Bonds between fibronectin and fibronectin-binding proteins on Staphylococcus aureus and Lactococcus lactis.

Bacterial cell-wall-associated fibronectin binding proteins A and B (FnBPA and FnBPB) form bonds with host fibronectin. This binding reaction is often the initial step in prosthetic device infections. Atomic force microscopy was used to evaluate binding interactions between a fibronectin-coated probe and laboratory-derived Staphylococcus aureus that are (i) defective in both FnBPA and FnBPB (fnbA fnbB double mutant, DU5883), (ii) capable of expressing only FnBPA (fnbA fnbB double mutant complemented with pFNBA4), or (iii) capable of expressing only FnBPB (fnbA fnbB double mutant complemented with pFNBB4). These experiments were repeated using Lactococcus lactis constructs expressing fnbA and fnbB genes from S. aureus. A distinct force signature was observed for those bacteria that expressed FnBPA or FnBPB. Analysis of this force signature with the biomechanical wormlike chain model suggests that parallel bonds form between fibronectin and FnBPs on a bacterium. The strength and covalence of bonds were evaluated via nonlinear regression of force profiles. Binding events were more frequent (p < 0.01) for S. aureus expressing FnBPA or FnBPB than for the S. aureus double mutant. The binding force, frequency, and profile were similar between the FnBPA and FnBPB expressing strains of S. aureus. The absence of both FnBPs from the surface of S. aureus removed its ability to form a detectable bond with fibronectin. By contrast, ectopic expression of FnBPA or FnBPB on the surface of L. lactis conferred fibronectin binding characteristics similar to those of S. aureus. These measurements demonstrate that fibronectin-binding adhesins FnBPA and FnBPB are necessary and sufficient for the binding of S. aureus to prosthetic devices that are coated with host fibronectin.

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.

The discriminatory power of MALDI-TOF mass spectrometry to differentiate between isogenic teicoplanin-susceptible and teicoplanin-resistant strains of methicillin-resistant Staphylococcus aureus.

To explore the discriminatory power of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for detecting subtle differences in isogenic isolates, we tested isogenic strains of Staphylococcus aureus differing in their expression of resistance to methicillin or teicoplanin. More important changes in MALDI-TOF MS spectra were found with strains differing in methicillin than in teicoplanin resistance. In comparison, very minor or no changes were recorded in pulsed-field gel electrophoresis profiles or peptidoglycan muropeptide digest patterns of these strains, respectively. MALDI-TOF MS might be useful to detect subtle strain-specific differences in ionizable components released from bacterial surfaces and not from their peptidoglycan network.

Efficacy of garenoxacin in treatment of experimental endocarditis due to Staphylococcus aureus or viridans group streptococci.

The activity of garenoxacin was investigated in rats with experimental endocarditis due to staphylococci and viridans group streptococci (VGS). The staphylococci tested comprised one ciprofloxacin-susceptible and methicillin-susceptible Staphylococcus aureus (MSSA) isolate (isolate 1112), one ciprofloxacin-susceptible but methicillin-resistant S. aureus (MRSA) isolate (isolate P8), and one ciprofloxacin-resistant mutant (grlA) of P8 (isolate P8-4). The VGS tested comprised one penicillin-susceptible isolate and one penicillin-resistant isolate (Streptococcus oralis 226 and Streptococcus mitis 531, respectively). To simulate the kinetics of drugs in humans, rats were infused intravenously with garenoxacin every 24 h (peak and trough levels in serum, 6.1 and 1.0 mg/liter, respectively; area under the concentration-time curve [AUC], 63.4 mg. h/liter) or levofloxacin every 12 h (peak and trough levels in serum, 7.3 and 1.5 mg/liter, respectively; AUC, 55.6 mg. h/liter) for 3 or 5 days. Flucloxacillin, vancomycin, and ceftriaxone were used as control drugs. Garenoxacin, levofloxacin, flucloxacillin, and vancomycin sterilized >/=70% of the vegetations infected with both ciprofloxacin-susceptible staphylococcal isolates (P < 0.05 versus the results for the controls). Garenoxacin and vancomycin also sterilized 70% of the vegetations infected with ciprofloxacin-resistant MRSA isolate P8-4, whereas treatment with levofloxacin failed against this organism (cure rate, 0%; P < 0.05 versus the results obtained with the comparator drugs). Garenoxacin did not select for resistant derivatives in vivo. In contrast, levofloxacin selected for resistant variants in four of six rats infected with MRSA isolate P8-4. Garenoxacin sterilized 90% of the vegetations infected with both penicillin-susceptible and penicillin-resistant isolates of VGS. Levofloxacin sterilized only 22 and 40% of the vegetations infected with penicillin-susceptible S. oralis 226 and penicillin-resistant S. mitis 531, respectively. Ceftriaxone sterilized only 40% of those infected with penicillin-resistant S. mitis 531 (P < 0.05 versus the results obtained with garenoxacin). No quinolone-resistant VGS were detected. In all the experiments successful quinolone treatment was predicted by specific pharmacodynamic criteria (D. R. Andes and W. A. Craig, Clin. Infect. Dis. 27:47-50, 1998). The fact that the activity of garenoxacin was equal or superior to those of the standard comparators against staphylococci and VGS indicates that it is a potential alternative for the treatment of infections caused by such bacteria.

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.

Impact of recombination on genetic variability within Staphylococcus aureus clonal complexes.

The population structure of Staphylococcus aureus is generally described as highly clonal and is consequently subdivided into several clonal complexes (CCs). Recent data suggested that recombination might occur more frequently within than among CCs. To test this hypothesis as well as to understand how genetic diversity is created in S. aureus, we analyzed a collection of 182 isolates with MLST and five highly variable core adhesion (ADH) genes. As expected the polymorphism of ADH genes was higher than MLST genes. However both categories of genes showed low within CCs diversity with a dominant haplotype and its single nucleotide variants. Several recombination events were detected but none involved intra-CC recombination. This did not confirm the hypothesis of higher recombination within CCs. Nevertheless, molecular analyses of variance indicated that these few recombination events have a significant impact on the genetic diversity within CCs. In addition, although most ADH genes were under purifying selection, signs of positive selection associated with a recombinant group were detected. These data highlight the importance of recombination on the evolution of the highly clonal S. aureus and suggest that recombination when combined with demographic mechanisms as well as selection might favor the rapid creation of new clonal complexes.

Treatment of experimental endocarditis due to erythromycin-susceptible or -resistant methicillin-resistant Staphylococcus aureus with RP 59500.

RP 59500 is a new injectable streptogramin composed of two synergistic components (quinupristin and dalfopristin) which are active against erythromycin-susceptible and -resistant gram-positive pathogens. The present experiments compared the therapeutic efficacy of RP 59500 with that of vancomycin against experimental endocarditis due to either of two erythromycin-susceptible or two constitutively erythromycin-resistant isolates of methicillin-resistant Staphylococcus aureus. RP 59500 had low MICs for the four test organisms as well as for 24 additional isolates (the MIC at which 90% of the isolates were inhibited was &lt; 1 mg/liter) which were mostly inducibly (47%) or constitutively (39%) erythromycin resistant. Aortic endocarditis in rats was produced with catheter-induced vegetations. Three-day therapy was initiated 12 h after infection, and the drugs were delivered via a computerized pump, which permitted the mimicking of the drug kinetics produced in human serum by twice-daily intravenous injections of 7 mg of RP 59500 per kg of body weight or 1 g of vancomycin. Both antibiotics reduced vegetation bacterial titers to below detection levels in ca. 70% of animals infected with the erythromycin-susceptible isolates (P &lt; 0.05 compared with titers in controls). Vancomycin was also effective against the constitutively resistant strains, but RP 59500 failed against these isolates. Further experiments proved that RP 59500 failures were related to the very short life span of dalfopristin in serum (&lt; or = 2 h, compared with &gt; or = 6 h for quinupristin), since successful treatment was restored by artificially prolonging the dalfopristin levels for 6 h. Thus, RP 59500 is a promising alternative to vancomycin against methicillin-resistant S. aureus infections, provided that pharmacokinetic parameters are adjusted to afford prolonged levels of both of its constituents in serum. This observation is also relevant to humans, in whom the life span of dalfopristin in serum is also shorter than that of quinupristin.

1.° M. T. Ciceronis de oratore libri tres ad Quintum fratrem : sub finem tertii nonnulla desiderantur. — 2.° Luc. Ann. Flori historiae Romanae epitome. — 3.° Titi Livii historiarum periochae, sive summaria. — 4.° Caii Plinii Secundi historiae naturalis liber primus : sub finem nonnulla desiderantur. — 5.° Probae Falconiae versus litteris Longobardicis. — Is codex, si Falconiae versiculos excipias, decimo tertio saeculo videtur exaratus.

Puteanus

Ecological temporal stability of Staphylococcus aureus nasal carriage.

We described the colonization dynamics of Staphylococcus aureus in a group of 266 healthy carriers over a period of approximately 1 year. We used precise genotyping methods, i.e., amplified fragment length polymorphism (AFLP), spa typing, and double-locus sequence typing (DLST), to detect changes in strain identity. Strain change took place rather rarely: out of 89 carriers who had initially been colonized, only 7 acquired a strain different from the original one. Approximately one-third of the carriers eliminated the colonization, and a similar number became newly colonized. Some of these events probably represent detection failure rather than genuine colonization loss or acquisition. Lower bacterial counts were associated with increased probability of eliminating the colonization. We have confirmed a high mutation rate in the spa locus: 6 out of 53 strains underwent mutation in the spa locus. There was no overall change in S. aureus genotype composition.

Staphylococcus aureus clfB and spa alleles of the repeat regions are segregated into major phylogenetic lineages.

To reliably differentiate among Staphylococcus aureus isolates we recently developed the Double Locus Sequence Typing (DLST) based on the analysis of partial sequences of clfB and spa genes. This method is highly discriminatory and gives unambiguous definition of types. The highly clonal population structure of S. aureus suggests that isolates with identical clfB or spa alleles belong to the same clonal complex (CC) defined by Multi-Locus Sequence Typing (MLST). To test this hypothesis as well as to investigate putative intra-CC genetic structure, we analyzed a total of 289 isolates (186 MSSA and 103 MRSA) with DLST-, spa- and MLST-typing. Among the 289 strains, 242 were clustered into 7 major MLST CCs, 40 into minor CCs and 7 were not grouped into CCs. A total of 205 DLST- and 129 spa-types were observed. With one exception, all DLST-clfB, DLST-spa and spa-type alleles were segregated into CCs. DLST-types sharing an identical allele (clfB or spa) were clustered using eBURST. Except for one strain, all isolates from each DLST cluster belonged to the same CC. However, using both DLST- and spa-typing we were not able to disclose a clear intra-CC structure. Nevertheless, the high diversity of these loci confirmed that they are good markers for local epidemiological investigations.

Comparative genomics of epidemic versus sporadic Staphylococcus aureus strains does not reveal molecular markers for epidemicity.

Staphylococcus aureus, especially when it is methicillin resistant, has been recognised as a major cause of nosocomial and community-acquired infections. It has also been shown that certain strains were able to cause clonal epidemics whereas others showed a more incidental occurrence. On the basis of this behavioural distinction, a genetic feature underlying this difference in epidemicity can be assumed. Understanding the difference will not only contribute to the development of markers for the identification of epidemic strains but will also shed light on the evolution of clones. Genomes of strains from two independent collections (n=18 and n=10 strains) were analysed. Both collections were composed of carefully selected, genetically diverse strains with clinically well-defined epidemic and sporadic behaviour. Comparative genome hybridisation (CGH) was performed using an Agilent array for one collection (up to 11 probes per open reading frame - ORF), and an Affymetrix array for the other (up to 30 probes per ORF). Presence and absence information of probe homologues and ORFs was taken for analysis of molecular variance (AMOVA) at the strain and behaviour levels. Not a single probe showed 100% concordant differences between epidemic and sporadic strains. Moreover, probe differences between groups were always smaller than those within groups. This was also true, when the analysis was focussed on presence versus absence of ORF's or when probe information was transformed into allelic profiles. These findings present strong evidence against the presence or absence of a single common specific genetic factor differentiating epidemic from sporadic S. aureus clones.

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.