Distinction between the Cfr Methyltransferase Conferring Antibiotic Resistance and the Housekeeping RlmN Methyltransferase

Atkinson, Gemma C.; Hansen, Lykke Haastrup; Tenson, Tanel; Rasmussen, Anders; Kirpekar, Finn; Vester, Birte

doi:10.1128/aac.00448-13

Cited by 36 publications

(61 citation statements)

References 37 publications

(52 reference statements)

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“…Cfr belongs to the S-adenosylmethionine (SAM) enzyme superfamily of proteins, which are widely distributed in nature (11). These proteins have similarities to the Cfr(B) protein described here, and many sequences belonging to this class observed in several bacterial species (e.g., E. faecium, P. difficile, Bacillus amyloliquefaciens, and Bacillus methylotrophicus) have been deposited in GenBank as putative chloramphenicol-florphenicol-resistance proteins.…”

Section: Discussionmentioning

confidence: 92%

“…The methylation at C8 protects the Cfr-producing bacteria from the action of five major classes of antibiotics, namely, phenicols, oxazolidinones, pleuromutilins, macrolides, and streptogramin A compounds (PhLOPS A phenotype) (9). Furthermore, it has been proposed that the cfr gene evolved from the naturally occurring rlmN methyltransferase (10), which is widely distributed in several organisms (11).…”

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confidence: 99%

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Detection of a New cfr -Like Gene, cfr (B), in Enterococcus faecium Isolates Recovered from Human Specimens in the United States as Part of the SENTRY Antimicrobial Surveillance Program

Deshpande

Ashcraft

Kahn

et al. 2015

Antimicrob Agents Chemother

125

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Two linezolid-resistant Enterococcus faecium isolates (MICs, 8 g/ml) from unique patients of a medical center in New Orleans were included in this study. Isolates were initially investigated for the presence of mutations in the V domain of 23S rRNA genes and L3, L4, and L22 ribosomal proteins, as well as cfr. Isolates were subjected to pulsed-field gel electrophoresis (just one band difference), and one representative strain was submitted to whole-genome sequencing. Gene location was also determined by hybridization, and cfr genes were cloned and expressed in a Staphylococcus aureus background. The two isolates had one out of six 23S rRNA alleles mutated (G2576T), had wild-type L3, L4, and L22 sequences, and were positive for a cfr-like gene. The sequence of the protein encoded by the cfr-like gene was most similar (99.7%) to that found in Peptoclostridium difficile, which shared only 74.9% amino acid identity with the proteins encoded by genes previously identified in staphylococci and non-faecium enterococci and was, therefore, denominated Cfr(B). When expressed in S. aureus, the protein conferred a resistance profile similar to that of Cfr. Two copies of cfr(B) were chromosomally located and embedded in a Tn6218 similar to the cfr-carrying transposon described in P. difficile. This study reports the first detection of cfr genes in E. faecium clinical isolates in the United States and characterization of a new cfr variant, cfr(B). cfr(B) has been observed in mobile genetic elements in E. faecium and P. difficile, suggesting potential for dissemination. However, further analysis is necessary to access the resistance levels conferred by cfr(B) when expressed in enterococci.

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Section: Discussionmentioning

confidence: 92%

mentioning

confidence: 99%

Detection of a New cfr -Like Gene, cfr (B), in Enterococcus faecium Isolates Recovered from Human Specimens in the United States as Part of the SENTRY Antimicrobial Surveillance Program

Deshpande

Ashcraft

Kahn

et al. 2015

Antimicrob Agents Chemother

125

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“…These genes belong to the rlmN family and are also able to methylate position A2503. Only a few studies have been addressed to determine the role of RlmN in the development of antimicrobial resistance, showing a null (Atkinson et al, 2013) or very modest role, with a maximum two-fold decrease in MIC , in the basal levels of ribosome-targeting antimicrobial agents such as phenicols, pleuromutilins, lincosamides or others. This different result has been related to its final product, which is a 2-methyladenosine, while that produced by the action of Cfr is an 8-methyladenosine (Atkinson et al, 2013).…”

Section: A745mentioning

confidence: 99%

Macrolide resistance mechanisms inEnterobacteriaceae: Focus on azithromycin

Gomes

Martínez-Puchol

Palma

et al. 2016

Critical Reviews in Microbiology

116

120

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“…Sequences of the relatively similar RlmN enzymes are included, and some of the proteins are misannotated (Cfr-like proteins as RlmN and vice versa). We previously investigated sequence differences to distinguish between Cfr-like proteins and RlmN (10). Unfortunately, there is not yet enough information to tell which Cfr-like proteins function as Cfr (making m 8 A2503 and causing resistance) and which do not.…”

mentioning

confidence: 99%

A cfr -Like Gene from Clostridium difficile Confers Multiple Antibiotic Resistance by the Same Mechanism as the cfr Gene

Hansen

Vester

2015

Antimicrob Agents Chemother

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The Cfr RNA methyltransferase causes multiple resistances to peptidyl transferase inhibitors by methylation of A2503 23S rRNA. Many cfr-like gene sequences in the databases code for unknown functions. This study confirms that a Cfr-like protein from a Peptoclostridium difficile (formerly Clostridium difficile) strain does function as a Cfr protein. The enzyme is expressed in Escherichia coli and shows elevated MICs for five classes of antibiotics. A primer extension stop indicates a modification at A2503 in 23S rRNA. The cfr gene was initially found on a plasmid in coagulasenegative staphylococci of animal origin (1). A very recent paper titled "Clostridium difficile isolates with high linezolid MICs harbor the multiresistance gene cfr" presented MIC data on linezolid susceptibility that indicate the presence of a cfr-like gene that may provide antibiotic resistance by the same mechanism as Cfr (2). As pointed out by Schwarz and Wang (3), such a statement requires proof that the gene actually expresses an enzyme with that function. We therefore introduced a gene coding for the same enzyme, herein named clcD, into a plasmid and transformed the plasmid into Escherichia coli to analyze for a Cfr-like function. The gene sequence was optimized to E. coli codon usage, synthesized, and inserted as described in reference 4. After transformation to E. coli AS19 (5) and E. coli JW2505-1 (⌬RlmN) (6), MICs and modification at A2503 23S rRNA were investigated as previously presented in a similar study with cfr-like genes from Bacillales (4).The MICs in Table 1 were determined as described in reference 4 and showed that IPTG (isopropyl-␤-D-thiogalactopyranoside) induction of expression of the clcD gene can provide resistance to representatives of five of the six antibiotic groups previously shown to be affected by Cfr (7,8). The resistance was not as strong as the one seen with Cfr in this setup, which was similar to that with the original cfr gene in pBglII (4), but the resistance was comparable to some of those seen from Bacillales Cfr-like proteins (4). To further establish the function of ClcD as similar to that of Cfr, which causes 8-methyladenosine (m 8 A) methylation at A2503 in 23S rRNA (9), we examined the A2503 position by primer extension. The pClcD plasmid was introduced into E. coli JW2505-1 (⌬RlmN) to remove interference from the weak primer extension stop known to result from RlmN m 2 A2503 methylation (9). After induction, total RNA was isolated and hybridized with a primer specific for a region downstream of A2503, followed by extension by reverse transcriptase and gel electrophoresis, all performed as described previously (4). An m 8 A modification will

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Distinction between the Cfr Methyltransferase Conferring Antibiotic Resistance and the Housekeeping RlmN Methyltransferase

Cited by 36 publications

References 37 publications

Detection of a New cfr -Like Gene, cfr (B), in Enterococcus faecium Isolates Recovered from Human Specimens in the United States as Part of the SENTRY Antimicrobial Surveillance Program

Detection of a New cfr -Like Gene, cfr (B), in Enterococcus faecium Isolates Recovered from Human Specimens in the United States as Part of the SENTRY Antimicrobial Surveillance Program

Macrolide resistance mechanisms inEnterobacteriaceae: Focus on azithromycin

A cfr -Like Gene from Clostridium difficile Confers Multiple Antibiotic Resistance by the Same Mechanism as the cfr Gene

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