title: Oxacillin sensitization of methicillin-resistant Staphylococcus aureus and methicillin-resistant Staphylococcus pseudintermedius by antisense peptide nucleic acids in vitro.
abstract: BACKGROUND
  Antibiotic resistance genes can be targeted by antisense agents, which can reduce their expression and thus restore cellular susceptibility to existing antibiotics. Antisense inhibitors can be gene and pathogen specific, or designed to inhibit a group of bacteria having conserved sequences within resistance genes. Here, we aimed to develop antisense peptide nucleic acids (PNAs) that could be used to effectively restore susceptibility to β-lactams in methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP).
  
  
  RESULTS
  Antisense PNAs specific for conserved regions of the mobilisable gene mecA, and the growth essential gene, ftsZ, were designed. Clinical MRSA and MRSP strains of high oxacillin resistance were treated with PNAs and assayed for reduction in colony forming units on oxacillin plates, reduction in target gene mRNA levels, and cell size. Anti-mecA PNA at 7.5 and 2.5 μM reduced mecA mRNA in MRSA and MRSP (p < 0.05). At these PNA concentrations, 66 % of MRSA and 92 % of MRSP cells were killed by oxacillin (p < 0.01). Anti-ftsZ PNA at 7.5 and 2.5 μM reduced ftsZ mRNA in MRSA and MRSP, respectively (p ≤ 0.05). At these PNA concentrations, 86 % of MRSA cells and 95 % of MRSP cells were killed by oxacillin (p < 0.05). Anti-ftsZ PNAs resulted in swelling of bacterial cells. Scrambled PNA controls did not affect MRSA but sensitized MRSP moderately to oxacillin without affecting mRNA levels.
  
  
  CONCLUSIONS
  The antisense PNAs effects observed provide in vitro proof of concept that this approach can be used to reverse β-lactam resistance in staphylococci. Further studies are warranted as clinical treatment alternatives are needed.
authors.affiliation: Pathology and Pathogen Biology, Royal Veterinary College, Hawkshead Lane, Hatfield, Hertfordshire, UK. sgoh@rvc.ac.uk.
authors.forename: Shan
authors.initials: S
authors.lastname: Goh
authors.affiliation: Clinical Sciences and Services, Royal Veterinary College, Hawkshead Lane, Hatfield, Hertfordshire, UK.
authors.forename: Anette
authors.initials: A
authors.lastname: Loeffler
authors.affiliation: Clinical Sciences and Services, Royal Veterinary College, Hawkshead Lane, Hatfield, Hertfordshire, UK.
authors.forename: David H
authors.initials: DH
authors.lastname: Lloyd
authors.affiliation: Department of Microbial Diseases, UCL Eastman Dental Institute, University College London, London, UK.
authors.forename: Sean P
authors.initials: SP
authors.lastname: Nair
authors.affiliation: Pathology and Pathogen Biology, Royal Veterinary College, Royal College Street, London, UK.
authors.forename: Liam
authors.initials: L
authors.lastname: Good
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D003598:Cytoskeletal Proteins / Q000037:antagonists & inhibitors*; D004357:Drug Synergism; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D066298:In Vitro Techniques; D016106:Methicillin Resistance / Q000187:drug effects*; D055624:Methicillin-Resistant Staphylococcus aureus / Q000187:drug effects / Q000235:genetics; D008826:Microbial Sensitivity Tests; D010068:Oxacillin / Q000494:pharmacology; D046915:Penicillin-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D020135:Peptide Nucleic Acids / Q000494:pharmacology*; D012329:RNA, Bacterial / Q000187:drug effects; D012333:RNA, Messenger / Q000187:drug effects; D013210:Staphylococcus / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D003598:Cytoskeletal Proteins; C071525:FtsZ protein, Bacteria; C548250:MecA protein, Staphylococcus pseudintermedius; D046915:Penicillin-Binding Proteins; D020135:Peptide Nucleic Acids; D012329:RNA, Bacterial; D012333:RNA, Messenger; C487789:mecA protein, Staphylococcus aureus; D010068:Oxacillin