Inhibitory and Bactericidal Activities of Daptomycin,Vancomycin, and Teicoplanin against Methicillin-Resistant Staphylococcus aureus Isolates Collected from 1985 to 2007

The inhibitory and bactericidal activities of daptomycin, vancomycin, and teicoplanin against a collection of 479 methicillin-resistant Staphylococcus aureus isolates were assessed. The isolates were collected from U.S. and European hospitals from 1985 to 2007 and were primarily from blood and abscess cultures. The MICs and minimum bactericidal concentrations (MBCs) of the three agents were determined, and the MBC/MIC ratios were calculated to determine the presence or absence of tolerance. Tolerance was defined as an MBC/MIC ratio of ≥32 or an MBC/MIC ratio of ≥16 when the MBC was greater than or equal to the breakpoint for resistance. Tolerance to vancomycin and teicoplanin was observed in 6.1% and 18.8% of the strains, respectively. Tolerance to daptomycin was not observed.Although vancomycin and teicoplanin are the standard therapies for staphylococcal bacteremia, tolerance to vancomycin and teicoplanin has been demonstrated in both coagulase-negative staphylococci and Staphylococcus aureus as well as in various Streptococcus species (2, 3, 7, 10, 13, 15, 20, 21, 23, 25). Daptomycin, a lipopeptide antibiotic, has been demonstrated to have rapid bactericidal activity against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), and tolerance to this drug has not been demonstrated (2, 9, 10, 19, 21, 24, 26, 28).The issue of antibiotic tolerance is a complicated one. Some studies have suggested that infections caused by tolerant strains may be more difficult to treat, especially when they cause complicated infections such as endocarditis, meningitis, or osteomyelitis or cause infections in immunocompromised patients (7, 8, 14, 15, 16, 18, 20, 22, 23, 25). Other investigators'' expert analyses do not agree that there is proof of a correlation between tolerant strains and treatment failures or that bactericidal activity is required for the treatment of serious MRSA infections (17, 25, 26, 27, 28). Controversy concerning the appropriate methods for the determination of tolerance in clinical isolates and in the practicality of testing isolates for tolerance in the clinical laboratory also exists.This study looked at MRSA isolates obtained primarily from blood and abscess cultures collected between 1985 and 2007. The main purpose of the study was to determine the in vitro inhibitory and bactericidal activities and the level of tolerance to the three drugs observed by standardized MIC and minimum bactericidal concentration (MBC) tests (4, 5, 19).(This study was presented in part at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 17 to 20 September 2007.)     

In Vitro Activity of Ceftaroline against Staphylococcus aureus Isolates Collected in 2012 from Latin American Countries as Part of the AWARE Surveillance Program

The in vitro activities of ceftaroline and comparators, using broth microdilution, were determined against 1,066 Staphylococcus aureus isolates from hospitalized patients. Seventeen medical centers from Latin American countries contributed isolates. Methicillin-resistant S. aureus (MRSA) percentages ranged from 46% (Brazil) to 62% (Argentina). All methicillin-susceptible S. aureus (MSSA) isolates were susceptible to ceftaroline. Ceftaroline activity against MRSA varied with MIC₉₀s of 0.5 (Venezuela) to 2 (Brazil, Chile, and Colombia) μg/ml, which was the highest MIC value. ST-5 was the most common sequence type.     

Comparison of the Antibiotic Activities of Daptomycin,Vancomycin, and the Investigational Fluoroquinolone Delafloxacin against Biofilms from Staphylococcus aureus Clinical Isolates

Biofilm-related infections remain a scourge. In an in vitro model of biofilms using Staphylococcus aureus reference strains, delafloxacin and daptomycin were found to be the most active among the antibiotics from 8 different pharmacological classes (J. Bauer, W. Siala, P. M. Tulkens, and F. Van Bambeke, Antimicrob. Agents Chemother. 57:2726–2737, 2013, doi:10.1128/AAC.00181-13). In this study, we compared delafloxacin to daptomycin and vancomycin using biofilms produced by 7 clinical strains (S. aureus epidemic clones CC5 and CC8) in order to rationalize the differences observed between the antibiotics and strains. The effects of the antibiotics on bacterial viability (resazurin reduction assay) and biomass (crystal violet staining) were measured and correlated with the proportion of polysaccharides in the matrix, the local microenvironmental pH (micro-pH), and the antibiotic penetration in the biofilm. At clinically meaningful concentrations, delafloxacin, daptomycin, and vancomycin caused a ≥25% reduction in viability against the biofilms formed by 5, 4, and 3 strains, respectively. The antibiotic penetration within the biofilms ranged from 0.6 to 52% for delafloxacin, 0.2 to 10% for daptomycin, and 0.2 to 1% for vancomycin; for delafloxacin, this was inversely related to the polysaccharide proportion in the matrix. Six biofilms were acidic, explaining the high potency of delafloxacin (lower MICs at acidic pH). Norspermidine and norspermine (disassembling the biofilm matrix) drastically increased delafloxacin potency and efficacy (50% reduction in viability for 6 biofilms at clinically meaningful concentrations) in direct correlation with its increased penetration within the biofilm, while they only modestly improved daptomycin efficacy (50% reduction in viability for 2 biofilms) and penetration, and they showed marginal effects with vancomycin. Delafloxacin potency and efficacy against biofilms are benefited by its penetration into the matrix and the local acidic micro-pH.     

In Vitro Activity of Ceftaroline against Community-Associated Methicillin-Resistant,Vancomycin-Intermediate,Vancomycin-Resistant,and Daptomycin-Nonsusceptible Staphylococcus aureus Isolates

This study assessed the in vitro activities of ceftaroline and five comparator agents against a collection of Staphylococcus aureus isolates. Ceftaroline demonstrated potent activity against community-associated methicillin-resistant S. aureus (CA-MRSA) isolates and showed bactericidal activity against vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA), heteroresistant VISA (hVISA), and daptomycin-nonsusceptible S. aureus (DNSSA) isolates. Ceftaroline may represent a bactericidal treatment option for infections caused by these pathogens.The increasing prevalence of resistant Staphylococcus aureus strains, including methicillin-resistant S. aureus (MRSA), community-associated MRSA (CA-MRSA), and S. aureus strains with reduced susceptibility to vancomycin, emphasizes the need for innovative antimicrobials with activity against these pathogens (19, 24). Although the prevalence of S. aureus strains with reduced vancomycin susceptibility remains low, such strains have been associated with vancomycin treatment failure, limiting the treatment options for patients with such infections (3, 8). Ceftaroline is a novel, parenteral, broad-spectrum cephalosporin exhibiting bactericidal activity against Gram-positive organisms, including MRSA and multidrug-resistant Streptococcus pneumoniae (MDRSP), as well as common Gram-negative pathogens (6, 9, 21). Ceftaroline is currently in phase 3 development for the treatment of complicated skin and skin structure infections and community-acquired bacterial pneumonia. The study described here evaluated the in vitro activities of ceftaroline and five comparator agents against CA-MRSA, vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA), heteroresistant VISA (hVISA), and daptomycin-nonsusceptible S. aureus (DNSSA) isolates.(A preliminary report of these results was presented at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy-Infectious Diseases Society of America Annual Meeting, Washington, DC, 25 to 28 October 2008.)A total of 132 MRSA strains were selected for evaluation. CA-MRSA strains (n = 92) were isolated from patients admitted to St. John Hospital and Medical Center in Detroit, MI. These patients had positive MRSA cultures within 48 h of admission, in accordance with the definition of CA-MRSA described by the Centers for Disease Control and Prevention (2). DNSSA strains (n = 7) and hVISA strains (n = 3) were obtained from blood collected from patients at the same hospital. The hVISA isolates were identified by a modified population analysis profile method (12). VISA isolates (n = 20) and VRSA isolates (n = 10) were obtained via the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) program, supported under NIAID/NIH contract HHSN272200700055C.All CA-MRSA isolates were typed by pulsed-field gel electrophoresis (PFGE) with the restriction enzyme SmaI, followed by visual interpretation and categorization. PCR methods were used to determine the staphylococcal cassette chromosome mec (SCCmec) type, the presence of Panton-Valentine leukocidin (PVL) genes lukS-PV and lukF-PV, and the presence of the arginine catabolic mobile element (ACME) via detection of the arcA locus.In vitro susceptibility tests were performed with the antimicrobials ceftaroline (lot CI 170-07; Forest Laboratories, Inc., New York, NY), vancomycin-HCl (Sigma), daptomycin (Cubist), clindamycin-HCl (Sigma), linezolid (Pfizer), and trimethoprim-sulfamethoxazole (Sigma), which were obtained individually and reconstituted to a 1:19 ratio. All antibiotics were received in powder form and were reconstituted according to guidelines of the Clinical and Laboratory Standards Institute (CLSI). MICs and minimum bactericidal concentrations (MBCs) were determined according to the guidelines of the CLSI (4, 5). Microdilution tests with cation-adjusted Mueller-Hinton broth were used to identify the MICs of all antimicrobial agents tested. The percentages of susceptible isolates were determined by using the CLSI breakpoints. For the testing of daptomycin, additional calcium was added to the broth for a final concentration of 50 mg/liter. The MICs were read visually and corresponded to the concentration in the well with the lowest drug concentration with no visible bacterial growth. The MBC was defined as the antibiotic concentration that reduced the number of viable cells by ≥99.9%. This was based on colony counts from the growth control well and rejection values determined by tables provided by Pearson (18).S. aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 were used as the control strains for the MIC determinations, and S. aureus ATCC 25923 was used as the control strain for the MBC determinations.Approximately 58% of the CA-MRSA isolates were SCCmec type IV/IVa, whereas the majority of the other isolates were SCCmec type II (Table ​(Table1).1). All SCCmec type IVa isolates were positive for the PVL and ACME genes, whereas isolates of all other SCCmec types were negative for these genes. Of all the other S. aureus strains, only two DNSSA isolates were SCCmec type IVa and positive for the PVL and ACME genes.

TABLE 1.

SCC mec elements, PVL and ACME genes, and ceftaroline MIC₉₀s and MBC₉₀s of CA-MRSA isolates^a

High Prevalence of Mupirocin Resistance in Staphylococcus aureus Isolates from a Pediatric Population

Topical mupirocin is used widely to treat skin and soft tissue infections and to eradicate nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA). Few studies to date have characterized the rates of S. aureus mupirocin resistance in pediatric populations. We retrospectively studied 358 unique S. aureus isolates obtained from 249 children seen in a predominantly outpatient setting by the Division of Pediatric Dermatology at a major academic center in New York City between 1 May 2012 and 17 September 2013. Mupirocin resistance rates and the associated risk factors were determined using a logistic regression analysis. In our patient population, 19.3% of patients had mupirocin-resistant S. aureus isolates at the time of their first culture, and 22.1% of patients with S. aureus infection had a mupirocin-resistant isolate at some time during the study period. Overall, 31.3% of all S. aureus isolates collected during the study period were resistant to mupirocin. Prior mupirocin use was strongly correlated (odds ratio [OR] = 26.5; P = <0.001) with mupirocin resistance. Additional risk factors for mupirocin resistance included methicillin resistance, atopic dermatitis (AD), epidermolysis bullosa (EB), immunosuppression, and residence in northern Manhattan and the Bronx. Resistance to mupirocin is widespread in children with dermatologic complaints in the New York City area, and given the strong association with mupirocin exposure, it is likely that mupirocin use contributes to the increased resistance. Routine mupirocin testing may be important for MRSA decolonization strategies or the treatment of minor skin infections in children.     

Telavancin In Vitro Activity against a Collection of Methicillin-Resistant Staphylococcus aureus Isolates,Including Resistant Subsets,from the United States

Telavancin had MIC₅₀, MIC₉₀, and MIC₁₀₀ values of 0.03, 0.06, and 0.12 μg/ml, respectively, against methicillin-susceptible Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), and non-multidrug-resistant (non-MDR) and MDR subsets. MRSA with elevated MIC values for vancomycin (2 to 4 μg/ml) or daptomycin (1 to 2 μg/ml) had telavancin MIC₅₀ (0.06 μg/ml) values 2-fold higher than those of isolates with lower MIC results (MIC₅₀, 0.03 μg/ml). However, telavancin had MIC₉₀ and MIC₁₀₀ results of 0.06 and 0.12 μg/ml (100% susceptible), respectively, regardless of the MRSA subset.     

PBP2a Mutations Causing High-Level Ceftaroline Resistance in Clinical Methicillin-Resistant Staphylococcus aureus Isolates

Ceftaroline is the first member of a novel class of cephalosporins approved for use in the United States. Although prior studies have identified eight ceftaroline-resistant methicillin-resistant Staphylococcus aureus (MRSA) isolates in Europe and Asia with MICs ranging from 4 to 8 mg/liter, high-level resistance to ceftaroline (>32 mg/liter) has not been described in MRSA strains isolated in the United States. We isolated a ceftaroline-resistant (MIC > 32 mg/liter) MRSA strain from the blood of a cystic fibrosis patient and five MRSA strains from the respiratory tract of this patient. Whole-genome sequencing identified two amino acid-altering mutations uniquely present in the ceftaroline-binding pocket of the transpeptidase region of penicillin-binding protein 2a (PBP2a) in ceftaroline-resistant isolates. Biochemical analyses and the study of isogenic mutant strains confirmed that these changes caused ceftaroline resistance. Thus, we identified the molecular mechanism of ceftaroline resistance in the first MRSA strain with high-level ceftaroline resistance isolated in the United States.     

In Vitro Activity of Human-Simulated Epithelial Lining Fluid Exposures of Ceftaroline,Ceftriaxone, and Vancomycin against Methicillin-Susceptible and -Resistant Staphylococcus aureus

Staphylococcus aureus, including methicillin-susceptible (MSSA) and -resistant (MRSA) strains, is an important pathogen of bacterial pneumonia. As antibiotic concentrations at the site of infection are responsible for killing, we investigated the activity of human-simulated epithelial lining fluid (ELF) exposures of three antibiotics (ceftaroline, ceftriaxone, and vancomycin) commonly used for treatment of S. aureus pneumonia. An in vitro pharmacodynamic model was used to simulate ELF exposures of vancomycin (1 g every 12 h [q12h]), ceftaroline (600 mg q12h and q8h), and ceftriaxone (2 g q24h and q12h). Four S. aureus isolates (2 MSSA and 2 MRSA) were evaluated over 72 h with a starting inoculum of ∼10⁶ CFU/ml. Time-kill curves were constructed, and microbiological response (change in log₁₀ CFU/ml from 0 h and the area under the bacterial killing and regrowth curve [AUBC]) was assessed in duplicate. The change in 72-h log₁₀ CFU/ml was largest for ceftaroline q8h (reductions of >3 log₁₀ CFU/ml against all strains). This regimen also achieved the lowest AUBC against all organisms (P < 0.05). Vancomycin produced reliable bacterial reductions of 0.9 to 3.3 log₁₀ CFU/ml, while the activity of ceftaroline q12h was more variable (reductions of 0.2 to 2.3 log₁₀ CFU/ml against 3 of 4 strains). Both regimens of ceftriaxone were poorly active against MSSA tested (0.1 reduction to a 1.8-log₁₀ CFU/ml increase). Against these S. aureus isolates, ELF exposures of ceftaroline 600 mg q8h exhibited improved antibacterial activity compared with ceftaroline 600 mg q12h and vancomycin, and therefore, this q8h regimen deserves further evaluation for the treatment of bacterial pneumonia. These data also suggest that ceftriaxone should be avoided for S. aureus pneumonia.     

Evaluation of Vancomycin in Combination with Piperacillin-Tazobactam or Oxacillin against Clinical Methicillin-Resistant Staphylococcus aureus Isolates and Vancomycin-Intermediate S. aureus Isolates In Vitro

Vancomycin with piperacillin-tazobactam is used as empirical therapy for critically ill patients. Studies of this combination against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-intermediate S. aureus (VISA) are limited, but β-lactams in combination with vancomycin have shown synergistic activity against MRSA and VISA. The goal of this study was to evaluate whether piperacillin-tazobactam and vancomycin were synergistic against MRSA and VISA in vitro. Bloodstream MRSA (n = 20) and VISA (n = 4) strains were selected. In vitro antimicrobial activities of piperacillin-tazobactam and oxacillin were evaluated by disk diffusion, and MICs were determined by Etest using Muller-Hinton agar with and without vancomycin at one-half the MIC. Time-kill studies evaluated 14 MRSA and all 4 VISA isolates using piperacillin-tazobactam at 300/35 mg/liter or oxacillin at 40 mg/liter alone and with vancomycin at one-half the MIC. Mean zones of inhibition for piperacillin-tazobactam and oxacillin increased with vancomycin against MRSA and VISA (P < 0.001 for all), and the MIC₉₀ decreased with vancomycin against MRSA and VISA to values meeting susceptibility criteria for S. aureus (P < 0.001 for both antibiotics against MRSA). In MRSA time-kill studies, the mean 24-h reductions in inoculum for piperacillin-tazobactam, piperacillin-tazobactam with vancomycin, and oxacillin with vancomycin were 3.53, 3.69, and 2.62 log₁₀ CFU/ml, respectively. The mean 24-h reductions in VISA inoculum for piperacillin-tazobactam, piperacillin-tazobactam with vancomycin, and oxacillin with vancomycin were 2.85, 2.93, and 3.45 log₁₀ CFU/ml, respectively. Vancomycin with piperacillin-tazobactam or oxacillin demonstrated synergistic activity against MRSA and VISA. The clinical implications of these combinations against MRSA and VISA should be investigated.     

In Vitro Activity of Dalbavancin against Drug-Resistant Staphylococcus aureus Isolates from a Global Surveillance Program

In over a decade (2002 to 2012) of Staphylococcus aureus surveillance testing on 62,195 isolates, dalbavancin was demonstrated to be active against isolates that were either susceptible or nonsusceptible to daptomycin, linezolid, or tigecycline. Nearly all (99.8%) multidrug-resistant methicillin-resistant S. aureus isolates were inhibited by dalbavancin at ≤0.12 μg/ml (MIC_50/90, 0.06/0.06 μg/ml), the current U.S. Food and Drug Administration (U.S. FDA) breakpoint. Overall, only 0.35% of the monitored S. aureus isolates had a dalbavancin MIC of either 0.25 or 0.5 μg/ml (i.e., were nonsusceptible).     

Evaluation of Ceftaroline,Vancomycin, Daptomycin,or Ceftaroline plus Daptomycin against Daptomycin-Nonsusceptible Methicillin-Resistant Staphylococcus aureus in an In Vitro Pharmacokinetic/Pharmacodynamic Model of Simulated Endocardial Vegetations

Infective endocarditis (IE) caused by methicillin-resistant Staphylococcus aureus (MRSA) with reduced susceptibility to vancomycin and daptomycin has few adequate therapeutic options. Ceftaroline (CPT) is bactericidal against daptomycin (DAP)-nonsusceptible (DNS) and vancomycin-intermediate MRSA, but supporting data are limited for IE. This study evaluated the activities of ceftaroline, vancomycin, daptomycin, and the combination of ceftaroline plus daptomycin against DNS MRSA in a pharmacokinetic/pharmacodynamic (PK/PD) model of simulated endocardial vegetations (SEVs). Simulations of ceftaroline-fosamil (600 mg) every 8 h (q8h) (maximum concentration of drug in serum [C_max], 21.3 mg/liter; half-life [t_1/2], 2.66 h), daptomycin (10 mg/kg of body weight/day) (C_max, 129.7 mg/liter; t_1/2, 8 h), vancomycin (1 g) q8h (minimum concentration of drug in serum [C_min], 20 mg/liter; t_1/2, 5 h), and ceftaroline plus daptomycin were evaluated against 3 clinical DNS, vancomycin-intermediate MRSA in a two-compartment, in vitro, PK/PD SEV model over 96 h with a starting inoculum of ∼8 log₁₀ CFU/g. Bactericidal activity was defined as a ≥3-log₁₀ CFU/g reduction from the starting inoculum. Therapeutic enhancement of combinations was defined as ≥2-log₁₀ CFU/g reduction over the most active agent alone. MIC values for daptomycin, vancomycin, and ceftaroline were 4 mg/liter, 4 to 8 mg/liter, and 0.5 to 1 mg/liter, respectively, for all strains. At simulated exposures, vancomycin was bacteriostatic, but daptomycin and ceftaroline were bactericidal. By 96 h, ceftaroline monotherapy offered significantly improved killing compared to other agents against one strain. The combination of DAP plus CPT demonstrated therapeutic enhancement, resulting in significantly improved killing versus either agent alone against 2/3 (67%) strains. CPT demonstrated bactericidal activity against DNS, vancomycin-intermediate MRSA at high bacterial densities. Ceftaroline plus daptomycin may offer more rapid and sustained activity against some MRSA in the setting of high-inoculum infections like IE and should also be considered.     

Activities of Daptomycin and Vancomycin Alone and in Combination with Rifampin and Gentamicin against Biofilm-Forming Methicillin-Resistant Staphylococcus aureus Isolates in an Experimental Model of Endocarditis

The findings of clinical and in vitro research support the theory that infective endocarditis (IE)-causing bacteria form biofilms and that biofilms negatively affect treatment outcomes. The purpose of the present study was to quantify the biofilm formation of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) isolates obtained from patients with IE and to evaluate the in vitro activities of daptomycin and vancomycin alone and in combination with rifampin (rifampicin) or gentamicin while monitoring the isolates for the development of resistance. A high-inoculum, stationary-phase infection model of IE was used to simulate the pharmacokinetics in humans of daptomycin at 6 mg/kg of body weight/day, vancomycin at 1.25 g every 12 h (q12h) alone and in combination with rifampin at 300 mg every 8 h, and gentamicin at 1.3 mg/kg q12h. Two randomly selected clinical MRSA isolates were obtained from patients with IE; both MRSA isolates quantitatively produced biofilms. The time to bactericidal activity in the presence of daptomycin was isolate dependent but was achieved by 24 h for both MRSA isolates. Vancomycin did not achieve bactericidal activity throughout the experiment. At 24, 48, and 72 h, daptomycin-containing regimens had significantly more activity (greater declines in the mean number of CFU/g) than any of the vancomycin-containing regimens (P = 0.03). Rifampin and gentamicin antagonized or delayed the bactericidal activity of daptomycin (against MRSA B346846 for rifampin and against both isolates for gentamicin) in the first 24 h. Increases in the daptomycin and vancomycin MICs were not observed. We conclude that in an IE model of biofilm-forming MRSA, daptomycin monotherapy has better in vitro activity than daptomycin in combination with rifampin or gentamicin or any vancomycin-containing regimen studied within the first 24 h. Further investigations are needed to understand the initial delay in bactericidal activity observed when gentamicin or rifampin is combined with daptomycin.Biofilm-forming Staphylococcus aureus isolates are frequently found on prosthetic devices and in deep tissue infections (21, 43), and both prosthetic devices and deep tissue infections serve as common sources for bacteremia. Clinical research supports the theory that infective endocarditis (IE)-causing bacteria form biofilms (14, 19) and that S. aureus isolates recovered from the blood of patients with IE tend to produce biofilms at a high inoculum and during the stationary phase of growth (24, 43). These isolates also typically carry accessory gene regulator (agr) groups I and II, which regulate the production of autolysins that promote biofilm formation (21, 43, 44). Despite this information, there are limited data about the biofilm-forming capabilities of S. aureus isolates that cause IE (SAIE).Until recently, effective antimicrobial therapy for methicillin (meticillin)-resistant S. aureus (MRSA) bacteremia and IE was limited to vancomycin. Although vancomycin has commonly been used since the 1980s for the treatment of MRSA infections, including endocarditis, several published studies indicate that it has limited efficacy because of bacterial resistance, bacterial tolerance, and poor tissue penetration (8, 22, 36). Patients with SAIE treated with vancomycin alone may still be bacteremic (as indicated by positive blood cultures) after 7 to 10 days of therapy (25, 26). Guidelines for the treatment of SAIE recommend the use of combination therapy with vancomycin plus gentamicin or rifampin (rifampicin) (2, 3, 41). However, these combinations can be problematic because gentamicin increases the risk of nephrotoxicity and rifampin increases the potential for drug interactions via its induction of cytochrome P450 metabolism. In addition, this recommendation is based on limited clinical data.Daptomycin, a novel lipopetide antimicrobial agent, received FDA approval in May 2006 for the treatment of bacteremia and right-sided SAIE caused by methicillin-susceptible and -resistant strains (12). The FDA indication is for monotherapy against gram-positive pathogens; however, the guidelines recommend combination therapy for SAIE. Data to support the optimal dose of daptomycin required for it to have activity when it is combined with commonly used synergistic agents such as gentamicin and rifampin are lacking. In addition, limited information about the activity of daptomycin in the presence of biofilm-forming S. aureus has been published (23, 32, 35, 37).The purpose of this study was to quantify the biofilm formation of S. aureus isolates obtained from patients with SAIE, to assess the in vitro activities of daptomycin and vancomycin alone and in combination with rifampin or gentamicin, and to evaluate the development of resistance in a high-inoculum, stationary-phase bacterial (biofilm) model of IE.(This work has been presented in part at the 108th General Meeting of the American Society for Microbiology, Boston, MA, 1 to 5 June 2008 [abstr. A-076].)     

Characterization of Erythromycin-Resistant Isolates of Staphylococcus aureus Recovered in the United States from 1958 through 1969

We tested 16 erythromycin-resistant clinical isolates of S. aureus, recovered from patients hospitalized in the United States from 1958 to 1969, for the presence of ermA, ermB, and ermC by using PCR. Fifteen of 16 isolates contained at least one copy of ermA; the remaining isolate, which was also clindamycin resistant, contained ermB. Eight of the 15 isolates harboring ermA, all of which were inducible, contained a single copy of the gene in the chromosome, while the remaining seven isolates had two copies of the gene. ermB was plasmid encoded and mediated constitutive resistance to erythromycin.     

In Vivo Activity of a Novel Anti-Methicillin-Resistant Staphylococcus aureus Cephalosporin,Ceftaroline, against Vancomycin-Susceptible and -Resistant Enterococcus faecalis Strains in a Rabbit Endocarditis Model: a Comparative Study with Linezolid and Vancomycin

We assessed the in vitro and in vivo efficacy of the novel parenteral broad-spectrum cephalosporin ceftaroline against Enterococcus faecalis in time-kill experiments and in a rabbit endocarditis model with simulated human dosing. Ceftaroline was more active than either vancomycin or linezolid against vancomycin-sensitive and -resistant isolates of E. faecalis.In recent years, enterococci have become significant pathogens due to their ability to resist most available antibiotics, including β-lactams, aminoglycosides, and glycopeptides, through intrinsic and/or acquired mechanisms of resistance (12). The lack of effective antimicrobial therapy for patients infected with glycopeptide-resistant enterococci has led to the need for new therapeutic agents. Ceftaroline is a broad-spectrum bactericidal cephalosporin that demonstrates time-dependent killing activity against gram-positive organisms, including methicillin-resistant Staphylococcus aureus and multidrug-resistant Streptococcus pneumoniae, as well as common gram-negative pathogens (6, 10, 16). Ceftaroline is currently in phase 3 trials for complicated skin and skin structure infections and community-acquired pneumonia. Ceftaroline is active in vitro against Enterococcus faecalis, including vancomycin-resistant (Van^r) strains, although like other cephalosporins, it is inactive against Enterococcus faecium (6, 16).In view of the limited number of antibiotics available for treating infections involving E. faecalis, it was of interest to us to investigate the efficacy of ceftaroline against this significant pathogen. The aim of the present study was to evaluate the in vitro and in vivo efficacy of ceftaroline, compared with that of linezolid and vancomycin, against E. faecalis strains by using time-kill experiments and a rabbit endocarditis model with simulated human dosing.The E. faecalis strains employed in these studies (EF 12704 and EF NJ1) were isolated from blood cultures. EF 12704 is susceptible to vancomycin (Van^s), and EF NJ1 exhibits a Van^r VanA phenotype. To assess in vitro activity, MICs were determined according to Clinical and Laboratory Standards Institute reference broth microdilution methods (1, 3). The bactericidal activity of each drug was evaluated by using time-kill experiments with an inoculum of 5 × 10⁶ CFU/ml (13).The in vivo efficacy of ceftaroline was assessed by using a rabbit endocarditis model (4, 14). All animal studies were approved by the Committee of Animal Ethics of the University of Nantes. Catheters were placed into the left ventricles of anesthetized New Zealand White rabbits. Twenty-four hours later, endocarditis was induced with an inoculum of 10⁸ CFU E. faecalis. Treatment was started 24 h after inoculation, with antibiotics delivered via the marginal ear vein by a computer-controlled pump as described elsewhere (2, 8). After 4 days of treatment, aortic valve vegetations were excised, weighed, homogenized in 0.5 ml of saline buffer, and used for quantitative cultures on agar for 24 h at 37°C. To evaluate whether ceftaroline, linezolid, or vancomycin could induce the selection of resistant variants, undiluted vegetation homogenates were spread on agar plates containing the study drugs at concentrations corresponding to four times the MIC. Bacterial counts were determined after 48 h of incubation at 37°C.The human pharmacokinetic profiles of linezolid and ceftaroline were simulated as previously described (8, 9). For ceftaroline, the study was designed to simulate pharmacokinetic parameters observed in healthy volunteers after a 1-h infusion of 600 mg ceftaroline fosamil (ca. 10 mg/kg) (7). For each E. faecalis strain, animals were randomly assigned to receive no treatment (control), ceftaroline mimicking a human dose of 10 mg/kg/12 h (600 mg every 12 h), linezolid mimicking a human dose of 10 mg/kg/12 h (600 mg every 12 h), or vancomycin by continuous intravenous infusion in order to reach a 20- to 25-mg/liter steady-state concentration in serum (17). Statistical analyses were performed with GraphPad Prism v4.0 (GraphPad Software, San Diego, CA). For each strain, analysis of variance was used to compare the effects between different groups, followed by Bonferroni''s test to compare treated groups two by two. A P value of ≤0.05 was considered significant.The MICs of ceftaroline, linezolid, and vancomycin for strains EF 12704 and EF NJ1 were 2 and 1 mg/liter, 2 and 1 mg/liter, and 2 and >256 mg/liter, respectively. As shown in the time-kill curves (Fig. ​(Fig.1),1), linezolid at 12 mg/liter reduced the initial inoculum by less than 1 log after 24 h. Vancomycin similarly failed to exhibit bactericidal activity against either strain. In contrast, ceftaroline showed time-dependent killing and bactericidal activity at a clinically achievable concentration of 20 mg/liter against both the Van^s and Van^r strains.Open in a separate windowFIG. 1.Killing curves for different concentrations of ceftaroline, linezolid, and vancomycin against strains EF 12704 and EF NJ1. Circles, control; white squares, ceftaroline at 4 mg/liter; black squares, ceftaroline at 20 mg/liter; white triangles, linezolid at 2 mg/liter; black triangles, linezolid at 12 mg/liter; crosses, vancomycin at 25 mg/liter.In the in vivo study, linezolid significantly reduced bacterial counts of both E. faecalis strains in aortic valve vegetations from rabbits after 4 days of treatment but failed to demonstrate bactericidal activity (a <3-log reduction in CFU counts; Table ​Table1)1) (15). Vancomycin achieved a significant reduction in bacterial counts compared with those of the untreated control group but was not bactericidal against EF 12704 (only a 2-log reduction), despite the susceptibility of this strain. Vancomycin was ineffective against Van^r (VanA phenotype) strain EF NJ1.

TABLE 1.

Bacterial titers in vegetations after 4 days of treatment

In Vitro Activities of Telavancin and Vancomycin against Biofilm-Producing Staphylococcus aureus,S. epidermidis,and Enterococcus faecalis Strains

We investigated the activities of telavancin and vancomycin against biofilm-producing Staphylococcus and Enterococcus strains. At clinically attainable concentrations, telavancin was active against bacteria embedded in biofilm (minimal biofilm eradication concentration [MBEC], 0.125 to 2 μg/ml) and inhibited biofilm formation at concentrations below the MIC. Vancomycin did not demonstrate the same activity (MBEC, ≥512 μg/ml) against Staphylococcus aureus and Enterococcus faecalis. Telavancin may have a unique role in biofilm-associated infections.Staphylococci and enterococci account for a large proportion of hospital-acquired infections, especially among patients with indwelling devices (17). These infections are often caused by biofilm-producing strains which are difficult to eradicate and which may cause bacteremia and metastatic infections (30).Telavancin is a new lipoglycopeptide antimicrobial agent with a core chemical structure similar to that of vancomycin yet modified to include a lipophilic side chain (15). Telavancin possesses a second mechanism of action that causes rapid depolarization and loss of the functional integrity of the bacterial membrane (1, 12). These two mechanisms of action may be implicated in the lower range of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) MICs observed with telavancin (MIC range, 0.06 to 1.0 μg/ml) compared to vancomycin (MIC range, 0.5 to 2 μg/ml) (8, 13).Previous reports have suggested that telavancin is more effective than vancomycin against biofilm-forming S. aureus in a pharmacokinetic filter model (9). We compared telavancin and vancomycin activities by using previously described in vitro biofilm activity assays. The first assay evaluated each agent''s activity in a preformed biofilm by using both a modified version and the standard version of the Calgary Biofilm Pin Lid Device (CBPD) (3, 14). The second assay evaluated activity in preventing biofilm formation by planktonic isolates (14).(This work was presented in part at the 48th annual Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 24 to 28 October 2008.)Telavancin and vancomycin analytical powder, freshly prepared each day, was obtained from Astellas Pharmaceuticals, Inc. (Audubon, PA), and Sigma-Aldrich (St. Louis, MO), respectively. Well-characterized biofilm-producing reference strains of S. aureus (ATCC 35556), Staphylococcus epidermidis (RP62A; ATCC 35984), and Enterococcus faecalis (ATCC 29212; vancomycin susceptible) were evaluated. A stable, slime-negative mutant, M7, from wild-type S. epidermidis RP62A served as a control (21). We also evaluated three randomly selected, biofilm-producing clinical isolates (methicillin-susceptible S. aureus [MSSA] L2, and MRSA L32 and L83) previously obtained from patients with catheter-related bloodstream infections at the Providence Veterans Affairs Medical Center.The medium used for biofilm growth was Bacto tryptic soy broth (TSB; Becton Dickinson, Sparks, MD) plus 1% glucose and 2% NaCl (14). All assays were run in quadruplicate, and cultures were incubated at 35°C. Conventional MICs and minimal bactericidal concentrations (MBCs) were determined in duplicate by using Clinical and Laboratory Standards Institute (CLSI) guidelines (5, 6).We used a modified version of the CBPD to determine the antimicrobial susceptibility of bacteria embedded in a 24-h biofilm to determine the minimum biofilm inhibitory concentration (MBIC) and the minimum biofilm eradication concentration (MBEC) (3). Briefly, a starting inoculum of 7 log₁₀ CFU/ml was established by the direct colony suspension method from a 24-h tryptic soy agar (TSA; Becton Dickinson, Sparks, MD) plate. This inoculum was then standardized with McFarland standards and validated by determination of viable counts on TSA plates. Biofilms developed on the pin lid (Immuno TSP; Nunc, Roskilde, Denmark) submerged in the inoculated broth at 35°C for 24 h on a rocking table (Boekel Shake and Bake; Boekel Scientific; Feasterville, PA). The pin lid was then rinsed three times in 1× phosphate-buffered saline (PBS) to remove sessile bacteria before placement into fresh uninoculated broth containing serial dilutions of each antibiotic and incubated for 24 h at 35°C. The next day, the pin lid was removed and the MBIC was recorded and defined as the last well in which there is no visible growth after incubation in the presence of biofilm and antibiotic. Next, to obtain the MBEC, the pin lid was rinsed in 1× PBS and then sonication (10 min) was performed on a low-output sonicator (45 Hz) in order to disperse the bacteria from the pin surface. After sonication, the broth was vortexed for 30 s. The fluid was serially diluted and plated in duplicate on TSA, and the number of CFU per milliliter was determined. The limit of detection was 2.4 CFU/ml. Viable counts were also obtained by measuring the turbidity at 570 nm (A₅₇₀) on a 96-well plate reader.Quantification of biofilm formation in the presence of telavancin (0 to 16 μg/ml) and vancomycin (0 to 16 μg/ml) was conducted with a previously described colorimetric microtiter plate assay (4, 14, 24). Wells with sterile TSB alone served as negative controls, and the mean optical density (OD) values of these wells was subtracted from the OD values of the test wells.Following incubation, the liquid was gently aspirated and replaced with sterile PBS (pH 7.3). Each well was rinsed three times and air dried. Adherent bacteria were then stained with crystal violet. The OD at 570 nm (OD₅₇₀) of stained adherent bacterial films was read with a spectrophotometer (Synergy 2; Bio-Tek Instruments, Inc., Winooski, VT). The ODs of bacterial films were classified into nonadherent, weakly, moderately, and strongly adherent categories based on multiples of the OD readings as described by Stepanović et al. The test was carried out in quadruplicate. Results were averaged, and standard deviations were calculated (23).Among planktonic bacteria, the ranges of telavancin MICs and MBCs were 0.03 to 0.25 and 0.125 to 1 μg/ml, respectively. The ranges of vancomycin MICs and MBCs were 1 to 2 and 2 to 16 μg/ml, respectively (Table ​(Table1).1). When evaluating the activity of sessile bacteria seeding the medium from formed biofilm, the MBICs of telavancin and vancomycin were 0.25 to 1 and 4 to 16 μg/ml, respectively (Table ​(Table1).1). Overall, the telavancin and vancomycin MBICs for each bacterial isolate were the same as the corresponding MICs or up to 3 dilutions higher.

TABLE 1.

Susceptibility testing results for planktonic and adherent (biofilm) strains

莫匹罗星、利奈唑胺、万古霉素和替加环素对MRSA的体外活性研究

目的：研究莫匹罗星、利奈唑胺、万古霉素和替加环素对甲氧西林耐药的金黄色葡萄球菌（MRSA）的体外活性。方法采用E-试验法对2010年1月～2011年6月我院分离的88株MRSA进行莫匹罗星、利奈唑胺、万古霉素和替加环素的MIC检测,并比较MIC50和MIC90的大小。结果莫匹罗星、利奈唑胺、万古霉素和替加环素对MRSA的体外活性均较好,全部敏感且表现为极低的MIC值。其中莫匹罗星和替加环素的MIC90均未超过0．25μg/mL。结论莫匹罗星、利奈唑胺、万古霉素和替加环素对MRSA活性较好,可作为MRSA感染治疗的有效选择,莫匹罗星是治疗MRSA相关的皮肤和浅表外伤感染的有效的局部外用抗菌药物。     

In Vitro Activity of Ceftaroline against Staphylococcus aureus Isolated in 2012 from Asia-Pacific Countries as Part of the AWARE Surveillance Program

Ceftaroline, the active metabolite of the prodrug ceftaroline-fosamil, is an advanced-generation cephalosporin with activity against methicillin-resistant Staphylococcus aureus (MRSA). This investigation provides in vitro susceptibility data for ceftaroline against 1,971 S. aureus isolates collected in 2012 from seven countries (26 centers) in the Asia-Pacific region as part of the Assessing Worldwide Antimicrobial Resistance and Evaluation (AWARE) program. Broth microdilution as recommended by the CLSI was used to determine susceptibility. In all, 62% of the isolates studied were MRSA, and the ceftaroline MIC₉₀ for all S. aureus isolates was 2 μg/ml (interpretive criteria: susceptible, ≤1 μg/ml). The overall ceftaroline susceptibility rate for S. aureus was 86.9%, with 100% of methicillin-sensitive S. aureus isolates and 78.8% of MRSA isolates susceptible to this agent. The highest percentages of ceftaroline-nonsusceptible MRSA isolates came from China (47.6%), all of which showed intermediate susceptibility, and Thailand (37.1%), where over half (52.8%) of isolates were resistant to ceftaroline (MIC, 4 μg/ml). Thirty-eight ceftaroline-nonsusceptible isolates (MIC values of 2 to 4 μg/ml) were selected for molecular characterization. Among the isolates analyzed, sequence type 5 (ST-5) was the most common sequence type encountered; however, all isolates analyzed from Thailand were ST-228. Penicillin-binding protein 2a (PBP2a) substitution patterns varied by country, but all isolates from Thailand had the Glu₂₃₉Lys substitution, and 12 of these also carried an additional Glu₄₄₇Lys substitution. Ceftaroline-fosamil is a useful addition to the antimicrobial agents that can be used to treat S. aureus infections. However, with the capability of this species to develop resistance to new agents, it is important to recognize and monitor regional differences in trends as they emerge.     

In Vitro Studies of Pharmacodynamic Properties of Vancomycin against Staphylococcus aureus and Staphylococcus epidermidis

The bactericidal activities of vancomycin against two reference strains and two clinical isolates of Staphylococcus aureus and Staphylococcus epidermidis were studied with five different concentrations ranging from 2× to 64× the MIC. The decrease in the numbers of CFU at 24 h was at least 3 log₁₀ CFU/ml for all strains. No concentration-dependent killing was observed. The postantibiotic effect (PAE) was determined by obtaining viable counts for two of the reference strains, and the viable counts varied markedly: 1.2 h for S. aureus and 6.0 h for S. epidermidis. The determinations of the PAE, the postantibiotic sub-MIC effect (PA SME), and the sub-MIC effect (SME) for all strains were done with BioScreen C, a computerized incubator for bacteria. The PA SMEs were longer than the SMEs for all strains tested. A newly developed in vitro kinetic model was used to expose the bacteria to continuously decreasing concentrations of vancomycin. A filter prevented the loss of bacteria during the experiments. One reference strain each of S. aureus and S. epidermidis and two clinical isolates of S. aureus were exposed to an initial concentration of 10× the MIC of vancomycin with two different half-lives (t_1/2s): 1 or 5 h. The post-MIC effect (PME) was calculated as the difference in time for the bacteria to grow 1 log₁₀ CFU/ml from the numbers of CFU obtained at the time when the MIC was reached and the corresponding time for an unexposed control culture. The difference in PME between the strains was not as pronounced as that for the PAE. Furthermore, the PME was shorter when a t_1/2 of 5 h (approximate terminal t_1/2 in humans) was used. The PMEs at t_1/2s of 1 and 5 h were 6.5 and 3.6 h, respectively, for S. aureus. The corresponding figures for S. epidermidis were 10.3 and less than 6 h. The shorter PMEs achieved with a t_1/2 of 5 h and the lack of concentration-dependent killing indicate that the time above the MIC is the parameter most important for the efficacy of vancomycin.