Log Drop Research Articles

Assessment of lyophilized microencapsulated multi strain probiotic for improved shelf life and stability

A Multi species Probiotics (MSP) of calf gut origin was encapsulated either with chitosan (ECMSP) or without chitosan (EMSP). Further, the probiotic was encapsulated and lyophilized (ELMSP) using three types of cryoprotectant solutions sucrose (ELMSP(S)), skim milk (ELMSP(SM)) and sucrose + skim milk combination (ELMSP (SSM)). The encapsulation yield was good and ranged from 75 % to 80 % for MSP products. These MSP products were stored at refrigeration temperature (4 °C), deep freeze and room temperature for 12 weeks. The shelf life (mean CFU) of encapsulated microcapsules was significantly better (P < 0.001) at the refrigeration temperature (9.44 Log10 CFU/g) than deep freeze (7.42 Log10 CFU/g) and room temperature (6.67 Log10 CFU/g). At the end of the 12 weeks storage period, EMSP maintained 25.1 %, 49.6 % and 32.6 % viable counts at room temperature, refrigeration and deep freeze condition respectively. For the lyophilized-encapsulated MSP, the mean probiotic counts of ELMSP(S) and ELMSP (SSM) were significantly (P < 0.001) higher (9.62 Log10 CFU/g) than those of ELMSP(SM) ((9.36 Log10 CFU/g) at room temperature. At refrigeration temperature, the mean probiotic counts for ELMSP (SSM. (10.1 Log10 CFU/g)) were significantly higher (P < 0.001) than ELMSP(S, (9.97 Log10 CFU/g) and ELMSP(SM, (9.86 Log10 CFU/g). Further, a 12 weeks storage study for ELMSP (SSM) showed that the microcapsules stored at refrigeration temperature (4°C) showed only 6.4 % drop in viability which was significantly lower (P < 0.001) than that of room temperature showing 15.3 % drop and deep freeze storage with 17.1 % drop in viability. It was appreciable that even at room temperature; there was only one log drop in the viability of MSP showing that it can be comfortably stored at room temperature for longer periods at field level.

An In-vitro Antioxidant and Antimicrobial Activity of Vitis vinifera Leaf Extract

Background: Vitis vinifera (Flame red grape) is known for its hepatoprotective, nephroprotective and cardioprotective activity of ethanolic and aqueous extracts of aerial parts has been reported but, due to presence of polyphenols, glycosides, saponins and alkaloid, the antioxidant and antimicrobial activity yet not to evaluated on salad plant and bacteria, respectively. Aim: This research examined grape (V. vinifera flaming red) leaf ethanolic extract (EE) as a bioactive resource. Methods: Ethanolic extract test, primarily to calculate the overall phenolic content by using the Folin-Ciocalteu technique and gallic acid taking as a reference. Similarly, flavonoid content measured using quercetin as a calibration curve. Antioxidant properties was confirmed by getting positive responses from different methods (DPPH, H2O2, FRAP and Phospho-Molybdenum). Antimicrobial activity tested against gram-positive bacteria (Lactobacillus cereus and Staphylococcus aureus) and gramnegative bacteria (Escherichia coli and Staphylococcus enterica ser. Typhimurium) in a broth microdilution test. The ethanolic extract (EE) are rich in antioxidant-rich phenolic compounds show minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) for every given bacterium on beetroot and spinach leaves. Results: We investigate this extract’s total phenol, total flavonoid, and antioxidant and antibacterial properties. We also identify its main phenolic components, revealing its phytochemical diversity. We found that the EE had 39.29 gGAkg-1 dw of phenol and 96.05 gQE kg-1 dw of flavonoids. The EE scavenges DPPH, H2O2, FRAP, and PM radicals at 142, 168, 275, and 172 g TE kg-1, respectively. We reveal the complex character of this extract by revealing catechins, flavonoids, tannins, malic acid, enzymes, resveratrol, phenolic acids, flavonols, procyanidins, and anthocyanins. Salmonella enterica subsp. enterica serovar Typhimurium, Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus are all inhibited by EE with a MIC range of 16 to 18 gL-1 (E. coli). The extract affects bacterial growth at different stages. The EE reduces spinach and beetroot leaf total bacterial load by 1.159 to 2.456 log drop when used as a sanitizer at 25 gL-1 (1.156–2.858 log reduction). Conclusion: This research work highlights grape leaf EE as a novel and appealing source of bioactive components with its strong antibacterial and antioxidant activities against common foodborne infectious agents. Our research reveals that leaf ethanolic extract could reduce human pathogenic microorganisms in fresh green vegetables, improving food safety.

The alginate polymer OligoG alters susceptibility of biofilm-embedded non-typeable Haemophilus influenzae to ampicillin and ciprofloxacin.

Treatment of respiratory infections with non-typeable Haemophilus influenzae (NTHi) in COPD patients is complicated by biofilm formation, protecting the bacteria against the hosts' immune response and antibiotics. We investigated the antibiofilm and antibacterial effects of the alginate polymer OligoG, alone or combined with ampicillin or ciprofloxacin, on mature NTHi biofilms. Two unrelated COPD strains with PBP3-mediated β-lactam resistance, with additional TEM-1 β-lactamase (Hi-022) or quinolone resistance due to altered GyrA and ParC (Hi-072) were used. Antibiofilm and antibacterial effects were assessed macroscopically, by measurement of biofilm biomass (OD), and by viable cell counts, with determination of minimum biofilm inhibitory concentration (MBIC) and the novel parameter 'minimum concentration for 2 log10 drop in viable cells in biofilm' (MB2LDC). Drug interactions between OligoG and antibiotics were assessed by comparing expected and observed inhibitory effects (percent inhibition of no-treatment control) of combined treatment. OligoG had dose-dependent biofilm disruptive abilities and a weak inhibitory effect on viable cells. Combination with OligoG (64 g/L) significantly lowered MBIC for ampicillin (both strains) and MB2LDC for ciprofloxacin (Hi-022). For Hi-022, there was significant synergism between OligoG and both antibiotics. For Hi-072, interactions were subtle, but a tendency in direction of antagonism was significant at two concentrations of ciprofloxacin. OligoG shows promise as a potential adjuvant to antibiotics in NTHi infections, but strain-specific factors appear to affect drug interactions and may lead to antagonism. More research is needed to clarify the mechanisms of action of OligoG and interactions with antibiotics.

Low Spontaneous Clearance Rates of Recently Acquired Hepatitis C Virus in Human Immunodeficiency Virus-Positive Men Who Have Sex With Men (PROBE-C Study).

Using direct-acting antivirals (DAAs) for recently acquired hepatitis C virus (RAHCV) infections, particularly in human immunodeficiency virus (HIV)-positive men who have sex with men (MSM), dramatically reduced the incidence of hepatitis C. However, implementation into clinical practice is challenging. The aim of this study was to analyze spontaneous clearance (SC) rates of RAHCV and to identify predictors of SC. The PROBE-C study is an observational European cohort on RAHCV infections in HIV-positive MSM. Between 2007 and 2017, RAHCV infections were documented with ≥12 months of follow-up. Fisher exact, χ2, and Mann-Whitney U tests were used for statistical analysis. A total of 464 RAHCV infections were documented; 457 of 464 patients (98%) were male, and the median age (interquartile range [IQR]) was 41 (38-46) years. The main risk group for hepatitis C virus (HCV) transmission was MSM (98.9%). Most participants were infected with HCV genotype 1 (78.3%). The median baseline HCV RNA level (IQR) was 230 000 (135 000-474 432) IU/mL, and the median CD4+ T-cell count was 574/µL (547-604/µL. Of all cases, 92% received combination antiretroviral therapy, with 91% showing suppressed HIV RNA levels (<200 copies/mL). The median maximum alanine aminotransferase level (IQR) was 445 (402-522) U/L. SC of RAHCV infection occurred in 55 of 464 cases (11.9%). A >2-log decline in HCV RNA levels 4 weeks after diagnosis of RAHCV infection was the strongest predictor of SC (P < .001; sensitivity, 96.4%; specificity, 97.5%; positive predictive value, 84.1%; negative predictive value, 99.5%). SC of RAHCV in HIV-positive MSM is found in only 11.9% of cases and a <2-log drop in HCV RNA level at week 4 after diagnosis should prompt early DAA-based treatment. However, immediate DAA treatment for RAHCV infection may also be favored in patients with ongoing transmission risk behavior.

Deciphering the antibacterial activity and mechanism of p-coumaric acid against Alicyclobacillus acidoterrestris and its application in apple juice

Alicyclobacillus acidoterrestris has been regarded as the main hazardous factor causing the spoilage of commercially pasteurized fruit juice due to its unique thermo-acidophilic properties. p-Coumaric acid, primarily isolated from plants and having high biological activity, is supposed to be a viable food additive. Herein, the antibacterial activity and mechanism of p-coumaric acid against A. acidoterrestris and its application in apple juice were investigated. The results showed that p-coumaric acid had an active inhibition against A. acidoterrestris vegetative cells, and both minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) were 0.2 mg/mL. MIC of p-coumaric acid against spores was also 0.2 mg/mL, while MBC was >1.6 mg/mL. Treatment of p-coumaric acid caused significant damage of cell integrity, decrease of intracellular ATP, hyperpolarization of cell membrane, degradation of whole cell protein, and malformation of cell morphology. Agarose gel retardation and fluorescence spectroscopy assays revealed that p-coumaric acid could interact with DNA and intercalate into the groove of DNA double helix to disturb normal cellular functions. Furthermore, the addition of p-coumaric acid at MIC caused 2.07 log drop in A. acidoterrestris vegetative cells and effectively inhibited the germination of spores in apple juice storage at 20 °C for 7 days. The low-temperature storage (4 °C) could inactivate A. acidoterrestris cells in apple juice, but the addition of p-coumaric acid further accelerated degradation of vegetative cells and the inactivation time was shortened from 5 days to 3 days compared with control. Importantly, the addition of p-coumaric acid could increase total phenolic content and ABTS radical scavenging activity and had no remarkable influence on pH, total soluble solids, color and sensory qualities of apple juice, regardless of storage temperature. These results revealed that p-coumaric acid could be expected to serve as an alternative or supplement agent for inhibiting the development of A. acidoterrestris in fruit juice.

BCR-ABL1 (p210) Transcript Kinetics.

Delta checks are a powerful technique for monitoring clinical assays in many disciplines but have not been routinely used in molecular testing. To determine if the biologically determined kinetics of BCR-ABL1's rise and fall could allow the development of a delta check in BCR-ABL1 testing. Nine years of BCR-ABL1 p210 results were evaluated, and patients with 3 or more results were selected for inclusion. The kinetics of these percentages of international standard values were plotted against time along with the median and the 90th and 95th percentile lines. A Monte Carlo simulation of a batch mix-up was performed for 6 months of data to determine the efficacy of the proposed cutoff. The median kinetics showed a 1-log drop of the percentage of international standard in 90 days, with less than 5% of cases showing faster than a 2-log drop in 90 days, and less than 2.5% showing a faster than 3-log drop in 90 days (extrapolated to 1 log in 30 days). The Monte Carlo simulation of a batch mix-up showed that an average batch mix-up of 23 samples could routinely be flagged by this cutoff, albeit with wide variance. These results suggest that using a drop in the percentage of international standard of greater than 1 log in 30 days can be a useful trigger in implementing a delta-check system for this molecular test.

Genotyping of Classical Hodgkin Lymphoma on the Liquid Biopsy

INTRODUCTION. In classical Hodgkin lymphoma (cHL), the low representation (~5%) of Hodgkin-Reed-Sternberg cells (HRS) challenged tumor genotyping on the tissue biopsy. Cell free DNA (cfDNA) is shed into the blood by tumor cells and can be used as source of tumor DNA for the identification of somatic mutations, track clonal evolution of tumors and detect minimal residual disease during therapy. AIMS. The study aimed at: i) showing that cfDNA mirrors the genetics of HRS cells in cHL patients; ii) characterizing the mutational profile of a large cohort of newly diagnosed and chemorefractory cHL; iii) identifying molecular prognostic subtypes; iv) early detecting residual disease during therapy; and v) longitudinally tracking tumor clonal evolution under different treatment modalities. METHODS. The study included 80 newly diagnosed cHL and 32 chemorefractory cHL. The following biological material was analyzed: i) cfDNA from plasma collected at diagnosis, during ABVD courses, at refractory progression, before and during therapy with brentuximab or nivolumab; and ii) normal germline genomic DNA (gDNA) from granulocytes. For comparative purposes, paired tumor gDNA from microdissected HRS cells of 13 cases was also analyzed. A targeted resequencing panel optimized to include the coding exons and splice sites of 77 genes recurrently mutated in B-cell lymphomas was used for genotyping. Ultra-deep next-generation sequencing (NGS) of the gene panel was performed on NexSeq 500 (Illumina) using the CAPP-seq library preparation strategy (NimbleGen). RESULTS. In cHL patients, cfDNA surrogated gDNA from HRS cells, since it harbored 87.5% of the tumor confirmed mutations. Genes recurrently affected by non-synonymous somatic mutations in >20% of cHL included STAT6 (37.5%), TNFAIP3 (35%), and ITPKB (27.5%) (Fig. 1A). Mutations clustered in major pathways, including NF-κB, PI3K-AKT, cytokine and NOTCH signaling, and immune evasion. ITPKB mutations: i) were quite specific for cHL, being rare or absent in other lymphomas; ii) caused the subcellular delocalization of the protein in primary HRS cells of mutated patients; iii) correlated with clues of PI3K-AKT signaling activation both at gene expression and protein levels; and iv) consistent with the positioning of ITPKB downstream PI3K in the pathway, associate with resistance to PI3K inhibitors. Mutations of CD58, encoding a co-stimulatory molecule for T-cells, associated with short PFS independent of interim PET/CT results, pointing to immune escape genetic lesions as biomarkers of aggressive disease (Fig. 1B). Newly diagnosed and chemorefractory cHL shared a largely overlapping mutational landscape. TP53 mutations were not enriched in refractory cHL as instead commonly found in other types of refractory B-cell tumors. Conversely, more TET2 mutations were documented in refractory cHL, including newly acquired mutation, thus signaling towards aberrant DNA methylation programming as a mechanism of resistance in cHL with potential therapeutic implications. By longitudinal analysis, in patients relapsing under/after chemotherapy or brentuximab vedotin, pre-treatment/relapse tumor pairs branched through the acquisition of phase specific mutations from an ancestral clone, that always persisted (Fig. 1C). Conversely, in patients maintained under nivolumab, clones were cyclically suppressed and replaced by completely novel clones. We utilized the change in circulating tumor cfDNA load from baseline to interim timepoint to predict the best response to ABVD and to complement interim PET/CT in anticipating cure. A drop of 100-fold or 2-log drop in tumor cfDNA after 2 ABVD courses associated with an eventual complete response and cure. All cured patients that were inconsistently judged as interim PET/CT positive turned out to have a >2 log drop in tumor cfDNA. A drop of less than 2-log in tumor cfDNA after 2 ABVD courses associated with an eventual progression. All relapsed patients that were inconsistently judged as interim PET/CT negative turned out to have a <2 log drop in tumor cfDNA (Fig. 1D). CONCLUSIONS. Circulating tumor cfDNA allows to noninvasively detect tumor-specific mutations: i) identify prognostic subtypes; ii) early detect residual disease during therapy; and iii) longitudinally track tumor clonal evolution under different treatment modalities. [Display omitted] DisclosuresStathis:Celgene: Research Funding; Pfizer: Research Funding; Merck: Research Funding; Roche: Consultancy, Other: Advisory board; Amgen: Honoraria. Bertoni:Acerta Pharma: Research Funding; Bayer: Research Funding; Cellestia: Research Funding; Menarini: Research Funding; Piqur: Research Funding; Immunogen: Research Funding. Santoro:Merck: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Zucca:Bayer: Consultancy, Other: Advisory Role; Jannsen: Consultancy, Honoraria, Other: Advisory role; Takeda: Consultancy, Other: Advisory role; Celltrion Healthcare: Consultancy, Other: Advisory Role; Celltrion Healthcare: Consultancy, Other: Advisory Role; Celgene: Honoraria, Research Funding; Sandoz: Consultancy, Other: Advisory role; Bayer: Consultancy, Other: Advisory Role; Gilead Science: Consultancy, Other: Advisory role; Roche: Honoraria, Research Funding; Jannsen: Consultancy, Honoraria, Other: Advisory role; Roche: Advisory role, Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Mundipharma: Research Funding; Mundipharma: Research Funding; Gilead Science: Consultancy, Other: Advisory role; Takeda: Consultancy, Other: Advisory role; Sandoz: Consultancy, Other: Advisory role. Gaidano:Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Carlo-Stella:ADC Therapeutics: Research Funding.

The pharmacodynamics of minocycline alone and in combination with rifampicin against Staphylococcus aureus studied in an in vitro pharmacokinetic model of infection.

Tetracyclines are widely used as oral therapy of MRSA infection, however, the pharmacodynamic underpinning is absent. We employed an in vitro pharmacokinetic model to study the pharmacodynamics of minocycline alone and in combination with rifampicin. An exposure-ranging design was used to establish fAUC/MIC targets for static, -1 log drop and -2 log drop effects against Staphylococcus aureus for minocycline and in combination with rifampicin. We then simulated 7-10 day human dosing of minocycline and the combination. The minocycline fAUC/MIC for 24 h static effect and -1 log drop in bacterial load were 12.5 ± 7.1 and 23.3 ± 12.4. fAUC/MIC targets for static and -1 log drop were greater at 48 and 72 h. The addition of simulated free rifampicin associated with dosing 300 mg q12h reduced the 24 h minocycline fAUC/MICs. Simulations performed over 7-10 days exposure indicated that for minocycline standard human doses there was a 1-3 log reduction in viable count and no changes in population profiles. Addition of rifampicin resulted in larger reductions in staphylococcal load but emergence of resistance to rifampicin. There was no resistance to minocycline. An fAUC/MIC minocycline target of 12-36 is appropriate for S. aureus. Addition of rifampicin decreases bacterial load but results in emergence of resistance to rifampicin. Unusually, there was no emergence of resistance to minocycline.

Improved neurocognitive performance in FIV infected cats following treatment with the p75 neurotrophin receptor ligand LM11A-31.

HIV rapidly infects the central nervous system (CNS) and establishes a persistent viral reservoir within microglia, perivascular macrophages and astrocytes. Inefficient control of CNS viral replication by antiretroviral therapy results in chronic inflammation and progressive cognitive decline in up to 50% of infected individuals with no effective treatment options. Neurotrophin based therapies have excellent potential to stabilize and repair the nervous system. A novel non-peptide ligand, LM11A-31, that targets the p75 neurotrophin receptor (p75NTR) has been identified as a small bioavailable molecule capable of strong neuroprotection with minimal side effects. To evaluate the neuroprotective effects of LM11A-31 in a natural infection model, we treated cats chronically infected with feline immunodeficiency virus (FIV) with 13mg/kg LM11A-31 twice daily over a period of 10weeks and assessed effects on cognitive functions, open field behaviors, activity, sensory thresholds, plasma FIV, cerebrospinal fluid (CSF) FIV, peripheral blood mononuclear cell provirus, CD4 and CD8 cell counts and general physiology. Between 12 and 18months post-inoculation, cats began to show signs of neural dysfunction in T maze testing and novel object recognition, which were prevented by LM11A-31 treatment. Anxiety-like behavior was reduced in the open field and no changes were seen in sensory thresholds. Systemic FIV titers were unaffected but treated cats exhibited a log drop in CSF FIV titers. No significant adverse effects were observed under all conditions. The data indicate that LM11A-31 is likely to be a potent adjunctive treatment for the control of neurodegeneration in HIV infected individuals.

Plasma Oncostatin M, TNF-α, IL-7, and IL-13 Network Predicts Crohn’s Disease Response to Infliximab, as Assessed by Calprotectin Log Drop

Background: Cytokines emerge as possible biomarkers of response in Crohn’s disease (CD). We aimed to determine the plasmatic cytokine profiles of active CD patients who started infliximab (IFX) treatment and their capacity to predict the response to IFX. Methods: A total of 30 active CD patients receiving an induction therapy of IFX were enrolled in the study. Peripheral blood samples pretreatment were collected. Concentrations of 15 cytokines were measured by Luminex technology. Responses to IFX were evaluated by the drop in fecal calprotectin based on its logarithm-transformed values. A random forest (RF) predictive model was used for data analyses. Results: Samples of 22 patients were analyzed. The RF model ranked the following cytokines as the top predictors of the response: tumor necrosis factor alpha (TNFα), interleukin (IL)-13, oncostatin M (OSM), and IL-7 (p < 0.005). Partial dependency plots showed that high levels of IL-13 pretreatment, low TNFα levels, and low IL-7 levels were associated with a favorable IFX response. Increased levels of OSM and TNFα predicted unfavorable responses to IFX. Conclusions: We here show that a log drop in calprotectin strongly correlates with clinical parameters and it can be proposed as a useful objective clinical response predictor. Plasma TNFα, IL-13, Il-7, and OSM network could predict CD response to IFX before induction therapy, as assessed by calprotectin log drop.

The pharmacodynamics of fosfomycin against Staphylococcus aureus studied in an in vitro model of infection

ObjectivesThe pharmacodynamics of intravenous fosfomycin have not been described for Gram-positive pathogens such as Staphylococcus aureus (S. aureus). This paper described the dominant pharmacodynamic index for fosfomycin against S. aureus and its size for antibacterial effect. MethodsA single-compartment dilutional in vitro pharmacokinetic model was used to provide fosfomycin exposures against S. aureus, three methicillin-susceptible S. aureus (MSSA), two methicillin-resistant S. aureus (MRSA); fosfomycin MICs were 2 mg/L (one strain), 4 mg/L (one strain), 8 mg/L (two strains) and 16 mg/L (one strain). For all simulations, a fosfomycin half-life of 2.5 hours was modelled. Cmax/MICs from 0–74.8, AUC/MICs from 0–750 and T>MIC 0–100% were simulated. The primary end-points were changes in bacterial load after 24 hours and changes in population profiles after 48 hours. ResultsLog AUC/MIC R2 = 0.55 and log Cmax/MIC R2 = 0.66 were related to S. aureus log reduction in viable count at 24 hours; T>MIC was poorly related. Cmax/MIC for a 24 hour static, –1 log drop and –2 log drop were 3.0 ± 1.7, 4.6 ± 2.4 and 6.6 ± 3.8, respectively. AUC/MIC for a 24 hour static, –1 log drop and –2 log drop were 26.4 ± 11.8, 42.8 ± 21.8 and 66.6 ± 39.1. Emergence of resistance as indicated by > 2 log growth on MICx8 recovery media was maximal at AUC/MIC ratios of 10–40 and was suppressed at AUC/MIC ratios of ≥ 250. ConclusionsThe dominant pharmacodynamic index for fosfomycin against S. aureus was Cmax/MIC in terms of reduction of bacterial load and AUC/MIC in terms of suppressing emergence of resistance. AUC/MIC ratios of 20–75 were associated with a –1 log reduction in bacterial load and AUC/MIC of 10–40 maximally increased emergence of resistance.

Pharmacodynamics of plazomicin and a comparator aminoglycoside, amikacin, studied in an in vitro pharmacokinetic model of infection

The new aminoglycoside plazomicin shows in vitro potency against multidrug-resistant Enterobacteriales. The exposure-response relationship of plazomicin and the comparator aminoglycoside amikacin was determined for Escherichia coli, while for Klebsiella pneumoniae only plazomicin was tested. An in vitro pharmacokinetic model was used. Five E. coli strains (two meropenem-resistant) and five K. pneumoniae strains (two meropenem-resistant) with plazomicin MICs of 0.5-4 mg/L were used. Antibacterial effect was assessed by changes in bacterial load and bacterial population profile. The correlation between change in initial inoculum after 24 h of drug exposure and the AUC/MIC ratio was good (plazomicin R2 ≥ 0.8302; amikacin R2 ≥ 0.9520). Escherichia coli plazomicin AUC/MIC ratios for 24-h static, -1, -2 and -3 log drop were 36.1 ± 18.4, 39.3 ± 20.9, 41.2 ± 21.9 and 44.8 ± 24.3, respectively, and for amikacin were 49.5 ± 12.7, 55.7 ± 14.8, 64.1 ± 19.2 and 73.3 ± 25.3. Klebsiella pneumoniae plazomicin AUC/MIC ratios for 24-h static, -1, -2 and -3 log drop were 34.0 ± 15.2, 46.8 ± 27.8, 67.4 ± 46.5 and 144.3 ±129.8. Plazomicin AUC/MIC ratios >66 and amikacin AUC/MIC ratios >57.7 were associated with suppression of E. coli growth on 4 × or 8 × MIC recovery plates. The equivalent plazomicin AUC/MIC to suppress resistance emergence with K. pneumoniae was >132. The plazomicin AUC/MIC for 24-h static effect and -1 log reduction in E. coli and K. pneumoniae bacterial load was in the range 30-60. Plazomicin AUC/MIC targets aligned with those of amikacin for E. coli.

Antibacterial effect of imipenem/relebactam on aerobic Gram-negative bacilli: in vitro simulations of 7 or 14 day human exposures.

We assessed the antibacterial effect of human simulations of dosing with imipenem/relebactam (with or without amikacin) on Enterobacteriaceae or Pseudomonas aeruginosa over 7 or 14 day antibiotic exposures. An in vitro pharmacokinetic model was used to assess changes in bacterial load and population profiles. Imipenem/relebactam produced an initial >4 log drop in viable counts followed by suppression for 7 days for Enterobacteriaceae whether the strain was WT, produced KPC enzymes or produced an AmpC enzyme with porin loss. Similarly, with the P. aeruginosa strains, there was an initial >4 log clearance over the first 24 h irrespective of whether the strain was WT, hyperexpressed AmpC or had OprD mutation with porin loss. However, with three of four strains there was modest regrowth over the 7 days. There were no changes in imipenem/relebactam MICs over the 7 days. Addition of amikacin in 7 day simulations resulted in more suppression of pseudomonal growth. In 14 day simulations with P. aeruginosa there was regrowth to 8 log10 by 14 days with imipenem/relebactam alone and associated increases in MICs. Addition of amikacin resulted in clearance from the model and prevented changes in population profiles. Imipenem/relebactam was highly effective at reducing the bacterial load of Enterobacteriaceae and there was no emergence of resistance. Against P. aeruginosa, the initial bacterial burden was also rapidly reduced, but there was subsequent regrowth, especially after 7 days of exposure. Addition of amikacin increased the clearance of P. aeruginosa and prevented emergence of resistance.

Reducing healthcare-associated infections incidence by a probiotic-based sanitation system: A multicentre, prospective, intervention study.

Healthcare Associated Infections (HAI) are a global concern, further threatened by the increasing drug resistance of HAI-associated pathogens. On the other hand, persistent contamination of hospital surfaces contributes to HAI transmission, and it is not efficiently controlled by conventional cleaning, which does not prevent recontamination, has a high environmental impact and can favour selection of drug-resistant microbial strains. In the search for effective approaches, an eco-sustainable probiotic-based cleaning system (Probiotic Cleaning Hygiene System, PCHS) was recently shown to stably abate surface pathogens, without selecting antibiotic-resistant species. The aim of this study was to determine whether PCHS application could impact on HAI incidence. A multicentre, pre-post interventional study was performed for 18 months in the Internal Medicine wards of six Italian public hospitals (January 1st 2016—June 30th 2017). The intervention consisted of the substitution of conventional sanitation with PCHS, maintaining unaltered any other procedure influencing HAI control. HAI incidence in the pre and post-intervention period was the main outcome measure. Surface bioburden was also analyzed in parallel. Globally, 11,842 patients and 24,875 environmental samples were surveyed. PCHS was associated with a significant decrease of HAI cumulative incidence from a global 4.8% (284 patients with HAI over 5,930 total patients) to 2.3% (128 patients with HAI over 5,531 total patients) (OR = 0.44, CI 95% 0.35–0.54) (P<0.0001). Concurrently, PCHS was associated with a stable decrease of surface pathogens, compared to conventional sanitation (mean decrease 83%, range 70–96.3%), accompanied by a concurrent up to 2 Log drop of surface microbiota drug-resistance genes (P<0.0001; Pc = 0.008). Our study provides findings which support the impact of a sanitation procedure on HAI incidence, showing that the use of a probiotic-based environmental intervention can be associated with a significant decrease of the risk to contract a HAI during hospitalization. Once confirmed in larger experiences and other target populations, this eco-sustainable approach might be considered as a part of infection control and prevention (IPC) strategies.Trial registration—ISRCTN International Clinical Trials Registry, ISRCTN58986947.

Population pharmacokinetics and pharmacodynamics of fosfomycin in non–critically ill patients with bacteremic urinary infection caused by multidrug-resistant Escherichia coli

ObjectivesTo describe the population pharmacokinetics of fosfomycin for patients with bacteraemic urinary tract infection (BUTI). The analysis identified optimal regimens on the basis of pharmacodynamic targets and assessed the adequacy of Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) susceptibility breakpoints for Escherichia coli. MethodsData of 16 patients with BUTI caused by multidrug-resistant E. coli (FOREST clinical trial) received intravenous fosfomycin (4 g every 6 hours) were analysed. A population pharmacokinetic analysis was performed, and Monte Carlo simulations were undertaken using 4 g every 6 hours and 8 g every 8 hours. The probability of pharmacodynamic target attainment was assessed using pharmacodynamic targets for E. coli for static effect, 1-log drop in bacterial burden and resistance suppression. ResultsSixty-four plasma samples were collected over a single dosing interval (day 2 or 3 after starting fosfomycin treatment). Fosfomycin concentrations were highly variable. Pharmacodynamic target attainment analysis showed mild improvement by increasing fosfomycin dosing (4 g every 6 hours vs. every 8 hours). These dosages showed success for decreasing 1-log bacterial burden in 89% to 96% (EUCAST breakpoints) and 33% to 54% (CLSI breakpoints) of patients, but they were unable to reach bacterial resistance suppression targets. ConclusionsFosfomycin concentrations are highly variable—a fact partially explained by renal impairment. The present work supports the use of 4 g every 6 hours as an effective regimen for the treatment of non–critically ill patients with BUTI caused by multidrug-resistant E. coli, as higher dosages might increase toxicity but may not significantly increase efficacy. The current information may suggest that fosfomycin susceptibility breakpoints need to be reappraised.

Pharmacodynamics of inhaled amikacin (BAY 41-6551) studied in an in vitro pharmacokinetic model of infection

The pharmacodynamics of inhaled antimicrobials are poorly studied. Amikacin is being developed for inhalational therapy as BAY 41-6551. We employed an in vitro pharmacokinetic model to study the pharmacokinetics/pharmacodynamics of amikacin. A dose-ranging design was used to establish fAUC/MIC and fCmax/MIC targets for static, -1 log drop and -2 log drop effects for strains of Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. We then modelled epithelial lining fluid (ELF) concentration associated with inhaled amikacin (400 mg every 12 h), over 5 days using mean human concentrations. The 24 h static effect fAUC/MIC targets and -1 log drop targets were 51.0 ± 26.7 and 71.6 ± 27.6 for all species of aerobic Gram-negative bacilli. fAUC/MIC targets for static effect, -1 log drop or -2 log drop were smaller than the 24 h values at 12 h and larger at 48 h. Emergence of resistance occurred maximally with E. coli in the fAUC/MIC range 12-60; K. pneumoniae 0-60 (48 h) and P. aeruginosa 12-80. When human ELF concentrations were modelled for strains with MIC ≤8 mg/L, there was rapid clearance and no regrowth. For strains with MIC ≥32 mg/L, there was initial clearance followed by regrowth. If MIC values were related to bacterial clearance then at least a static effect or -1 log drop in count would be expected for bacterial strains with MICs of ≤180 mg/L (static effect) or ≤148 mg/L (-1 log drop effect). An fAUC/MIC amikacin target of 50-80 is appropriate for aerobic Gram-negative bacilli and mean ELF concentrations of BAY 41-6551 would produce a static to -1 log clearance with strains up to 128 mg/L.

Genotyping of Classical Hodgkin Lymphoma on the Liquid Biopsy

Abstract INTRODUCTION. In classical Hodgkin lymphoma (cHL), the low representation (~5%) of Hodgkin-Reed-Sternberg cells (HRS) challenged tumor genotyping on the tissue biopsy. Cell free DNA (cfDNA) is shed into the blood by tumor cells and can be used as source of tumor DNA for the identification of somatic mutations, track clonal evolution of tumors and detect minimal residual disease during therapy. AIMS. The study aimed at: i) showing that cfDNA mirrors the genetics of HRS cells in cHL patients; ii) characterizing the mutational profile of a large cohort of newly diagnosed and chemorefractory cHL; iii) identifying molecular prognostic subtypes; iv) early detecting residual disease during therapy; and v) longitudinally tracking tumor clonal evolution under different treatment modalities. METHODS. The study included 80 newly diagnosed cHL and 32 chemorefractory cHL. The following biological material was analyzed: i) cfDNA from plasma collected at diagnosis, during ABVD courses, at refractory progression, before and during therapy with brentuximab or nivolumab; and ii) normal germline genomic DNA (gDNA) from granulocytes. For comparative purposes, paired tumor gDNA from microdissected HRS cells of 13 cases was also analyzed. A targeted resequencing panel optimized to include the coding exons and splice sites of 77 genes recurrently mutated in B-cell lymphomas was used for genotyping. Ultra-deep next-generation sequencing (NGS) of the gene panel was performed on NexSeq 500 (Illumina) using the CAPP-seq library preparation strategy (NimbleGen). RESULTS. In cHL patients, cfDNA surrogated gDNA from HRS cells, since it harbored 87.5% of the tumor confirmed mutations. Genes recurrently affected by non-synonymous somatic mutations in >20% of cHL included STAT6 (37.5%), TNFAIP3 (35%), and ITPKB (27.5%) (Fig. 1A). Mutations clustered in major pathways, including NF-κB, PI3K-AKT, cytokine and NOTCH signaling, and immune evasion. ITPKB mutations: i) were quite specific for cHL, being rare or absent in other lymphomas; ii) caused the subcellular delocalization of the protein in primary HRS cells of mutated patients; iii) correlated with clues of PI3K-AKT signaling activation both at gene expression and protein levels; and iv) consistent with the positioning of ITPKB downstream PI3K in the pathway, associate with resistance to PI3K inhibitors. Mutations of CD58, encoding a co-stimulatory molecule for T-cells, associated with short PFS independent of interim PET/CT results, pointing to immune escape genetic lesions as biomarkers of aggressive disease (Fig. 1B). Newly diagnosed and chemorefractory cHL shared a largely overlapping mutational landscape. TP53 mutations were not enriched in refractory cHL as instead commonly found in other types of refractory B-cell tumors. Conversely, more TET2 mutations were documented in refractory cHL, including newly acquired mutation, thus signaling towards aberrant DNA methylation programming as a mechanism of resistance in cHL with potential therapeutic implications. By longitudinal analysis, in patients relapsing under/after chemotherapy or brentuximab vedotin, pre-treatment/relapse tumor pairs branched through the acquisition of phase specific mutations from an ancestral clone, that always persisted (Fig. 1C). Conversely, in patients maintained under nivolumab, clones were cyclically suppressed and replaced by completely novel clones. We utilized the change in circulating tumor cfDNA load from baseline to interim timepoint to predict the best response to ABVD and to complement interim PET/CT in anticipating cure. A drop of 100-fold or 2-log drop in tumor cfDNA after 2 ABVD courses associated with an eventual complete response and cure. All cured patients that were inconsistently judged as interim PET/CT positive turned out to have a >2 log drop in tumor cfDNA. A drop of less than 2-log in tumor cfDNA after 2 ABVD courses associated with an eventual progression. All relapsed patients that were inconsistently judged as interim PET/CT negative turned out to have a Download : Download high-res image (196KB) Download : Download full-size image Figure 1 . Disclosures Stathis: Celgene: Research Funding; Pfizer: Research Funding; Merck: Research Funding; Roche: Consultancy, Other: Advisory board; Amgen: Honoraria. Bertoni: Acerta Pharma: Research Funding; Bayer: Research Funding; Cellestia: Research Funding; Menarini: Research Funding; Piqur: Research Funding; Immunogen: Research Funding. Santoro: Merck: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Zucca: Bayer: Consultancy, Other: Advisory Role; Jannsen: Consultancy, Honoraria, Other: Advisory role; Takeda: Consultancy, Other: Advisory role; Celltrion Healthcare: Consultancy, Other: Advisory Role; Celltrion Healthcare: Consultancy, Other: Advisory Role; Celgene: Honoraria, Research Funding; Sandoz: Consultancy, Other: Advisory role; Bayer: Consultancy, Other: Advisory Role; Gilead Science: Consultancy, Other: Advisory role; Roche: Honoraria, Research Funding; Jannsen: Consultancy, Honoraria, Other: Advisory role; Roche: Advisory role, Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Mundipharma: Research Funding; Mundipharma: Research Funding; Gilead Science: Consultancy, Other: Advisory role; Takeda: Consultancy, Other: Advisory role; Sandoz: Consultancy, Other: Advisory role. Gaidano: Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Carlo-Stella: ADC Therapeutics: Research Funding.

Enhancement of photodynamic inactivation of Staphylococcus aureus biofilms by disruptive strategies.

Photodynamic inactivation (PDI) has been used to inactivate microorganisms through the use of photosensitizers and visible light. On the one hand, near-infrared treatment (NIRT) has also bactericidal and dispersal effects on biofilms. In addition, dispersal biological tools such as enzymes have also been employed in antibiotic combination treatments. The aim of this work was to use alternative approaches to increase the PDI efficacy, employing combination therapies aimed at the partial disruption of the biofilms, thus potentially increasing photosensitizer or oxygen penetration and interaction with bacteria. To that end, we applied toluidine blue (TB)-PDI treatment to Staphylococcus aureus biofilms previously treated with NIRT or enzymes and investigated the outcome of the combined therapies. TB employed at 0.5mM induced per se 2-log drop in S. aureus RN6390 biofilm viability. Each NIRT (980-nm laser) and PDI (635-nm laser) treatment induced a further reduction of 1-log of viable counts. The combination of successive 980- and 635-nm laser treatments on TB-treated biofilms induced additive effects, leading to a 4.5-log viable count decrease. Proteinase K treatment applied to S. aureus of the Newman strain induced an additive effect on PDI mortality, leading to an overall 4-log decrease in S. aureus viability. Confocal scanning laser microscopy after biofilm staining with a fluorescent viability test and scanning electron microscopy observations were correlated with colony counts. The NIRT dose employed (227J/cm2) led to an increase from 21 to 47°C in the buffer temperature of the biofilm system, and this NIRT dose also induced 100% keratinocyte death. Further work is needed to establish conditions under which biofilm dispersal occurs at lower NIRT doses.

Development of a Novel Collagen Wound Model To Simulate the Activity and Distribution of Antimicrobials in Soft Tissue during Diabetic Foot Infection.

Diabetes has major implications for public health, with diabetic foot ulcers (DFUs) being responsible for significant morbidity and mortality. A key factor in the development of nonhealing ulcers is infection, which often leads to the development of biofilm, gangrene, and amputation. A novel approach to treating DFUs is the local release of antibiotics from calcium sulfate beads. We have developed a novel model system to study and compare the release and efficacy of antibiotics released locally, using collagen as a substrate for biofilm growth and incorporating serum to mimic the biochemical complexity of the wound environment. We found that our soft-tissue model supports the growth of a robust Pseudomonas aeruginosa biofilm, and that this was completely eradicated by the introduction of calcium sulfate beads loaded with tobramycin or gentamicin. The model also enabled us to measure the concentration of these antibiotics at different distances from the beads and in simulated wound fluid bathing the collagen matrix. We additionally found that a multidrug-resistant Staphylococcus aureus biofilm, nonsusceptible to antibiotics, nonetheless showed an almost 1-log drop in viable counts when exposed to calcium sulfate beads combined with antibiotics. Together, these data suggest that locally applied antibiotics combined with calcium sulfate provide surprising efficacy in diabetic foot infections and offer an effective alternative approach to infection management. Our study additionally establishes our new system as a biochemically and histologically relevant model that may be used to study the effectiveness of a range of therapies locally or systemically for infected DFUs.

The Use of Kosher Phenotyping for Mapping QTL Affecting Susceptibility to Bovine Respiratory Disease.

Bovine respiratory disease (BRD) is the leading cause of morbidity and mortality in feedlot cattle, caused by multiple pathogens that become more virulent in response to stress. As clinical signs often go undetected and various preventive strategies failed, identification of genes affecting BRD is essential for selection for resistance. Selective DNA pooling (SDP) was applied in a genome wide association study (GWAS) to map BRD QTLs in Israeli Holstein male calves. Kosher scoring of lung adhesions was used to allocate 122 and 62 animals to High (Glatt Kosher) and Low (Non-Kosher) resistant groups, respectively. Genotyping was performed using the Illumina BovineHD BeadChip according to the Infinium protocol. Moving average of -logP was used to map QTLs and Log drop was used to define their boundaries (QTLRs). The combined procedure was efficient for high resolution mapping. Nineteen QTLRs distributed over 13 autosomes were found, some overlapping previous studies. The QTLRs contain polymorphic functional and expression candidate genes to affect kosher status, with putative immunological and wound healing activities. Kosher phenotyping was shown to be a reliable means to map QTLs affecting BRD morbidity.