Impaired alanine transport or exposure to D-cycloserine increases the susceptibility of MRSA to β-lactam antibiotics

Abstract

Running title: Re-sensitization of MRSA to b-lactams 23 33 DCS re-sensitised MRSA to β-lactams in vitro and significantly enhanced MRSA eradication by 34 oxacillin in a mouse bacteraemia model. These findings reveal alanine transport as a new 35 therapeutic target to enhance the susceptibility of MRSA to β-lactam antibiotics. 36 37 Abstract word count: 161 38 3 Manuscript word count: 3,494 39 40 58 resistance (HoR) [5-7]. Exposure of HeR isolates to b-lactam antibiotics induces expression of 59 mecA, which encodes the alternative penicillin binding protein 2a (PBP2a) and can select for 60 mutations in accessory genes resulting in a HoR phenotype, including mutations that affect 61 the stringent response and c-di-AMP signalling [8-12]. Because accessory genes can influence 62 the expression of methicillin resistance in MRSA, targeting the pathways associated with such 63 genes may identify new ways to increase the susceptibility of MRSA to β-lactams. To pursue 64 this, we performed a forward genetic screen to identify loci that impact the expression of 65 resistance to β-lactam antibiotics in MRSA. Using the Nebraska Transposon Mutant Library, 66 which comprises 1,952 sequence-defined transposon insertion mutants [13], inactivation of 67 a putative amino acid permease gene, cycA, was found to reduce resistance to cefoxitin, the 68 β-lactam drug recommended by the Clinical and Laboratory Standards Institute for measuring 69 mecA-mediated methicillin resistance in MRSA isolates. Amino acid transport and 70

Mutation of lipoprotein processing pathway genelspAor inhibition of LspA activity by globomycin increases MRSA resistance to β-lactam antibiotics

“…Mutants identified by this screen included NE869 (yjbH) (37), NE1909 (sagA) (38) and NE810 (cycA) (39), all of which have previously been implicated in β-lactam resistance. A new mutant identified in this screen was NE1757 (lspA::Tn), which exhibited increased resistance to cefoxitin (Fig 1A, B).…”

“…The Nebraska Transposon Mutant Library (NTML) (36) was screened to identify mutants exhibiting altered susceptibility to cefoxitin, which is recommended as a surrogate for measuring mecA- mediated oxacillin resistance in clinical laboratories, in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines for disk diffusion susceptibility assays. Mutants identified by this screen included NE869 (yjbH) (37), NE1909 (sagA) (38) and NE810 ( cycA )(39), all of which have previously been implicated in β-lactam resistance. A new mutant identified in this screen was NE1757 ( lspA ::Tn), which exhibited increased resistance to cefoxitin (Fig 1A, B).…”

“…Genetic manipulation of S. aureus. Phage 80α transduction was used to verify the association between antibiotic resistance phenotypes and transposon insertion-marked mutations from the NTML as described previously (39). Transductants were verified by PCR amplification of the target locus using primers listed in Table S3.…”

“…Peptidoglycan analysis. For each strain and growth condition tested, independent quadruplicate 50 ml cultures were grown to an OD600 of 0.5, harvested and resuspended in 5 ml PBS before peptidoglycan was extracted as described previously(39,62). Mass spectrometry was performed on a Waters XevoG2-XS QTof mass spectrometer.…”

Feline Otitis Externa Caused by Methicillin-Resistant Staphylococcus aureus with Mixed Hemolytic Phenotype and Overview of Possible Genetic Backgrounds

“…A single missense mutation differentiating the studied MRSA isolates was identified in cycA gene. The disruption of this gene has been shown to increase the susceptibility to β-lactams [ 32 ]; however, its role in hemolysis is currently unknown. None of the identified intergenic mutations were located in the agr locus.…”