Efficacy of clarithromycin versus that of clindamycin for single-dose prophylaxis of experimental streptococcal endocarditis.

Clarithromycin is compared with clindamycin for single-dose prophylaxis of streptococcal endocarditis in rats. Human-like kinetics of the two antibiotics prevented endocarditis in animals challenged with both small and large amounts of bacterial inocula. Clarithromycin was marginally superior to clindamycin against small amounts of inocula. Clarithromycin may be considered for endocarditis chemoprophylaxis in human.

Tigecycline in combination with other antimicrobials: a review of in vitro, animal and case report studies.

Tigecycline has been investigated in combination with other antibacterials against a wide range of susceptible and multiresistant Gram-positive and Gram-negative bacteria. Combinations have been analysed in vitro, in animal models and in human case reports. In vitro, tigecycline combined with other antimicrobials produces primarily an indifferent response (neither synergy nor antagonism). Nevertheless, synergy occurred when tigecycline was combined with rifampicin against 64-100% of Enterococcus spp., Streptococcus pneumoniae, Enterobacter spp. and Brucella melitensis isolates. Combinations of tigecycline with amikacin also showed synergy for 40-100% of Enterobacter spp., Klebsiella pneumoniae, Proteus spp. and Stenotrophomonas maltophilia isolates. Moreover, bactericidal synergisms occurred with tigecycline plus amikacin against problematic Acinetobacter baumannii and Proteus vulgaris, and with colistin against K. pneumoniae. Data from animal experiments and case reports, although limited, displayed consistent beneficial activity of tigecycline in combination with other antibacterials against multiresistant organisms, including vancomycin against penicillin-resistant S. pneumoniae in experimental meningitis, gentamicin against Pseudomonas aeruginosa in experimental pneumonia, daptomycin against Enterococcus faecium endocarditis, and colistin against K. pneumoniae bacteraemia and P. aeruginosa osteomyelitis. Antagonism was extremely rare in vitro and was not reported in vivo. Thus, tigecycline may be combined with a second antimicrobial as part of a combination regimen.

BAL9141, a novel extended-spectrum cephalosporin active against methicillin-resistant Staphylococcus aureus in treatment of experimental endocarditis.

The therapeutic efficacy of BAL9141 (formerly Ro 63-9141), a novel cephalosporin with broad in vitro activity that also has activity against methicillin-resistant Staphylococcus aureus (MRSA), was investigated in rats with experimental endocarditis. The test organisms were homogeneously methicillin-resistant S. aureus strain COL transformed with the penicillinase-encoding plasmid pI524 (COL Bla+) and homogeneously methicillin-resistant, penicillinase-producing isolate P8-Hom, selected by serial exposure of parent strain P8 to methicillin. The MICs of BAL9141 for these organisms (2 mg/liter) were low, and BAL9141was bactericidal in time-kill curve studies after 24 h of exposure to either two, four, or eight times the MIC. Rats with experimental endocarditis were treated in a three-arm study with a continuous infusion of BAL5788 (formerly Ro 65-5788), a carbamate prodrug of BAL9141, or with amoxicillin-clavulanate or vancomycin. The rats were administered BAL9141 to obtain steady-state target levels of 20, 10, and 5 mg of per liter or were administered either 1.2 g of amoxicillin-clavulanate (ratio 5:1) every 6 h or 1 g of vancomycin every 12 h at changing flow rates to simulate the pharmacokinetics produced in humans by intermittent intravenous treatment. Treatment was started 12 h after bacterial challenge and lasted for 3 days. BAL9141 was successful in the treatment of experimental endocarditis due to either MRSA isolate COL Bla+ or MRSA isolate P8-Hom at the three targeted steady-state concentrations and sterilized >90% of cardiac vegetations (P < 0.005 versus controls; P < 0.05 versus amoxicillin-clavulanate and vancomycin treatment groups). These promising in vivo results with BAL9141 correlated with the high affinity of the drug for PBP 2a and its stability to penicillinase hydrolysis observed in vitro.

Importance of genotypic and phenotypic tolerance in the treatment of experimental endocarditis due to Streptococcus gordonii.

Genotypic and phenotypic tolerance was studied in penicillin treatment of experimental endocarditis due to nontolerant and tolerant Streptococcus gordonii and to their backcross transformants. The organisms were matched for in vitro and in vivo growth rates. Rats with aortic endocarditis were treated for 3 or 5 days, starting 12, 24, or 48 h after inoculation. When started at 12 h, during fast intravegetation growth, 3 days of treatment cured 80% of the nontolerant parent compared with <30% of the tolerant derivative (P < .005). When started at 24 or 48 h and if intravegetation growth had reached a plateau, 3 days of treatment failed against both bacteria. However, a significant difference between the 2 organisms was restored when treatment was extended to 5 days. Thus, genotypic tolerance conferred a survival advantage in both fast- and slow-growing bacteria, demonstrating that the in vitro-defined tolerant phenotype also carried the risk of treatment failure in vivo.

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.

In vitro prevention of the emergence of daptomycin resistance in Staphylococcus aureus and enterococci following combination with amoxicillin/clavulanic acid or ampicillin.

Daptomycin is bactericidal against meticillin-resistant Staphylococcus aureus (MRSA), glycopeptide-intermediate-resistant S. aureus (GISA) and vancomycin-susceptible and -resistant enterococci. However, selection for daptomycin-resistant derivatives has occasionally been reported during therapy in humans. Here we evaluate whether selection for daptomycin-resistant S. aureus or enterococci could be prevented in vitro by combining daptomycin with amoxicillin/clavulanic acid, ampicillin, gentamicin or rifampicin. Six strains of S. aureus (four MRSA and two GISA) and four strains of enterococci (two Enterococcus faecalis and two Enterococcus faecium) were serially exposed in broth to two-fold stepwise increasing concentrations of daptomycin alone or in combination with a fixed concentration [0.25x minimum inhibitory concentration (MIC)] of either of the second agents. The daptomycin MIC was examined after each cycle. Exposure to daptomycin alone gradually selected for S. aureus and enterococci with an increased MIC. Gentamicin did not prevent the emergence of daptomycin-resistant bacteria. Rifampicin was also unable to prevent daptomycin resistance, although resistance was slightly delayed. In contrast, amoxicillin/clavulanic acid or ampicillin prevented or greatly delayed the selection of daptomycin-resistant mutants in S. aureus and enterococci, respectively. Addition of amoxicillin/clavulanic acid or ampicillin to daptomycin prevents, or greatly delays, daptomycin resistance in vitro. Future studies in animal models are needed to predict the utility of these combinations in humans.

High-dose daptomycin plus fosfomycin is safe and effective in treating methicillin-susceptible and methicillin-resistant Staphylococcus aureus endocarditis.

We describe 3 patients with left-sided staphylococcal endocarditis (1 with methicillin-susceptible Staphylococcus aureus [MSSA] prosthetic aortic valve endocarditis and 2 with methicillin-resistant S. aureus [MRSA] native-valve endocarditis) who were successfully treated with high-dose intravenous daptomycin (10 mg/kg/day) plus fosfomycin (2 g every 6 h) for 6 weeks. This combination was tested in vitro against 7 MSSA, 5 MRSA, and 2 intermediately glycopeptide-resistant S. aureus isolates and proved to be synergistic against 11 (79%) strains and bactericidal against 8 (57%) strains. This combination deserves further clinical study.

Meropenem prevents levofloxacin-induced resistance in penicillin-resistant pneumococci and acts synergistically with levofloxacin in experimental meningitis.

The aim of the present study was to investigate the potential synergy between meropenem and levofloxacin in vitro and in experimental meningitis and to determine the effect of meropenem on levofloxacin-induced resistance in vitro. Meropenem increased the efficacy of levofloxacin against the penicillin-resistant pneumococcal strain KR4 in time-killing assays in vitro and acted synergistically against a second penicillin-resistant strain WB4. In the checkerboard, only an additive effect (FIC indices: 1.0) was observed for both strains. In cycling experiments in vitro, levofloxacin alone led to a 64-fold increase in the MIC for both strains after 12 cycles. Addition of meropenem in sub-MIC concentrations (0.25 x MIC) completely inhibited the selection of levofloxacin-resistant mutants in WB4 after 12 cycles. In KR4, the addition of meropenem led to just a twofold increase in the MIC for levofloxacin after 12 cycles. Mutations detected in the genes encoding for topoisomerase IV (parC) and gyrase (gyrA) confirmed the levofloxacin-induced resistance in both strains. Addition of meropenem was able to completely suppress levofloxacin-induced mutations in WB4 and led to only one mutation in parE in KR4. In experimental meningitis, meropenem, given in two doses (2 x 125 mg/kg), produced a good bactericidal activity (-0.45 Deltalog10 cfu/ml.h) comparable to one dose (1 x 10 mg/kg) of levofloxacin (-0.44 Deltalog10 cfu/ml.h) against the penicillin-resistant strain WB4. Meropenem combined with levofloxacin acted synergistically (-0.93 Deltalog10 cfu/ml.h), sterilizing the CSF of all rabbits.

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 microg/ml, respectively. In time-kill studies, daptomycin showed rapid and concentration-dependent killing of MRSA in stationary growth phase. At concentrations above 20 microg/ml, daptomycin reduced the counts by >3 log(10) CFU/ml in 2 to 4 h. In sterile cage fluid, daptomycin peak concentrations of 23.1, 46.3, and 53.7 microg/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 log(10) CFU/ml, whereas in combination with rifampin, a reduction in the counts of >6 log(10) 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.

Structure-function characterization and optimization of a plant-derived antibacterial peptide.

Crushed seeds of the Moringa oleifera tree have been used traditionally as natural flocculants to clarify drinking water. We previously showed that one of the seed peptides mediates both the sedimentation of suspended particles such as bacterial cells and a direct bactericidal activity, raising the possibility that the two activities might be related. In this study, the conformational modeling of the peptide was coupled to a functional analysis of synthetic derivatives. This indicated that partly overlapping structural determinants mediate the sedimentation and antibacterial activities. Sedimentation requires a positively charged, glutamine-rich portion of the peptide that aggregates bacterial cells. The bactericidal activity was localized to a sequence prone to form a helix-loop-helix structural motif. Amino acid substitution showed that the bactericidal activity requires hydrophobic proline residues within the protruding loop. Vital dye staining indicated that treatment with peptides containing this motif results in bacterial membrane damage. Assembly of multiple copies of this structural motif into a branched peptide enhanced antibacterial activity, since low concentrations effectively kill bacteria such as Pseudomonas aeruginosa and Streptococcus pyogenes without displaying a toxic effect on human red blood cells. This study thus identifies a synthetic peptide with potent antibacterial activity against specific human pathogens. It also suggests partly distinct molecular mechanisms for each activity. Sedimentation may result from coupled flocculation and coagulation effects, while the bactericidal activity would require bacterial membrane destabilization by a hydrophobic loop.

Multivalent display of the antimicrobial peptides BP100 and BP143

Carbohydrates are considered as promising templates for the display of multiple copies of antimicrobial peptides. Herein, wedescribe the design and synthesis of chimeric structures containing two or four copies of the antimicrobial peptidesKKLFKKILKYL-NH2 (BP100) and KKLfKKILKYL-NH2 (BP143) attached to the carbohydrate template cyclodithioerythritol(cDTE) or α-D-galactopyranoside (Galp). The synthesis involved the preparation of the corresponding peptide aldehyde followedby coupling to an aminooxy-functionalized carbohydrate template. After purification, the multivalent display systems were obtainedin high purities (90–98%) and in good yields (42–64%). These compounds were tested against plant and human pathogenic bacteriaand screened for their cytotoxicity on eukaryotic cells. They showed lower MIC values than the parent peptides against the bacteriaanalyzed. In particular, the carbopeptides derived from cDTE and Galp, which contained two or four copies of BP100, respectively,were 2- to 8-fold more active than the monomeric peptide against the phytopathogenic bacteria. These results suggest thatpreassembling antimicrobial peptides to multimeric structures is not always associated with a significant improvement of theactivity. In contrast, the carbopeptides synthesized were active against human red blood cells pointing out that peptide preassemblyis critical for the hemolytic activity. Notably, peptide preassembly resulted in an enhanced bactericidal effect

Molecular mechanisms of penicillin-induced killing and penicillin tolerance in Streptococcus gordonii

Abstract The main thesis topic relates to the 'molecular mechanisms of penicillin-induced bacterial death. Indeed, bacteria have developed two principal mechanisms to escape the killing effect of ß-lactam antibiotics: resistance and tolerance. Resistant bacteria are characterized by their ability to grow in the presence of drug concentrations higher than the one inhibiting the growth of susceptible members of the same species. Hence, resistant bacteria have an increased minimal inhibitory concentration (MIC) of the drug. Nevertheless, when exposed to antibiotic concentrations exceeding their new MIC, resistant bacteria remain sensitive to the antibiotic killing effect. In contrast, tolerant bacteria have an unchanged MIC. However, they have a considerably increased ability to survive drug-induced killing, even at concentrations exceeding their MIC by several orders of magnitude. In other words, in the presence of the antibiotic, tolerant bacteria become persister cells which stop growing but are not killed. In the present thesis, it is shown that the survival phenotype of a tolerant Streptococcus gordonii strain depends on two components belonging to sugar metabolism pathways. First, the transcription factor CcpA which mediates a global regulatory mechanism allowing bacteria to utilize the most efficient sugar source for their growth. We show that the inactivation of the ccpA gene leads to a partial loss of penicillin tolerance both in vitro and in a rat model of experimental endocarditis. Second, the Enzyme I of the phosphotransferase system which is involved in the uptake and phosphorylation of sugars. Here, we -show that a single nucleotide mutation in ptsI, the gene encoding the Enzyme I, is sufficient to confer a fully tolerant phenotype in S. gordonii both in vivo and in vivo. The mutation results in a radical proline to arginine substitution in the C-terminal domain of the protein, probably leading to a decrease in its homodimerization and subsequent activity. Taken together our results prove that tolerance is a global survival mechanism linked to sugar metabolism. We hypothesize that, in the presence of the antibiotic, the already altered metabolic processes of the tolerant strain are completely inactivated. Hence, bacteria may enter in a dormant state and become insensitive to the bactericidal effect of ß-lactams, which depends on actively dividing cells. This thesis manuscript also contains two other side-projects. The first one establishes that the ability to form a biofilm is not a requisite for the successful establishment of endocarditis due to S. gordonii. The second one characterizes the S. gordonii a-phosphoglucomutase gene, and shows that its inactivation results in a loss of in vitro fitness and in vivo virulence. Résumé Le sujet principal de cette thèse concerne les mécanismes moléculaires de la mort bactérienne induite par la pénicilline. En effet, les bactéries ont développé deux mécanismes principaux pour échapper à l'effet bactéricide des ß-lactamines : la résistance et la tolérance. Les bactéries résistantes sont caractérisées par leur capacité de croître en présence de concentration d'antibiotiques plus élevées que celles inhibant la croissance des organismes sensibles de la même espèce. Les bactéries résistantes ont donc une augmentation de leur concentration minimale inhibitrice (CMI) à l'antibiotique. Néanmoins, quand elles sont exposées à des concentrations dépassant leur nouvelle CMI, elles restent sensibles à l'effet bactéricide. Au contraire, les bactéries tolérantes ont une CMI inchangée. Toutefois, elles ont une très importante capacité à survivre à l'effet bactéricide des ß-lactamines, ceci même à des concentrations excédant leur CMI de plusieurs ordres de grandeur. En d'autres termes, en présence de l'antibiotique, les bactéries tolérantes deviennent des cellules persistantes qui arrêtent leur croissance mais ne sont pas tuées. Dans la présente thèse, il est montré que le phénotype de survie d'un Streptococcus gordonii tolérant dépend de deux composants appartenant aux voies du métabolisme des sucres. Premièrement, le facteur de transcription CcpA qui contrôle un système global de régulation permettant à la bactérie d'utiliser les sources de sucre les plus efficaces pour sa croissance. Il est montré que l'inactivation du gène ccpA résulte en la perte partielle de la tolérance à la pénicilline aussi bien in vitro que dans un modèle d'endocardite expérimentale chez le rat. Deuxièmement, l'Enzyme I du système de phosphotransfert impliqué dans l'import et la phosphorylation des sucres. Nous montrons qu'une mutation ponctuelle d'un nucléotide dans ptsl, le gène codant pour l'Enzyme I, suffit à complètement conférer un phénotype tolérant chez S. gordonii aussi bien in vitro qu'in vivo. La mutation induit la substitution radicale d'une proline en une arginine dans le domaine C-terminal de la protéine, résultant probablement en une diminution de sa capacité d'homodimérisation et donc d'activité. Dans leur ensemble, nos résultats prouvent que la tolérance est un mécanisme global de survie lié au métabolisme des sucres. Nous présentons l'hypothèse que, en présence de l'antibiotique, les processus métaboliques déjà altérés de la souche tolérante deviennent complètement inactifs. En conséquence, les bactéries entreraient dans un état dormant nonréplicatif, devenant ainsi insensibles à l'effet bactéricide des ß-lactamines qui nécessite des cellules en cours de division active. Le manuscrit de cette thèse contient également deux projets secondaires. Le premier montre que la capacité de former un biofilm n'est pas un prérequis pour le succès de l'initiation de l'endocardite à S. gordonii. Le second caractérise le gène de l'a-phosphoglucomutase de S. gordonii et montre que son inactivation résulte en une perte de fitness in vitro et de virulence in vivo.

Role of rifampin against Propionibacterium acnes biofilm in vitro and in an experimental foreign-body infection model.

Propionibacterium acnes is an important cause of orthopedic-implant-associated infections, for which the optimal treatment has not yet been determined. We investigated the activity of rifampin, alone and in combination, against planktonic and biofilm P. acnes in vitro and in a foreign-body infection model. The MIC and the minimal bactericidal concentration (MBC) were 0.007 and 4 μg/ml for rifampin, 1 and 4 μg/ml for daptomycin, 1 and 8 μg/ml for vancomycin, 1 and 2 μg/ml for levofloxacin, 0.03 and 16 μg/ml for penicillin G, 0.125 and 512 μg/ml for clindamycin, and 0.25 and 32 μg/ml for ceftriaxone. The P. acnes minimal biofilm eradication concentration (MBEC) was 16 μg/ml for rifampin; 32 μg/ml for penicillin G; 64 μg/ml for daptomycin and ceftriaxone; and ≥128 μg/ml for levofloxacin, vancomycin, and clindamycin. In the animal model, implants were infected by injection of 10⁹ CFU P. acnes in cages. Antimicrobial activity on P. acnes was investigated in the cage fluid (planktonic form) and on explanted cages (biofilm form). The cure rates were 4% for daptomycin, 17% for vancomycin, 0% for levofloxacin, and 36% for rifampin. Rifampin cured 63% of the infected cages in combination with daptomycin, 46% with vancomycin, and 25% with levofloxacin. While all tested antimicrobials showed good activity against planktonic P. acnes, for eradication of biofilms, rifampin was needed. In combination with rifampin, daptomycin showed higher cure rates than with vancomycin in this foreign-body infection model.

Polymorphisms in toll-like receptor 4 and toll-like receptor 9 influence viral load in a seroincident cohort of HIV-1-infected individuals.

OBJECTIVES: Toll-like receptors (TLRs) are innate immune sensors that are integral to resisting chronic and opportunistic infections. Mounting evidence implicates TLR polymorphisms in susceptibilities to various infectious diseases, including HIV-1. We investigated the impact of TLR single nucleotide polymorphisms (SNPs) on clinical outcome in a seroincident cohort of HIV-1-infected volunteers. DESIGN: We analyzed TLR SNPs in 201 antiretroviral treatment-naive HIV-1-infected volunteers from a longitudinal seroincident cohort with regular follow-up intervals (median follow-up 4.2 years, interquartile range 4.4). Participants were stratified into two groups according to either disease progression, defined as peripheral blood CD4(+) T-cell decline over time, or peak and setpoint viral load. METHODS: Haplotype tagging SNPs from TLR2, TLR3, TLR4, and TLR9 were detected by mass array genotyping, and CD4(+) T-cell counts and viral load measurements were determined prior to antiretroviral therapy initiation. The association of TLR haplotypes with viral load and rapid progression was assessed by multivariate regression models using age and sex as covariates. RESULTS: Two TLR4 SNPs in strong linkage disequilibrium [1063 A/G (D299G) and 1363 C/T (T399I)] were more frequent among individuals with high peak viral load compared with low/moderate peak viral load (odds ratio 6.65, 95% confidence interval 2.19-20.46, P < 0.001; adjusted P = 0.002 for 1063 A/G). In addition, a TLR9 SNP previously associated with slow progression was found less frequently among individuals with high viral setpoint compared with low/moderate setpoint (odds ratio 0.29, 95% confidence interval 0.13-0.65, P = 0.003, adjusted P = 0.04). CONCLUSION: This study suggests a potentially new role for TLR4 polymorphisms in HIV-1 peak viral load and confirms a role for TLR9 polymorphisms in disease progression.

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 log(10) CFU/ml, whereas in combination with colistin, tigecycline, or gentamicin, it reduced counts by >6 log(10) 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.