Antimicrobial efficacy in orthopedic device infections is diminished because of bacterial biofilms which express tolerance to antibiotics. Recently, the use of high doses of levofloxacin with rifampin has been recommended for staphylococcal infections. In the present study, we evaluated the efficacy of levofloxacin at doses of 50 mg/kg/day and 100 mg/kg/day (mimicking the usual and high human doses of 500 mg/day and 750 to 1,000 mg/day, respectively) and compared it to that of to linezolid, cloxacillin, vancomycin, and rifampin in a rat tissue cage model of experimental foreign-body infection by Staphylococcus aureus. The antimicrobial efficacy in vitro (by MIC, minimum bactericidal concentration, and kill curves) for logarithmic-and stationary-phase bacteria was compared with the in vivo efficacy. In vitro bactericidal activity at clinically relevant concentrations was reached by all drugs except rifampin and linezolid in the log-phase studies but only by levofloxacin in the stationary-phase studies. The bacterial count decreases from in vivo tissue cage fluids (means) for levofloxacin at 50 and 100 mg/kg/day, rifampin, cloxacillin, vancomycin, linezolid, and controls, respectively, were: ؊1.24, ؊2.26, ؊2.1, ؊1.56, ؊1.47, ؊1.15, and 0.33 (all groups versus controls, P < 0.05). Levofloxacin at 100 mg/kg/day (area under the concentration-time curve/MIC ratio, 234) was the most active therapy (P ‫؍‬ 0.03 versus linezolid). Overall, in vivo efficacy was better predicted by stationary-phase studies, in which it reached a high correlation coefficient even if the rifampin group was excluded (r ‫؍‬ 0.96; P < 0.05). Our results, including in vitro studies with nongrowing bacteria, pharmacodynamic parameters, and antimicrobial efficacy in experimental infection, provide good evidence to support the use of levofloxacin at high doses (750 to 1,000 mg/day), as recently recommended for treating patients with orthopedic prosthesis infections.Patients suffering from orthopedic device infections will have usually undergone surgical interventions and received antibiotic therapies over a long period of time, these being major clinical issues. It is very difficult to eradicate such infections using antibiotics because of the formation of biofilm, a protein matrix including bacteria with reduced metabolism and with tolerance to antimicrobials (2,9,11,38).These infections are frequently caused by Staphylococcus aureus, and rifampin has been shown to be the most effective antimicrobial agent in such cases, in in vitro and experimental studies (26, 48) and in clinical practice (14,15,46). Since this drug should not be given alone due to the development of early bacterial resistance, antibiotic combinations are required. The combination of rifampin and fluoroquinolones has been found to be particularly efficacious and is thus usually recommended (10,14,44).In recent years, the experimental foreign-body infection model developed by Lucet et al. (26) has provided relevant data regarding the antimicrobial efficacy of several antibiotics ...

Pseudomonas savastanoi pv. savastanoi causes olive knot disease, which is present in most countries where olive trees are grown. Although the use of cultivars with low susceptibility may be one of the most appropriate methods of disease control, little information is available from inoculation assays, and cultivar susceptibility assessments have been limited to few cultivars. We have evaluated the effects of pathogen virulence, plant age, the dose/response relationship, and the induction of secondary tumors in olive inoculation assays. Most P. savastanoi pv. savastanoi strains evaluated were highly virulent to olive plants, but interactions between cultivars and strains were found. The severity of the disease in a given cultivar was strongly dependent of the pathogen dose applied at the wound sites. Secondary tumors developed in noninoculated wounds following inoculation at another position on the stem, suggesting the migration of the pathogen within olive plants. Proportion and weight of primary knots and the presence of secondary knots were evaluated in 29 olive cultivars inoculated with two pathogen strains at two inoculum doses, allowing us to rate most of the cultivars as having either high, medium, or low susceptibility to olive knot disease. None of the cultivars were immune to the disease.

The sequence of the gene iaaL of Pseudomonas savastanoi EW2009 was used to design primers for PCR amplification. The iaaL-derived primers directed the amplification of a 454-bp fragment from genomic DNA isolated from 70 strains of P. savastanoi, whereas genomic DNA from 93 non-P. savastanoi isolates did not yield this amplified product. A previous bacterial enrichment in the semiselective liquid medium PVF-1 improved the PCR sensitivity level, allowing detection of 10 to 100 CFU/ml of plant extract. P. savastanoi was detected by the developed enrichment-PCR method in knots from different varieties of inoculated and naturally infected olive trees. Moreover, P. savastanoi was detected in symptomless stem tissues from naturally infected olive plants. This enrichment-PCR method is more sensitive and less cumbersome than the conventional isolation methods for detection of P. savastanoi.Pseudomonas savastanoi and its pathovars savastanoi, fraxini, and nerii incite a disease of olive (Olea europaea L.), ash (Fraxinus excelsior L.), other Oleaceae plants and oleander (Nerium oleander L.) that is characterized by tumorous outgrowths (4,19). This development of knots is dependent on bacterial production of the phytohormone indoleacetic acid (IAA) and cytokinins (3,7,16,18). P. savastanoi can conjugate IAA with lysine to form 3-indoleacetyl-ε-L-lysine (IAA-lysine) (6). The two enzymes involved in IAA biosynthesis are tryptophan monooxygenase, which converts tryptophan to indoleacetamide, and indoleacetamide hydrolase, which catalyzes the conversion of indoleacetamide to IAA (12). The enzyme involved in the conversion of IAA to IAA-lysine is (indole-3-acetyl)-L-lysine synthethase (5). The genes for tryptophan monooxygenase (iaaM), indoleacetamide hydrolase (iaaH), and IAA-lysine synthethase (iaaL) reside on the 52-kb plasmid pIAA1 in the oleander P. savastanoi strain EW2009, and they have been sequenced (3,5,14,20). Sequence analysis revealed that iaaM and iaaH have significant similarity with homologous genes of other plant-associated bacteria (13,20). In contrast, to date, no nucleotide homologies have been found with the iaaL gene.Detection of P. savastanoi is currently based on bacterial isolation followed by pathogenicity tests and biochemical or serological techniques (2,8,17,21). These conventional methods are time-consuming and expensive, requiring bacterial isolation. We used the published sequence of iaaL (14) to design specific primers for amplification of this gene. We report here the development of a new sensitive and specific detection method for P. savastanoi based on amplification of iaaL after a bacterial enrichment. The developed enrichment-PCR assay can be applied to specifically detect low levels of P. savastanoi in inoculated and naturally infected plants.Specificity of the PCR assay. Seventy strains of P. savastanoi isolated from olive, oleander, ash, and jasmine (Jasminus officinalis L.) plants from different countries, 23 outgroup strains, and 70 saprophytic isolates from olive plants were used to te...

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