Oxacillin sensitization of methicillin-resistant Staphylococcus aureus and methicillin-resistant Staphylococcus pseudintermedius by antisense peptide nucleic acids in vitro.

Shan Goh,David H Lloyd,Liam Good,Anette Loeffler,Sean P Nair

doi:10.1186/s12866-015-0599-x

Abstract

BackgroundAntibiotic 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).ResultsAntisense 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0599-x) contains supplementary material, which is available to authorized users.

Highlights

Antibiotic resistance genes can be targeted by antisense agents, which can reduce their expression and restore cellular susceptibility to existing antibiotics
As we were interested in the major UK epidemic strain NCTC 13142 (EMRSA-15, ST22, SCCmecIV) [23, 24], and the first genetically well-characterized clinical methicillin-resistant Staphylococcus pseudintermedius (MRSP) strain, HH-1 (ST71, SCCmecII-III) [19], we based our antisense designs on sequence alignments on these strains
Initial screening at low and sub-optimal concentrations of the four antimecA peptide nucleic acids (PNAs) showed that A73 was the most effective and sensitized NCTC 13142 and HH-1 broth cultures to oxacillin by 4–8 fold at 2.5–5 μM (Additional file 1: Table S2)

Summary

Introduction

Antibiotic resistance genes can be targeted by antisense agents, which can reduce their expression and restore cellular susceptibility to existing antibiotics. Given limited success in the development of new antimicrobial drugs, there is great interest in targeting resistance genes to re-establish susceptibility to existing antibiotics [3, 4]. The predominant staphylococcal species involved with disease would typically be susceptible to most classes of antimicrobial agents, e.g. S_aureus in humans and S. pseudintermedius in pets Their methicillinresistant counterparts, MRSA and MRSP, tend to be resistant to most clinically relevant antimicrobial drugs and both species have zoonotic potential [9, 10]. It may be possible to develop a common strategy for inhibiting mecA expression in these pathogens and re-sensitize staphylococci

Objectives

Methods

Results

Discussion

Conclusion