A positive interaction between inhibitors of protein synthesis and cefepime in the fight against methicillin-resistant Staphylococcus aureus.

Quinupristin-dalfopristin (Q-D) synergizes with cefepime for the treatment of methicillin-resistant Staphylococcus aureus (MRSA). Here, we studied whether the synergism was restricted to MRSA and if it extended to non-beta-lactam cell wall inhibitors or to other inhibitors of protein synthesis. Three MRSA and two methicillin-susceptible S. aureus (MSSA) strains were tested, including an isogenic pair of mecA (-)/mecA (+) S. aureus Newman. The drug interactions were determined by fractional inhibitory concentration (FIC) indices and population analysis profiles. The antibacterial drugs that we used included beta-lactam (cefepime) and non-beta-lactam cell wall inhibitors (D-cycloserine, fosfomycin, vancomycin, teicoplanin), inhibitors of protein synthesis (Q-D, erythromycin, chloramphenicol, tetracycline, linezolid, fusidic acid), and polynucleotide inhibitors (cotrimoxazole, ciprofloxacin). The addition of each protein inhibitor to cefepime was synergistic (FIC ≤ 0.5) or additive (FIC > 0.5 but < 1) against MRSA, but mostly indifferent against MSSA (FIC ≥ 1 but ≤ 4). This segregation was not observed after adding cotrimoxazole or ciprofloxacin to cefepime. Population analysis profiles were performed on plates in the presence of increasing concentrations of the cell wall inhibitors plus 0.25 × minimum inhibitory concentration (MIC) of Q-D. Cefepime combined with Q-D was synergistic against MRSA, but D-cycloserine and glycopeptides were not. Thus, the synergism was specific to beta-lactam antibiotics. Moreover, the synergism was not lost against fem mutants, indicating that it acted at another level. The restriction of the beneficial effect to MRSA suggests that the functionality of penicillin-binding protein 2A (PBP2A) was affected, either directly or indirectly. Further studies are necessary in order to provide a mechanism for this positive interaction.

In vivo synergism of ceftobiprole and vancomycin against experimental endocarditis due to vancomycin-intermediate Staphylococcus aureus.

The efficacy of ceftobiprole combined with vancomycin was tested against two vancomycin-intermediate Staphylococcus aureus (VISA) strains, PC3 and Mu50, in rats with experimental endocarditis. Animals with infected aortic vegetations were treated for 3 days with doses simulating the kinetics after intravenous administration in humans of (i) the standard dose of ceftobiprole of 500 mg every 12 h (b.i.d.) (SD-ceftobiprole), (ii) a low dose of ceftobiprole of 250 mg b.i.d. (LD-ceftobiprole), (iii) a very low dose of ceftobiprole of 125 mg b.i.d. (VLD-ceftobiprole), (iv) SD-vancomycin of 1 g b.i.d., or (v) LD- or VLD-ceftobiprole combined with SD-vancomycin. Low dosages of ceftobiprole were purposely used to highlight positive drug interactions. Treatment with SD-ceftobiprole sterilized 12 of 14 (86%) and 10 of 13 (77%) vegetations infected with PC3 and Mu50, respectively (P < 0.001 versus controls). In comparison, LD-ceftobiprole sterilized 10 of 11 (91%) vegetations infected with PC3 (P < 0.01 versus controls) but only 3 of 12 (25%) vegetations infected with Mu50 (P > 0.05 versus controls). VLD-ceftobiprole and SD-vancomycin alone were ineffective against both strains (≤8% sterile vegetations). In contrast, the combination of VLD-ceftobiprole and SD-vancomycin sterilized 7 of 9 (78%) and 6 of 14 (43%) vegetations infected with PC3 and Mu50, respectively, and the combination of LD-ceftobiprole and SD-vancomycin sterilized 5 of 6 (83%) vegetations infected with Mu50 (P < 0.05 versus controls and monotherapy). Thus, ceftobiprole monotherapy simulating standard therapeutic doses was active against VISA experimental endocarditis. Moreover, subtherapeutic LD- and VLD-ceftobiprole synergized with ineffective vancomycin to restore efficacy. Hence, combining ceftobiprole with vancomycin broadens the therapeutic margin of these two compounds against VISA infections.

Induction of experimental endocarditis by continuous low-grade bacteremia mimicking spontaneous bacteremia in humans.

Transient high-grade bacteremia following invasive procedures carries a risk of infective endocarditis (IE). This is supported by experimental endocarditis. On the other hand, case-control studies showed that IE could be caused by cumulative exposure to low-grade bacteremia occurring during daily activities. However, no experimental demonstration of this latter possibility exists. This study investigated the infectivity in animals of continuous low-grade bacteremia compared to that of brief high-grade bacteremia. Rats with aortic vegetations were inoculated with Streptococcus intermedius, Streptococcus gordonii or Staphylococcus aureus (strains Newman and P8). Animals were challenged with 10(3) to 10(6) CFU. Identical bacterial numbers were given by bolus (1 ml in 1 min) or continuous infusion (0.0017 ml/min over 10 h). Bacteremia was 50 to 1,000 times greater after bolus than during continuous inoculation. Streptococcal bolus inoculation of 10(5) CFU infected 63 to 100% vegetations compared to 30 to 71% infection after continuous infusion (P > 0.05). When increasing the inoculum to 10(6) CFU, bolus inoculation infected 100% vegetations and continuous infusion 70 to 100% (P > 0.05). S. aureus bolus injection of 10(3) CFU infected 46 to 57% valves. This was similar to the 53 to 57% infection rates produced by continuous infusion (P > 0.05). Inoculation of 10(4) CFU of S. aureus infected 80 to 100% vegetations after bolus and 60 to 75% after continuous infusion (P > 0.05). These results show that high-level bacteremia is not required to induce experimental endocarditis and support the hypothesis that cumulative exposure to low-grade bacteremia represents a genuine risk of IE in humans.

Failure of vancomycin continuous infusion against experimental endocarditis due to vancomycin-intermediate Staphylococcus aureus.

Continuous infusion of vancomycin was evaluated against experimental endocarditis due to heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) and VISA. Animals were infected with hVISA PC1 (vancomycin MIC, 2 mg/liter) or VISA PC3 (vancomycin MIC, 8 mg/liter) and treated for 5 days with constant serum levels of 20 or 40 mg/liter. Vancomycin continuous infusion was unsuccessful, as 20 mg/liter was barely active against PC1 (6 of 13 sterile vegetations) and 40 mg/liter failed against PC3 (2 of 9 sterile vegetations).

Genealogy of wine grape cultivars: "Pinot" is related to "Syrah".

Since the domestication of wild grapes ca 6000 years ago, numerous cultivars have been generated by spontaneous or deliberate crosses, and up to 10 000 are still in existence today. Just as in human paternity analysis, DNA typing can reveal unexpected parentage of grape cultivars. In this study, we have analysed 89 grape cultivars with 60 microsatellite markers in order to accurately calculate the identity-by-descent (IBD) and relatedness (r) coefficients among six putatively related cultivars from France ("Pinot", "Syrah" and "Dureza") and northern Italy ("Teroldego", "Lagrein" and "Marzemino"). Using a recently developed likelihood-based approach to analyse kinship in grapes, we provide the first evidence of a genetic link between grapes across the Alps: "Dureza" and "Teroldego" turn out to be full-siblings (FS). For the first time in grapevine genetics we were able to detect FS without knowing one of the parents and identify unexpected second-degree relatives. We reconstructed the most likely pedigree that revealed a third-degree relationship between the worldwide-cultivated "Pinot" from Burgundy and "Syrah" from the Rhone Valley. Our finding was totally unsuspected by classical ampelography and it challenges the commonly assumed independent origins of these grape cultivars. Our results and this new approach in grape genetics will (a) help grape breeders to avoid choosing closely related varieties for new crosses, (b) provide pedigrees of cultivars in order to detect inheritance of disease-resistance genes and (c) open the way for future discoveries of first- and second-degree relationships between grape cultivars in order to better understand viticultural migrations.

Localization of saturated karst aquifer with magnetic resonance sounding and resistivity imagery.

To answer one of the main questions of hydrogeologists implementing boreholes or working on pollution questions in a karst environment--i.e., where is the ground water?--numerous tools including geophysics are used. However, the contribution of geophysics differs from one method to the other. The magnetic resonance sounding (MRS) method has the advantage of direct detection of ground water over other geophysical methods. Eight MRSs were implemented over a known karst conduit explored and mapped by speleologists to estimate the MRS ability to localize ground water. Two direct current resistivity imageries (DC-2D imagery) were also implemented to check their capability to map a known cave. We found that the MRS is a useful tool to locate ground water in karst as soon as the quantity of water is enough to be detected. The threshold quantity is a function of depth and it was estimated by forward modeling to propose a support graph to hydrogeologists. The measured MRS's signals could be used to calculate transmissivity and permeability estimators. These estimators were used to map and to draw a cross section of the case study site, which underline accurately the known karst conduit location and depth. We also found that the DC-2D imagery could underline the karst structures: It was able to detect the known cave through its associated faults. We prepared a computer simulation to check the depth of such a cave to induce resistivity anomaly which could be measured in similar conditions.

Microsatellite analysis of ancient alpine grape cultivars: pedigree reconstruction of Vitis vinifera L. 'Cornalin du Valais'.

Ancient and closely related grape cultivars from the Alps were analyzed with 50 microsatellite markers: 'Cornalin', 'Humagne Rouge' and 'Goron' from Valais (Switzerland); 'Cornalin', 'Petit Rouge' and 'Mayolet' from the Aosta Valley (Italy). Our results confirmed previous studies showing that the 'Cornalin' cultivars from Switzerland and Italy are distinct, and that 'Humagne Rouge' is identical to 'Cornalin' from the Aosta Valley. We propose the nomenclature 'Cornalin du Valais' and 'Cornalin d'Aoste' in order to prevent further confusion. At each locus, 'Goron', 'Petit Rouge', 'Mayolet' and 'Cornalin d'Aoste' all share at least one allele with 'Cornalin du Valais', strongly suggesting parent/offspring relationships. Alleles at 49 out of 50 microsatellite loci are consistent with 'Cornalin du Valais' being the progeny of 'Petit Rouge' and 'Mayolet'. The exception is a 10-base pair discrepancy at one locus, most likely the result of somatic mutation in one of the parents, since this parentage is supported by high likelihood ratios and historical data. We hypothesize that 'Cornalin du Valais' originated in the Aosta Valley through a natural cross and was then introduced into Valais centuries ago, probably via the Great St. Bernard Pass. Furthermore, 'Cornalin du Valais' is likely to be one of the parents of both 'Goron' and 'Cornalin d'Aoste', the respective second parents remaining unknown. This pedigree provides a convincing explanation for the allele-sharing patterns and is strongly supported by historical data. The present work is the first grapevine parentage study to deal with a multiple repeat unit discrepancy at a microsatellite locus. We suggest that the use of increasingly large numbers of loci in making parentage determinations leads to a corresponding increase in the probability of encountering a locus with intra-cultivar variability during the analysis. We therefore assume that a sole multiple repeat unit discrepancy is not sufficient to discard a parentage hypothesis.

Efficacies of moxifloxacin, ciprofloxacin, and vancomycin against experimental endocarditis due to methicillin-resistant Staphylococcus aureus expressing various degrees of ciprofloxacin resistance.

The new 8-methoxyquinolone moxifloxacin was tested against two ciprofloxacin-susceptible Staphylococcus aureus strains (strains P8 and COL) and two ciprofloxacin-resistant derivatives of strain P8 carrying a single grlA mutation (strain P8-4) and double grlA and gyrA mutations (strain P8-128). All strains were resistant to methicillin. The MICs of ciprofloxacin and moxifloxacin were 0.5 and 0.125 mg/liter, respectively, for P8; 0.25 and 0.125 mg/liter, respectively, for COL; 8 and 0.25 mg/liter, respectively, for P8-4; and >or=128 and 2 mg/liter, respectively, for P8-128. In vitro, the rate of spontaneous resistance of P8 and COL was 10(-7) on agar plates containing ciprofloxacin at two times the MIC, whereas it was

Effect of compost in phytoremediation of diesel-contaminated soils.

The effect of compost on phytoremediation of diesel-contaminated soils was investigated using 130 small (200 g) containers in two screening tests. The experiments were conducted in a controlled environment using ryegrass from seed. Containers were destructively sampled at various times and analyzed for plant mass and total petroleum hydrocarbons. The results indicate that the presence of diesel reduces grass growth, and that compost helps reduced the impact of diesel on grass growth. The addition of compost helps increase diesel loss from the soils both with and without grass, though the addition of grass leads to lower diesel levels compared with controls. A second set of experiments indicates that the compost helps in phytoremediation of diesel-contaminated soil independent of the dilution effect that compost addition has. The results indicate that the compost addition allowed diesel loss down to 200 mg TPH/kg even though the compost would be expected to hold the diesel more tightly in the soil/compost mixture. The simplicity of the screening tests led to difficulties in controlling moisture content and germination rates. The conclusion of the research is that the tilling of compost into soils combined with grass seeding appears to be a valuable option for treating petroleum-contaminated soils.

Quinupristin-dalfopristin combined with beta-lactams for treatment of experimental endocarditis due to Staphylococcus aureus constitutively resistant to macrolide-lincosamide-streptogramin B antibiotics.

Quinupristin-dalfopristin (Q-D) is an injectable streptogramin active against most gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). In experimental endocarditis, however, Q-D was less efficacious against MRSA isolates constitutively resistant to macrolide-lincosamide-streptogram B (C-MLS(B)) than against MLS(B)-susceptible isolates. To circumvent this problem, we used the checkerboard method to screen drug combinations that would increase the efficacy of Q-D against such bacteria. beta-Lactams consistently exhibited additive or synergistic activity with Q-D. Glycopeptides, quinolones, and aminoglycosides were indifferent. No drugs were antagonistic. The positive Q-D-beta-lactam interaction was independent of MLS(B) or beta-lactam resistance. Moreover, addition of Q-D at one-fourth the MIC to flucloxacillin-containing plates decreased the flucloxacillin MIC for MRSA from 500 to 1,000 mg/liter to 30 to 60 mg/liter. Yet, Q-D-beta-lactam combinations were not synergistic in bactericidal tests. Rats with aortic vegetations were infected with two C-MLS(B)-resistant MRSA isolates (isolates AW7 and P8) and were treated for 3 or 5 days with drug dosages simulating the following treatments in humans: (i) Q-D at 7 mg/kg two times a day (b.i.d.) (a relatively low dosage purposely used to help detect positive drug interactions), (ii) cefamandole at constant levels in serum of 30 mg/liter, (iii) cefepime at 2 g b.i.d., (iv) Q-D combined with either cefamandole or cefepime. Any of the drugs used alone resulted in treatment failure. In contrast, Q-D plus either cefamandole or cefepime significantly decreased valve infection compared to the levels of infection for both untreated controls and those that received monotherapy (P < 0.05). Importantly, Q-D prevented the growth of highly beta-lactam-resistant MRSA in vivo. The mechanism of this beneficial drug interaction is unknown. However, Q-D-beta-lactam combinations might be useful for the treatment of complicated infections caused by multiple organisms, including MRSA.

Efficacy of trovafloxacin in treatment of experimental staphylococcal or streptococcal endocarditis.

The efficacy of trovafloxacin against Staphylococcus aureus and viridans group streptococci was investigated in vitro and in an experimental model of endocarditis. The MICs at which trovafloxacin and ciprofloxacin inhibited 90% of clinical isolates of such bacteria (MIC90s) were (i) 0.03 and 2 mg/liter, respectively, for 30 ciprofloxacin-susceptible S. aureus isolates, (ii) 32 and 128 mg/liter, respectively, for 20 ciprofloxacin-resistant S. aureus isolates, and (iii) 0.25 and 8 mg/liter, respectively, for 28 viridans group streptococci. Rats with aortic vegetations were infected with either of two ciprofloxacin-susceptible but methicillin-resistant S. aureus strains (strains COL and P8), one penicillin-susceptible Streptococcus sanguis strain, or one penicillin-resistant Streptococcus mitis strain. Rats were treated for 3 or 5 days with doses that resulted in kinetics that simulated those achieved in humans with trovafloxacin (200 mg orally once a day), ciprofloxacin (750 mg orally twice a day), vancomycin (1 g intravenously twice a day), or ceftriaxone (2 g intravenously once a day). Against the staphylococci, the activities of both trovafloxacin and ciprofloxacin were equivalent to that of vancomycin, and treatment of endocarditis with these drugs was successful (P < 0.05). However, ciprofloxacin selected for resistant derivatives in vitro and in vivo, whereas trovafloxacin was 10 to 100 times less prone than ciprofloxacin to select for resistance in vitro and did not select for resistance in vivo. Against the two streptococcal isolates, trovafloxacin significantly (P < 0.05) decreased bacterial counts in the vegetations but was less effective than the control drug, ceftriaxone. Thus, a simulated oral dose of trovafloxacin (200 mg per day) was effective against ciprofloxacin-susceptible staphylococci and was less likely than ciprofloxacin to select for resistance. The simulated oral dose of trovafloxacin also had some activity against streptococcal endocarditis, but optimal treatment of infections caused by such organisms might require higher doses of the drug.

Levofloxacin versus ciprofloxacin, flucloxacillin, or vancomycin for treatment of experimental endocarditis due to methicillin-susceptible or -resistant Staphylococcus aureus.

Levofloxacin is the L isomer of ofloxacin, a racemic mixture in which the L stereochemical form carries the antimicrobial activity. Levofloxacin is more active than former quinolones against gram-positive bacteria, making it potentially useful against such pathogens. In this study, levofloxacin was compared to ciprofloxacin, flucloxacillin, and vancomycin for the treatment of experimental endocarditis due to two methicillin-susceptible Staphylococcus aureus (MSSA) and two methicillin-resistant S. aureus (MRSA) isolates. The four test organisms were susceptible to ciprofloxacin, the levofloxacin MICs for the organisms were low (0.12 to 0.25 mg/liter), and the organisms were killed in vitro by drug concentrations simulating both the peak and trough levels achieved in human serum (5 and 0.5 mg/liter, respectively) during levofloxacin therapy. Rats with aortic endocarditis were treated for 3 days. Antibiotics were injected with a programmable pump to simulate the kinetics of either levofloxacin (350 mg orally once a day), ciprofloxacin (750 mg orally twice a day), flucloxacillin (2 g intravenously four times a day), or vancomycin (1 g intravenously twice a day). Levofloxacin tended to be superior to ciprofloxacin in therapeutic experiments (P = 0.08). More importantly, levofloxacin did not select for resistance in the animals, in contrast to ciprofloxacin. The lower propensity of levofloxacin than ciprofloxacin to select for quinolone resistance was also clearly demonstrated in vitro. Finally, the effectiveness of this simulation of oral levofloxacin therapy was at least equivalent to that of standard treatment for MSSA or MRSA endocarditis with either flucloxacillin or vancomycin. This is noteworthy, because oral antibiotics are not expected to succeed in the treatment of severe staphylococcal infections. These good results obtained with animals suggest that levofloxacin might deserve consideration for further study in the treatment of infections due to ciprofloxacin-susceptible staphylococci in humans.

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.