 BSAC - Antimicrobial Susceptibility Testing User Days

4 Exceptional resistance? 4 BSAC - Antimicrobial Susceptibility Testing User Days

5 Mechanism of action Resistance mechanisms inhibits the bacterial ribosomal translation process by binding to a site on the 23S ribosomal RNA of the 50S subunit chromosomal mutations in 23S ribosomal RNA of the 50S subunit (G2576T mutation) alterations in the L3 and L4 ribosomal proteins cfr gene (chloramphenicol, florfenicol) : methyltransferase(a2503) PhLOPSa Phenicols, Lincosamides, Oxazolidinones, Pleuromutilines, Streptogramins A optra gene (oxazolidinones, phenicols) : ATPbinding cassette transporter linezolid R, tedizolid R, chloramphenicol R, florfenicol R 5 BSAC - Antimicrobial Susceptibility Testing User Days

6 Mutations Among staphylococci and enterococci; G2576T (E. coli numbering), G2447T or T2504A in 23S rrna other mutations found in clinical isolates and in vitro mutations differ according to organisms not transmissible between species associated with previous exposure to linezolid mutations in domain V of 23S rrna and ribosomal proteins L3 and L4 due to prolonged exposure to linezolid From: Linezolid: a promising option in the treatment of Gram-positives J Antimicrob Chemother. 2016;72(2): BSAC - Antimicrobial Susceptibility Testing User Days

7 cfr (chloramphenicol/florfenicol resistance) initially identified in a bovine S. sciuri in the late 1990s cfr encodes for a methyl transferase catalysing methylation of the C8 atom of A2503 in the 23S rrna conferring resistance to Phenicols, Lincosamides, Oxazolidinones, Pleuromutilin, Streptogramin A initially on a 17kb multiresistance plasmid pscfs1, mostly associated with mobile elements such as insertion sequences and mobilization genes located on plasmids and transposons 2005 Colombian MRSA isolate CM05: first report of clinical cfr positive strain described in enterococci, staphylococci, in Bacillus, Macrococcus, Jeotgalicoccus, in Proteus vulgaris from a porcine nasal swab and in one Escherichia coli isolated from the nasal swab of a pig at slaughterhouse cfr gene reported worldwide : U.S., U.K., China, Germany, Denmark, Italy, Mexico and Ireland 7 BSAC - Antimicrobial Susceptibility Testing User Days

8 cfr (chloramphenicol/florfenicol resistance) cfr(b) in E. faecium in U.S. and Germany also described in Peptoclostridium difficile all human origin cfr(c) in Campylobacter coli isolated from cattle cfr(c) transferrable under laboratory conditions from C. coli to C. jejuni cfr(c) shares 55% identity with cfr and cfr(b) cfr can be a silent gene 8 BSAC - Antimicrobial Susceptibility Testing User Days

9 optra (oxazolidinone and phenicols transferable resistance) identified in 2015 in E. faecium and E. faecalis isolated in human and animal origin encodes for an ATP-binding cassette (ABC) transporter conferring resistance to oxazolidinone and phenicols located on plasmid optra in China 2 years before linezolid in 2005 in enterococci, in methicillin resistant S. sciuri, in E. faecalis from poultry meat, in Streptococci suis optra reported in China, Italy, Columbia, UK 9 BSAC - Antimicrobial Susceptibility Testing User Days

10 Detection phenotypic methods linezolid: bacteriostatic so susceptibility patterns can be challenging to interpret MIC by broth microdilution: trailing can be observed reading the MIC at the first well where the trailing began without regard for pinpoint trailing, according to CLSI M07-A10 and M100-S26 document instructions for reading linezolid disk diffusion requires the use of transmitted light gradient strips requires that 90% inhibition needs to be considered 10 BSAC - Antimicrobial Susceptibility Testing User Days

11 Detection phenotypic methods 11 BSAC - Antimicrobial Susceptibility Testing User Days

12 Detection molecular methods Detection of G2576T mutation - sequencing of 23S RNA product - PCR-RFLP - amplification of 23S + digestion with NheI - RT-PCR allelic discrimination L1 L2 L3 L2 heterozygous L1 homozygous L3 wild-type 12 BSAC - Antimicrobial Susceptibility Testing User Days

13 Detection molecular methods Detection of transferable resistance to linezolid -multiplex PCR for detection of cfr and optra genes NCTC S. epidermidis cfr + G2576T positive NCTC E. faecium optra positive 13 BSAC - Antimicrobial Susceptibility Testing User Days

14 Epidemiology worldwide - US LEADER Surveillance program ( ): 6,741 Gram-positive pathogens from 60 U.S. sites In 2015: linezolid inhibits 99.9% S. aureus, 99.2% of CNS, 99.7% enterococci and 100% streptococci ( Streptococcus pneumoniae, viridans group and - haemolytic streptococci) Overall linezolid resistance rate 1% from 2011 to % (9/14) S. aureus with decreased susceptibility MIC 4 mg/l have cfr 21.9% (7/32) of linezolid-resistant staphylococci have cfr 2/4 E. faecalis harbour optra 16/16 E. faecium linezolid resistant have mutations in the 23S rrna (G2576T) Pfaller MA, Mendes RE, Streit JM, Hogan PA, Flamm RK. Antimicrob Agents Chemother BSAC - Antimicrobial Susceptibility Testing User Days