Targeting of PBP1 by ß-lactams determines recA/SOS response activation in heterogeneous MRSA clinical strains.

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 ß-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 ß-lactam and non-ß-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 ß-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 ß-lactam-mediated HeR/HoR selection and provides new insights, based on ß-lactam synergistic combinations, that mitigate drug resistance for the treatment of MRSA infections.

ß-Lactams increase the antibacterial activity of daptomycin against clinical methicillin-resistant Staphylococcus aureus strains and prevent selection of daptomycin-resistant derivatives.

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged to be one of the most important pathogens both in health care and in community-onset infections. Daptomycin (DAP) is a cyclic anionic lipopeptide recommended for treatment of skin infections, bacteremia, and right-sided endocarditis caused by MRSA. Resistance to DAP (DAP(r)) has been reported in MRSA and is mostly accompanied by a parallel decrease in oxacillin resistance, a process known as the "seesaw effect." Our study provides evidence that the seesaw effect applies to other ß-lactams and carbapenems of clinical use, including nafcillin (NAF), cefotaxime (CTX), amoxicillin-clavulanic (AMC), and imipenem (IMP), in heterogeneous DAP(r) MRSA strains but not in MRSA strains expressing homogeneous ß-lactam resistance. The antibacterial efficacy of DAP in combination with ß-lactams was evaluated in isogenic DAP-susceptible (DAP(s))/Dap(r) MRSA strains originally obtained from patients that failed DAP monotherapy. Both in vitro (MIC, synergy-kill curve) and in vivo (wax worm model) approaches were used. In these models, DAP and a ß-lactam proved to be highly synergistic against both heterogeneous and homogeneous clinical DAP(r) MRSA strains. Mechanistically, ß-lactams induced a reduction in the cell net positive surface charge, reverting the increased repulsion provoked by DAP alone, an effect that may favor the binding of DAP to the cell surface. The ease of in vitro mutant selection was observed when DAP(s) MRSA strains were exposed to DAP. Importantly, the combination of DAP and a ß-lactam prevented the selection of DAP(r) variants. In summary, our data show that the DAP-ß-lactam combination may significantly enhance both the in vitro and in vivo efficacy of anti-MRSA therapeutic options against DAP(r) MRSA infections and represent an option in preventing DAP(r) selection in persistent or refractory MRSA infections.

Characteristics of biofilm on tunneled cuffed hemodialysis catheters in the presence and absence of clinical infection.

BACKGROUND: Biofilm occurring on the surface of tunneled hemodialysis (HD) catheters is difficult to eradicate and often is associated with recurrent bacteremia. We studied biofilm formation on catheters from patients with and without bacteremia to identify the location of bacterial growth and measure biofilm thickness. STUDY DESIGN: Prospective observational study. SETTING & PARTICIPANTS: 76 adult HD patients; 26 had HD catheters removed for bacteremia and 50 had catheters removed for reasons other than infection. PREDICTORS: Segment of catheter, reason for catheter removal. OUTCOMES & MEASUREMENTS: Microbiological growth and biofilm thickness on the outer and luminal surfaces of extravascular and intravascular catheter segments. RESULTS: Catheter cultures were positive in 16 (62%) patients with bacteremia and 15 (30%) when the catheter was removed for non-infection-related reasons. In catheters with positive cultures, the outer surface of the extravascular segment was the most common site of bacterial growth (15/16 [94%] and 11/15 [73%] for bacteremic and nonbacteremic patients, respectively). Bacteremic patients had significantly thicker biofilm on all catheter surfaces, and in bacteremic patients, the biofilm was significantly thicker on the outer compared with the luminal surface for both extravascular (14.53 ± 6.17 vs 11.97 ± 5.01 µm; P < 0.001) and intravascular (12.21 ± 5.3 vs 9.46 ± 3.71 µm; P < 0.001) segments. Extravascular segments had significantly thicker biofilm compared with intravascular segments on both the outer (P < 0.001) and luminal (P < 0.001) surfaces. Similarly, in patients for whom the catheter was removed for non-infection-related reasons, the catheter had thicker biofilm on the outer compared with the inner surface in both extravascular (2.19 ± 2.84 vs 1.62 ± 2.33 µm; P < 0.001) and intravascular (1.92 ± 2.62 vs 1.29 ± 2.33 µm; P < 0.001) segments. Similar to catheters from bacteremic patients, the outer and luminal surfaces of the extravascular segments of the catheters had significantly thicker biofilm compared with their corresponding surfaces on the intravascular segments. LIMITATIONS: Observational study. CONCLUSIONS: The outer surface of the extravascular segment of tunneled dialysis catheters in both bacteremic and nonbacteremic HD patients has the thickest biofilm and highest microbiological yield, and biofilm is thicker in patients with bacteremia. This knowledge is important for designing preventive strategies and also in the management of patients with catheter infection.

VraSR two-component regulatory system contributes to mprF-mediated decreased susceptibility to daptomycin in in vivo-selected clinical strains of methicillin-resistant Staphylococcus aureus.

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 Ca-dependent 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.

Increased expression of type-1 fimbriae by nonpathogenic Escherichia coli 83972 results in an increased capacity for catheter adherence and bacterial interference.

BACKGROUND: In vitro, urinary catheter colonization by avirulent Escherichia coli 83972 impedes subsequent catheter colonization by a variety of uropathogenic organisms. However, E. coli 83972 shows a low efficacy of adherence to silicone urinary catheter material, possibly because the fim operon encoding adhesive type 1 fimbriae is incomplete. We hypothesized that improving the catheter adherence of E. coli 83972 would improve its bacterial interference properties. METHODS: We created adhesive mutants by transforming wild-type E. coli 83972 with fim(+) plasmids. Adherence to urinary catheters and ability to prevent uropathogenic E. coli from colonizing urinary catheters were studied by use of a sonication assay. RESULTS: The addition of a single-copy fim(+) plasmid increased adherence to urinary catheters 10-fold, and addition of an 18-copy fim(+) plasmid increased adherence 100-fold. The more adherent 18-copy fim(+) plasmid strain was more effective at blocking catheter colonization by pathogenic E. coli than was the wild-type parental strain. Neither Deltafim nor fim(+) E. coli 83972 adhered to shed urinary epithelial cells. CONCLUSIONS: Our results indicate that improving urinary catheter adherence augments the bacterial interference capabilities of benign E. coli 83972. Increased expression of type-1 fimbriae may enhance bacterial interference without conferring virulence on E. coli 83972.