Daptomycin (DAP) is a new class of cyclic lipopeptide antibiotic highly active against methicillin-resistant Staphylococcus aureus (MRSA) infections. Proposed mechanisms involve disruption of the functional integrity of the bacterial membrane in a Cadependent manner. In the present work, we investigated the molecular basis of DAP resistance in a group of isogenic MRSA clinical strains obtained from patients with S. aureus infections after treatment with DAP. Different point mutations were found in the mprF gene in DAP-resistant (DR) strains. Investigation of the mprF L826F mutation in DR strains was accomplished by inactivation and transcomplementation of either full-length wild-type or mutated mprF in DAP-susceptible (DS) strains, revealing that they were mechanistically linked to the DR phenotype. However, our data suggested that mprF was not the only factor determining the resistance to DAP. Differential gene expression analysis showed upregulation of the two-component regulatory system vraSR. Inactivation of vraSR resulted in increased DAP susceptibility, while complementation of vraSR mutant strains restored DAP resistance to levels comparable to those observed in the corresponding DR wild-type strain. Electron microscopy analysis showed a thicker cell wall in DR CB5012 than DS CB5011, an effect that was related to the impact of vraSR and mprF mutations in the cell wall. Moreover, overexpression of vraSR in DS strains resulted in both increased resistance to DAP and decreased resistance to oxacillin, similar to the phenotype observed in DR strains. These results support the suggestion that, in addition to mutations in mprF, vraSR contributes to DAP resistance in the present group of clinical strains. Staphylococcus aureus is the most common Gram-positive pathogen among skin and soft tissue infections (2). Methicillin resistance in S. aureus is mediated by the acquisition of a penicillin-binding protein (PBP), PBP 2a, which has decreased affinity for ␤-lactam antibiotics but can continue to cross-link the cell wall once the native PBPs (i.e., PBPs 1 to 4) have been inactivated (23). A distinctive feature for most methicillin-resistant S. aureus (MRSA) strains is the heterogeneous expression of resistance, characterized by a small proportion (Յ0.1%) of the population expressing a high level of homogeneous resistance while most of the other isolates in the population express resistance to 10 g/ml (12, 15, 43). Daptomycin (DAP) is a cyclic anionic lipopeptide antibiotic that is produced by Streptomyces roseosporus (3) and is approved for treatment of skin and skin structure infections as well as treatment of bacteremia and right-side endocarditis caused by MRSA (1). The mechanism of action involves disruption of cytoplasmic membrane function, resulting in depolarization and cell death due to disruption of critical metabolic functions, such as protein, DNA, and RNA synthesis (2).The incidence of DAP resistance in clinical isolates is very low, and resistant strains display small increases in MIC (2). The exact m...

Methicillin-resistant

Study Design Non-randomized pilot trial Objectives Determine whether Escherichia coli 83972-coated urinary catheters in persons with spinal cord injury (SCI) practicing an intermittent catheterization program (ICP) could (1) achieve bladder colonization with this benign organism, and (2) decrease the rate of symptomatic urinary tract infection (UTI). Setting Outpatient SCI clinic in a Veterans Affairs hospital (USA) Methods Participants had neurogenic bladders secondary to SCI, were practicing ICP, had experienced at least 1 UTI, and had documented bacteruria within the past year. All subjects received a urinary catheter that had been pre-inoculated with E. coli 83972. The catheter was left in place for 3 days then removed. Subjects were followed with urine cultures and telephone calls weekly for 28 days and then monthly until E. coli 83972 was lost from the urine. Outcome measures were (1) the rate of successful bladder colonization, defined as the detection (≥102 cfu/ml) of E. coli 83972 in urine cultures for > 3 days after catheter removal and (2) the rate of symptomatic UTI while colonized with E. coli 83972. Results Thirteen subjects underwent 19 insertions of study catheters. Eight subjects (62%) became successfully colonized for > 3 days after catheter removal. In these 8 subjects, the rate of UTI while colonized was 0.77 per patient-year, in comparison to the rate of 2.27 UTI per patient-year prior to enrollment. Conclusions E. coli 83972-coated urinary catheters are a viable means to achieve bladder colonization with this potentially protective strain in persons practicing ICP.

The SOS response, a conserved regulatory network in bacteria that is induced in response to DNA damage, has been shown to be associated with the emergence of resistance to antibiotics. Previously, we demonstrated that heterogeneous (HeR) MRSA strains, when exposed to sub-inhibitory concentrations of oxacillin, were able to express a homogeneous high level of resistance (HoR). Moreover, we showed that oxacillin appeared to be the triggering factor of a β-lactam-mediated SOS response through lexA/recA regulators, responsible for an increased mutation rate and selection of a HoR derivative. In this work, we demonstrated, by selectively exposing to β-lactam and non-β-lactam cell wall inhibitors, that PBP1 plays a critical role in SOS-mediated recA activation and HeR-HoR selection. Functional analysis of PBP1 using an inducible PBP1-specific antisense construct showed that PBP1 depletion abolished both β-lactam-induced recA expression/activation and increased mutation rates during HeR/HoR selection. Furthermore, based on the observation that HeR/HoR selection is accompanied by compensatory increases in the expression of PBP1,-2, -2a, and -4, our study provides evidence that a combination of agents simultaneously targeting PBP1 and either PBP2 or PBP2a showed both in-vitro and in-vivo efficacy, thereby representing a therapeutic option for the treatment of highly resistant HoR-MRSA strains. The information gathered from these studies contributes to our understanding of β-lactam-mediated HeR/HoR selection and provides new insights, based on β-lactam synergistic combinations, that mitigate drug resistance for the treatment of MRSA infections.