The clinical burden of infections caused by antimicrobial resistant (AMR) pathogens is a leading threat to public health. Maintaining the effectiveness of existing antimicrobial drugs or finding ways to reintroduce drugs to which resistance is widespread is an important part of efforts to address the AMR crisis.

mecA -dependent methicillin resistance in MRSA is subject to regulation by numerous accessory factors involved in cell wall biosynthesis, nucleotide signaling, and central metabolism. Here, we report that mutations in the TCA cycle gene, sucC , increased susceptibility to β-lactam antibiotics and was accompanied by significant accumulation of succinyl-CoA, which in turn perturbed lysine succinylation in the proteome.

Regulation of icaADBC-encoded polysaccharide intercellular adhesin (PIA)/poly-N-acetylglucosasmine (PNAG) production in staphylococci plays an important role in biofilm-associated medical-device-related infections. Here, we report that the AraCtype transcriptional regulator Rbf activates icaADBC operon transcription and PIA production in Staphylococcus epidermidis. Purified recombinant Rbf did not bind to the ica operon promoter region in electrophoretic mobility shift assays (EMSAs), indicating that Rbf regulates ica transcription indirectly. To identify the putative transcription factor(s) involved in Rbf-mediated icaADBC regulation, the ability of recombinant Rbf to interact with the promoter sequences of known icaADBC regulators was investigated. Recombinant Rbf bound to the sarR promoter and not the sarX, sarA, sarZ, spx, and srrA promoters. Reverse transcription (RT)-PCR demonstrated that Rbf acts as a repressor of sarR transcription. PIA expression and biofilm production were restored to wild-type levels in an rbf sarR double mutant grown in brain heart infusion (BHI) medium supplemented with NaCl, which is known to activate the ica locus, but not in BHI medium alone. RT-PCR further demonstrated that although Rbf does not bind the sarX promoter, it nevertheless exerted a negative effect on sarX expression. Apparently, direct downregulation of the SarR repressor by Rbf has a dominant effect over indirect repression of the SarX activator by Rbf in the control of S. epidermidis PIA production and biofilm formation. IMPORTANCEThe importance of Staphylococcus epidermidis as an opportunistic pathogen in hospital patients with implanted medical devices derives largely from its capacity to form biofilm. Expression of the icaADBC-encoded extracellular polysaccharide is the predominant biofilm mechanism in S. epidermidis clinical isolates and is tightly regulated. Here, we report that the transcriptional regulator Rbf promotes icaADBC expression by negatively regulating expression of sarR, which encodes an ica operon repressor. Furthermore, Rbf indirectly represses the ica operon activator, SarX. The data reveal complicated interplay between Rbf and two Sar family proteins in fine-tuning regulation of the biofilm phenotype and indicate that in the hierarchy of biofilm regulators, IcaR is dominant over the Rbf-SarR-SarX axis. Staphylococci are responsible for the majority of biofilm-mediated device-related infections (1). Biofilms assist in the evasion of the host immune response and offer increased resistance to antimicrobial drugs. Many hospital patients undergo procedures involving the insertion of foreign biomaterials, ranging from simple intravascular catheters to more sophisticated ventricular assist devices. Vulnerable hospital patients with underlying medical conditions are particularly susceptible to device-related infections frequently caused by antibiotic-resistant pathogens. Thus, devicerelated infections represent a serious clinical problem, which in turn underpins the importance of understandin...

Prolonging the clinical effectiveness of β-lactams, which remain first-line antibiotics for many infections, is an important part of efforts to address antimicrobial resistance. We report here that inactivation of the predicted d-cycloserine (DCS) transporter gene cycA resensitized methicillin-resistant Staphylococcus aureus (MRSA) to β-lactam antibiotics. The cycA mutation also resulted in hypersusceptibility to DCS, an alanine analogue antibiotic that inhibits alanine racemase and d-alanine ligase required for d-alanine incorporation into cell wall peptidoglycan. Alanine transport was impaired in the cycA mutant, and this correlated with increased susceptibility to oxacillin and DCS. The cycA mutation or exposure to DCS were both associated with the accumulation of muropeptides with tripeptide stems lacking the terminal d-ala-d-ala and reduced peptidoglycan cross-linking, prompting us to investigate synergism between β-lactams and DCS. DCS resensitized MRSA to β-lactams in vitro and significantly enhanced MRSA eradication by oxacillin in a mouse bacteremia model. These findings reveal alanine transport as a new therapeutic target to enhance the susceptibility of MRSA to β-lactam antibiotics.

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