Clinical and Microbiological Aspects of Linezolid Resistance Mediated by the cfr Gene Encoding a 23S rRNA Methyltransferase

ABSTRACT Abilities of amoxicillin-clavulanate, cefpodoxime, cefprozil, azithromycin, and clarithromycin to select resistant mutants of Haemophilus influenzae were tested by multistep and single-step methodologies. For multistep studies, 10 random strains were tested: 5 of these were β-lactamase positive. After 50 daily subcultures in amoxicillin-clavulanate, MICs did not increase more than fourfold. However, cefprozil MICs increased eightfold for one strain. Clarithromycin and azithromycin gave a >4-fold increase in 8 and 10 strains after 14 to 46 and 20 to 50 days, respectively. Mutants selected by clarithromycin and azithromycin were associated with mutations in 23S rRNA and ribosomal proteins L4 and L22. Three mutants selected by clarithromycin or azithromycin had alterations in ribosomal protein L4, while five had alterations in ribosomal protein L22. Two mutants selected by azithromycin had mutations in the gene encoding 23S rRNA: one at position 2058 and the other at position 2059 (Escherichia coli numbering), with replacement of A by G. One clone selected by clarithromycin became hypersusceptible to macrolides. In single-step studies azithromycin and clarithromycin had the highest mutation rates, while amoxicillin-clavulanate had the lowest. All resistant clones were identical to parents as observed by pulsed-field gel electrophoresis. The MICs of azithromycin for azithromycin-resistant clones were 16 to >128 μg/ml, and those of clarithromycin for clarithromycin-resistant clones were 32 to >128 μg/ml in multistep studies. For strains selected by azithromycin, the MICs of clarithromycin were high and vice versa. After 50 daily subcultures in the presence of drugs, MICs of amoxicillin-clavulanate and cefpodoxime against H. influenzae did not rise more than fourfold, in contrast to cefprozil, azithromycin, and clarithromycin, whose MICs rose to variable degrees.

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Recommendations To Address the Difficulties Encountered When Determining Linezolid Resistance from Whole-Genome Sequencing Data

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00613-18 ◽

2018 ◽

Vol 62 (8) ◽

Cited By ~ 2

Author(s):

Alicia G. Beukers ◽

Henrik Hasman ◽

Kristin Hegstad ◽

Sebastiaan J. van Hal

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

23S Rrna ◽

Whole Genome Sequencing Data ◽

Rrna Gene ◽

Whole Genome ◽

Sequencing Data ◽

Content Type ◽

Numbering System ◽

Linezolid Resistance

ABSTRACT Mutations associated with linezolid resistance within the V domain of 23S rRNA are annotated using an Escherichia coli numbering system. The 23S rRNA gene varies in length, nucleotide sequence, and copy number among bacterial species. Consequently, this numbering system is not intuitive and can lead to confusion when mutation sites are being located using whole-genome sequencing data. Using the mutation G2576T as an example, we demonstrate the difficulties associated with using the E. coli numbering system.

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Resistance to Linezolid Caused by Modifications at Its Binding Site on the Ribosome

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05702-11 ◽

2011 ◽

Vol 56 (2) ◽

pp. 603-612 ◽

Cited By ~ 192

Author(s):

Katherine S. Long ◽

Birte Vester

Keyword(s):

Binding Site ◽

Ribosomal Proteins ◽

Resistance Mechanism ◽

Ribosomal Subunit ◽

Resistance Mechanisms ◽

Drug Binding ◽

23S Rrna ◽

Detailed Knowledge ◽

Linezolid Resistance ◽

Almost All

ABSTRACTLinezolid is an oxazolidinone antibiotic in clinical use for the treatment of serious infections of resistant Gram-positive bacteria. It inhibits protein synthesis by binding to the peptidyl transferase center on the ribosome. Almost all known resistance mechanisms involve small alterations to the linezolid binding site, so this review will therefore focus on the various changes that can adversely affect drug binding and confer resistance. High-resolution structures of linezolid bound to the 50S ribosomal subunit show that it binds in a deep cleft that is surrounded by 23S rRNA nucleotides. Mutation of 23S rRNA has for some time been established as a linezolid resistance mechanism. Although ribosomal proteins L3 and L4 are located further away from the bound drug, mutations in specific regions of these proteins are increasingly being associated with linezolid resistance. However, very little evidence has been presented to confirm this. Furthermore, recent findings on the Cfr methyltransferase underscore the modification of 23S rRNA as a highly effective and transferable form of linezolid resistance. On a positive note, detailed knowledge of the linezolid binding site has facilitated the design of a new generation of oxazolidinones that show improved properties against the known resistance mechanisms.

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Changes in plastid biogenesis leading to the formation of albino regenerants in barley microspore culture

10.21203/rs.3.rs-42246/v1 ◽

2020 ◽

Author(s):

Monika Gajecka ◽

Marek Marzec ◽

Beata Chmielewska ◽

Janusz Jelonek ◽

Justyna Zbieszczyk ◽

...

Keyword(s):

Microspore Culture ◽

Doubled Haploids ◽

Microspore Embryogenesis ◽

Rrna Genes ◽

23S Rrna ◽

Gene Encoding ◽

Plastid Differentiation ◽

Barley Cultivars ◽

Albino Plants ◽

Genes Encoding

Abstract Background: Microspore embryogenesis is potentially the most effective method of obtaining doubled haploids (DH), which are utilized in breeding programs to accelerate production of new cultivars. However, the regeneration of albino plants significantly limits the exploitation of androgenesis for DH production in cereals. Despite many efforts, the precise mechanisms leading to development of albino regenerants have not yet been elucidated. The objective of this study was to reveal the genotype-dependent molecular differences in chloroplast differentiation that lead to the formation of green and albino regenerants in microspore culture of barley.Results: We performed a detailed analysis of plastid differentiation at successive stages of androgenesis in two barley cultivars, ‘Jersey’ and ‘Mercada’ that differed in their ability to produce green regenerants. We demonstrated the lack of transition from the NEP-dependent to PEP-dependent transcription in plastids of ‘Mercada’ that produced mostly albino regenerants in microspore culture. The failed NEP-to-PEP transition was associated with the lack of activity of Sig2 gene encoding a sigma factor necessary for transcription of plastid rRNA genes. The impaired PEP activity caused a very low level of 16S and 23S rRNA transcripts, lack of plastid translation machinery and inhibition of photomorphogenesis in regenerating embryos and albino regenerants. Furthermore, the plastids present in differentiating ‘Mercada’ embryos contained a low number of plastome copies whose replication was not always completed. Contrary to ‘Mercada’, ‘Jersey’ that produced 90% green regenerants, showed the high activity of PEP, the highly increased expression of Sig2, plastid rRNA and tRNAGlu transcripts, which indicated the NEP inhibition. The increased expression of GLKs genes encoding transcription factors required for induction of photomorphogenesis was also observed in ‘Jersey’ regenerants. Conclusions: Proplastids present in microspore-derived embryos of albino-producing genotypes did not pass the early checkpoint of their development that are required for induction of further light-dependent differentiation of chloroplasts. The failed activation of plastid-encoded RNA polymerase during differentiation of embryos was the main cause of the genotype-dependent inability to regenerate green plants in barley microspore culture. The better understanding of molecular mechanism underlying formation of albino regenerants may be helpful in overcoming the problem of albinism in cereal androgenesis.

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Changes in plastid biogenesis leading to the formation of albino regenerants in barley microspore culture

10.21203/rs.3.rs-42246/v3 ◽

2020 ◽

Author(s):

Monika Gajecka ◽

Marek Marzec ◽

Beata Chmielewska ◽

Janusz Jelonek ◽

Justyna Zbieszczyk ◽

...

Keyword(s):

Microspore Culture ◽

Doubled Haploids ◽

Microspore Embryogenesis ◽

Rrna Genes ◽

23S Rrna ◽

Gene Encoding ◽

Plastid Differentiation ◽

Barley Cultivars ◽

Albino Plants ◽

Genes Encoding

Abstract Background: Microspore embryogenesis is potentially the most effective method of obtaining doubled haploids (DH) which are utilized in breeding programs to accelerate production of new cultivars. However, the regeneration of albino plants significantly limits the exploitation of androgenesis for DH production in cereals. Despite many efforts, the precise mechanisms leading to development of albino regenerants have not yet been elucidated. The objective of this study was to reveal the genotype-dependent molecular differences in chloroplast differentiation that lead to the formation of green and albino regenerants in microspore culture of barley.Results: We performed a detailed analysis of plastid differentiation at successive stages of androgenesis in two barley cultivars, ‘Jersey’ and ‘Mercada’ that differed in their ability to produce green regenerants. We demonstrated the lack of transition from the NEP-dependent to PEP-dependent transcription in plastids of cv. ‘Mercada’ that produced mostly albino regenerants in microspore culture. The failed NEP-to-PEP transition was associated with the lack of activity of Sig2 gene encoding a sigma factor necessary for transcription of plastid rRNA genes. A very low level of 16S and 23S rRNA transcripts and impaired plastid translation machinery resulted in the inhibition of photomorphogenesis in regenerating embryos and albino regenerants. Furthermore, the plastids present in differentiating ‘Mercada’ embryos contained a low number of plastome copies whose replication was not always completed. Contrary to ‘Mercada’, cv. ‘Jersey’ that produced 90% green regenerants, showed the high activity of PEP polymerase, the highly increased expression of Sig2, plastid rRNAs and tRNAGlu, which indicated the NEP inhibition. The increased expression of GLKs genes encoding transcription factors required for induction of photomorphogenesis was also observed in ‘Jersey’ regenerants. Conclusions: Proplastids present in microspore-derived embryos of albino-producing genotypes did not pass the early checkpoint of their development that are required for induction of further light-dependent differentiation of chloroplasts. The failed activation of plastid-encoded RNA polymerase during differentiation of embryos was associated with the genotype-dependent inability to regenerate green plants in barley microspore culture. The better understanding of molecular mechanism underlying formation of albino regenerants may be helpful in overcoming the problem of albinism in cereal androgenesis.

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Identification of the rrmA Gene Encoding the 23S rRNA m1G745 Methyltransferase in Escherichia coli and Characterization of an m1G745-Deficient Mutant

Journal of Bacteriology ◽

10.1128/jb.180.2.359-365.1998 ◽

1998 ◽

Vol 180 (2) ◽

pp. 359-365 ◽

Cited By ~ 70

Author(s):

Claes Gustafsson ◽

Britt C. Persson

Keyword(s):

Escherichia Coli ◽

Polypeptide Chain ◽

Chain Elongation ◽

23S Rrna ◽

Drastic Reduction ◽

Gene Encoding ◽

E Coli ◽

Loosely Coupled ◽

Rich Media

ABSTRACT An Escherichia coli mutant lacking the modified nucleotide m1G in rRNA has previously been isolated (G. R. Björk and L. A. Isaksson, J. Mol. Biol. 51:83–100, 1970). In this study, we localize the position of the m1G to nucleotide 745 in 23S rRNA and characterize a mutant deficient in this modification. This mutant shows a 40% decreased growth rate in rich media, a drastic reduction in loosely coupled ribosomes, a 20% decreased polypeptide chain elongation rate, and increased resistance to the ribosome binding antibiotic viomycin. TherrmA gene encoding 23S rRNA m1G745 methyltransferase was mapped to bp 1904000 on the E. colichromosome and identified to be identical to the previously sequenced gene yebH.

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Inactivation of the Indigenous Methyltransferase RlmN in Staphylococcus aureus Increases Linezolid Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00183-11 ◽

2011 ◽

Vol 55 (6) ◽

pp. 2989-2991 ◽

Cited By ~ 26

Author(s):

Jacqueline M. LaMarre ◽

Benjamin P. Howden ◽

Alexander S. Mankin

Keyword(s):

Staphylococcus Aureus ◽

23S Rrna ◽

Potential Mechanism ◽

Content Type ◽

Clinical Strains ◽

Linezolid Resistance

ABSTRACTThe indigenous methyltransferase RlmN modifies A2503 in 23S rRNA. A recently describedrlmNmutation in a clinicalStaphylococcus aureusisolate decreases susceptibility to linezolid and was thought to increase the extent of A2503 modification. However, we show that the mutation in fact abolishes RlmN activity, resulting in a lack of A2503 modification. Since many mutations could inactivate therlmNgene, our findings unveil a potential mechanism for future linezolid resistance in clinical strains.

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Methylation of 23S rRNA Nucleotide G748 by RlmAIIMethyltransferase Renders Streptococcus pneumoniae Telithromycin Susceptible

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00164-13 ◽

2013 ◽

Vol 57 (8) ◽

pp. 3789-3796 ◽

Cited By ~ 11

Author(s):

Akiko Takaya ◽

Yoshiharu Sato ◽

Tatsuma Shoji ◽

Tomoko Yamamoto

Keyword(s):

Streptococcus Pneumoniae ◽

Stop Codon ◽

Regulatory Region ◽

Dimethyl Sulfate ◽

23S Rrna ◽

Gene Encoding ◽

Content Type ◽

Resistant Mutants ◽

High Level ◽

Domain V

ABSTRACTSeveral posttranscriptional modifications of bacterial rRNAs are important in determining antibiotic resistance or sensitivity. In all Gram-positive bacteria, dimethylation of nucleotide A2058, located in domain V of 23S rRNA, by the dimethyltransferase Erm(B) results in low susceptibility and resistance to telithromycin (TEL). However, this is insufficient to produce high-level resistance to TEL inStreptococcus pneumoniae. Inactivation of the methyltransferase RlmAII, which methylates the N-1 position of nucleotide G748, located in hairpin 35 of domain II of 23S rRNA, results in increased resistance to TEL inerm(B)-carryingS. pneumoniae. Sixteen TEL-resistant mutants (MICs, 16 to 32 μg/ml) were obtained from a clinically isolatedS. pneumoniaestrain showing low TEL susceptibility (MIC, 2 μg/ml), with mutation resulting in constitutive dimethylation of A2058 because of nucleotide differences in the regulatory region oferm(B) mRNA. Primer extension analysis showed that the degree of methylation at G748 in all TEL-resistant mutants was significantly reduced by a mutation in the gene encoding RlmAIIto create a stop codon or change an amino acid residue. Furthermore, RNA footprinting with dimethyl sulfate and a molecular modeling study suggested that methylation of G748 may contribute to the stable interaction of TEL with domain II of 23S rRNA, even after dimethylation of A2058 by Erm(B). This novel finding shows that methylation of G748 by RlmAIIrendersS. pneumoniaeTEL susceptible.

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Isolation and first draft genome sequence of a linezolid-dependent Staphylococcus aureus clinical strain

Future Microbiology ◽

10.2217/fmb-2020-0068 ◽

2020 ◽

Vol 15 (12) ◽

pp. 1123-1129

Author(s):

Víctor A. García-Angulo ◽

Beatrice Herve ◽

Joel Melo ◽

Camila Sanhueza ◽

Sebastián De la Fuente ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Genome Sequence ◽

Draft Genome ◽

Draft Genome Sequence ◽

Aureus Strain ◽

23S Rrna ◽

Clinical Strain ◽

Antibiotics Resistance ◽

Type I ◽

Linezolid Resistance

Background: Antibiotic-dependent pathogenic bacteria are sporadically isolated from patients that received prolonged antibiotic treatments. Evolution of antibiotics dependence and its clinical implications are scarcely studied. Materials & methods: A linezolid-dependent Staphylococcus aureus strain was isolated from a cystic fibrosis patient. A draft genome sequence was obtained and searched for known antibiotics resistance determinants and virulence factors. Results: The genome was assembled into 79 contigs for a total of 2.83 Mbp. This strain is a sequence type 5 methicillin-resistant Staphylococcus aureus with a type I SCC mec cassette also conserving the Panton–Valentine leukocidin. The G2576T substitution, conferring linezolid resistance, was harbored by all five copies of the 23S rRNA. Conclusion: The linezolid-dependent strain is related to a strain circulating in Latin America that acquired a mutation conferring linezolid resistance.

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