In Vitro and In Vivo Effectiveness of an Innovative Silver-Copper Nanoparticle Coating of Catheters To Prevent Methicillin-Resistant Staphylococcus aureus Infection.

In this study, silver/copper (Ag/Cu)-coated catheters were investigated for their efficacy in preventing methicillin-resistant Staphylococcus aureus (MRSA) infection in vitro and in vivo Ag and Cu were sputtered (67/33% atomic ratio) on polyurethane catheters by direct-current magnetron sputtering. In vitro, Ag/Cu-coated and uncoated catheters were immersed in phosphate-buffered saline (PBS) or rat plasma and exposed to MRSA ATCC 43300 at 10(4) to 10(8) CFU/ml. In vivo, Ag/Cu-coated and uncoated catheters were placed in the jugular vein of rats. Directly after, MRSA (10(7) CFU/ml) was inoculated in the tail vein. Catheters were removed 48 h later and cultured. In vitro, Ag/Cu-coated catheters preincubated in PBS and exposed to 10(4) to 10(7) CFU/ml prevented the adherence of MRSA (0 to 12% colonization) compared to uncoated catheters (50 to 100% colonization; P < 0.005) and Ag/Cu-coated catheters retained their activity (0 to 20% colonization) when preincubated in rat plasma, whereas colonization of uncoated catheters increased (83 to 100%; P < 0.005). Ag/Cu-coating protection diminished with 10(8) CFU/ml in both PBS and plasma (50 to 100% colonization). In vivo, Ag/Cu-coated catheters reduced the incidence of catheter infection compared to uncoated catheters (57% versus 79%, respectively; P = 0.16) and bacteremia (31% versus 68%, respectively; P < 0.05). Scanning electron microscopy of explanted catheters suggests that the suboptimal activity of Ag/Cu catheters in vivo was due to the formation of a dense fibrin sheath over their surface. Ag/Cu-coated catheters thus may be able to prevent MRSA infections. Their activity might be improved by limiting plasma protein adsorption on their surfaces.

Bactericidal activity and mechanism of action of copper-sputtered flexible surfaces against multidrug-resistant pathogens.

Using direct current magnetron sputtering (DCMS), we generated flexible copper polyester surfaces (Cu-PES) and investigated their antimicrobial activity against a range of multidrug-resistant (MDR) pathogens including eight Gram-positive isolates (three methicillin-resistant Staphylococcus aureus [MRSA], four vancomycin-resistant enterococci, one methicillin-resistant Staphylococcus epidermidis) and four Gram-negative strains (one extended-spectrum ß-lactamase-producing [ESBL] Escherichia coli, one ESBL Klebsiella pneumoniae, one imipenem-resistant Pseudomonas aeruginosa, and one ciprofloxacin-resistant Acinetobacter baumannii). Bactericidal activity (≥3 log10 CFU reduction of the starting inoculum) was reached within 15-30 min exposure to Cu-PES. Antimicrobial activity of Cu-PES persisted in the absence of oxygen and against both Gram-positive and Gram-negative bacteria containing elevated levels of catalases, indicating that reactive oxygen species (ROS) do not play a primary role in the killing process. The decrease in cell viability of MRSA ATCC 43300 and Enterococcus faecalis V583 correlated with the progressive loss of cytoplasmic membrane integrity both under aerobic and anaerobic conditions, suggesting that Cu-PES mediated killing is primarily induced by disruption of the cytoplasmic membrane function. Overall, we here present novel antimicrobial copper surfaces with improved stability and sustainability and provide further insights into their mechanism of killing.

Loss of Antibiotic Tolerance in Sod-Deficient Mutants Is Dependent on the Energy Source and Arginine Catabolism in Enterococci.

UNLABELLED: Enterococci are naturally tolerant to typically bactericidal cell wall-active antibiotics, meaning that their growth is inhibited but they are not killed even when exposed to a high concentration of the drug. The molecular reasons for this extraordinary tolerance are still incompletely understood. Previous work showed that resistance to killing collapsed specifically in mutants affected in superoxide dismutase (Sod) activity, arguing that bactericidal antibiotic treatment led to induction of a superoxide burst. In the present work, we show that loss of antibiotic tolerance in ΔsodA mutants of pathogenic enterococci is dependent on the energy source present during antibiotic treatment. Hexoses induce greater killing than the pentose ribose, and no killing was observed with glycerol as the energy source. These results point to glycolytic reactions as crucial for antibiotic-mediated killing of ΔsodA mutants. A transposon mutant library was constructed in Enterococcus faecalis ΔsodA mutants and screened for restored tolerance of vancomycin. Partially restored tolerance was observed in mutants with transposon integrations into intergenic regions upstream of regulators implicated in arginine catabolism. In these mutants, the arginine deiminase operon was highly upregulated. A model for the action of cell wall-active antibiotics in tolerant and nontolerant bacteria is proposed. IMPORTANCE: Antibiotic tolerance is a serious clinical concern, since tolerant bacteria have considerably increased abilities to resist killing by bactericidal drugs. Using enterococci as models for highly antibiotic-tolerant pathogens, we showed that tolerance of these bacteria is linked to their superoxide dismutase (Sod), arguing that bactericidal antibiotics induce generation of reactive oxygen species inside cells. Wild-type strains are tolerant because they detoxify these deleterious molecules by the activity of Sod, whereas Sod-deficient strains are killed. This study showed that killing depends on the energy source present during treatment and that an increase in arginine catabolism partially restored tolerance of the Sod mutants. These results are used to propose a mode-of-action model of cell wall-active antibiotics in tolerant and nontolerant bacteria.

Impact of matrix-assisted laser desorption ionization time-of-flight mass spectrometry on the clinical management of patients with Gram-negative bacteremia: a prospective observational study.

BACKGROUND: Early identification of pathogens from blood cultures using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry may optimize the choice of empirical antibiotic therapy in the setting of bloodstream infections. We aimed to assess the impact of this new technology on the use of antibiotic treatment in patients with gram-negative bacteremia. METHODS: We conducted a prospective observational study from January to December 2010 to evaluate the sequential and separate impacts of Gram stain reporting and MALDI-TOF bacterial identification performed on blood culture pellets in patients with gram-negative bacteremia. The primary outcome was the impact of MALDI-TOF on empirical antibiotic choice. RESULTS: Among 202 episodes of gram-negative bacteremia, Gram stain reporting had an impact in 42 cases (20.8%). MALDI-TOF identification led to a modification of empirical therapy in 71 of all 202 cases (35.1%), and in 16 of 27 cases (59.3%) of monomicrobial bacteremia caused by AmpC-producing Enterobacteriaceae. The most frequently observed impact was an early appropriate broadening of the antibiotic spectrum in 31 of 71 cases (43.7%). In total, 143 of 165 episodes (86.7%) of monomicrobial bacteremia were correctly identified at genus level by MALDI-TOF. CONCLUSIONS: In a low prevalence area for extended spectrum betalactamases (ESBL) and multiresistant gram-negative bacteria, MALDI-TOF performed on blood culture pellets had an impact on the clinical management of 35.1% of all gram-negative bacteremia cases, demonstrating a greater impact than Gram stain reporting. Thus, MALDI-TOF could become a vital second step beside Gram stain in guiding the empirical treatment of patients with bloodstream infection.

Mutations in msrA and msrB, encoding epimer-specific methionine sulfoxide reductases, affect expression of glycerol-catabolic operons in Enterococcus faecalis differently.

This study aims to define the cellular roles of methionine sulfoxide reductases A and B, evolutionarily highly conserved enzymes able to repair oxidized methionines in proteins. msrA and msrB mutants were exposed to an internal oxidative stress by growing them under aerobic conditions on glycerol. Interestingly, the msr mutants behave completely differently under these conditions. The msrA mutant is inhibited, whereas the msrB mutant is stimulated in its growth in comparison with the parent strain. Glycerol can be catabolized by either the GlpK or DhaK pathways in Enterococcus faecalis. Our results strongly suggest that in the msrA mutant, glycerol is catabolized via the GlpK pathway leading to increased synthesis of H2O2, which accumulates to concentrations inhibitory to growth in comparison with the parent strain. In contrast in the msrB mutant, glycerol is metabolized via the DhaK pathway which is not accompanied by the synthesis of H2O2. The molecular basis for the differences in glycerol flux seems to be due to expression differences of the two glycerol-catabolic operons in the msr mutants.

Analysis of the tolerance of pathogenic enterococci and Staphylococcus aureus to cell wall active antibiotics.

OBJECTIVES: Tolerance refers to the phenomenon that bacteria do not significantly die when exposed to bactericidal antibiotics. Enterococci are known for their high tolerance to these drugs, but the molecular reasons why they resist killing are not understood. In a previous study we showed that the superoxide dismutase (SOD) is implicated in this tolerance. This conclusion was based on the results obtained with one particular strain of Enterococcus faecalis and therefore the objective of the present communication was to analyse whether dependence of tolerance on active SOD is a general phenomenon for enterococci and another Gram-positive pathogen, Staphylococcus aureus. METHODS: Mutants deficient in SOD activity were constructed in pathogenic enterococci. The wild-type sodA gene was cloned into an expression vector and transformed into SOD-deficient strains for complementation with varying levels of SOD activity. Previously constructed SOD-deficient strains of S. aureus were also included in this study. Tolerance to vancomycin and penicillin was then tested. RESULTS: We demonstrated that the dependence on SOD of tolerance to vancomycin and penicillin is a common trait of antibiotic-susceptible pathogenic enterococci. By varying the levels of expression we could also show that tolerance to vancomycin is directly correlated to SOD activity. Interestingly, deletion of the sodA gene in a non-tolerant Enterococcus faecium strain did not further sensitize the mutant to bactericidal antibiotics. Finally, we showed that the SOD enzymes of S. aureus are also implicated in tolerance to vancomycin. CONCLUSION: High tolerance of enterococci to cell wall active antibiotics can be reversed by SOD deficiency.

Force volume and stiffness tomography investigation on the dynamics of stiff material under bacterial membranes.

The determination of the characteristics of micro-organisms in clinical specimens is essential for the rapid diagnosis and treatment of infections. A thorough investigation of the nanoscale properties of bacteria can prove to be a fundamental tool. Indeed, in the latest years, the importance of high resolution analysis of the properties of microbial cell surfaces has been increasingly recognized. Among the techniques available to observe at high resolution specific properties of microscopic samples, the Atomic Force Microscope (AFM) is the most widely used instrument capable to perform morphological and mechanical characterizations of living biological systems. Indeed, AFM can routinely study single cells in physiological conditions and can determine their mechanical properties with a nanometric resolution. Such analyses, coupled with high resolution investigation of their morphological properties, are increasingly used to characterize the state of single cells. In this work, we exploit the capabilities and peculiarities of AFM to analyze the mechanical properties of Escherichia coli in order to evidence with a high spatial resolution the mechanical properties of its structure. In particular, we will show that the bacterial membrane is not mechanically uniform, but contains stiffer areas. The force volume investigations presented in this work evidence for the first time the presence and dynamics of such structures. Such information is also coupled with a novel stiffness tomography technique, suggesting the presence of stiffer structures present underneath the membrane layer that could be associated with bacterial nucleoids.

Vancomycin-intermediate Staphylococcus aureus selected during vancomycin therapy of experimental endocarditis are not detected by culture-based diagnostic procedures and persist after treatment arrest.

OBJECTIVES: Laboratory detection of vancomycin-intermediate Staphylococcus aureus (VISA) and their heterogeneous VISA (hVISA) precursors is difficult. Thus, it is possible that vancomycin failures against supposedly vancomycin-susceptible S. aureus are due to undiagnosed VISA or hVISA. We tested this hypothesis in experimental endocarditis. METHODS: Rats with aortic valve infection due to the vancomycin-susceptible (MIC 2 mg/L), methicillin-resistant S. aureus M1V2 were treated for 2 days with doses of vancomycin that mimicked the pharmacokinetics seen in humans following intravenous administration of 1 g of the drug every 12 h. Half of the treated animals were killed 8 h after treatment arrest and half 3 days thereafter. Population analyses were done directly on vegetation homogenates or after one subculture in drug-free medium to mimic standard diagnostic procedures. RESULTS: Vancomycin cured 14 of 26 animals (54%; P<0.05 versus controls) after 2 days of treatment. When vegetation homogenates were plated directly on vancomycin-containing plates, 6 of 13 rats killed 8 h after treatment arrest had positive cultures, 1 of which harboured hVISA. Likewise, 6 of 13 rats killed 3 days thereafter had positive valve cultures, 5 of which harboured hVISA. However, one subculture of vegetations in drug-free broth was enough to revert all the hVISA phenotypes to the susceptible pattern of the parent. Thus, vancomycin selected for hVISA during therapy of experimental endocarditis due to vancomycin-susceptible S. aureus. These hVISA were associated with vancomycin failure. The hVISA phenotype persisted in vivo, even after vancomycin arrest, but was missed in vitro after a single passage of the vegetation homogenate on drug-free medium. CONCLUSIONS: hVISA might escape detection in clinical samples if they are subcultured before susceptibility tests.

Comparison of methods for evaluation of the bactericidal activity of copper-sputtered surfaces against methicillin-resistant Staphylococcus aureus.

Bacteria can survive on hospital textiles and surfaces, from which they can be disseminated, representing a source of health care-associated infections (HCAIs). Surfaces containing copper (Cu), which is known for its bactericidal properties, could be an efficient way to lower the burden of potential pathogens. The antimicrobial activity of Cu-sputtered polyester surfaces, obtained by direct-current magnetron sputtering (DCMS), against methicillin-resistant Staphylococcus aureus (MRSA) was tested. The Cu-polyester microstructure was characterized by high-resolution transmission electron microscopy to determine the microstructure of the Cu nanoparticles and by profilometry to assess the thickness of the layers. Sputtering at 300 mA for 160 s led to a Cu film thickness of 20 nm (100 Cu layers) containing 0.209% (wt/wt) polyester. The viability of MRSA strain ATCC 43300 on Cu-sputtered polyester was evaluated by four methods: (i) mechanical detachment, (ii) microcalorimetry, (iii) direct transfer onto plates, and (iv) stereomicroscopy. The low efficacy of mechanical detachment impeded bacterial viability estimations. Microcalorimetry provided only semiquantitative results. Direct transfer onto plates and stereomicroscopy seemed to be the most suitable methods to evaluate the bacterial inactivation potential of Cu-sputtered polyester surfaces, since they presented the least experimental bias. Cu-polyester samples sputtered for 160 s by DCMS were further tested against 10 clinical MRSA isolates and showed a high level of bactericidal activity, with a 4-log(10) reduction in the initial MRSA load (10(6) CFU) within 1 h. Cu-sputtered polyester surfaces might be of use to prevent the transmission of HCAI pathogens.

Glycerol is metabolized in a complex and strain-dependent manner in Enterococcus faecalis.

Enterococcus faecalis is equipped with two pathways of glycerol dissimilation. Glycerol can either first be phosphorylated by glycerol kinase and then oxidized by glycerol-3-phosphate oxidase (the glpK pathway) or first be oxidized by glycerol dehydrogenase and then phosphorylated by dihydroxyacetone kinase (the dhaK pathway). Both pathways lead to the formation of dihydroxyacetone phosphate, an intermediate of glycolysis. It was assumed that the glpK pathway operates during aerobiosis and that the dhaK pathway operates under anaerobic conditions. Because this had not been analyzed by a genetic study, we constructed mutants of strain JH2-2 affected in both pathways. The growth of these mutants on glycerol under aerobic and anaerobic conditions was monitored. In contrast to the former model, results strongly suggest that glycerol is catabolized simultaneously by both pathways in the E. faecalis JH2-2 strain in the presence of oxygen. In accordance with the former model, glycerol is metabolized by the dhaK pathway under anaerobic conditions. Comparison of different E. faecalis isolates revealed an impressive diversity of growth behaviors on glycerol. Analysis by BLAST searching and real-time reverse transcriptase PCR revealed that this diversity is based not on different gene contents but rather on differences in gene expression. Some strains used preferentially the glpK pathway whereas others probably exclusively the dhaK pathway under aerobic conditions. Our results demonstrate that the species E. faecalis cannot be represented by only one model of aerobic glycerol catabolism.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry for direct bacterial identification from positive blood culture pellets.

An ammonium chloride erythrocyte-lysing procedure was used to prepare a bacterial pellet from positive blood cultures for direct matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry analysis. Identification was obtained for 78.7% of the pellets tested. Moreover, 99% of the MALDI-TOF identifications were congruent at the species level when considering valid scores. This fast and accurate method is promising.

Streptococcus anginosus and Coxiella burnetii vascular graft co-infection.

Vascular graft infections are rare complications, usually associated with a monomicrobial pyogenic culture. We report a case of vascular graft co-infection with Streptococcus anginosus and Coxiella burnetii, complicated by an aorto-duodenal fistula. Screening for chronic C. burnetii co-infection in at-risk patients might prevent adverse long-term outcomes.

The Enterococcus faecalis superoxide dismutase is essential for its tolerance to vancomycin and penicillin.

OBJECTIVES: Enterococcus faecalis is a human commensal that has the ability to become a pathogen. Because of its ruggedness, it can persist in the hospital setting and cause serious nosocomial infections. E. faecalis can acquire multiple drug resistance determinants but is also intrinsically tolerant to a number of antibiotics, such as penicillin or vancomycin, meaning that these usually bactericidal drugs only exhibit a bacteriostatic effect. Recently, evidence has been presented that exposure to bactericidal antibiotics induced the production of reactive oxygen species in bacteria. Here, we studied the role of enzymes involved in the oxidative stress response in the survival of E. faecalis after antibiotic treatment. METHODS: Mutants defective in genes encoding oxidative stress defence activities were tested by time-kill curves for their contribution to antibiotic tolerance in comparison with the E. faecalis JH2-2 wild-type (WT). RESULTS: In killing assays, WT cultures lost 0.2 +/- 0.1 and 1.3 +/- 0.2 log(10) cfu/mL after 24 h of vancomycin or penicillin exposure, respectively. A deletion mutant of the superoxide dismutase gene (DeltasodA) exhibited a lack of tolerance as cultures lost 4.1 +/- 0.5 and 4.8 +/- 0.7 log(10) cfu/mL after 24 h of exposure to the same drugs. Complementation of DeltasodA re-established the tolerant phenotype. Bacterial killing was an oxygen-dependent process and a model is presented implicating the superoxide anion as the mediator of this killing. As predicted from the model, a mutant defective in peroxidase activities excreted hydrogen peroxide at an elevated rate. CONCLUSIONS: SodA is central to the intrinsic ability of E. faecalis to withstand drug-induced killing, and the superoxide anion seems to be the key effector of bacterial death.

Fungicidal activity of copper-sputtered flexible surfaces under dark and actinic light against azole-resistant Candida albicans and Candida glabrata.

Candida spp. are able to survive on hospital surfaces and causes healthcare-associated infections (HCAIs). Since surface cleaning and disinfecting interventions are not totally effective to eliminate Candida spp., new approaches should be devised. Copper (Cu) has widely recognized antifungal activity and the use of Cu-sputtered surfaces has recently been proposed to curb the spread of HCAIs. Moreover, the activity of Cu under the action of actinic light remains underexplored. We investigated the antifungal activity of Cu-sputtered polyester surfaces (Cu-PES) against azole-resistant Candida albicans and Candida glabrata under dark and low intensity visible light irradiation (4.65mW/cm2). The surface properties of Cu-PES photocatalysts were characterized by diffuse reflectance spectroscopy (DRS) and X-ray fluorescence (XRF). Under dark, Cu-PES showed a fungicidal activity (≥3log10CFU reduction of the initial inoculum) against both C. albicans DSY296 and C. glabrata DSY565 leading to a reduction of the starting inoculum of 3.1 and 3.0log10CFU, respectively, within 60min of exposure. Under low intensity visible light irradiation, Cu-PES exhibited an accelerated fungicidal activity against both strains with a reduction of 3.0 and 3.4log10CFU, respectively, within 30min of exposure. This effect was likely due to the semiconductor Cu2O/CuO charge separation. The decrease in cell viability of the two Candida strains under dark and light conditions correlated with the progressive loss of membrane integrity. These results indicate that Cu-PES represent a promising strategy for decreasing the colonization of surfaces by yeasts and that actinic light can improve its self-disinfecting activity.

Lack of in vitro biofilm formation does not attenuate the virulence of Streptococcus gordonii in experimental endocarditis.

The ability to induce experimental endocarditis of biofilm-deficient mutants of Streptococcus gordonii was studied in an isogenic background. Strains were inactivated in either comD, fruK or pbp2b genes, which are involved in biofilm formation. These strains were clearly impaired (>75% reduction) in biofilm production in vitro. However, this did not result in a decreased severity of infection in vivo.

Evaluation of a new serological test for the detection of anti-Coxiella and anti-Rickettsia antibodies.

Coxiella burnetii and members of the genus Rickettsia are obligate intracellular bacteria. Since cultivation of these organisms requires dedicated techniques, their diagnosis usually relies on serological or molecular biology methods. Immunofluorescence is considered the gold standard to detect antibody-reactivity towards these organisms. Here, we assessed the performance of a new automated epifluorescence immunoassay (InoDiag) to detect IgM and IgG against C. burnetii, Rickettsia typhi and Rickettsia conorii. Samples were tested with the InoDiag assay. A total of 213 sera were tested, of which 63 samples from Q fever, 20 from spotted fever rickettsiosis, 6 from murine typhus and 124 controls. InoDiag results were compared to micro-immunofluorescence. For acute Q fever, the sensitivity of phase 2 IgG was only of 30% with a cutoff of 1 arbitrary unit (AU). In patients with acute Q fever with positive IF IgM, sensitivity reached 83% with the same cutoff. Sensitivity for chronic Q fever was 100% whereas sensitivity for past Q fever was 65%. Sensitivity for spotted Mediterranean fever and murine typhus were 91% and 100%, respectively. Both assays exhibited a good specificity in control groups, ranging from 79% in sera from patients with unrelated diseases or EBV positivity to 100% in sera from healthy patients. In conclusion, the InoDiag assay exhibits an excellent performance for the diagnosis of chronic Q fever but a very low IgG sensitivity for acute Q fever likely due to low reactivity of phase 2 antigens present on the glass slide. This defect is partially compensated by the detection of IgM. Because it exhibits a good negative predictive value, the InoDiag assay is valuable to rule out a chronic Q fever. For the diagnosis of rickettsial diseases, the sensitivity of the InoDiag method is similar to conventional immunofluorescence.

Acute schistosomiasis: a risk underestimated by travelers and a diagnosis frequently missed by general practitioners-a cluster analysis of 42 travelers.

BACKGROUND: In 2011, a patient was admitted to our hospital with acute schistosomiasis after having returned from Madagascar and having bathed at the Lily waterfalls. On the basis of this patient's indication, infection was suspected in 41 other subjects. This study investigated (1) the knowledge of the travelers about the risks of schistosomiasis and their related behavior to evaluate the appropriateness of prevention messages and (2) the diagnostic workup of symptomatic travelers by general practitioners to evaluate medical care of travelers with a history of freshwater exposure in tropical areas. METHODS: A questionnaire was sent to the 42 travelers with potential exposure to schistosomiasis. It focused on pre-travel knowledge of the disease, bathing conditions, clinical presentation, first suspected diagnosis, and treatment. RESULTS: Of the 42 questionnaires, 40 (95%) were returned, among which 37 travelers (92%) reported an exposure to freshwater, and 18 (45%) were aware of the risk of schistosomiasis. Among these latter subjects, 16 (89%) still reported an exposure to freshwater. Serology was positive in 28 (78%) of 36 exposed subjects at least 3 months after exposure. Of the 28 infected travelers, 23 (82%) exhibited symptoms and 16 (70%) consulted their general practitioner before the information about the outbreak had spread, but none of these patients had a serology for schistosomiasis done during the first consultation. CONCLUSIONS: The usual prevention message of avoiding freshwater contact when traveling in tropical regions had no impact on the behavior of these travelers, who still went swimming at the Lily waterfalls. This prevention message should, therefore, be either modified or abandoned. The clinical presentation of acute schistosomiasis is often misleading. General practitioners should at least request an eosinophil count, when confronted with a returning traveler with fever. If eosinophilia is detected, it should prompt the search for a parasitic disease.

The effect of inoculum size on selection of in vitro resistance to vancomycin, daptomycin, and linezolid in methicillin-resistant Staphylococcus aureus.

OBJECTIVES: The inoculum effect (IE) is an increase in the minimum inhibitory concentration (MIC) at high bacterial densities. The effect of three inoculum sizes on the selection of resistance to vancomycin, daptomycin, and linezolid was investigated in methicillin-resistant Staphylococcus aureus (MRSA). METHODS: Low (10(4) CFU/ml), medium (10(6) CFU/ml), and high (10(8) CFU/ml) inocula of MRSA were exposed to twofold increasing concentrations of either drug during 15 days of cycling. MICs for low (MICL), medium (MICM), and high (MICH) inocula were determined daily. Conventional MICs were measured at days 1, 5, 10, and 15. Experiments were performed in triplicate. RESULTS: At the beginning of the experiment a small IE was observed for vancomycin (MICL=1 µg/ml, MICM=1-2 µg/ml, and MICH=2 µg/ml) and a significant IE for daptomycin (MICL=0.25 µg/ml, MICM=0.25-0.5 µg/ml, and MICH=2 µg/ml). Linezolid exhibited no IE at low and medium inocula (MICL=1 µg/ml and MICM=1-2 µg/ml), but with the high inoculum, concentrations up to 2,048 µg/ml did not fully inhibit visual growth. During cycling, increase of MIC was observed for all antibiotics. At day 15, MICL, MICM, and MICH of vancomycin were 2-4, 4-8, and 4-16 µg/ml and of daptomycin were 0.5-2, 8-128, and 64-256 µg/ml, respectively. MICL and MICM of linezolid were 1 and 2-4 µg/ml, respectively. Conventional MICs showed vancomycin and daptomycin selection of resistance since day 5 depending on the inocula. No selection of linezolid resistance was observed. CONCLUSIONS: Our results showed the importance of the inoculum size in the development of resistance. Measures aimed at lowering the inoculum at the site of infection should be used whenever possible in parallel to antimicrobial therapy.

Antibiotic-induced modifications of the stiffness of bacterial membranes.

In the latest years the importance of high resolution analysis of the microbial cell surface has been increasingly recognized. Indeed, in order to better understand bacterial physiology and achieve rapid diagnostic and treatment techniques, a thorough investigation of the surface modifications induced on bacteria by different environmental conditions or drugs is essential. Several instruments are nowadays available to observe at high resolution specific properties of microscopic samples. Among these, AFM can routinely study single cells in physiological conditions, measuring the mechanical properties of their membrane at a nanometric scale (force volume). Such analyses, coupled with high resolution investigation of their morphological properties, are increasingly used to characterize the state of single cells. In this work we exploit such technique to characterize bacterial systems. We have performed an analysis of the mechanical properties of bacteria (Escherichia coli) exposed to different conditions. Such measurements were performed on living bacteria, by changing in real-time the liquid environment: standard phosphate buffered saline, antibiotic (ampicillin) in PBS and growth medium. In particular we have focused on the determination of the membrane stiffness modifications induced by these solutions, in particular between stationary and replicating phases and what is the effect of the antibiotic on the bacterial structure.