Impact of sarA on Antibiotic Susceptibility of Staphylococcus aureus in a Catheter-Associated In Vitro Model of Biofilm Formation

Mutation of the staphylococcal accessory regulator (sarA) in Staphylococcus aureus limits but does not abolish the capacity of the organism to form a biofilm. As a first step toward determining whether this limitation is therapeutically relevant, we carried out in vitro studies comparing the relative susceptibility of an S. aureus clinical isolate (UAMS-1) and its isogenic sarA mutant (UAMS-929) in the specific context of a catheter-associated biofilm. The antibiotics tested were daptomycin, linezolid, and vancomycin, all of which were evaluated by using concentrations based on the MIC defined as the breakpoint for a susceptible strain of S. aureus (≤1.0, ≤2.0, and ≤4.0 μg/ml for daptomycin, vancomycin, and linezolid, respectively). Mutation of sarA had no significant impact on the MIC of UAMS-1 for any of the targeted antibiotics, as defined by Etest antimicrobial susceptibility testing. However, mutation of sarA did result in a significant increase in antimicrobial susceptibility to all targeted antibiotics when they were tested in the specific context of a biofilm. Additionally, whether susceptibility was assessed by using UAMS-1 or its sarA mutant, daptomycin was found to be more effective against established S. aureus biofilms than either linezolid or vancomycin.Staphylococcus aureus is a devastating human pathogen, with the death toll from invasive S. aureus infections recently having passed that from AIDS in the United States (9, 18). These infections range from acute toxemias and septic shock to more chronic infections, including osteomyelitis and infections associated with indwelling medical devices. The latter are characterized by the formation of a biofilm, which has a significant impact not only on the disease process itself but also on the ability of clinicians to effectively treat the infection. This is true because biofilm-associated infections are recalcitrant to antimicrobial therapy, irrespective of the resistance status of the offending strain or the ability to achieve what would otherwise be therapeutic serum levels of antibiotic (10, 21, 30). For this reason, the resolution of biofilm-associated staphylococcal infections often requires surgical debridement to remove infected tissues and/or indwelling devices (5, 20).An alternative approach to the therapeutic problem of biofilm-associated infection would be the development of methods that specifically prevent or at least limit biofilm formation. This could be done by targeting either the substrate (e.g., by developing novel biomaterials that are less conducive to biofilm formation) or the bacterium (e.g., by developing novel agents capable of limiting biofilm formation). The latter approach requires the identification of those bacterial targets that are most relevant in the specific context of a biofilm. Studies focusing on the staphylococci have led to the identification of many potential targets (24). However, in almost every case, there are conflicting reports about the roles of different genes and gene products in biofilm formation (12, 24, 35).The single exception is the staphylococcal accessory regulator (sarA), the mutation of which has been shown to limit biofilm formation in both S. aureus and S. epidermidis (2, 7, 15, 25, 28, 31, 32, 33, 34, 37). This is true with respect to both in vitro and in vivo models of biofilm formation (3, 6). However, in no case has mutation of sarA eradicated biofilm formation. This suggests that the therapeutic relevance of any antibiofilm approach directed at sarA would be dependent on the ability to enhance conventional antimicrobial therapy. The studies reported here were aimed at addressing this possibility by determining whether the reduced capacity of an S. aureus sarA mutant to form a biofilm can be correlated with increased susceptibility to specific antibiotics in the context of an established, catheter-associated biofilm.     

In Vivo Efficacy of Daptomycin against Methicillin-Resistant Staphylococcus aureus in a Mouse Model of Hematogenous Pulmonary Infection

Daptomycin is inactivated by pulmonary surfactant, but its effectiveness in hematogenous pulmonary infection has been poorly studied. The potential therapeutic application was evaluated in a methicillin-resistant Staphylococcus aureus (MRSA) hematogenous pulmonary infection mouse model. Compared with control results, daptomycin improved survival (P < 0.001) and decreased the number of abscesses and bacteria in the lungs (P < 0.01). Daptomycin may be an effective therapeutic option for MRSA hematogenous pulmonary infection.     

Correlation between Reduced Daptomycin Susceptibility and Vancomycin Resistance in Vancomycin-Intermediate Staphylococcus aureus

We present here findings of a strong positive correlation between reduced daptomycin susceptibility and vancomycin resistance in vancomycin-intermediate Staphylococcus aureus (VISA). This correlation is related to cell wall thickening, suggesting that, similar to the case with vancomycin resistance in VISA, the physical barrier of a thickened cell wall may contribute to daptomycin resistance in S. aureus.     

Impact of Glycopeptide Resistance in Staphylococcus aureus on the Dalbavancin In Vivo Pharmacodynamic Target

Dalbavancin is a novel lipoglycopeptide with activity against Staphylococcus aureus, including glycopeptide-resistant isolates. The in vivo investigation reported here tested the effects of this antibiotic against seven S. aureus isolates with higher MICs, including several vancomycin-intermediate strains. Results of 1-log kill and 2-log kill were achieved against seven and six of the isolates, respectively. The mean free-drug area under the concentration-time curve (fAUC)/MIC values for net stasis, 1-log kill, and 2-log kill were 27.1, 53.3, and 111.1, respectively.     

Staphylococcus aureus Metabolic Adaptations during the Transition from a Daptomycin Susceptibility Phenotype to a Daptomycin Nonsusceptibility Phenotype

Staphylococcus aureus is a major cause of nosocomial and community-acquired infections. The success of S. aureus as a pathogen is due in part to its many virulence determinants and resistance to antimicrobials. In particular, methicillin-resistant S. aureus has emerged as a major cause of infections and led to increased use of the antibiotics vancomycin and daptomycin, which has increased the isolation of vancomycin-intermediate S. aureus and daptomycin-nonsusceptible S. aureus strains. The most common mechanism by which S. aureus acquires intermediate resistance to antibiotics is by adapting its physiology and metabolism to permit growth in the presence of these antibiotics, a process known as adaptive resistance. To better understand the physiological and metabolic changes associated with adaptive resistance, six daptomycin-susceptible and -nonsusceptible isogenic strain pairs were examined for changes in growth, competitive fitness, and metabolic alterations. Interestingly, daptomycin nonsusceptibility coincides with a slightly delayed transition to the postexponential growth phase and alterations in metabolism. Specifically, daptomycin-nonsusceptible strains have decreased tricarboxylic acid cycle activity, which correlates with increased synthesis of pyrimidines and purines and increased carbon flow to pathways associated with wall teichoic acid and peptidoglycan biosynthesis. Importantly, these data provided an opportunity to alter the daptomycin nonsusceptibility phenotype by manipulating bacterial metabolism, a first step in developing compounds that target metabolic pathways that can be used in combination with daptomycin to reduce treatment failures.     

In Vivo Effect of Flucloxacillin in Experimental Endocarditis Caused by mecC-positive Staphylococcus aureus Showing Temperature-Dependent Susceptibility In Vitro

Methicillin-resistant Staphylococcus aureus (MRSA) carrying the mecC gene (mecC-MRSA) exhibited at 37°C MICs of oxacillin close to those of methicillin-susceptible S. aureus (MSSA). We investigated whether at this temperature, mecC-MRSA strains respond to flucloxacillin treatment like MSSA strains, using a rat model of endocarditis. Flucloxacillin (human-like kinetics of 2 g intravenously every 6 h) cured 80 to 100% of aortic vegetations infected with five different mecC-MRSA strains. These results suggest that mecC-MRSA infections may successfully respond to treatment with β-lactams.     

Activity of Gallidermin on Staphylococcus aureus and Staphylococcus epidermidis Biofilms

Due to their abilities to form strong biofilms, Staphylococcus aureus and Staphylococcus epidermidis are the most frequently isolated pathogens in persistent and chronic implant-associated infections. As biofilm-embedded bacteria are more resistant to antibiotics and the immune system, they are extremely difficult to treat. Therefore, biofilm-active antibiotics are a major challenge. Here we investigated the effect of the lantibiotic gallidermin on two representative biofilm-forming staphylococcal species. Gallidermin inhibits not only the growth of staphylococci in a dose-dependent manner but also efficiently prevents biofilm formation by both species. The effect on biofilm might be due to repression of biofilm-related targets, such as ica (intercellular adhesin) and atl (major autolysin). However, gallidermin''s killing activity on 24-h and 5-day-old biofilms was significantly decreased. A subpopulation of 0.1 to 1.0% of cells survived, comprising “persister” cells of an unknown genetic and physiological state. Like many other antibiotics, gallidermin showed only limited activity on cells within mature biofilms.     

Daptomycin treatment of Staphylococcus aureus experimental chronic osteomyelitis

BACKGROUND: Infection due to methicillin-resistant Staphylococcus aureus (MRSA) is increasingly common in nosocomial and community settings. Daptomycin is a cyclic lipopeptide anti-infective with activity against MRSA, approved for treatment of complicated skin and skin structure infections. Daptomycin may be useful in systemic or local treatment of chronic osteomyelitis. METHODS: We measured mechanical strength of daptomycin- and vancomycin-loaded polymethylmethacrylate (PMMA), assayed in vivo release of daptomycin and vancomycin from daptomycin- and vancomycin-loaded PMMA, respectively, and compared the efficacy of two systemic doses of daptomycin with that of vancomycin, each with or without the respective anti-infective loaded into PMMA, using a rat model of MRSA chronic osteomyelitis. RESULTS: Neither tensile nor compressive strength of PMMA was impacted by impregnation with these antimicrobials at a concentration of 7.5% by weight. The peak concentrations of daptomycin and vancomycin in rat tibial bone surrounding a 7.5% daptomycin- and vancomycin-loaded 3 mm PMMA bead were 178 and 49 mg/L, respectively. In the treatment of experimental osteomyelitis, rats assigned to no treatment, daptomycin 50 mg/kg subcutaneously twice daily, daptomycin 60 mg/kg subcutaneously twice daily, and vancomycin 50 mg/kg intraperitoneally twice daily had 6.4, 4.1, 4.0 and 4.5 median log10 cfu/g of bone at the end of 21 days of therapy. All systemic anti-infectives studied were more active than was no treatment. Daptomycin- or vancomycin-loaded PMMA did not, however, exhibit microbiological efficacy alone or adjunctively, as assessed 21 days after implantation. CONCLUSIONS: Daptomycin is released from PMMA in vivo at a rate similar to that of vancomycin. Systemic daptomycin is as active as vancomycin in a rat model of chronic MRSA experimental osteomyelitis.     

Impact of the Combination of Daptomycin and Trimethoprim-Sulfamethoxazole on Clinical Outcomes in Methicillin-Resistant Staphylococcus aureus Infections

Complicated Staphylococcus aureus infections, including bacteremia, are often associated with treatment failures, prolonged hospital stays, and the emergence of resistance to primary and even secondary therapies. Daptomycin is commonly used as salvage therapy after vancomycin failure for the treatment of methicillin-resistant S. aureus (MRSA) infections. Unfortunately, the emergence of daptomycin resistance, especially in deep-seated infections, has been reported, prompting the need for alternative or combination therapy. Numerous antibiotic combinations with daptomycin have been investigated clinically and in vitro. Of interest, the combination of daptomycin and trimethoprim-sulfamethoxazole (TMP-SMX) has proved to be rapidly bactericidal in vitro to strains that are both susceptible and nonsusceptible to daptomycin. However, to date, there is limited clinical evidence supporting the use of this combination. This was a multicenter, retrospective case series of patients treated with the combination of daptomycin and TMP-SMX for at least 72 h. The objective of this study was to describe the safety and effectiveness of this regimen in clinical practice. The most commonly stated reason that TMP-SMX was added to daptomycin was persistent bacteremia and/or progressive signs and symptoms of infection. After the initiation of combination therapy, the median time to clearance of bacteremia was 2.5 days. Microbiological eradication was demonstrated in 24 out of 28 patients, and in vitro synergy was demonstrated in 17 of the 17 recovered isolates. Further research with this combination is necessary to describe the optimal role and its impact on patient outcomes.     

A Combined Pharmacodynamic Quantitative and Qualitative Model Reveals the Potent Activity of Daptomycin and Delafloxacin against Staphylococcus aureus Biofilms

Biofilms are associated with persistence of Staphylococcus aureus infections and therapeutic failures. Our aim was to set up a pharmacodynamic model comparing antibiotic activities against biofilms and examining in parallel their effects on viability and biofilm mass. Biofilms of S. aureus ATCC 25923 (methicillin-sensitive S. aureus [MSSA]) or ATCC 33591 (methicillin-resistant S. aureus [MRSA]) were obtained by culture in 96-well plates for 6 h/24 h. Antibiotic activities were assessed after 24/48 h of exposure to concentrations ranging from 0.5 to 512 times the MIC. Biofilm mass and bacterial viability were quantified using crystal violet and the redox indicator resazurin. Biofilms stained with Live/Dead probes were observed by using confocal microscopy. Concentration-effect curves fitted sigmoidal regressions, with a 50% reduction toward both matrix and viability obtained at sub-MIC or low multiples of MICs against young biofilms for all antibiotics tested. Against mature biofilms, maximal efficacies and potencies were reduced, with none of the antibiotics being able to completely destroy the matrix. Delafloxacin and daptomycin were the most potent, reducing viability by more than 50% at clinically achievable concentrations against both strains, as well as reducing biofilm depth, as observed in confocal microscopy. Rifampin, tigecycline, and moxifloxacin were effective against mature MRSA biofilms, while oxacillin demonstrated activity against MSSA. Fusidic acid, vancomycin, and linezolid were less potent overall. Antibiotic activity depends on biofilm maturity and bacterial strain. The pharmacodynamic model developed allows ranking of antibiotics with respect to efficacy and potency at clinically achievable concentrations and highlights the potential utility of daptomycin and delafloxacin for the treatment of biofilm-related infections.     

Daptomycin exerts bactericidal activity without lysis of Staphylococcus aureus

The ability of daptomycin to produce bactericidal activity against Staphylococcus aureus while causing negligible cell lysis has been demonstrated using electron microscopy and the membrane integrity probes calcein and ToPro3. The formation of aberrant septa on the cell wall, suggestive of impairment of the cell division machinery, was also observed.     

Daptomycin disrupts membrane potential in growing Staphylococcus aureus.

Daptomycin (LY146032) caused a calcium-dependent dissipation of the membrane potential (delta psi) in Staphylococcus aureus without noticeably affecting the chemical gradient (delta pH) across the membrane. The effect of daptomycin on membrane energization may account for many of the inhibitory effects on macromolecular biosyntheses and membrane function reported for this antibiotic. Our evidence indicates that the bactericidal activity of daptomycin is dependent on an available delta psi.     

Effects of Low-Dose Amoxicillin on Staphylococcus aureus USA300 Biofilms

Previous studies showed that sub-MIC levels of β-lactam antibiotics stimulate biofilm formation in most methicillin-resistant Staphylococcus aureus (MRSA) strains. Here, we investigated this process by measuring the effects of sub-MIC amoxicillin on biofilm formation by the epidemic community-associated MRSA strain USA300. We found that sub-MIC amoxicillin increased the ability of USA300 cells to attach to surfaces and form biofilms under both static and flow conditions. We also found that USA300 biofilms cultured in sub-MIC amoxicillin were thicker, contained more pillar and channel structures, and were less porous than biofilms cultured without antibiotic. Biofilm formation in sub-MIC amoxicillin correlated with the production of extracellular DNA (eDNA). However, eDNA released by amoxicillin-induced cell lysis alone was evidently not sufficient to stimulate biofilm. Sub-MIC levels of two other cell wall-active agents with different mechanisms of action—d-cycloserine and fosfomycin—also stimulated eDNA-dependent biofilm, suggesting that biofilm formation may be a mechanistic adaptation to cell wall stress. Screening a USA300 mariner transposon library for mutants deficient in biofilm formation in sub-MIC amoxicillin identified numerous known mediators of S. aureus β-lactam resistance and biofilm formation, as well as novel genes not previously associated with these phenotypes. Our results link cell wall stress and biofilm formation in MRSA and suggest that eDNA-dependent biofilm formation by strain USA300 in low-dose amoxicillin is an inducible phenotype that can be used to identify novel genes impacting MRSA β-lactam resistance and biofilm formation.     

Intra- and Extracellular Activities of Dicloxacillin against Staphylococcus aureus In Vivo and In Vitro

Antibiotic treatment of Staphylococcus aureus infections is often problematic due to the slow response and recurrences. The intracellular persistence of the staphylococci offers a plausible explanation for the treatment difficulties because of the impaired intracellular efficacies of the antibiotics. The intra- and extracellular time- and concentration-kill relationships were examined in vitro with THP-1 cells and in vivo by use of a mouse peritonitis model. The in vivo model was further used to estimate the most predictive pharmacokinetic/pharmacodynamic (PK/PD) indices (the ratio of the maximum concentration of drug in plasma/MIC, the ratio of the area under the concentration-time curve/MIC, or the cumulative percentage of a 24-h period that the free [f] drug concentration exceeded the MIC under steady-state pharmacokinetic conditions [fT_MIC]) for dicloxacillin (DCX) intra- and extracellularly. In general, DCX was found to have similar intracellular activities, regardless of the model used. Both models showed (i) the relative maximal efficacy (1-log-unit reduction in the numbers of CFU) of DCX intracellularly and (ii) the equal relative potency of DCX intra- and extracellularly, with the MIC being a good indicator of the overall response in both situations. Discordant results, based on data obtained different times after dosing, were obtained from the two models when the extracellular activity of DCX was measured, in which the in vitro model showed a considerable reduction in the number of CFU from that in the original inoculum (3-log-unit decrease in the number of CFU after 24 h), whereas the extracellular CFU reduction achieved in vivo after 4 h did not exceed 1 log unit. Multiple dosing of DCX in vivo revealed increased extra- and intracellular efficacies (2.5 log and 2 log units of reduction in the numbers of CFU after 24 h, respectively), confirming that DCX is a highly active antistaphylococcal antibiotic. PK/PD analysis revealed that fT_MIC is the index that is the most predictive of the outcome of infection both intra- and extracellularly.Staphylococcus aureus is a major cause of both community- and hospital-acquired infections (28, 30), which range from simple and uncomplicated skin and wound infections (2, 24) to more serious and life-threatening situations such as pneumonia (15, 36), endocarditis (16, 37), osteomyelitis (13, 25), and meningitis (34). S. aureus infections often show poor and slow responses to therapy, with recurrences and ensuing mortality (8, 9, 27, 37, 38, 46). These responses could be caused by the ability of the bacteria to invade and survive inside cells (5, 10, 21, 22, 31, 32). Intracellular antimicrobial activity depends on both drug- and bacterium-related factors (penetration, accumulation, subcellular bioavailability, expression of activity in the local environment, and the state of responsiveness of the organisms [42, 44]). In general, intracellular antimicrobial activity is markedly impaired compared to the activity seen in broth or the extracellular milieu (3, 39, 45), although we know about situations in which the opposite is true (7). Thus, the direct assessment of antibiotic activity in the pertinent models is warranted. Several in vitro models with either human or animal cells have been developed to study the intracellular activities of antibiotics (3, 6, 14, 21, 35, 41), and a corresponding in vivo model (a modified version of a murine peritonitis model) has recently been described (39). We have now combined these models and report here our results obtained by using dicloxacillin (DCX) as a prototype of antistaphylococcal β-lactam antibiotics. Isoxazolyl penicillins have usually been preferred for the treatment of methicillin-susceptible S. aureus (MSSA) infections (2, 20, 26, 30). DCX has been the main choice in Denmark and many other countries due to its stability against penicillinases, low level of toxicity, and availability for both oral and intravenous administration (19). We examined the intra- and extracellular time- and concentration-kill relationships for two MSSA strains in vitro using macrophages and performed corresponding intra- and extracellular dose-kill studies with the murine peritonitis model. In combination with pharmacokinetic (PK) analysis and measurement of the amount of free drug (f) versus protein-bound drug, this allowed us to estimate which PK/pharmacodynamic (PD) index best predicts the efficacy of DCX intra- and extracellularly.(Part of this study was presented at the 46th Interscience Conference on Antimicrobial Agents and Chemotherapy, September 2006, San Francisco, CA.)     

Effect of Temperature on the In Vitro Susceptibility of Staphylococcus aureus to Penicillinase-Resistant Penicillins

Heteroresistant (methicillin-resistant) and nonheteroresistant strains of Staphylococcus aureus were tested for their susceptibility to penicillinase-resistant penicillins at incubation temperatures of 37, 35, and 30 C. Susceptibilities were determined by agar dilution and by the standard Kirby-Bauer agar diffusion tests. Minimal inhibitory concentrations were higher at 35 and 30 C than at 37 C. Heteroresistance could be detected with the Kirby-Bauer test if the incubation temperature was 30 or 35 C instead of 37 C, when tests were performed against methicillin, oxacillin, or nafcillin, because the resistant organisms grew up to the disks even though the susceptible organisms were inhibited. At 37 C, the resistance was detectable with some strains but not with others. When cloxacillin disks were used, the temperature effect was not seen. The incubation temperature did not affect results with nonheteroresistant strains. Therefore, it is recommended that all Kirby-Bauer tests be incubated at a temperature of 35 C to insure detection of methicillin-resistant S. aureus strains. Detection of these strains is of increasing importance because the incidence of infections with these organisms is increasing, particularly in hospitalized patients.     

Daptomycin inoculum effects and mutant prevention concentration with Staphylococcus aureus

BACKGROUND: Antimutant activity of antimicrobials can be estimated by comparing drug pharmacokinetics with mutant prevention concentration (MPC). Large bacterial inocula known to reduce susceptibility have not been studied for effects on MPC determination. METHODS: Staphylococcus aureus inoculum size was varied with solid and liquid media containing daptomycin and Ca(2+), a cation expected to lower inoculum effects, to assess effects on MIC and MPC. RESULTS: With drug-containing agar, individual colonies were obtained over a narrow range of inoculum size that shifted to higher inoculum size as daptomycin concentration increased. Increasing Ca(2+) supplementation from 1 to 50 mM lowered MIC by 2-fold and MPC from 20 to 3 mg/L, the latter determined by extrapolation of population analysis profiles to an inoculum size of 10(10) cfu. Cells of colonies recovered from daptomycin-containing agar had wild-type MIC. With liquid medium, supplemented with 1 mM Ca(2+)and containing 10(10) cfu, MPC was between 2.5 and 5 mg/L at an inoculum density of 10(7) cfu/mL. Bacteria recovered from liquid assays exhibited a 4- to 8-fold increase in MIC and contained point mutations in mprF. CONCLUSIONS: Inoculum effects on MPC can be reduced by measurement with low-density (large volume) liquid bacterial cultures. Retesting putative mutants for susceptibility can be important: stable mutants having genetic variations in the mprF gene were recovered from liquid medium, but not from agar. Daptomycin MPC with S. aureus was below minimal plasma drug concentration with approved doses, which is consistent with resistance to daptomycin arising rarely.