Activities of Fosfomycin and Rifampin on Planktonic and Adherent Enterococcus faecalis Strains in an Experimental Foreign-Body Infection Model

Enterococcal implant-associated infections are difficult to treat because antibiotics generally lack activity against enterococcal biofilms. We investigated fosfomycin, rifampin, and their combinations against planktonic and adherent Enterococcus faecalis (ATCC 19433) in vitro and in a foreign-body infection model. The MIC/MBC_log values were 32/>512 μg/ml for fosfomycin, 4/>64 μg/ml for rifampin, 1/2 μg/ml for ampicillin, 2/>256 μg/ml for linezolid, 16/32 μg/ml for gentamicin, 1/>64 μg/ml for vancomycin, and 1/5 μg/ml for daptomycin. In time-kill studies, fosfomycin was bactericidal at 8× and 16× MIC, but regrowth of resistant strains occurred after 24 h. With the exception of gentamicin, no complete inhibition of growth-related heat production was observed with other antimicrobials on early (3 h) or mature (24 h) biofilms. In the animal model, fosfomycin alone or in combination with daptomycin reduced planktonic counts by ≈4 log₁₀ CFU/ml below the levels before treatment. Fosfomycin cleared planktonic bacteria from 74% of cage fluids (i.e., no growth in aspirated fluid) and eradicated biofilm bacteria from 43% of cages (i.e., no growth from removed cages). In combination with gentamicin, fosfomycin cleared 77% and cured 58% of cages; in combination with vancomycin, fosfomycin cleared 33% and cured 18% of cages; in combination with daptomycin, fosfomycin cleared 75% and cured 17% of cages. Rifampin showed no activity on planktonic or adherent E. faecalis, whereas in combination with daptomycin it cured 17% and with fosfomycin it cured 25% of cages. Emergence of fosfomycin resistance was not observed in vivo. In conclusion, fosfomycin showed activity against planktonic and adherent E. faecalis. Its role against enterococcal biofilms should be further investigated, especially in combination with rifampin and/or daptomycin treatment.     

High Activity of Fosfomycin and Rifampin against Methicillin-Resistant Staphylococcus aureus Biofilm In Vitro and in an Experimental Foreign-Body Infection Model

Increasing antimicrobial resistance reduces treatment options for implant-associated infections caused by methicillin-resistant Staphylococcus aureus (MRSA). We evaluated the activity of fosfomycin alone and in combination with vancomycin, daptomycin, rifampin, and tigecycline against MRSA (ATCC 43300) in a foreign-body (implantable cage) infection model. The MICs of the individual agents were as follows: fosfomycin, 1 μg/ml; daptomycin, 0.125 μg/ml; vancomycin, 1 μg/ml; rifampin, 0.04 μg/ml; and tigecycline, 0.125 μg/ml. Microcalorimetry showed synergistic activity of fosfomycin and rifampin at subinhibitory concentrations against planktonic and biofilm MRSA. In time-kill curves, fosfomycin exhibited time-dependent activity against MRSA with a reduction of 2.5 log₁₀ CFU/ml at 128 × the MIC. In the animal model, planktonic bacteria in cage fluid were reduced by <1 log₁₀ CFU/ml with fosfomycin and tigecycline, 1.7 log₁₀ with daptomycin, 2.2 log₁₀ with fosfomycin-tigecycline and fosfomycin-vancomycin, 3.8 log₁₀ with fosfomycin-daptomycin, and >6.0 log₁₀ with daptomycin-rifampin and fosfomycin-rifampin. Daptomycin-rifampin cured 67% of cage-associated infections and fosfomycin-rifampin cured 83%, whereas all single drugs (fosfomycin, daptomycin, and tigecycline) and rifampin-free fosfomycin combinations showed no cure of MRSA cage-associated infections. No emergence of fosfomycin resistance was observed in animals; however, a 4-fold increase in fosfomycin MIC (from 2 to 16 μg/ml) occurred in the fosfomycin-vancomycin group. In summary, the highest eradication of MRSA cage-associated infections was achieved with fosfomycin in combination with rifampin (83%). Fosfomycin may be used in combination with rifampin against MRSA implant-associated infections, but it cannot replace rifampin as an antibiofilm agent.     

Efficacy of Daptomycin in Implant-Associated Infection Due to Methicillin-Resistant Staphylococcus aureus: Importance of Combination with Rifampin

Limited treatment options are available for implant-associated infections caused by methicillin (meticillin)-resistant Staphylococcus aureus (MRSA). We compared the activity of daptomycin (alone and with rifampin [rifampicin]) with the activities of other antimicrobial regimens against MRSA ATCC 43300 in the guinea pig foreign-body infection model. The daptomycin MIC and the minimum bactericidal concentration in logarithmic phase and stationary growth phase of MRSA were 0.625, 0.625, and 20 μg/ml, respectively. In time-kill studies, daptomycin showed rapid and concentration-dependent killing of MRSA in stationary growth phase. At concentrations above 20 μg/ml, daptomycin reduced the counts by >3 log₁₀ CFU/ml in 2 to 4 h. In sterile cage fluid, daptomycin peak concentrations of 23.1, 46.3, and 53.7 μg/ml were reached 4 to 6 h after the administration of single intraperitoneal doses of 20, 30, and 40 mg/kg of body weight, respectively. In treatment studies, daptomycin alone reduced the planktonic MRSA counts by 0.3 log₁₀ CFU/ml, whereas in combination with rifampin, a reduction in the counts of >6 log₁₀ CFU/ml was observed. Vancomycin and daptomycin (at both doses) were unable to cure any cage-associated infection when they were given as monotherapy, whereas rifampin alone cured the infections in 33% of the cages. In combination with rifampin, daptomycin showed cure rates of 25% (at 20 mg/kg) and 67% (at 30 mg/kg), vancomycin showed a cure rate of 8%, linezolid showed a cure rate of 0%, and levofloxacin showed a cure rate of 58%. In addition, daptomycin at a high dose (30 mg/kg) completely prevented the emergence of rifampin resistance in planktonic and adherent MRSA cells. Daptomycin at a high dose, corresponding to 6 mg/kg in humans, in combination with rifampin showed the highest activity against planktonic and adherent MRSA. Daptomycin plus rifampin is a promising treatment option for implant-associated MRSA infections.Implants are increasingly used in modern medicine to replace a compromised biological function or missing anatomical structure. Periprosthetic infections represent a devastating complication, causing high rates of morbidity and consuming considerable health care resources. Implant-associated infections are caused by microorganisms growing adherent to the device surface and embedded in an extracellular polymeric matrix, a complex three-dimensional structure called a microbial biofilm (8). Bacterial communities in biofilms cause persistent infection due to increased resistance to antibiotics and the immune system and the difficulty with eradicating them from the implant (6).Staphylococcus aureus is one of the leading pathogens causing implant-associated infections. Successful treatment requires the use of bactericidal drugs acting on surface-adhering microorganisms, which predominantly exist in the stationary growth phase. Previous in vitro, experimental, and clinical studies demonstrated that rifampin (rifampicin)-containing antimicrobial regimens were able to eradicate staphylococcal biofilms and cure implant-associated infections (23, 25). Quinolones are often used in combination with rifampin in order to prevent the emergence of rifampin resistance (4, 19, 21). However, methicillin (meticillin)-resistant S. aureus (MRSA) strains are often resistant to quinolones. In addition, MRSA strains were recently shown to have decreased susceptibility to vancomycin, reducing the efficacy of this drug. Therefore, alternative drugs for use in combination with rifampin against implant-associated infections are needed (12, 20).Daptomycin is a negatively charged cyclic lipopeptide with bactericidal activity against gram-positive organisms, including MRSA (17). The drug inserts into the bacterial cytoplasmic membrane in a calcium-dependent fashion, leading to rapid cell death without lysis, and causing only minimal inflammation (15). Daptomycin has been well tolerated in healthy volunteers dosed with up to 12 mg/kg of body weight intravenously for 14 days (2). Only limited data on the use of daptomycin in combination with rifampin against staphylococcal implant-associated infections are available.In this study, we investigated the activity of daptomycin against MRSA ATCC 43300 in vitro. In addition, we evaluated the activity of daptomycin in combination with rifampin in a cage-associated infection model in guinea pigs and compared the efficacy of the treatment with the efficacies of three other antibiotics commonly used against MRSA, vancomycin, linezolid, and levofloxacin (alone and in combination with rifampin).(Part of the results of the present study were presented at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 24 to 29 October 2008 [abstr. B-1000].)     

Fosfomycin-Daptomycin and Other Fosfomycin Combinations as Alternative Therapies in Experimental Foreign-Body Infection by Methicillin-Resistant Staphylococcus aureus

The efficacy of daptomycin, imipenem, or rifampin with fosfomycin was evaluated and compared with that of daptomycin-rifampin in a tissue cage model infection caused by methicillin-resistant Staphylococcus aureus (MRSA). Strain HUSA 304 was used. The study yielded the following results for MICs (in μg/ml): fosfomycin, 4; daptomycin, 1; imipenem, 0.25; and rifampin, 0.03. The study yielded the following results for minimum bactericidal concentration (MBC) (in μg/ml): fosfomycin, 8; daptomycin, 4; imipenem, 32; and rifampin, 0.5. Daptomycin-rifampin was confirmed as the most effective therapy against MRSA foreign-body infections. Fosfomycin combinations with high doses of daptomycin and rifampin were efficacious alternative therapies in this setting. Fosfomycin-imipenem was relatively ineffective and did not protect against resistance.     

Prospective, open-label investigation of the pharmacokinetics of daptomycin during cardiopulmonary bypass surgery

As methicillin-resistant Staphylococcus aureus (MRSA) becomes more prevalent, vancomycin is becoming increasingly used as a prophylaxis against surgical-site infections for cardiothoracic surgeries. However, vancomycin administration can be challenging, and the pharmacokinetics of alternative antibiotics in this setting are poorly understood. The primary objective of this investigation was to describe the pharmacokinetics of daptomycin in patients undergoing coronary artery bypass graft surgery. We enrolled 15 patients undergoing coronary artery bypass surgery requiring cardiopulmonary bypass. Each subject was administered a single open-label dose of daptomycin (8 mg/kg of body weight) for surgical prophylaxis. Fourteen daptomycin plasma samples were collected. Safety outcomes between subjects who received daptomycin and 15 control subjects who received the standard-of-care antibiotic were compared. The mean maximal concentration of daptomycin (C_max) was 84.4 ± 27.1 μg/ml; the mean daptomycin concentration during the cardiopulmonary bypass procedure was 33.2 ± 11.4 μg/ml and was 30.9 ± 12.7 μg/ml at sternum closure. Mean daptomycin concentrations at 12, 18, 24, and 48 h were 22.7 ± 9.7, 16.2 ± 8.2, 12.0 ± 4.7, and 3.5 ± 2.3 μg/ml, respectively. Mean daptomycin concentrations were consistently above the MIC at which 90% of the tested isolates are inhibited (MIC₉₀) for S. aureus and S. epidermidis during the cardiopulmonary bypass procedure. Daptomycin was not associated with surgical-site infections or differences in adverse events compared to findings for control subjects. We found that a single dose of daptomycin at 8 mg/kg was well tolerated and achieved adequate plasma concentrations against common pathogens associated with surgical-site infections after cardiothoracic surgery. Daptomycin may be considered an alternative surgical prophylaxis antibiotic for patients undergoing cardiothoracic bypass surgery who are unable to receive vancomycin.     

Efficacy of usual and high doses of daptomycin in combination with rifampin versus alternative therapies in experimental foreign-body infection by methicillin-resistant Staphylococcus aureus

The treatment of prosthetic joint infections caused by methicillin-resistant Staphylococcus aureus (MRSA) continues to be a challenge for the clinician. The aim of this study was to evaluate the efficacies of daptomycin at usual and high doses (equivalent to 6 and 10 mg/kg of body weight/day, respectively, in humans) and in combination with rifampin and to compare the activities to those of conventional anti-MRSA therapies. We used MRSA strain HUSA 304, with the following MICs and minimal bactericidal concentrations (MBCs), respectively: daptomycin, 1 μg/ml and 4 μg/ml; vancomycin, 2 μg/ml and 4 μg/ml; linezolid, 2 μg/ml and >32 μg/ml; and rifampin, 0.03 μg/ml and 0.5 μg/ml. In time-kill curves, only daptomycin and its combinations with rifampin achieved a bactericidal effect in log and stationary phases. For in vivo studies, we used a rat foreign-body infection model. Therapy was administered for 7 days with daptomycin at 100 mg/kg/day and 45/mg/kg/day, vancomycin at 50 mg/kg/12 h, rifampin at 25 mg/kg/12 h, and linezolid at 35 mg/kg/12 h, and each antibiotic was also combined with rifampin. Among monotherapies, daptomycin at 100 mg/kg/day and rifampin performed better than vancomycin and linezolid. In combination with rifampin, both dosages of daptomycin were significantly better than all other combinations, but daptomycin at 100 mg/kg/day plus rifampin achieved better cure rates at day 11 (P < 0.05) than daptomycin at 45 mg/kg/day plus rifampin. Resistant strains were found in monotherapies with rifampin and daptomycin at 45 mg/kg/day. In conclusion, daptomycin at high doses was the most effective monotherapy and also improved the efficacy of the combination with rifampin against foreign-body infections by MRSA. Clinical studies should confirm whether this combination may be considered the first-line treatment for foreign-body infections by MRSA in humans.     

Antimicrobial Activity of Ceftaroline Tested against Staphylococci with Reduced Susceptibility to Linezolid,Daptomycin, or Vancomycin from U.S. Hospitals, 2008 to 2011

Vancomycin, linezolid, and daptomycin are very active against staphylococci, but isolates with decreased susceptibility to these antimicrobial agents are isolated sporadically. A total of 19,350 Staphylococcus aureus isolates (51% methicillin resistant [MRSA]) and 3,270 coagulase-negative staphylococci (CoNS) were collected consecutively from 82 U.S. medical centers from January 2008 to December 2011 and tested for susceptibility against ceftaroline and comparator agents by the reference broth microdilution method. Among S. aureus strains, 14 isolates (0.07%) exhibited decreased susceptibility to linezolid (MIC, ≥8 μg/ml), 18 (0.09%) to daptomycin (MIC, ≥2 μg/ml), and 369 (1.9%) to vancomycin (MIC, ≥2 μg/ml; 368 isolates at 2 μg/ml and 1 at 4 μg/ml). Fifty-one (1.6%) CoNS were linezolid resistant (MIC, ≥8 μg/ml), and four (0.12%) were daptomycin nonsusceptible (MIC, ≥2 μg/ml). Ceftaroline was very active against S. aureus overall (MIC_50/90, 0.5/1 μg/ml; 98.5% susceptible), including MRSA (MIC_50/90, 0.5/1 μg/ml; 97.2% susceptible). All daptomycin-nonsusceptible and 85.7% of linezolid-resistant S. aureus isolates were susceptible to ceftaroline. Against S. aureus isolates with a vancomycin MIC of ≥2 μg/ml, 91.9, 96.2, and 98.9% were susceptible to ceftaroline, daptomycin, and linezolid, respectively. CoNS strains were susceptible to ceftaroline (MIC_50/90, 0.25/0.5 μg/ml; 99.1% inhibited at ≤1 μg/ml), including methicillin-resistant (MIC_50/90, 0.25/0.5 μg/ml), linezolid-resistant (MIC_50/90, 0.5/0.5 μg/ml), and daptomycin-nonsusceptible (4 isolates; MIC range, 0.03 to 0.12 μg/ml) strains. In conclusion, ceftaroline demonstrated potent in vitro activity against staphylococci with reduced susceptibility to linezolid, daptomycin, or vancomycin, and it may represent a valuable treatment option for infections caused by these multidrug-resistant staphylococci.     

In Vitro Activity of Rifampin Alone and in Combination with Nafcillin and Vancomycin Against Pathogenic Strains of Staphylococcus aureus

Twenty strains of Staphylococcus aureus isolated from patients with endocarditis were examined in vitro for susceptibility to rifampin, nafcillin, and vancomycin and to combinations of rifampin with nafcillin or vancomycin. Minimum bactericidal concentrations of rifampin ranged from 0.0031 to 0.0125 μg/ml, of nafcillin ranged from 0.078 to 0.312 μg/ml, and of vancomycin ranged from 0.312 to 1.25 μg/ml. The combination of rifampin with nafcillin was synergistic for 12 strains; the combination of rifampin plus vancomycin was synergistic for 5 of the isolates.     

In vitro activities of dalbavancin and nine comparator agents against anaerobic gram-positive species and corynebacteria

Dalbavancin is a novel semisynthetic glycopeptide with enhanced activity against gram-positive species. Its comparative in vitro activities and those of nine comparator agents, including daptomycin, vancomycin, linezolid, and quinupristin-dalfopristin, against 290 recent gram-positive clinical isolates strains, as determined by the NCCLS agar dilution method, were studied. The MICs of dalbavancin at which 90% of various isolates tested were inhibited were as follows: Actinomyces spp., 0.5 μg/ml; Clostridium clostridioforme, 8 μg/ml; C. difficile, 0.25 μg/ml; C. innocuum, 0.25 μg/ml; C. perfringens, 0.125 μg/ml; C. ramosum, 1 μg/ml; Eubacterium spp., 1 μg/ml; Lactobacillus spp., >32 μg/ml, Propionibacterium spp., 0.5 μg/ml; and Peptostreptococcus spp., 0.25 μg/ml. Dalbavancin was 1 to 3 dilutions more active than vancomycin against most strains. Dalbavancin exhibited excellent activity against gram-positive strains tested and warrants clinical evaluation.     

In vitro activity of TD-1792, a multivalent glycopeptide-cephalosporin antibiotic, against 377 strains of anaerobic bacteria and 34 strains of Corynebacterium species

TD-1792 is a multivalent glycopeptide-cephalosporin heterodimer antibiotic with potent activity against Gram-positive bacteria. We tested TD-1792 against 377 anaerobes and 34 strains of Corynebacterium species. Against nearly all Gram-positive strains, TD-1792 had an MIC₉₀ of 0.25 μg/ml and was typically 3 to 7 dilutions more active than vancomycin and daptomycin.     

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.     

Oritavancin Activity against Staphylococcus aureus Causing Invasive Infections in U.S. and European Hospitals: a 5-Year International Surveillance Program

In this study, oritavancin had modal MIC, MIC₅₀, and MIC₉₀ values of 0.03, 0.03, and 0.06 μg/ml, respectively, against Staphylococcus aureus. Similar results (MIC_50/90, 0.03/0.06 μg/ml) were observed against methicillin-resistant and -susceptible isolates and those demonstrating multidrug-resistant (MDR) and non-MDR phenotypes. When oritavancin (MIC_50/90, 0.06/0.12 mg/ml) was tested against S. aureus with elevated MIC values for daptomycin (i.e., 1 to 4 mg/ml) and vancomycin (i.e., 2 mg/ml), it showed MIC results 2-fold higher than those for the more susceptible vancomycin or daptomycin counterparts (MIC_50/90, 0.03/0.06 mg/ml), yet it inhibited these isolates at ≤0.25 mg/ml.     

Activities of Fluconazole,Caspofungin, Anidulafungin,and Amphotericin B on Planktonic and Biofilm Candida Species Determined by Microcalorimetry

We investigated the activities of fluconazole, caspofungin, anidulafungin, and amphotericin B against Candida species in planktonic form and biofilms using a highly sensitive assay measuring growth-related heat production (microcalorimetry). C. albicans, C. glabrata, C. krusei, and C. parapsilosis were tested, and MICs were determined by the broth microdilution method. The antifungal activities were determined by isothermal microcalorimetry at 37°C in RPMI 1640. For planktonic Candida, heat flow was measured in the presence of antifungal dilutions for 24 h. Candida biofilm was formed on porous glass beads for 24 h and exposed to serial dilutions of antifungals for 24 h, and heat flow was measured for 48 h. The minimum heat inhibitory concentration (MHIC) was defined as the lowest antifungal concentration reducing the heat flow peak by ≥50% (≥90% for amphotericin B) at 24 h for planktonic Candida and at 48 h for Candida biofilms (measured also at 24 h). Fluconazole (planktonic MHICs, 0.25 to >512 μg/ml) and amphotericin B (planktonic MHICs, 0.25 to 1 μg/ml) showed higher MHICs than anidulafungin (planktonic MHICs, 0.015 to 0.5 μg/ml) and caspofungin (planktonic MHICs, 0.125 to 0.5 μg/ml). Against Candida species in biofilms, fluconazole''s activity was reduced by >1,000-fold compared to its activity against the planktonic counterparts, whereas echinocandins and amphotericin B mainly preserved their activities. Fluconazole induced growth of planktonic C. krusei at sub-MICs. At high concentrations of caspofungin (>4 μg/ml), paradoxical growth of planktonic C. albicans and C. glabrata was observed. Microcalorimetry enabled real-time evaluation of antifungal activities against planktonic and biofilm Candida organisms. It can be used in the future to evaluate new antifungals and antifungal combinations and to study resistant strains.     

Dalbavancin is active in vitro against biofilms formed by dalbavancin-susceptible enterococci

We tested the in vitro activity of dalbavancin, vancomycin and daptomycin against 83 enterococcal isolates in planktonic and biofilm states. The MIC₉₀ for vancomycin-susceptible Enterococcus faecalis was 0.125 and 4 μg/mL for dalbavancin and daptomycin, respectively. For vancomycin-resistant Enterococcus faecium, the MIC₉₀ was >16 and 2 μg/mL for dalbavancin and daptomycin, respectively. Dalbavancin minimum biofilm inhibitory concentrations (MBICs) for vancomycin-susceptible and -resistant isolates were ≤0.25 and >16 μg/mL, respectively. The daptomycin MBIC₉₀ for all isolates was 4 μg/mL. For E. faecalis and E. faecium, dalbavancin minimum biofilm bactericidal concentrations (MBBCs) for vancomycin-susceptible and -resistant isolates were ≤4 and >16 μg/mL, respectively, whereas vancomycin MBBCs were >128 μg/mL for all isolates, and daptomycin MBBC₉₀ values for both species were 128 μg/mL. In summary, dalbavancin exhibited in vitro activity against all tested isolates of vancomycin-susceptible, but not against vancomycin-resistant enterococci; activity was observed in both the planktonic and biofilm states.     

Eravacycline (TP-434) Is Active In Vitro against Biofilms Formed by Uropathogenic Escherichia coli

Eravacycline (formerly TP-434) was evaluated in vitro against pre-established biofilms formed by a uropathogenic Escherichia coli strain. Biofilms were eradicated by 0.5 μg/ml eravacycline, which was within 2-fold of the MIC for planktonic cells. In contrast, colistin and meropenem disrupted biofilms at 32 and 2 μg/ml, respectively, concentrations well above their respective MICs of 0.5 and 0.03 μg/ml. Gentamicin and levofloxacin eradicated biofilms at concentrations within 2-fold of their MICs.     

In Vitro Activity of Retapamulin against Staphylococcus aureus Resistant to Various Antimicrobial Agents

Retapamulin and six other antimicrobial agents were evaluated against 155 methicillin-resistant Staphylococcus aureus (MRSA) isolates, including strains resistant to vancomycin, linezolid, daptomycin, and mupirocin by microdilution tests. Time-kill assays were performed against representative MRSA, vancomycin-intermediate S. aureus (VISA), and vancomycin-resistant S. aureus (VRSA) isolates. Retapamulin and mupirocin demonstrated MIC₉₀s of 0.12 μg/ml and 8 μg/ml, respectively, with resistance seen in 2.6% and 10% of isolates, respectively. Retapamulin maintained good activity against 94% (15/16) of mupirocin-resistant isolates.     

Activities of Fosfomycin,Tigecycline, Colistin,and Gentamicin against Extended-Spectrum-β-Lactamase-Producing Escherichia coli in a Foreign-Body Infection Model

Limited antimicrobial agents are available for the treatment of implant-associated infections caused by fluoroquinolone-resistant Gram-negative bacilli. We compared the activities of fosfomycin, tigecycline, colistin, and gentamicin (alone and in combination) against a CTX-M15-producing strain of Escherichia coli (Bj HDE-1) in vitro and in a foreign-body infection model. The MIC and the minimal bactericidal concentration in logarithmic phase (MBC_log) and stationary phase (MBC_stat) were 0.12, 0.12, and 8 μg/ml for fosfomycin, 0.25, 32, and 32 μg/ml for tigecycline, 0.25, 0.5, and 2 μg/ml for colistin, and 2, 8, and 16 μg/ml for gentamicin, respectively. In time-kill studies, colistin showed concentration-dependent activity, but regrowth occurred after 24 h. Fosfomycin demonstrated rapid bactericidal activity at the MIC, and no regrowth occurred. Synergistic activity between fosfomycin and colistin in vitro was observed, with no detectable bacterial counts after 6 h. In animal studies, fosfomycin reduced planktonic counts by 4 log₁₀ CFU/ml, whereas in combination with colistin, tigecycline, or gentamicin, it reduced counts by >6 log₁₀ CFU/ml. Fosfomycin was the only single agent which was able to eradicate E. coli biofilms (cure rate, 17% of implanted, infected cages). In combination, colistin plus tigecycline (50%) and fosfomycin plus gentamicin (42%) cured significantly more infected cages than colistin plus gentamicin (33%) or fosfomycin plus tigecycline (25%) (P < 0.05). The combination of fosfomycin plus colistin showed the highest cure rate (67%), which was significantly better than that of fosfomycin alone (P < 0.05). In conclusion, the combination of fosfomycin plus colistin is a promising treatment option for implant-associated infections caused by fluoroquinolone-resistant Gram-negative bacilli.     

Gentamicin improves the activities of daptomycin and vancomycin against Enterococcus faecalis in vitro and in an experimental foreign-body infection model

For enterococcal implant-associated infections, the optimal treatment regimen has not been defined. We investigated the activity of daptomycin, vancomycin, and gentamicin (and their combinations) against Enterococcus faecalis in vitro and in a foreign-body infection model. Antimicrobial activity was investigated by time-kill and growth-related heat production studies (microcalorimetry) as well as with a guinea pig model using subcutaneously implanted cages. Infection was established by percutaneous injection of E. faecalis in the cage. Antibiotic treatment for 4 days was started 3 h after infection. Cages were removed 5 days after end of treatment to determine the cure rate. The MIC, the minimal bactericidal concentration (MBC) in the logarithmic phase, and the MBC in the stationary phase were 1.25, 5, and >20 μg/ml for daptomycin, 1, >64, and >64 μg/ml for vancomycin, and 16, 32, and 4 μg/ml for gentamicin, respectively. In vitro, gentamicin at subinhibitory concentrations improved the activity against E. faecalis when combined with daptomycin or vancomycin in the logarithmic and stationary phases. In the animal model, daptomycin cured 25%, vancomycin 17%, and gentamicin 50% of infected cages. In combination with gentamicin, the cure rate for daptomycin increased to 55% and that of vancomycin increased to 33%. In conclusion, daptomycin was more active than vancomycin against adherent E. faecalis, and its activity was further improved by the addition of gentamicin. Despite a short duration of infection (3 h), the cure rates did not exceed 55%, highlighting the difficulty of eradicating E. faecalis from implants already in the early stage of implant-associated infection.     

A High-Affinity Native Human Antibody Disrupts Biofilm from Staphylococcus aureus Bacteria and Potentiates Antibiotic Efficacy in a Mouse Implant Infection Model

Many serious bacterial infections are difficult to treat due to biofilm formation, which provides physical protection and induces a sessile phenotype refractory to antibiotic treatment compared to the planktonic state. A key structural component of biofilm is extracellular DNA, which is held in place by secreted bacterial proteins from the DNABII family: integration host factor (IHF) and histone-like (HU) proteins. A native human monoclonal antibody, TRL1068, has been discovered using single B-lymphocyte screening technology. It has low-picomolar affinity against DNABII homologs from important Gram-positive and Gram-negative bacterial pathogens. The disruption of established biofilm was observed in vitro at an antibody concentration of 1.2 μg/ml over 12 h. The effect of TRL1068 in vivo was evaluated in a murine tissue cage infection model in which a biofilm is formed by infection with methicillin-resistant Staphylococcus aureus (MRSA; ATCC 43300). Treatment of the established biofilm by combination therapy of TRL1068 (15 mg/kg of body weight, intraperitoneal [i.p.] administration) with daptomycin (50 mg/kg, i.p.) significantly reduced adherent bacterial count compared to that after daptomycin treatment alone, accompanied by significant reduction in planktonic bacterial numbers. The quantification of TRL1068 in sample matrices showed substantial penetration of TRL1068 from serum into the cage interior. TRL1068 is a clinical candidate for combination treatment with standard-of-care antibiotics to overcome the drug-refractory state associated with biofilm formation, with potential utility for a broad spectrum of difficult-to-treat bacterial infections.     

Nine-Hospital Study Comparing Broth Microdilution and Etest Method Results for Vancomycin and Daptomycin against Methicillin-Resistant Staphylococcus aureus

Vancomycin and daptomycin MIC results for 1,800 randomly selected oxacillin (methicillin [meticillin])-resistant Staphylococcus aureus (MRSA) bloodstream isolates from nine U.S. hospitals (collected from 2002 to 2006) were determined by a reference broth microdilution (BMD) method using frozen-form panels with precise incremental dilutions and by the Etest technique. The Etest provided vancomycin and daptomycin MIC results that were consistently higher (0.5 to 1.5 log₂ dilution steps) than those provided by the reference BMD method. The dominant MRSA population (91.2% of MRSA isolates) would be categorized as vancomycin nonsusceptible by the MIC results from the Etest method if the susceptibility breakpoint was adjusted downward to ≤1 μg/ml, as suggested by clinical outcome studies.Vancomycin is still used extensively for the treatment of oxacillin (methicillin [meticillin])-resistant Staphylococcus aureus (MRSA) bacteremia, as well as other less serious MRSA infections. However, vancomycin treatment failure is not uncommon, even when the MRSA strains are fully susceptible to vancomycin according to the criterion (breakpoint MIC, ≤2 μg/ml) used by the Clinical and Laboratory Standards Institute (CLSI). A reduction in the efficacy of vancomycin against MRSA strains for which vancomycin MICs are elevated (to 1 to 2 μg/ml) has been widely reported, suggesting that modest elevations in MICs may explain some suboptimal clinical outcomes (10, 13).The vast majority of clinical laboratories use automated systems and a distinct minority use the disk diffusion method as the routine susceptibility testing technique. However, the disk diffusion method and some automated systems do not accurately detect vancomycin-intermediate S. aureus (7). In addition, there have been a growing number of reports showing discrepancies between in vitro susceptibility test results for vancomycin and clinical outcomes of MRSA infections treated with this glycopeptide (6, 11). Thus, many laboratories are being requested to assess exact MICs by reference or alternative methods, such as that of Etest (AB Biodisk, Solna, Sweden).Although daptomycin remains very active against S. aureus, an association between reduced susceptibility to vancomycin and reduced susceptibility to daptomycin has been reported by some investigators (1). S. aureus isolates classified as daptomycin nonsusceptible according to MICs (≥2 μg/ml) are still rare, and no definitive resistance mechanism has been identified. However, genetic mutations and increases in daptomycin MICs after prolonged vancomycin and/or daptomycin treatment of infections associated with septic arthritis, osteomyelitis, septic thrombophlebitis, and endocarditis, especially in the presence of intravenous catheters and other prosthetic devices, have been reported previously (1). Thus, microbiology laboratories may be required to perform proper daptomycin susceptibility testing. Since a daptomycin disk diffusion test has not been developed, daptomycin susceptibility has to be evaluated by reference methods (2, 3) or the Etest method. In the present study, we evaluated the correlation between vancomycin and daptomycin MICs obtained by the Etest technique and the CLSI reference broth microdilution (BMD) method.(This study was presented in part at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy-46th Infectious Diseases Society of America meeting, Washington, DC, 25 to 28 October 2008.)A total of 1,800 MRSA strains from nine hospitals, one in each of the U.S. census regions, were tested. Each medical center contributed 200 MRSA strains collected from patients with bloodstream infections from 2002 through 2006 (target, 40 strains per year per center). The participant centers were as follows: Tufts Medical Center, Boston, MA; New York Hospital Queens, New York, NY; Ochsner Clinic Foundation, New Orleans, LA; University of Colorado Denver, Aurora, CO; University of Nebraska Medical Center, Omaha, NE; University of Washington, Seattle, WA; University of Alabama at Birmingham, Birmingham, AL; Wayne State University, Detroit, MI; and the Medical University of South Carolina, Charleston, SC.MICs of daptomycin and vancomycin were determined by the reference BMD method (using frozen-form panels), with the appropriate medium variation (the addition of 50 mg/liter of calcium) for testing daptomycin (2). Thirty-six precise incremental dilutions of vancomycin between 64 and 0.06 μg/ml (64, 32, 16, 8, 4, 3, 2.5, 2, 1.875, 1.75, 1.625, 1.5, 1.375, 1.25, 1.125, 1, 0.938, 0.875, 0.813, 0.75, 0.688, 0.625, 0.563, 0.5, 0.469, 0.438, 0.406, 0.375, 0.344, 0.313, 0.281, 0.25, 0.188, 0.12, 0.094, and 0.06 μg/ml) were tested, and 20 dilutions of daptomycin between 16 and 0.06 μg/ml (16, 8, 4, 2, 1.5, 1, 0.875, 0.75, 0.625, 0.5, 0.438, 0.375, 0.313, 0.25, 0.219, 0.188, 0.156, 0.12, 0.094, and 0.06 μg/ml) were tested. Isolates were also evaluated by Etest for susceptibilities to daptomycin and vancomycin according to the recommendations of the Etest manufacturer (AB Biodisk). To compare the results obtained with the BMD method and those obtained with the Etest technique, BMD results were rounded up to the next dilution provided for the Etest method. Resistance to oxacillin was confirmed by the reference BMD method (dilution range tested, 0.5 to 4 μg/ml) and cefoxitin susceptibilities tested by disk diffusion (2, 3). Quality control strains S. aureus ATCC 25923 and Enterococcus faecalis ATCC 29212 were evaluated concurrently with every set of tests.MICs of vancomycin obtained by the Etest method were consistently higher (+0.5 to 1.5 log₂ dilutions) than those obtained by the BMD method (Fig. ​(Fig.1a).1a). For strains from all centers combined, the overall vancomycin MIC mode determined by the BMD method was 0.75 μg/ml (corresponding to 75.3% of values, including those rounded up from measured 0.563-, 0.625-, and 0.688-μg/ml MICs) and 96.9% of strains exhibited vancomycin MIC results of ≤1 μg/ml. In contrast, when tested by the Etest, MICs for 58.3 and 32.1% of MRSA strains were 1.5 and 2 μg/ml, respectively (Fig. ​(Fig.1a1a).Open in a separate windowFIG. 1.Scattergrams showing the correlations between vancomycin (a) and daptomycin (b) MICs obtained by the BMD method and the Etest. Solid lines indicate CLSI susceptibility breakpoints, and broken lines indicate complete agreement between results from the two methods.For all strains except one, the Etest yielded higher vancomycin MICs than the reference method. Among 1,628 strains for which the Etest MIC was 1.5 μg/ml (1,050 strains) or 2 μg/ml (578 strains), only 44 (2.7%) had BMD MIC results of >1 μg/ml. Furthermore, among 13 strains considered to be nonsusceptible (intermediate) to vancomycin (MIC, 3 or 4 μg/ml) according to the Etest results, only 1 had a vancomycin MIC result of >2 μg/ml by the reference method, demonstrating a positive predictive value of only 7.7%.Etest MICs of daptomycin were also slightly elevated (+0.5 to 1 log₂ dilution) compared to BMD MICs (Fig. ​(Fig.1b).1b). When tested by the BMD method, the daptomycin MIC mode was 0.19 μg/ml (corresponding to 55.4% of values, including MICs of 0.156 and 0.188 μg/ml) and 92.9% of strains exhibited daptomycin MIC results of ≤0.25 μg/ml. In contrast, when tested by the Etest, the MIC mode was 0.38 μg/ml (corresponding to 37.2% of values), which is 1 doubling dilution higher than that obtained by the BMD method. Among nine strains considered to be daptomycin nonsusceptible according to the Etest results, only two had a MIC result of >1 μg/ml when tested by the reference BMD method, demonstrating a positive predictive value of 22.2% (Fig. ​(Fig.1b1b).Although the emergence of vancomycin-intermediate S. aureus strains and that of vancomycin-resistant S. aureus strains are reasons for concern, these organisms still are extremely rare (12). Nevertheless, a number of studies have demonstrated increased clinical failure with MRSA isolates for which vancomycin MICs are increased (>1 μg/ml) but still within the CLSI-defined susceptibility range (≤2 μg/ml) (3, 11, 13). In addition, there are reports of the increase of vancomycin MICs over time (designated “MIC creep”) at individual institutions, although this trend has not been validated by large, multi-institutional studies (14). Thus, it is becoming clear to physicians that an “S” result for vancomycin is not sufficient to appropriately guide therapy and that the clinical laboratory should provide accurate and reliable MICs.Since most clinical laboratories use automated systems to perform susceptibility testing and these systems do not provide a precise vancomycin MIC, many laboratories are using alternative methods for testing vancomycin in selected cases (6). The Etest method is an attractive option for alternative vancomycin testing since it is easy to perform and cost-effective for testing only one drug-bug combination. However, the results of the present study clearly show that the Etest provides vancomycin and daptomycin MIC results consistently higher (by 0.5 to 1.5 log₂ dilutions) than those provided by precisely performed reference BMD tests. Furthermore, the fact that the higher vancomycin MIC results provided by the Etest appear to be more reliable in predicting vancomycin treatment responses (6) indicates that the vancomycin susceptibility breakpoint should be reevaluated.The susceptibility testing method used may have a large impact on physician decisions for vancomycin and daptomycin treatment of MRSA infections. According to the information presented here, the dominant MRSA population (91.2% of strains) would be categorized as vancomycin nonsusceptible by the Etest method if the susceptibility breakpoint was adjusted to ≤1 μg/ml, as suggested by some published clinical outcome studies on recognizing isolates for which vancomycin MICs are 2 μg/ml (4, 5, 8-10, 14). Daptomycin MICs were also affected by the method used, but to a lesser degree (MICs were found to be 0.5 to 1 dilution step higher by the Etest method than by the reference method, with 0.4% false-nonsusceptible results), and may also suffer from the perception of declining daptomycin potency.