Isothermal Microcalorimetry Detects the Presence of Persister Cells in a Staphylococcus aureus Biofilm After Vancomycin Treatment

Staphylococcus aureus biofilm plays a major role in implant-associated infections. Here, the susceptibility of biofilm S. aureus to daptomycin, fosfomycin, vancomycin, trimethoprim/sulfamethoxazole, linezolid, and rifampicin was investigated by isothermal microcalorimetry (IMC). Moreover, the persister status of cells isolated from S. aureus biofilm after treatment with vancomycin was also analyzed. S. aureus biofilm was tolerant to all the antibiotics tested [minimum biofilm bactericidal concentration (MBBC) 7> 256 mu g/ml], except to daptomycin [MBBC and minimum biofilm eradicating concentration (MBEC) = 32 mu g/ml] and rifampin (MBBC and MBEC = 128 mu g/ml). After the treatment of MRSA biofilm with 1024 mu g/ml vancomycin, similar to 5% cells survived, although metabolically inactive (persisters). Interestingly, IMC revealed that persister bacteria reverted to a normal-growing phenotype when inoculated into fresh medium without antibiotics. A staggered treatment of MRSA biofilm with vancomycin to kill all the metabolically active cells and daptomycin to kill persister cells eradicated the whole bacterial population. These results support the use in the clinical practice of a therapeutic regimen based on the use of two antibiotics to kill persister cells and eradicate MRSA biofilms. IMC represents a suitable technique to characterize in real-time the reversion from persister to metabolically-active cells

Comparison of Sonication With Chemical Biofilm Dislodgement Methods Using Chelating and Reducing Agents: Implications for the Microbiological Diagnosis of Implant Associated Infection

The diagnosis of implant-associated infections is hampered due to microbial adherence and biofilm formation on the implant surface. Sonication of explanted devices was shown to improve the microbiological diagnosis by physical removal of biofilms. Recently, chemical agents have been investigated for biofilm dislodgement such as the chelating agent ethylenediaminetetraacetic acid (EDTA) and the reducing agent dithiothreitol (DTT). We compared the activity of chemical methods for biofilm dislodgement to sonication in an established in vitro model of artificial biofilm. Biofilm-producing laboratory strains of Staphylococcus epidermidis (ATCC 35984), S. aureus (ATCC 43300), E. coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 53278) were used. After 3 days of biofilm formation, porous glass beads were exposed to control (0.9% NaCl), sonication or chemical agents. Quantitative and qualitative biofilm analyses were performed by colony counting, isothermal microcalorimetry and scanning electron microscopy. Recovered colony counts after treatment with EDTA and DTT were similar to those after exposure to 0.9% NaCl for biofilms of S. epidermidis (6.3 and 6.1 vs. 6.0 log10 CFU/mL, S. aureus (6.4 and 6.3 vs. 6.3 log10 CFU/mL), E. coli (5.2 and 5.1 vs. 5.1 log10 CFU/mL and P. aeruginosa (5.1 and 5.2 vs. 5.0 log10 CFU/mL, respectively). In contrast, with sonication higher CFU counts were detected with all tested microorganisms (7.5, 7.3, 6.2 and 6.5 log10 CFU/mL, respectively) (p <0.05). Concordant results were observed with isothermal microcalorimetry and scanning electron microscopy. In conclusion, sonication is superior to both tested chemical methods (EDTA and DTT) for dislodgement of S. epidermidis, S. aureus, E. coli and P. aeruginosa biofilms. Future studies may evaluate potential additive effect of chemical dislodgement to sonication

Isothermal Microcalorimetry Detects the Presence of Persister Cells in a Staphylococcus aureus Biofilm After Vancomycin Treatment

Staphylococcus aureus biofilm plays a major role in implant-associated infections. Here, the susceptibility of biofilm S. aureus to daptomycin, fosfomycin, vancomycin, trimethoprim/sulfamethoxazole, linezolid, and rifampicin was investigated by isothermal microcalorimetry (IMC). Moreover, the persister status of cells isolated from S. aureus biofilm after treatment with vancomycin was also analyzed. S. aureus biofilm was tolerant to all the antibiotics tested [minimum biofilm bactericidal concentration (MBBC) &gt; 256 μg/ml], except to daptomycin [MBBC and minimum biofilm eradicating concentration (MBEC) = 32 μg/ml] and rifampin (MBBC and MBEC = 128 μg/ml). After the treatment of MRSA biofilm with 1024 μg/ml vancomycin, ∼5% cells survived, although metabolically inactive (persisters). Interestingly, IMC revealed that persister bacteria reverted to a normal-growing phenotype when inoculated into fresh medium without antibiotics. A staggered treatment of MRSA biofilm with vancomycin to kill all the metabolically active cells and daptomycin to kill persister cells eradicated the whole bacterial population. These results support the use in the clinical practice of a therapeutic regimen based on the use of two antibiotics to kill persister cells and eradicate MRSA biofilms. IMC represents a suitable technique to characterize in real-time the reversion from persister to metabolically-active cells

Nutritionally enriched tomatoes (Solanum lycopersicum L.) grown with wood distillate: chemical and biological characterization for quality assessment

: Bio-based products are nowadays useful tools able to affect the productivity and quality of conventionally cultivated crops. Several bio-based products are currently on the market; one of the newest and most promising is the wood distillate (WD) derived from the pyrolysis process of waste biomass after timber. Its foliar application has been widely investigated and shown to promote the antioxidant profile of cultivated crops. WD was used here as additive for the cultivation of tomato (Solanum lycopersicum L.) plants. The application improved quality (chemical) parameters, minerals, polyphenols, and lycopene contents of tomato fruits. The extracts of WD-treated and untreated tomatoes have been chemically and biologically characterized. The 1 H-NMR and ESI-mass spectrometry analyses of the extracts revealed the presence of different fatty acids, amino acids and sugars. In particular, the WD-treated tomatoes showed the presence of pyroglutamic acid and phloridzin derivatives, but also dihydrokaempferol, naringenin glucoside, cinnamic acid, and kaempferol-3-O-glucoside. When tested in cells, the extracts showed a promising anti-inflammatory profile in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Furthermore, the extracts displayed a slight vasorelaxant activity on rat aorta rings (either endothelium-denuded or endothelium-intact) pre-contracted with phenylephrine or potassium chloride. PRACTICAL APPLICATION: Wood distillate has been used for tomato plant growth. Tomatoes showed improved nutritional parameters, and their extracts displayed antioxidant and anti-inflammatory activities

Extra virgin olive oil extracts of indigenous Southern Tuscany cultivar act as anti-inflammatory and vasorelaxant nutraceuticals

Extra virgin olive oil (EVOO) is the typical source of fats in the Mediterranean diet. While fatty acids are essential for the EVOO nutraceutical properties, multiple biological activities are also due to the presence of polyphenols. In this work, autochthonous Tuscany EVOOs were chemically characterized and selected EVOO samples were extracted to obtain hydroalcoholic phytocomplexes, which were assayed to establish their anti-inflammatory and vasorelaxant properties. The polar extracts were characterized via 1H-NMR and UHPLC-HRMS to investigate the chemical composition and assayed in CaCo-2 cells exposed to glucose oxidase or rat aorta rings contracted by phenylephrine. Apigenin and luteolin were found as representative flavones; other components were pinoresinol, ligstroside, and oleuropein. The extracts showed anti-inflammatory and antioxidant properties via modulation of NF-κB and Nrf2 pathways, respectively, and good vasorelaxant activity, both in the presence and absence of an intact endothelium. In conclusion, this study evaluated the nutraceutical properties of autochthonous Tuscany EVOO cv., which showed promising anti-inflammatory and vasorelaxant effects

Traceability and authentication in agri-food production: a multivariate approach to the characterization of the Italian food excellence elephant garlic (Allium ampeloprasum L.), a vasoactive nutraceutical

A research platform for food authentication was set up by combining stable isotope ratio analysis, metabolomics by gas and liquid mass-spectrometry and NMR investigations, chemometric analyses for food excellences. This multi-analytical approach was tested on samples of elephant garlic (Allium ampeloprasum L.), a species belonging to the same genus of common garlic (Allium ampeloprasum L.), mainly produced in southern Tuscany-(Allium ampeloprasum). The isotopic composition allowed the product to be geographically characterized. Flavonoids, like (+)-catechin, cinnamic acids, quercetin glycosides were identified. The samples showed also a significant amount of dipeptides, sulphur-containing metabolites and glutathione, the latter of which could be considered a molecular marker of the analyzed elephant garlic. For nutraceutical profiling to reach quality labels, extracts were investigated in specific biological assays, displaying interesting vasorelaxant properties in rat aorta by mediating nitric oxide release from the endothelium and exhibited positive inotropic and negative chronotropic effects in rat perfused hear

Methodologic quality and risk-of-bias in systematic reviews of healthcare interventions: a review of methods

OBJECTIVE: To compare the characteristics of systematic reviews of healthcare interventions that assessed or did not methodologic quality/risk-of-bias of included studies. Additionally, to analyze methodologic features of those assessing the methodologic quality/risk-of-bias. METHODS: PubMed database was searched. From 25,571 systematic reviews retrieved, a random sample of 1,025 was screened. Frequencies were used to describe outcomes. Unadjusted and adjusted logistic regressions were performed to test the associations with methodologic quality/risk-of-bias results assessment. In a second analysis, systematic reviews that assessed methodologic quality/risk of bias were dichotomized according to the design of included studies (randomized clinical trials-only versus non-randomized studies of intervention or a combination of both). RESULTS: 303 systematic reviews were fully analyzed. Methodologic quality/risk of bias was assessed by 278 (92%). Methodologic quality/risk-of-bias assessment was associated with a higher number of databases searched (>4, P= 0.008), the presence of meta-analysis (P= 0.005), and the design of included studies (randomized clinical trials-only, P= 0.042). The chance of using a suitable tool and a tool designed for risk-of-bias assessment rather than methodologic quality was higher for randomized clinical trials-only systematic reviews (P4). The most used tool was Cochrane’s RoB Tool, with no clearly defined rating system. Methodologic quality/risk-of-bias assessment methods description, results, and impacts on meta-analysis, the certainty of evidence, and systematic reviews’ results are still to be consistently addressed.

Molecular analysis of hepatitis B virus (HBV) in an HIV co-infected patient with reactivation of occult HBV infection following discontinuation of lamivudine-including antiretroviral therapy

Abstract Background Occult hepatitis B virus (HBV) infection (OBI) is characterized by HBV DNA persistence even though the pattern of serological markers indicates an otherwise resolved HBV infection. Although OBI is usually clinically silent, immunocompromised patients may experience reactivation of the liver disease. Case presentation We report the case of an individual with human immunodeficiency virus (HIV) infection and anti-HBV core antibody positivity, who experienced severe HBV reactivation after discontinuation of lamivudine-including antiretroviral therapy (ART). HBV sequencing analysis showed a hepatitis B surface antigen escape mutant whose presence in an earlier sample excluded reinfection. Molecular sequencing showed some differences between two isolates collected at a 9-year interval, indicating HBV evolution. Resumption of ART containing an emtricitabine/tenofovir combination allowed control of plasma HBV DNA, which fell to undetectable levels. Conclusion This case stresses the ability of HBV to evolve continuously, even during occult infection, and the effectiveness of ART in controlling OBI reactivation in HIV-infected individuals.</p

A Prognostic Model for Estimating the Time to Virologic Failure in HIV-1 Infected Patients Undergoing a New Combination Antiretroviral Therapy Regimen

<p>Abstract</p> <p>Background</p> <p>HIV-1 genotypic susceptibility scores (GSSs) were proven to be significant prognostic factors of fixed time-point virologic outcomes after combination antiretroviral therapy (cART) switch/initiation. However, their relative-hazard for the time to virologic failure has not been thoroughly investigated, and an expert system that is able to predict how long a new cART regimen will remain effective has never been designed.</p> <p>Methods</p> <p>We analyzed patients of the Italian ARCA cohort starting a new cART from 1999 onwards either after virologic failure or as treatment-naïve. The time to virologic failure was the endpoint, from the 90^thday after treatment start, defined as the first HIV-1 RNA > 400 copies/ml, censoring at last available HIV-1 RNA before treatment discontinuation. We assessed the relative hazard/importance of GSSs according to distinct interpretation systems (Rega, ANRS and HIVdb) and other covariates by means of Cox regression and random survival forests (RSF). Prediction models were validated via the bootstrap and c-index measure.</p> <p>Results</p> <p>The dataset included 2337 regimens from 2182 patients, of which 733 were previously treatment-naïve. We observed 1067 virologic failures over 2820 persons-years. Multivariable analysis revealed that low GSSs of cART were independently associated with the hazard of a virologic failure, along with several other covariates. Evaluation of predictive performance yielded a modest ability of the Cox regression to predict the virologic endpoint (c-index≈0.70), while RSF showed a better performance (c-index≈0.73, p < 0.0001 vs. Cox regression). Variable importance according to RSF was concordant with the Cox hazards.</p> <p>Conclusions</p> <p>GSSs of cART and several other covariates were investigated using linear and non-linear survival analysis. RSF models are a promising approach for the development of a reliable system that predicts time to virologic failure better than Cox regression. Such models might represent a significant improvement over the current methods for monitoring and optimization of cART.</p

Development and Validation of a Risk Score for Chronic Kidney Disease in HIV Infection Using Prospective Cohort Data from the D:A:D Study

Ristola M. on työryhmien DAD Study Grp ; Royal Free Hosp Clin Cohort ; INSIGHT Study Grp ; SMART Study Grp ; ESPRIT Study Grp jäsen.Background Chronic kidney disease (CKD) is a major health issue for HIV-positive individuals, associated with increased morbidity and mortality. Development and implementation of a risk score model for CKD would allow comparison of the risks and benefits of adding potentially nephrotoxic antiretrovirals to a treatment regimen and would identify those at greatest risk of CKD. The aims of this study were to develop a simple, externally validated, and widely applicable long-term risk score model for CKD in HIV-positive individuals that can guide decision making in clinical practice. Methods and Findings A total of 17,954 HIV-positive individuals from the Data Collection on Adverse Events of Anti-HIV Drugs (D:A:D) study with >= 3 estimated glomerular filtration rate (eGFR) values after 1 January 2004 were included. Baseline was defined as the first eGFR > 60 ml/min/1.73 m2 after 1 January 2004; individuals with exposure to tenofovir, atazanavir, atazanavir/ritonavir, lopinavir/ritonavir, other boosted protease inhibitors before baseline were excluded. CKD was defined as confirmed (>3 mo apart) eGFR In the D:A:D study, 641 individuals developed CKD during 103,185 person-years of follow-up (PYFU; incidence 6.2/1,000 PYFU, 95% CI 5.7-6.7; median follow-up 6.1 y, range 0.3-9.1 y). Older age, intravenous drug use, hepatitis C coinfection, lower baseline eGFR, female gender, lower CD4 count nadir, hypertension, diabetes, and cardiovascular disease (CVD) predicted CKD. The adjusted incidence rate ratios of these nine categorical variables were scaled and summed to create the risk score. The median risk score at baseline was -2 (interquartile range -4 to 2). There was a 1: 393 chance of developing CKD in the next 5 y in the low risk group (risk score = 5, 505 events), respectively. Number needed to harm (NNTH) at 5 y when starting unboosted atazanavir or lopinavir/ritonavir among those with a low risk score was 1,702 (95% CI 1,166-3,367); NNTH was 202 (95% CI 159-278) and 21 (95% CI 19-23), respectively, for those with a medium and high risk score. NNTH was 739 (95% CI 506-1462), 88 (95% CI 69-121), and 9 (95% CI 8-10) for those with a low, medium, and high risk score, respectively, starting tenofovir, atazanavir/ritonavir, or another boosted protease inhibitor. The Royal Free Hospital Clinic Cohort included 2,548 individuals, of whom 94 individuals developed CKD (3.7%) during 18,376 PYFU (median follow-up 7.4 y, range 0.3-12.7 y). Of 2,013 individuals included from the SMART/ESPRIT control arms, 32 individuals developed CKD (1.6%) during 8,452 PYFU (median follow-up 4.1 y, range 0.6-8.1 y). External validation showed that the risk score predicted well in these cohorts. Limitations of this study included limited data on race and no information on proteinuria. Conclusions Both traditional and HIV-related risk factors were predictive of CKD. These factors were used to develop a risk score for CKD in HIV infection, externally validated, that has direct clinical relevance for patients and clinicians to weigh the benefits of certain antiretrovirals against the risk of CKD and to identify those at greatest risk of CKD.Peer reviewe